Ampelopsis grossedentata fermentation liquor as well as preparation method and application thereof in cosmetics

Abstract

The invention provides an optimized preparation method of ampelopsis grossedentata fermentation liquor, which comprises the following steps: fermenting by using Aspergillus fungi, and culturing a seed solution of the fungi by using an optimized liquid culture medium for fermentation. The invention also provides the ampelopsis grossedentata fermentation liquor prepared by the preparation method and cosmetics or medicines containing the ampelopsis grossedentata fermentation liquor. The invention further provides application of the ampelopsis grossedentata fermentation liquor in preparation of cosmetics or medicines. The invention also relates to a novel Aspergillus niger strain MicFAn3127. The invention also relates to the novel Aspergillus niger strain MicFAn3127

Description

Technical Field

[0001] The present invention relates to a method for preparing an optimized fermented liquid of Ampelopsis grossedentata. The present invention also relates to the fermented liquid of Ampelopsis grossedentata prepared by the said preparation method and cosmetics or drugs containing the said fermented liquid of Ampelopsis grossedentata. The present invention further relates to the use of the said fermented liquid of Ampelopsis grossedentata in the preparation of cosmetics or drugs. Background Art

[0002] Ampelopsis grossedentata, also known as Maoyanmei tea, Ganlu tea, Fairy Grass, etc. among the people, with the scientific name Ampelopsis grossedentata, belongs to the tender stems and leaves of plants in the genus Ampelopsis of the Vitaceae family, and is a plant resource with rich medicinal value and health care functions. Ampelopsis grossedentata is mainly distributed in provinces south of the Yangtze River in China, especially in Hunan, Hubei, Guangdong, Guangxi, Yunnan and other places, where it has a relatively long history of cultivation and utilization. Ampelopsis grossedentata is a plant with both medicinal and edible uses, rich in a large number of flavonoid compounds. The main chemical components other than flavonoids include polysaccharides, phenols, amino acids, steroids, volatile oils, etc. Ampelopsis grossedentata is the plant with the highest flavonoid content among all the plants discovered so far (about 40.3%), and is therefore known as the "king of flavonoids". These flavonoid compounds include dihydromyricetin (DMY), myricetin, taxifolin, etc., among which the content of dihydromyricetin dominates in the total flavonoids of Ampelopsis grossedentata, accounting for about 41.25%. Flavonoid components play an important role in antioxidant, anti-inflammatory, hypoglycemic and lipid-lowering, antithrombotic, liver protection, anti-tumor, antibacterial, immune regulation and other aspects. There are significant differences in the flavonoid content in different parts of Ampelopsis grossedentata. Flavonoid substances mainly exist in its leaf and stem parts, and the flavonoid content in the leaves is 3 to 4 times that in the stems.

[0003] In recent years, the development speed and market popularity of plant fermentation extract products such as Chinese herbal medicines have been continuously increasing. Plant fermentation is a process in which microorganisms ferment using plants as substrates. Microbial fermentation of Chinese herbal medicines has many advantages, such as: microbial fermentation of Chinese herbal medicines can effectively promote the release of active substances in Chinese herbal medicines, reduce the toxicity of Chinese herbal medicines and reduce side effects. In addition, microbial fermentation of Chinese herbal medicines combines the characteristics of Chinese herbal medicines, probiotics and enzyme preparations, and has advantages such as "high efficiency, green, safe, and residue-free", and is expected to be used for alternative applications of antibiotics. However, at present, although the fermentation of Chinese herbal medicines is gradually developing in a good direction, it is still in a relatively primary stage and needs to be developed and improved using more advanced fermentation technologies.

[0004] The existing technologies CN102018083A, CN102578335A, CN117898352A, CN102018083B, etc. that have been reported and involve or contain the fermentation of Ampelopsis grossedentata by Aspergillus niger mainly have the food category as their main application field. Aspergillus niger or a microbial community including Aspergillus niger is used for piling / solid-state fermentation to improve the color and taste of Ampelopsis grossedentata. Similar to other fermented teas, the intuitive manifestation is an increase in tea pigments such as theabrownine, and the degradation of phenolic substances, resulting in a darker color and a more mellow and gentle taste. If the above technologies are applied in the cosmetics field, there are difficulties in production and application such as too long fermentation time, an increase in subsequent steps for extracting effective ingredients, and too dark color. Moreover, along with the traditional tea fermentation process, the original efficacy of Ampelopsis grossedentata may be lost, which goes against the original purpose of being a cosmetic raw material.

[0005] In addition, the existing technology CN 115212154A ferments Ampelopsis grossedentata using cellulase and yeast. The purpose is to decompose plant cellulose and the components of plant cell walls, enrich more flavonoids and polyphenols, and improve the precipitation of these effective substances. To a certain extent, this technology improves the utilization rate of Ampelopsis grossedentata, but key application problems such as instability and easy precipitation still exist.

[0006] Moreover, when Ampelopsis grossedentata is used as an external efficacy raw material, its extracts have always had extremely unstable problems, such as fluctuations in pH, deepening of color at low, normal, and high temperatures. In addition, the poor solubility of the extracts will inevitably consume a large amount of organic reagents. As a very effective raw material, Ampelopsis grossedentata urgently needs to solve the above problems to further solve its application in cosmetics. Summary of the Invention

[0007] The present invention relates to a method for preparing a fermentation broth of Ampelopsis grossedentata and a fermentation broth of Ampelopsis grossedentata prepared by the method. The preparation method of the present invention and the fermentation broth of Ampelopsis grossedentata prepared by the method have the following advantages:

[0008] (1) The efficacy of the fermentation broth of Ampelopsis grossedentata prepared by the fermentation method of the present invention has been significantly improved in terms of oil control, acne removal, antioxidant, anti-wrinkle, and barrier repair;

[0009] (2) The fermentation broth of Ampelopsis grossedentata prepared by the fermentation method of the present invention has a whitening effect, while the unfermented Ampelopsis grossedentata does not have a whitening effect;

[0010] (3) The cytotoxicity of the fermentation broth of Ampelopsis grossedentata prepared by the fermentation method of the present invention has been significantly reduced;

[0011] (4) The fermentation broth of Ampelopsis grossedentata prepared by the fermentation method of the present invention has a lighter color and a fresh smell;

[0012] (5) The solubility of the Ampelopsis grossedentata fermented liquid prepared by the fermentation method of the present invention is good, and it is not easy to precipitate;

[0013] (6) The stability of the Ampelopsis grossedentata fermented liquid prepared by the fermentation method of the present invention is better, and the pH fluctuation is extremely small after long-term placement at different temperatures;

[0014] (7) By improving the culture medium for the fungal seed liquid used for fermentation in the present invention, the fermentation time is shortened from 3 days to 17 hours;

[0015] (8) The production process of the fermentation method in the present invention is simple, the energy consumption is very low, the process is easy to control, it is more conducive to improving the efficacy and application adaptability of Ampelopsis grossedentata leaves than the conventional fermentation method, and the fermented liquid has a lighter color, better stability, and a significant improvement in efficacy. Applied to the production of cosmetic raw materials, the cost is low, and it can bring important economic value to the cosmetic industry.

[0016] The first aspect of the present invention relates to a method for preparing an Ampelopsis grossedentata fermented liquid, and the method comprises the following steps:

[0017] (a) Culturing a fungus of the genus Aspergillus on a solid medium to obtain spore powder of the fungus;

[0018] (b) Inoculating the spore powder of the fungus obtained in step (a) onto a liquid medium and culturing to obtain a seed liquid of the fungus;

[0019] (c) Inoculating the seed liquid obtained in step (b) into an Ampelopsis grossedentata fermentation substrate for fermentation,

[0020] wherein the liquid medium in step (b) comprises glucose, corncob powder, Ampelopsis grossedentata powder, yeast extract powder, (NH4)2SO4, KH2PO4, CaCl2, KCl, MgSO4, MnSO4 and vitamin B12,

[0021] wherein the Ampelopsis grossedentata fermentation substrate is a solution of Ampelopsis grossedentata in water.

[0022] The second aspect of the present invention relates to an Ampelopsis grossedentata fermented liquid, which is prepared by the method described in the first aspect of the present invention.

[0023] The third aspect of the present invention relates to the use of the Ampelopsis grossedentata fermented liquid described in the second aspect of the present invention in the preparation of cosmetics / drugs.

[0024] The fourth aspect of the present invention relates to a cosmetic or drug, which comprises the Ampelopsis grossedentata fermented liquid described in the second aspect of the present invention, wherein the Ampelopsis grossedentata fermented liquid accounts for 0.1%-99% by weight of the weight of the cosmetic or drug.

[0025] The fifth aspect of the present invention relates to an organism, which is Aspergillus niger deposited at the China Center for Type Culture Collection (CCTCC) under the accession number M20242388. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a diagram of the appearance of the samples after standing at 4°C for 24 hours for the samples in Examples 1.1 to 1.5 and Comparative Example 1 according to the present invention. Detailed Description of the Invention

[0028] Definition

[0029] The terms "s", "min" and "h" used herein represent "second", "minute" and "hour" respectively.

[0030] As used herein, the term "comprising", which is synonymous with "including", "containing" or "characterized by", is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. However, in this text, each mention of "including" is intended to cover the alternative embodiments of "consisting essentially of..." and "consisting of...", where "consisting of..." excludes any element or step not specified, and "consisting essentially of..." allows the inclusion of other elements or steps that do not substantially affect the essential or basic features and novelty features of the composition or method being discussed.

[0031] The term "about" used herein means ±10% of the value modified by the term, more preferably ±5%, and most preferably ±2%. Therefore, those of ordinary skill in the art can clearly determine the scope of the term "about" based on the value being modified.

[0032] Preparation Method of Ampelopsis grossedentata Fermentation Broth

[0033] The first aspect of the present invention relates to a method for preparing a fermented liquid of Ampelopsis grossedentata, the method comprising the following steps:

[0034] (a) Culturing a fungus of the genus Aspergillus on a solid medium to obtain spore powder of the fungus;

[0035] (b) Inoculating the spore powder of the fungus obtained in step (a) onto a liquid medium and culturing to obtain a seed liquid of the fungus;

[0036] (c) Inoculating the seed liquid obtained in step (b) into an Ampelopsis grossedentata fermentation substrate for fermentation,

[0037] Among them, the liquid medium in step (b) contains glucose, corncob powder, tengcha powder, yeast extract powder, (NH4)2SO4, KH2PO4, CaCl2, KCl, MgSO4, MnSO4 and vitamin B12.

[0038] Among them, the tengcha fermentation substrate is a solution of tengcha in water.

[0039] In some embodiments, the tengcha is derived from a raw material selected from Ampelopsis Grossedentata, Ampelopsis chaffanjoni, Ampelopsis megalophylla, Ampelopsis cantoniensis or a combination thereof.

[0040] In some embodiments, the Aspergillus fungus is selected from Aspergillus niger, Aspergillus candidus, Aspergillus oryzae or a combination of one or more thereof, preferably Aspergillus niger, and most preferably Aspergillus MicF_An3127.

[0041] In some embodiments, the solid medium in step (a) is a potato dextrose agar medium. In some embodiments, the potato dextrose agar medium contains potatoes, glucose, agar and water. In some embodiments, the potato dextrose agar medium contains 5 - 50 g / L of glucose. In some embodiments, the potato dextrose agar medium contains 5 - 10 g / L, 10 - 15 g / L, 25 - 20 g / L, 20 - 25 g / L, 25 - 30 g / L, 30 - 35 g / L, 35 - 40 g / L, 40 - 45 g / L or 45 - 50 g / L of glucose. In some embodiments, the potato dextrose agar medium contains 20 g / L of glucose.

[0042] In some embodiments, the solid medium in step (a) is a potato dextrose agar medium. In some embodiments, the potato dextrose agar medium contains potatoes, glucose, agar, and water. In some embodiments, the potato dextrose agar medium contains 5 - 50 g / L of agar. In some embodiments, the potato dextrose agar medium contains 5 - 10 g / L, 10 - 15 g / L, 15 - 20 g / L, 20 - 25 g / L, 25 - 30 g / L, 30 - 35 g / L, 35 - 40 g / L, 40 - 45 g / L, or 45 - 50 g / L of agar. In some embodiments, the potato dextrose agar medium contains 15 g / L of agar.

[0043] In some embodiments, the solid medium in step (a) is a potato dextrose agar medium. In some embodiments, the potato dextrose agar medium contains potatoes, glucose, agar, and water. In some embodiments, the potato dextrose agar medium contains the filtrate obtained by filtering the solution obtained by boiling an aqueous potato solution of 50 - 500 g / L. In some embodiments, the potato dextrose agar medium contains the filtrate obtained by filtering the solution obtained by boiling an aqueous potato solution of 50 - 100 g / L, 100 - 150 g / L, 150 - 200 g / L, 200 - 250 g / L, 250 - 300 g / L, 300 - 350 g / L, 350 - 400 g / L, 400 - 450 g / L, or 450 - 500 g / L. In some embodiments, the potato dextrose agar medium contains the filtrate obtained by filtering the solution obtained by boiling an aqueous potato solution of 200 g / L.

[0044] In some embodiments, the solid culture in step (a) is cultured at a temperature of 25 - 35°C. In some embodiments, the solid culture in step (a) is cultured at a temperature of 25 - 26°C, 26 - 27°C, 27 - 28°C, 28 - 29°C, 29 - 30°C, 30 - 31°C, 31 - 32°C, 32 - 33°C, 33 - 34°C, or 34 - 35°C. In some embodiments, the solid culture in step (a) is cultured at a temperature of 28°C.

[0045] In some embodiments, the culture time of the solid culture in step (a) is 60 - 90 h. In some embodiments, the culture time of the solid culture in step (a) is 60 - 62 h, 62 - 64 h, 64 - 66 h, 66 - 68 h, 68 - 70 h, 70 - 72 h, 72 - 74 h, 74 - 76 h, 76 - 78 h, 78 - 80 h, 80 - 82 h, 82 - 84 h, 84 - 86 h, 86 - 88 h, or 88 - 90 h. In some embodiments, the culture time of the solid culture in step (a) is 72 h.

[0046] In some embodiments, in step (a), after the culture is completed, the spore powder solution obtained in step (a) is collected by adding physiological saline to the culture medium for use in step (b). In some embodiments, in step (a), after the culture is completed, physiological saline is added to the culture medium until the spore powder concentration is (0.1 - 5) * 10 7 CFU / mL to collect the spore powder solution obtained in step (a) for use in step (b). In some embodiments, in step (a), after the culture is completed, physiological saline is added to the culture medium until the spore powder concentration is (0.1 - 0.5) * 10 7 CFU / mL, (0.5 - 1) * 10 7 CFU / mL, (1 - 1.5) * 10 7 CFU / mL, (1.5 - 2) * 10 7 CFU / mL, (2 - 2.5) * 10 7 CFU / mL, (2.5 - 3) * 10 7 CFU / mL, (3 - 3.5) * 10 7 CFU / mL, (3.5 - 4) * 10 7 CFU / mL, (4 - 4.5) * 10 7 CFU / mL, or (4.5 - 5) * 10 7 CFU / mL to collect the spore powder solution obtained in step (a) for use in step (b). In some embodiments, in step (a), after the culture is completed, physiological saline is added to the culture medium until the spore powder concentration is 1 * 10 7 CFU / mL to collect the spore powder solution obtained in step (a) for use in step (b).

[0047] In some embodiments, the liquid medium in step (b) comprises glucose, corncob powder, Ampelopsis grossedentata powder, yeast extract powder, (NH4)2SO4, KH2PO4, CaCl2, KCl, MgSO4, MnSO4, and vitamin B12. In some embodiments, the liquid medium in step (b) comprises 2 - 10 g / L of glucose. In some embodiments, the liquid medium in step (b) comprises 2 - 3 g / L, 3 - 4 g / L, 4 - 5 g / L, 5 - 6 g / L, 6 - 7 g / L, 7 - 8 g / L, 8 - 9 g / L, or 9 - 10 g / L of glucose. In some embodiments, the liquid medium in step (b) comprises 5 g / L of glucose.

[0048] In some embodiments, the liquid medium in step (b) comprises 20 - 30 g / L of corncob powder. In some embodiments, the liquid medium in step (b) comprises 20 - 21 g / L, 21 - 22 g / L, 22 - 23 g / L, 23 - 24 g / L, 24 - 25 g / L, 25 - 26 g / L, 26 - 27 g / L, 27 - 28 g / L, 28 - 29 g / L, or 29 - 30 g / L of corncob powder. In some embodiments, the liquid medium in step (b) comprises 25 g / L of corncob powder.

[0049] In some embodiments, the liquid medium in step (b) comprises 5 - 20 g / L of Ampelopsis grossedentata powder. In some embodiments, the liquid medium in step (b) comprises 5 - 6 g / L, 6 - 7 g / L, 7 - 8 g / L, 8 - 9 g / L, 9 - 10 g / L, 10 - 11 g / L, 11 - 12 g / L, 12 - 13 g / L, 13 - 14 g / L, 14 - 15 g / L, 15 - 16 g / L, 16 - 17 g / L, 17 - 18 g / L, 18 - 19 g / L, or 19 - 20 g / L of Ampelopsis grossedentata powder. In some embodiments, the liquid medium in step (b) comprises 10 g / L of Ampelopsis grossedentata powder.

[0050] In some embodiments, the liquid medium in step (b) comprises 2 - 10 g / L of yeast extract powder. In some embodiments, the liquid medium in step (b) comprises 2 - 3 g / L, 3 - 4 g / L, 4 - 5 g / L, 5 - 6 g / L, 6 - 7 g / L, 7 - 8 g / L, 8 - 9 g / L, or 9 - 10 g / L of yeast extract powder. In some embodiments, the liquid medium in step (b) comprises 5 g / L of yeast extract powder.

[0051] In some embodiments, the liquid medium in step (b) contains 1 - 5 g / L of (NH4)2SO4. In some embodiments, the liquid medium in step (b) contains 1 - 1.5 g / L, 1.5 - 2 g / L, 2 - 2.5 g / L, 2.5 - 3 g / L, 3 - 3.5 g / L, 3.5 - 4 g / L, 4 - 4.5 g / L, or 4.5 - 5 g / L of (NH4)2SO4. In some embodiments, the liquid medium in step (b) contains 2.6 g / L of (NH4)2SO4.

[0052] In some embodiments, the liquid medium in step (b) contains 1 - 5 g / L of KH2PO4. In some embodiments, the liquid medium in step (b) contains 1 - 1.5 g / L, 1.5 - 2 g / L, 2 - 2.5 g / L, 2.5 - 3 g / L, 3 - 3.5 g / L, 3.5 - 4 g / L, 4 - 4.5 g / L, or 4.5 - 5 g / L of KH2PO4. In some embodiments, the liquid medium in step (b) contains 3.5 g / L of KH2PO4.

[0053] In some embodiments, the liquid medium in step (b) contains 0.1 - 2 g / L of CaCl2. In some embodiments, the liquid medium in step (b) contains 0.1 - 0.2 g / L, 0.2 - 0.3 g / L, 0.3 - 0.4 g / L, 0.4 - 0.5 g / L, 0.5 - 0.6 g / L, 0.6 - 0.7 g / L, 0.7 - 0.8 g / L, 0.8 - 0.9 g / L, 0.9 - 1.0 g / L, 1.0 - 1.1 g / L, 1.1 - 1.2 g / L, 1.2 - 1.3 g / L, 1.3 - 1.4 g / L, 1.4 - 1.5 g / L, 1.5 - 1.6 g / L, 1.6 - 1.7 g / L, 1.7 - 1.8 g / L, 1.8 - 1.9 g / L, or 1.9 - 2.0 g / L of CaCl2. In some embodiments, the liquid medium in step (b) contains 0.6 g / L of CaCl2.

[0054] In some embodiments, the liquid medium in step (b) contains 0.01 - 0.5 g / L of KCl. In some embodiments, the liquid medium in step (b) contains 0.01 - 0.05 g / L, 0.05 - 0.1 g / L, 0.1 - 0.15 g / L, 0.15 - 0.2 g / L, 0.2 - 0.25 g / L, 0.25 - 0.3 g / L, 0.3 - 0.35 g / L, 0.35 - 0.4 g / L, 0.4 - 0.45 g / L, or 0.45 - 0.5 g / L of KCl. In some embodiments, the liquid medium in step (b) contains 0.1 g / L of KCl.

[0055] In some embodiments, the liquid medium in step (b) contains 0.1 - 1 g / L of MgSO4. In some embodiments, the liquid medium in step (b) contains 0.1 - 0.2 g / L, 0.2 - 0.3 g / L, 0.3 - 0.4 g / L, 0.4 - 0.5 g / L, 0.5 - 0.6, 0.6 - 0.7, 0.7 - 0.8, 0.8 - 0.9 or 0.9 - 1 g / L of MgSO4. In some embodiments, the liquid medium in step (b) contains 0.5 g / L of MgSO4.

[0056] In some embodiments, the liquid medium in step (b) contains 0.01 - 0.5 g / L of MnSO4. In some embodiments, the liquid medium in step (b) contains 0.01 - 0.05 g / L, 0.05 - 0.1 g / L, 0.1 - 0.15 g / L, 0.15 - 0.2 g / L, 0.2 - 0.25 g / L, 0.25 - 0.3 g / L, 0.3 - 0.35 g / L, 0.35 - 0.4 g / L, 0.4 - 0.45 g / L or 0.45 - 0.5 g / L of MnSO4. In some embodiments, the liquid medium in step (b) contains 0.1 g / L of MnSO4.

[0057] In some embodiments, the liquid medium in step (b) contains 0.1 - 2 mg / mL of vitamin B12. In some embodiments, the liquid medium in step (b) contains 0.1 - 0.2 mg / mL, 0.2 - 0.3 mg / mL, 0.3 - 0.4 mg / mL, 0.4 - 0.5 mg / mL, 0.5 - 0.6 mg / mL, 0.6 - 0.7 mg / mL, 0.7 - 0.8 mg / mL, 0.8 - 0.9 mg / mL, 0.9 - 1.0 mg / mL, 1.0 - 1.1 mg / mL, 1.1 - 1.2 mg / mL, 1.2 - 1.3 mg / mL, 1.3 - 1.4 mg / mL, 1.4 - 1.5 mg / mL, 1.5 - 1.6 mg / mL, 1.6 - 1.7 mg / mL, 1.7 - 1.8 mg / mL, 1.8 - 1.9 mg / mL or 1.9 - 2 mg / mL of vitamin B12. In some embodiments, the liquid medium in step (b) contains 0.5 mg / mL of vitamin B12.

[0058] In some embodiments, the liquid medium in step (b) contains 2 - 10 g / L glucose, 20 - 30 g / L corncob powder, 5 - 20 g / L Ampelopsis grossedentata powder, 2 - 10 g / L yeast extract powder, 1 - 5 g / L (NH4)2SO4, 1 - 5 g / L KH2PO4, 0.1 - 2 g / L CaCl2, 0.01 - 0.5 g / L KCl, 0.1 - 1 g / L MgSO4, 0.01 - 0.5 g / L MnSO4, and 0.1 - 2 mg / mL vitamin B12. In some embodiments, the liquid medium in step (b) contains 5 g / L glucose, 25 g / L corncob powder, 10 g / L Ampelopsis grossedentata powder, 5 g / L yeast extract powder, 2.6 g / L (NH4)2SO4, 3.5 g / L KH2PO4, 0.6 g / L CaCl2, 0.1 g / L KCl, 0.5 g / L MgSO4, 0.1 g / L MnSO4, and 0.5 mg / mL vitamin B12.

[0059] In some embodiments, the pH of the liquid medium in step (b) is 4.5 - 6. In some embodiments, the pH of the liquid medium in step (b) is 4.5 - 4.6, 4.6 - 4.7, 4.7 - 4.8, 4.8 - 4.9, 4.9 - 5.0, 5.0 - 5.1, 5.1 - 5.2, 5.2 - 5.3, 5.3 - 5.4, 5.4 - 5.5, 5.5 - 5.6, 5.6 - 5.7, 5.7 - 5.8, 5.8 - 5.9, or 5.9 - 6.0. In some embodiments, the pH of the liquid medium in step (b) is 5.0 - 5.4.

[0060] In some embodiments, the ratio between the spore powder inoculated in step (b) and the liquid medium is (0.5 - 5) * 10 5 CFU / mL. In some embodiments, the ratio between the spore powder inoculated in step (b) and the liquid medium is (0.5 - 1) * 10 5 CFU / mL, (1 - 1.5) * 10 5 CFU / mL, (1.5 - 2) * 10 5 CFU / mL, (2 - 2.5) * 10 5 CFU / mL, (2.5 - 3) * 10 5 CFU / mL, (3 - 3.5) * 10 5 CFU / mL, (3.5 - 4) * 10 5 CFU / mL, (4 - 4.5) * 10 5 CFU / mL, or (4.5 - 5) * 10 5 CFU / mL. In some embodiments, the ratio between the spore powder inoculated in step (b) and the liquid medium is 2 * 105 CFU / mL.

[0061] In some embodiments, the liquid culture in step (b) is carried out at a temperature of 25 - 35°C. In some embodiments, the liquid culture in step (b) is carried out at a temperature of 25 - 26°C, 26 - 27°C, 27 - 28°C, 28 - 29°C, 29 - 30°C, 30 - 31°C, 31 - 32°C, 32 - 33°C, 33 - 34°C or 34 - 35°C. In some embodiments, the liquid culture in step (b) is carried out at a temperature of 28°C.

[0062] In some embodiments, the culture time of the liquid culture in step (b) is 60 - 90 h. In some embodiments, the culture time of the liquid culture in step (b) is 60 - 62 h, 62 - 64 h, 64 - 66 h, 66 - 68 h, 68 - 70 h, 70 - 72 h, 72 - 74 h, 74 - 76 h, 76 - 78 h, 78 - 80 h, 80 - 82 h, 82 - 84 h, 84 - 86 h, 86 - 88 h or 88 - 90 h. In some embodiments, the culture time of the liquid culture in step (b) is 72 h.

[0063] In some embodiments, the liquid culture in step (b) is carried out at a rotation speed of 50 - 500 rmp. In some embodiments, the liquid culture in step (b) is carried out at a rotation speed of 50 - 100 rmp, 100 - 150 rmp, 150 - 200 rmp, 200 - 250 rmp, 250 - 300 rmp, 300 - 350 rmp, 350 - 400 rmp, 400 - 450 rmp or 450 - 500 rmp. In some embodiments, the liquid culture in step (b) is carried out at a rotation speed of 200 rmp.

[0064] In some embodiments, the Ampelopsis grossedentata fermentation matrix in step (c) is a solution of 5 - 20 g / L of Ampelopsis grossedentata in water. In some embodiments, the Ampelopsis grossedentata fermentation matrix in step (c) is a solution of 5 - 6 g / L, 6 - 7 g / L, 7 - 8 g / L, 8 - 9 g / L, 9 - 10 g / L, 10 - 11 g / L, 11 - 12 g / L, 12 - 13 g / L, 13 - 14 g / L, 14 - 15 g / L, 15 - 16 g / L, 16 - 17 g / L, 17 - 18 g / L, 18 - 19 g / L or 19 - 20 g / L of Ampelopsis grossedentata in water. In some embodiments, the Ampelopsis grossedentata fermentation matrix in step (c) is a solution of 10 g / L of Ampelopsis grossedentata in water.

[0065] In some embodiments, in step (c), before inoculating the seed liquid obtained in step (b) into the Ampelopsis grossedentata fermentation substrate, the Ampelopsis grossedentata fermentation substrate is sterilized. In some embodiments, the sterilization is by high-temperature sterilization. In some embodiments, the sterilization is by sterilization at a high temperature of 100-130 °C. In some embodiments, the sterilization is by sterilization at a high temperature of 100-105 °C, 105-110 °C, 110-115 °C, 115-120 °C, 120-125 °C or 125-130 °C. In some embodiments, the high-temperature sterilization lasts for 10-60 min. In some embodiments, the high-temperature sterilization lasts for 10-15 min, 15-20 min, 20-25 min, 25-30 min, 30-35 min, 35-40 min, 40-45 min, 45-50 min, 50-55 min or 55-60 min.

[0066] In some embodiments, the amount of the seed liquid inoculated in step (c) is 5%-15% by volume of the volume of the Ampelopsis grossedentata fermentation substrate. In some embodiments, the amount of the seed liquid inoculated in step (c) is 5%-6%, 6%-7%, 7%-8%, 8%-9%, 9%-10%, 10%-11%, 11%-12%, 12%-13%, 13%-14% or 14%-15% by volume of the volume of the Ampelopsis grossedentata fermentation substrate. In some embodiments, the amount of the seed liquid inoculated in step (c) is 8% by volume of the volume of the Ampelopsis grossedentata fermentation substrate.

[0067] In some embodiments, the fermentation time in step (c) is 15-36 h. In some embodiments, the fermentation time in step (c) is 15-16 h, 16-17 h, 17-18 h, 18-19 h, 19-20 h, 20-21 h, 21-22 h, 22-23 h, 23-24 h, 24-25 h, 25-26 h, 26-27 h, 27-28 h, 28-29 h, 29-30 h, 30-31 h, 31-32 h, 32-33 h, 33-34 h, 34-35 h or 35-36 h. In some embodiments, the fermentation time in step (c) is 18 h.

[0068] In some embodiments, the fermentation temperature in step (c) is 25-40 °C. In some embodiments, the fermentation temperature in step (c) is 25-26 °C, 26-27 °C, 27-28 °C, 28-29 °C, 29-30 °C, 30-31 °C, 31-32 °C, 32-33 °C, 33-34 °C, 34-35 °C, 35-36 °C, 36-37 °C, 37-38 °C, 38-39 °C or 39-40 °C. In some embodiments, the fermentation temperature in step (c) is 32 °C.

[0069] In some embodiments, the rotation speed during fermentation in step (c) is 150 - 250 rpm. In some embodiments, the rotation speed during fermentation in step (c) is 150 - 160 rpm, 160 - 170 rpm, 170 - 180 rpm, 180 - 190 rpm, 190 - 200 rpm, 200 - 210 rpm, 210 - 220 rpm, 220 - 230 rpm, 230 - 240 rpm, or 240 - 250 rpm. In some embodiments, the rotation speed during fermentation in step (c) is 200 rpm.

[0070] In some embodiments, the pressure during fermentation in step (c) is 0.01 - 0.05 MPa. In some embodiments, the pressure during fermentation in step (c) is 0.01 - 0.015 MPa, 0.015 - 0.02 MPa, 0.02 - 0.025 MPa, 0.025 - 0.03 MPa, 0.03 - 0.035 MPa, 0.035 - 0.04 MPa, 0.04 - 0.045 MPa, or 0.045 - 0.05 MPa. In some embodiments, the pressure during fermentation in step (c) is 0.02 MPa.

[0071] In some embodiments, the ventilation rate during fermentation in step (c) is 0.1 - 5 L / min. In some embodiments, the ventilation rate during fermentation in step (c) is 0.1 - 0.5 L / min, 0.5 - 1 L / min, 1 - 1.5 L / min, 1.5 - 2 L / min, 2 - 2.5 L / min, 2.5 - 3 L / min, 3 - 3.5 L / min, 3.5 - 4 L / min, 4 - 4.5 L / min, or 4.5 - 5 L / min. In some embodiments, the ventilation rate during fermentation in step (c) is 1 L / min.

[0072] In some embodiments, after the fermentation in step (c) is completed, the fermentation system is sterilized. In some embodiments, after the fermentation in step (c) is completed, the fermentation system is sterilized at a high temperature. In some embodiments, after the fermentation in step (c) is completed, the fermentation system is sterilized at a high temperature of 80 - 120 °C. In some embodiments, after the fermentation in step (c) is completed, the fermentation system is sterilized at a high temperature of 80 - 85 °C, 85 - 90 °C, 90 - 95 °C, 95 - 100 °C, 100 - 105 °C, 105 - 110 °C, 110 - 115 °C or 115 - 120 °C. In some embodiments, after the fermentation in step (c) is completed, the fermentation system is sterilized at a high temperature for 10 - 40 min. In some embodiments, after the fermentation in step (c) is completed, the fermentation system is sterilized at a high temperature for 10 - 15 min, 15 - 20 min, 20 - 25 min, 25 - 30 min, 30 - 35 min or 35 - 40 min.

[0073] In some embodiments, the method further includes step (d): filtering and subjecting the Ampelopsis grossedentata fermentation broth obtained in step (c) to membrane separation to obtain an Ampelopsis grossedentata fermentation filtrate. In some embodiments, the membrane used in step (d) is a membrane capable of filtering out bacteria and fungi. In some embodiments, the membrane used in step (d) is a membrane with a pore size less than 1 μm. In some embodiments, the membrane used in step (d) is a membrane with a pore size less than 0.2 μm. In some embodiments, the membrane used in step (d) is a membrane with a pore size of 0.22 μm. In some embodiments, the membrane used in step (d) is a membrane with a pore size of 0.45 μm. In some embodiments, the membrane used in step (d) is a membrane with a pore size of 50 nm.

[0074] In some embodiments, the method further includes step (d): filtering and subjecting the Ampelopsis grossedentata fermentation broth obtained in step (c) to membrane separation to obtain an Ampelopsis grossedentata fermentation filtrate, wherein the filtering is by filtration through a gauze.

[0075] Ampelopsis grossedentata Fermentation Broth

[0076] The second aspect of the present invention relates to an Ampelopsis grossedentata fermentation broth, which is prepared by the method described in the first aspect of the present invention.

[0077] In some embodiments, the storage stability of the Ampelopsis grossedentata fermentation broth is higher than that of the unfermented Ampelopsis grossedentata. In some embodiments, the storage stability of the Ampelopsis grossedentata fermentation broth is higher than that of the unfermented Ampelopsis grossedentata without inoculating Aspergillus fungi during the fermentation process.

[0078] In some embodiments, the toxicity of the fermented Ampelopsis grossedentata liquid to cells (such as human immortalized sebaceous gland cells, etc.) is lower than that of unfermented Ampelopsis grossedentata. In some embodiments, the toxicity of the fermented Ampelopsis grossedentata liquid to cells (such as human immortalized sebaceous gland cells, etc.) is lower than that of unfermented Ampelopsis grossedentata without inoculating Aspergillus fungi during the fermentation process.

[0079] In some embodiments, the fermented Ampelopsis grossedentata liquid has an oil-control effect. In some embodiments, the oil-control effect of the fermented Ampelopsis grossedentata liquid is stronger than that of unfermented Ampelopsis grossedentata. In some embodiments, the oil-control effect of the fermented Ampelopsis grossedentata liquid is stronger than that of unfermented Ampelopsis grossedentata without inoculating Aspergillus fungi during the fermentation process.

[0080] In some embodiments, the fermented Ampelopsis grossedentata liquid has an antibacterial effect. In some embodiments, the fermented Ampelopsis grossedentata liquid has an anti-inflammatory effect.

[0081] In some embodiments, the fermented Ampelopsis grossedentata liquid has an antioxidant effect. In some embodiments, the antioxidant effect of the fermented Ampelopsis grossedentata liquid is stronger than that of unfermented Ampelopsis grossedentata. In some embodiments, the antioxidant effect of the fermented Ampelopsis grossedentata liquid is stronger than that of unfermented Ampelopsis grossedentata without inoculating Aspergillus fungi during the fermentation process.

[0082] In some embodiments, the fermented Ampelopsis grossedentata liquid has a whitening effect, while unfermented Ampelopsis grossedentata has no whitening effect. In some embodiments, the fermented Ampelopsis grossedentata liquid has a whitening effect, while unfermented Ampelopsis grossedentata without inoculating Aspergillus fungi during the fermentation process has no whitening effect.

[0083] In some embodiments, the fermented Ampelopsis grossedentata liquid has an anti-wrinkle effect. In some embodiments, the anti-wrinkle effect of the fermented Ampelopsis grossedentata liquid is stronger than that of unfermented Ampelopsis grossedentata. In some embodiments, the anti-wrinkle effect of the fermented Ampelopsis grossedentata liquid is stronger than that of unfermented Ampelopsis grossedentata without inoculating Aspergillus fungi during the fermentation process.

[0084] In some embodiments, the fermented Ampelopsis grossedentata liquid has a barrier repair effect. In some embodiments, the barrier repair effect of the fermented Ampelopsis grossedentata liquid is stronger than that of unfermented Ampelopsis grossedentata. In some embodiments, the barrier repair effect of the fermented Ampelopsis grossedentata liquid is stronger than that of unfermented Ampelopsis grossedentata without inoculating Aspergillus fungi during the fermentation process.

[0085] Use of the Ampelopsis grossedentata Fermentation Broth of the Present Invention in the Preparation of Cosmetics / Drugs

[0086] The third aspect of the present invention relates to the use of the fermented Ampelopsis grossedentata liquid according to the second aspect of the present invention in the preparation of cosmetics / drugs.

[0087] The cosmetic / drug has the functions of oil control, antibacterial, anti-inflammatory, acne removal, antioxidant, whitening, anti-wrinkle, anti-aging and / or barrier repair.

[0088] Cosmetics and Drugs

[0089] The fourth aspect of the present invention relates to a cosmetic or drug, which comprises the Ampelopsis grossedentata fermentation broth according to the second aspect of the present invention, wherein the Ampelopsis grossedentata fermentation broth accounts for 0.1%-99% by weight of the weight of the cosmetic or drug.

[0090] In some embodiments, the Ampelopsis grossedentata fermentation broth accounts for 0.1%-0.5%, 0.5%-1%, 1%-1.5%, 1.5%-2%, 2%-2.5%, 2.5%-3%, 3%-3.5%, 3.5%-4%, 4%-4.5%, 4.5%-5%, 5%-5.5%, 5.5%-6, 6%-6.5%, 6.5%-7%, 7%-7.5%, 7.5%-8%, 8%-8.5%, 8.5%-9%, 9%-9.5%, 9.5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-99% or any combination of these ranges by weight of the weight of the cosmetic or drug.

[0091] In some embodiments, the cosmetic or drug is used for skin care. In some embodiments, the drug and cosmetic are used for oil control, antibacterial, anti-inflammatory, acne removal, antioxidant, whitening, anti-wrinkle, anti-aging and / or barrier repair. In some embodiments, the drug and cosmetic comprise the composition according to the third aspect of the present invention and one or more pharmaceutically or cosmetically acceptable carriers, diluents or excipients. In some embodiments, the drug and cosmetic are creams, lotions, pastes, ointments, masks, gels, lotions or serums. In some embodiments, the drugs and cosmetics of the present invention may further contain one or more other ingredients, such as plant extracts, nutritional additives, surfactants, perfumes and fragrances, pigments, preservatives, antioxidants, moisturizers, ultraviolet absorbers, astringents, penetration enhancers, pH regulators, etc. Those skilled in the art can make selections according to their common sense and specific needs.

[0092] In some embodiments, the cosmetic or drug further comprises one or more other cosmetic active ingredients, such as, but not limited to, humectants, horny repair agents, sebum control agents, anti-oil agents, moisturizing agents, anti-allergy agents, anti-aging and anti-wrinkle agents, etc.

[0093] Organism

[0094] The fifth aspect of the present invention relates to an organism, which is Aspergillus niger deposited in the China Center for Type Culture Collection (CCTCC) under the accession number M20242388.

[0095] In some embodiments, the organism is an Aspergillus niger strain. In some embodiments, the Aspergillus niger strain is named Aspergillus niger MicF_An3217.

[0096] In some embodiments, the organism consists of an Aspergillus niger strain deposited in the China Center for Type Culture Collection (CCTCC) under the accession number M20242388. Detailed implementation manners

[0097] The present invention can be implemented through the following embodiments, but the present invention is not limited thereto.

[0098] The instrument and equipment used in the embodiments of the present invention are all conventional instrument and equipment in the art and can be replaced by instrument and equipment meeting the corresponding standards.

[0099] The Chinese herbal medicine raw materials used in the embodiments of the present invention are commercially available without clear indication, and the reagents used in the embodiments of the present invention are all commercially available analytical pure chemical reagents without clear indication.

[0100] The raw materials and instrument and equipment mentioned in the present invention are all common raw materials and instrument and equipment in the art. Only for example, it is not intended to limit the protection scope of the present invention. Those skilled in the art can select equivalent raw materials and related instrument and equipment based on the disclosed content of the present invention.

[0101] Experimental Materials, Equipment and Statistical Analysis Methods

[0102] Sources of strains in the examples: Lactobacillus plantarum (strain number: CICC 24809) and Saccharomyces cerevisiae (strain number: CICC 1270) were purchased from the China Center for Industrial Culture Collection, and Bacillus subtilis (strain number: CGMCC1.15792) was purchased from the China General Microbiological Culture Collection Center. Aspergillus niger MicF_An3127 and Kluyveromyces thermotolerans are both strains isolated in this laboratory. Among them, Aspergillus niger MicF_An3127 used in this patent has been deposited in the China Center for Type Culture Collection (CCTCC), with the deposit number of CCTCC NO: M20242388, the address of the deposit unit being Wuhan University, Wuhan, China, and the postal code being 430072. The relevant deposit information is as follows:

[0103]

[0104] Vine tea raw materials: Produced in Zhangjiajie, Hunan.

[0105] Experimental reagents: MRS broth and potato dextrose water were purchased from Haibo Biotechnology, and yeast extract powder and glucose were purchased from OXOID, UK, 500 g / bottle; the rest of the reagents were purchased from Shanghai Titan Technology Co., Ltd., with the packaging specification of 500 g / bottle.

[0106] Experimental equipment: (1) Constant temperature oscillator, Shanghai Zhicheng ZWY-2102C; (2) Clean bench, Sujing Antai HVS-1300-U; (3) High-speed crusher, Zhejiang Hongjingtian Industry and Trade Co., Ltd. DE-300g.

[0107] Examples

[0108] Example 1: Screening of Strains for Fermenting Ampelopsis grossedentata

[0109] Preparation of Ampelopsis grossedentata Fermentation Broth with Different Strains

[0110] Example 1.1: Fermentation of vine tea by Saccharomyces cerevisiae

[0111] a. Take 200 μL of the cryopreserved Saccharomyces cerevisiae cryopreservation solution and inoculate it into 50 mL of YPD medium on a clean bench, and culture it in a constant temperature shaker at 28 °C and 200 rpm for 24 h. The composition of the YPD medium is: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract powder, pH = 5.8 ± 0.2.

[0112] b. Take 4 mL of the Saccharomyces cerevisiae seed solution cultured for 24 h and inoculate it into 50 mL of vine tea fermentation substrate, and continuously ferment at 28 °C and 200 rpm for 72 h. The vine tea fermentation substrate is a solution of 10 g / L of vine tea powder in deionized water, and the vine tea powder is obtained by sieving through a 100-mesh sieve after the vine tea is crushed by a high-speed crusher.

[0113] Example 1.2: Fermentation of Ampelopsis grossedentata by Kluyveromyces thermotolerans

[0114] a. Take 200 μL of the cryopreserved Kluyveromyces thermotolerans cryopreservation solution on a clean bench and inoculate it into 50 mL of YPD medium. Incubate it in a constant temperature shaker at 28 °C and 200 rpm for 24 h. The composition of the YPD medium is: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract powder, pH = 5.8 ± 0.2.

[0115] b. Take 4 mL of the Kluyveromyces thermotolerans seed solution cultured for 24 h and inoculate it into 50 mL of Ampelopsis grossedentata fermentation substrate. Continuously ferment at 28 °C and 200 rpm for 72 h. The Ampelopsis grossedentata fermentation substrate is a solution of 10 g / L Ampelopsis grossedentata powder in deionized water. The Ampelopsis grossedentata powder is obtained by sieving through a 100-mesh sieve after being crushed by a high-speed crusher.

[0116] Example 1.3: Fermentation of Ampelopsis grossedentata by Lactobacillus plantarum

[0117] a. Take 400 μL of the cryopreserved Lactobacillus plantarum cryopreservation solution on a clean bench and inoculate it into 50 mL of MRS broth medium. Incubate it in a constant temperature shaker at 37 °C and 150 rpm for 24 h.

[0118] b. Take 4 mL of the Lactobacillus plantarum seed solution cultured for 24 h and inoculate it into 50 mL of Ampelopsis grossedentata fermentation substrate. Continuously ferment at 37 °C and 150 rpm for 72 h. The Ampelopsis grossedentata fermentation substrate is a solution of 10 g / L Ampelopsis grossedentata powder in deionized water. The Ampelopsis grossedentata powder is obtained by sieving through a 100-mesh sieve after being crushed by a high-speed crusher.

[0119] Example 1.4: Fermentation of Ampelopsis grossedentata by Bacillus subtilis

[0120] a. Take 400 μL of the cryopreserved Bacillus subtilis cryopreservation solution on a clean bench and inoculate it into 50 mL of LB medium. Incubate it in a constant temperature shaker at 37 °C and 150 rpm for 24 h. The composition of the LB medium is: 10 g / L peptone, 5 g / L yeast extract powder, 10 g / L sodium chloride, pH = 7.0 ± 0.2.

[0121] b. Take 4 mL of the Bacillus subtilis seed solution cultured for 24 h and inoculate it into 50 mL of Ampelopsis grossedentata fermentation substrate. Continuously ferment in a constant temperature shaker at 37 °C and 150 rpm for 72 h. The Ampelopsis grossedentata fermentation substrate is a solution of 10 g / L Ampelopsis grossedentata powder in deionized water. The Ampelopsis grossedentata powder is obtained by sieving through a 100-mesh sieve after being crushed by a high-speed crusher.

[0122] Example 1.5: Fermentation of Ampelopsis grossedentata by Aspergillus niger MicF_An3127

[0123] a. Take 100 μL of the cryopreservation solution of Aspergillus niger MicF_An3127 stored in a cryogenic freezer and spread it on a potato dextrose agar culture dish. Place it in an incubator at 28 °C for 72 h. Add an appropriate amount of sterile normal saline to the culture dish to collect the Aspergillus niger MicF_An3127 spore powder, and dilute it to a spore powder concentration of 1×10 7 CFU / mL to obtain an Aspergillus niger MicF_An3127 spore powder solution. The preparation method of the potato dextrose agar medium is as follows: Wash and peel the potatoes, cut them into small pieces, weigh 200 grams, add 1000 mL of deionized water, boil for 20 min, filter with a gauze, add 20 g of glucose and 15 g of agar powder to the filtrate, make up the volume to 1000 mL with water, dispense and sterilize for later use.

[0124] b. Take 2 mL of the Aspergillus niger MicF_An3127 spore powder with a concentration of 1×10 7 CFU / mL and inoculate it into the Ampelopsis grossedentata fermentation substrate. Continuously ferment at 28 °C and 200 rpm in a constant temperature shaker for 72 h. The Ampelopsis grossedentata fermentation substrate is a solution of 10 g / L Ampelopsis grossedentata powder in deionized water, and the Ampelopsis grossedentata powder is obtained by sieving through a 100-mesh sieve after being crushed by a high-speed crusher.

[0125] Comparative Example 1: Prepare 50 mL of a solution of 10 g / L Ampelopsis grossedentata powder in deionized water, and place it in a constant temperature shaker at 28 °C and 200 rpm for 72 h. No microorganisms were inoculated in this comparative example.

[0126] Evaluation of Ampelopsis grossedentata Fermentation Broth with Different Strains

[0127] 1. Experimental scheme

[0128] Centrifuge the Ampelopsis grossedentata fermentation broth obtained in Examples 1.1 to 1.5 and Comparative Example 1, take the supernatant and filter it through a 0.22 μM aqueous filter membrane to remove bacteria. Place the filtered supernatant in a 4 °C refrigerator and observe the solution state after 24 h.

[0129] 2. Experimental results

[0130] After standing at 4 °C for 24 h, the sample states of Examples 1.1 to 1.5 and Comparative Example 1 Figure 1 As shown, a large amount of precipitate was precipitated in Examples 1.1 to 1.4 (the second to fifth bottles from the left), and there was no improvement compared with Comparative Example 1 (the first bottle from the left). However, the precipitation situation of Example 1.5 was significantly improved, almost clear, and the color was also the lightest. Therefore, using Aspergillus niger MicF_An3127 as the Ampelopsis grossedentata fermentation strain (i.e., Example 1.5) can better solve the problem of Ampelopsis grossedentata precipitation.

[0131] Example 2: Screening of Different Material-Liquid Ratios of Ampelopsis grossedentata

[0132] 1. Preparation of Fermentation Broth

[0133] a. Take 100 μL of the frozen Aspergillus niger MicF_An3127 cryopreservation solution on a clean bench and spread it on a potato dextrose agar culture dish, and place it in an incubator at 28 °C for 72 h. Add an appropriate amount of sterile normal saline to the culture dish to collect the Aspergillus niger MicF_An3127 spore powder, and dilute it to a spore powder concentration of 1*10 7 CFU / mL to obtain an Aspergillus niger MicF_An3127 spore powder solution.

[0134] b. Take 2 mL of Aspergillus niger MicF_An3127 spore powder at 1*10 7 CFU / mL and inoculate it into Ampelopsis grossedentata substrates at 5 g / L, 10 g / L, and 20 g / L respectively, and continuously ferment at 28 °C and 200 rpm on a constant temperature shaker for 48 h, 72 h, and 144 h respectively.

[0135] 2. Evaluation Method

[0136] Under the fermentation of Aspergillus niger MicF_An3127, take Ampelopsis grossedentata at 20 g / L, 10 g / L, and 5 g / L (i.e., the solution of Ampelopsis grossedentata powder at the corresponding concentration in deionized water), collect the fermentation filtrates at different fermentation time periods, quickly cool them at -20 °C, observe the precipitation of substances, record the fermentation time without precipitation as the fermentation termination time, and observe the color and detect the DPPH free radical scavenging rate.

[0137] The DPPH free radical scavenging rate is determined by the following method: Take an appropriate amount of DPPH solid powder and dissolve it in absolute ethanol until the absorbance at 517 nm is 1.6. Take 100 μL of the sample / deionized water and add it to a 96-well plate, set 3 replicates, and add 100 μL of the prepared DPPH solution in ethanol to each well to obtain the sample group / blank group; take 100 μL of the sample / deionized water and add it to a 96-well plate, set 1 well, and add 100 μL of absolute ethanol to obtain the sample control group / blank control group. Mix well with a multichannel pipette and place it in the dark at room temperature for 30 min. Then measure the absorbance at 517 nm.

[0138] Inhibition rate % = [1 - (sample group - sample control group) / (blank group - blank control group)] * 100%

[0139] 3. Experimental Results

[0140] With the increase of the material-liquid ratio in the fermentation of Ampelopsis grossedentata by Aspergillus niger MicF_An3127, the fermentation time will also be extended. Among them, the fermentation time is the longest under the material-liquid ratio of 20 g / L. The precipitation of substances at low temperature only improves after 144 h, but too long cultivation time will make the color of the fermentation broth darker. The difference in the fermentation end time between 10 g / L and 5 g / L is only 1 day, and the colors are both light yellow. The IC 50 values of 10 g / L and 20 g / L only differ by 1.1%. Considering the time, color and IC 50 together, fermenting Ampelopsis grossedentata with a material-liquid ratio of 10 g / L is the best choice.

[0141] Table 1

[0142]

[0143]

[0144] Example 3: Screening of Different Seed-Liquid Culture Media

[0145] Preparation of Ampelopsis grossedentata Fermentation Broth Using Different Seed-Liquid Culture Media

[0146] Example 3.1

[0147] a. Take 100 μL of the frozen stock solution of Aspergillus niger MicF_An3127 on the ultra-clean workbench and spread it on a potato dextrose agar culture dish, and place it in a constant temperature incubator at 28 °C for 72 h. Add an appropriate amount of sterile normal saline to the culture dish to collect the spore powder of Aspergillus niger MicF_An3127, and dilute it to a spore powder concentration of 1*10 7 CFU / mL to obtain the spore powder solution of Aspergillus niger MicF_An3127.

[0148] b. Take 1 mL of the spore powder solution in a and inoculate it into 50 mL of No. 1 medium, and culture it in a constant temperature shaker at 28 °C and 200 rpm for 72 h to obtain the No. 1 seed liquid of Aspergillus niger MicF_An3127. The composition of the No. 1 medium is: 12 g / L potato leaching powder, 20 g / L glucose, 0.5 g / L magnesium sulfate heptahydrate, 0.1 g / L manganese sulfate, pH = 5.6 ± 0.2.

[0149] c. Take 4 mL of the No. 1 seed liquid and inoculate it into 50 mL of Ampelopsis grossedentata fermentation matrix, and continuously culture it at 28 °C and 200 rpm under a constant temperature shaker until precipitation does not occur, and record the fermentation time. The Ampelopsis grossedentata fermentation matrix is a solution of 10 g / L Ampelopsis grossedentata powder in deionized water.

[0150] Example 3.2

[0151] According to the scheme of Example 3.1, replace the No. 1 culture medium with the No. 2 culture medium. The composition of the No. 2 culture medium is: glucose 5 g / L, wheat bran 35 g / L, yeast extract powder 5 g / L, (NH4)2SO4 2.6 g / L, KH2PO4 3.5 g / L, CaCl2 0.6 g / L, KCl 0.1 g / L, MgSO4 0.5 g / L, MnSO4 0.1 g / L, vitamin B12 0.5 mg / L, pH = 5.2 ± 0.2.

[0152] Example 3.3

[0153] According to the scheme of Example 3.1, replace the No. 1 culture medium with the No. 3 culture medium. The composition of the No. 3 culture medium is: glucose 5 g / L, wheat bran 25 g / L, tengcha powder 10 g / L, yeast extract powder 5 g / L, (NH4)2SO4 2.6 g / L, KH2PO4 3.5 g / L, CaCl2 0.6 g / L, KCl 0.1 g / L, MgSO4 0.5 g / L, MnSO4 0.1 g / L, vitamin B12 0.5 mg / L, pH = 5.2 ± 0.2.

[0154] Example 3.4

[0155] According to the scheme of Example 3.1, replace the No. 1 culture medium with the No. 4 culture medium. The composition of the No. 4 culture medium is: glucose 5 g / L, corncob powder 35 g / L, yeast extract powder 5 g / L, (NH4)2SO4 2.6 g / L, KH2PO4 3.5 g / L, CaCl2 0.6 g / L, KCl 0.1 g / L, MgSO4 0.5 g / L, MnSO4 0.1 g / L, vitamin B12 0.5 mg / L, pH = 5.2 ± 0.2.

[0156] Example 3.5

[0157] According to the scheme of Example 3.1, replace the No. 1 culture medium with the No. 5 culture medium. The composition of the No. 5 culture medium is: glucose 5 g / L, corncob powder 25 g / L, tengcha powder 10 g / L, yeast extract powder 5 g / L, (NH4)2SO4 2.6 g / L, KH2PO4 3.5 g / L, CaCl2 0.6 g / L, KCl 0.1 g / L, MgSO4 0.5 g / L, MnSO4 0.1 g / L, vitamin B12 0.5 mg / L, pH = 5.2 ± 0.2.

[0158] Evaluation of Ampelopsis grossedentata Fermentation Broth Using Different Seed-Liquid Culture Media

[0159] 1. Experimental scheme

[0160] During the fermentation processes of Examples 3.1 to 3.5, the fermentation broth was collected at different time intervals. After centrifugation and filtration, it was placed at -20°C for rapid cooling to observe the precipitation at low temperature. The fermentation time when no precipitation occurred at low temperature in each example was recorded as the fermentation end time.

[0161] 2. Experimental results

[0162] As shown in Table 2, the fermentation end times of Examples 3 - 5 were the shortest, that is, the required fermentation time was the shortest. Therefore, the optimal composition of the seed liquid medium was: glucose 5 g / L, corncob powder 25 g / L, tengcha powder 10 g / L, yeast extract powder 5 g / L, (NH4)2SO4 2.6 g / L, KH2PO4 3.5 g / L, CaCl2 0.6 g / L, KCl 0.1 g / L, MgSO4 0.5 g / L, MnSO4 0.1 g / L, vitamin B1 20.5 mg / L, pH = 5.2 ± 0.2.

[0163] Table 2

[0164]

[0165]

[0166] Example 4: Preparation of Ampelopsis grossedentata Fermentation Broth

[0167] a. Resuscitation of Aspergillus niger MicF_An3127: Take 100 μL of the frozen stock solution of Aspergillus niger MicF_An3127 on the ultra-clean workbench and spread it on a potato dextrose agar culture dish. Place it in a constant temperature incubator at 28°C for 72 h. Add an appropriate amount of sterile normal saline to the culture dish to collect the Aspergillus niger MicF_An3127 spore powder, and dilute it to a spore powder concentration of 1*10 7 CFU / mL to obtain an Aspergillus niger MicF_An3127 spore powder solution.

[0168] b. Take 4 mL of the spore powder solution and inoculate it into 200 mL of the self-prepared medium. Culture it in a constant temperature oscillator at 28°C and 200 rpm for 72 h to obtain an Aspergillus niger MicF_An3127 fermentation seed liquid. The composition of the self-prepared medium was: glucose 5 g / L, corncob powder 25 g / L, tengcha powder 10 g / L, yeast extract powder 5 g / L, (NH4)2SO4 2.6 g / L, KH2PO4 3.5 g / L, CaCl2 0.6 g / L, KCl 0.1 g / L, MgSO4 0.5 g / L, MnSO4 0.1 g / L, vitamin B1 20.5 mg / mL, pH = 5.2 ± 0.2.

[0169] c. The 5L tank fermentation experiment was carried out using a 5L glass fermentation tank (Shanghai Baoxing BIOTECH-5JGY). 25g of Ampelopsis grossedentata powder was poured into the 5L tank, and 2.5L of deionized water was added to obtain an Ampelopsis grossedentata fermentation substrate with a material-liquid ratio of 10g / L. The 5L fermentation tank was sterilized at 115°C for 30 minutes. After cooling, 200mL of the seed liquid obtained in step b was inoculated. The 5L tank fermentation process: rotation speed 200rpm, temperature 32°C, aeration 1.0L / min, pressure 0.02MPa, fermentation cycle 18h. After fermentation, it was sterilized at 95°C for 20 minutes.

[0170] d. After filtering the fermentation broth obtained in step c through a filter cloth, it was separated using a membrane separation device SY-UNR-Lab (50nm) to obtain the Ampelopsis grossedentata fermentation filtrate.

[0171] Comparative Example 2: Blank Control of Example 4 Without Fermentation

[0172] 25g of Ampelopsis grossedentata powder was poured into the 5L tank, and 2.5L of deionized water was added, resulting in a material-liquid ratio of 10g / L. It was sterilized at 115°C for 30 minutes. After cooling, the inoculation step was omitted, and the fermentation process parameters in step c of Example 4 were followed. After completion, it was sterilized at 95°C for 20 minutes. After filtering through a filter cloth, it was separated using a membrane separation device SY-UNR-Lab (50nm) to obtain a blank control group (unfermented) of Ampelopsis grossedentata fermentation filtrate.

[0173] Example 5: Stability Evaluation of Ampelopsis grossedentata Fermentation Broth

[0174] 1. Test method

[0175] 10mL each of the Ampelopsis grossedentata fermentation broth and the unfermented Ampelopsis grossedentata blank control prepared in Example 4 and Comparative Example 2 were added to transparent sample bottles, and 4 groups were prepared. They were placed in a constant temperature incubator at 4°C, 25°C, 43°C, and 4 - 43°C for 2 weeks respectively to examine the color, pH, precipitation, etc. of the samples.

[0176] 2. Test results

[0177] According to the stability investigation data shown in Table 3, compared with Comparative Example 2, Example 4 had smaller fluctuations in pH changes under different temperature conditions, a weaker degree of color change (darkening), and completely solved the problem of easy precipitation. Therefore, after fermentation using the technology of this patent, the stability and compatibility of Ampelopsis grossedentata in cosmetics can be greatly improved.

[0178] Table 3

[0179]

[0180] *Note: "#" indicates the degree of color depth: "#" indicates a slight darkening, "##" indicates a moderate darkening, "0" indicates the initial color; "+" indicates the precipitation situation: "+++" indicates very severe precipitation, "++" indicates severe precipitation, and "-" indicates no precipitation.

[0181] Example 6: Evaluation of Oil-Control Efficacy

[0182] Example 6.1: Studying the cytotoxicity of Ampelopsis grossedentata fermented liquid using sebaceous gland cells

[0183] 1. Experimental protocol

[0184] a. Cell seeding: Adjust the density of the suspension of immortalized human sebaceous gland cells SZ95 to 1×10 ^5 cells / mL and then seed them in a 96-well plate, 100 μL / well, and culture them in an incubator at 37°C and 5% CO2 for 24 h;

[0185] b. Experimental groups:

[0186] Negative control group (NT): Normal cell culture medium;

[0187] Model group (M): Normal cell culture medium + 100 μM linoleic acid + 10 μM dihydrotestosterone;

[0188] Positive control group (PC): 20 μM EGCG + normal cell culture medium + 100 μM linoleic acid + 10 μM dihydrotestosterone;

[0189] Sample group (TA): Samples at gradient concentrations (10%, 5%, 1%, 0.5%) + normal cell culture medium + 100 μM linoleic acid + 10 μM dihydrotestosterone;

[0190] c. Take 100 μL of complete medium (DMEM / F-12 medium / Gibco / 11320033) and 2× the detection concentration of the sample / positive control sample and add them to each well of the cells respectively. Add 200 μL of complete medium to the negative control group; add 100 μL of complete medium and 100 μL of 2× concentration stimulator (200 μM linoleic acid + 20 μM dihydrotestosterone) to the model group. The number of replicate wells n = 5. After adding the samples, place the 96-well plate in an incubator at 37°C and 5% CO2 and culture for 24 h;

[0191] CCK8 detection: Discard the supernatant in the 96-well plate and wash it twice with PBS. Under light-proof conditions, prepare the CCK8 reaction solution (CCK8:DMEM high-glucose complete medium = 10:1). After preparation, add 100 μL of the CCK8 reaction solution to each well, place the 96-well plate in an incubator at 37°C and 5% CO2 and react fully for 2 h, and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value at 450 nm of each well;

[0192] Calculation: After subtracting the background absorption from the absorbance values of each group, taking the absorbance value of the cell control group as 100%, calculate the percentage of cell viability of the cell groups with different sample concentrations added. The calculation formula is as follows:

[0193] Percentage of cell viability (%) = Average absorbance value of the sample well / Average absorbance value of the negative control well × 100%

[0194] 2. Experimental results

[0195] Measure the maximum addition amount of the samples in Example 4 and Comparative Example 2 when maintaining the cell viability greater than 80%. The measured results are shown in Table 4 below. It can be seen from the results in Table 4 that when maintaining the cell viability of human immortalized sebaceous gland cells SZ95 greater than 80%, the maximum addition amount of unfermented Ampelopsis grossedentata (Comparative Example 2) is 1%, while the maximum addition amount of Ampelopsis grossedentata fermented by the method of the present invention (Example 4) can be as high as 5% under this condition, indicating that after fermentation by the method of the present invention, the cytotoxicity of the Ampelopsis grossedentata fermentation broth is significantly reduced.

[0196] Table 4

[0197]

[0198] Example 6.2: Determination of oil secretion inhibition rate

[0199] 1. Experimental protocol

[0200] a. Cell seeding: Adjust the density of the suspension of human immortalized sebaceous gland cells SZ95 to 1×10 5 cells / mL and then seed them in a 96-well plate, 100 μL / well. After seeding, place them in a CO2 incubator for cultivation for 24 h ± 2 h.

[0201] b. Drug administration treatment: After the cells in the 96-well plate reach an appropriate confluence, discard the culture medium in the 96-well plate and perform drug administration operations according to the following experimental groups.

[0202] Negative control group (NT): Normal cell culture medium;

[0203] Model group (M): Normal cell culture medium + 100 μM linoleic acid + 10 μM dihydrotestosterone;

[0204] Positive control group (PC): 20 μM EGCG + Normal cell culture medium + 100 μM linoleic acid + 10 μM dihydrotestosterone;

[0205] Sample group (TA): Gradient concentration samples (1%, 0.5%, 0.1%) + Normal cell culture medium + 100 μM linoleic acid + 10 μM dihydrotestosterone;

[0206] Add the culture medium containing the test substance to the test substance wells; add normal cell culture medium to the blank control wells, with a liquid addition volume of 200 μL per well. After administration, place the 96-well plate in a CO2 incubator and incubate for 48 h ± 2 h.

[0207] c. Staining detection: After incubating for 48 h, discard the old liquid in the 96-well plate, rinse the well plate 2 times with PBS buffer solution, add 100 μL of 10 μg / mL nile red dye (Shanghai Yuanye Bio-Technology Co., Ltd., S02N11G129713) to each well, place the 96-well plate in an incubator at 37 °C and 5% CO2 for a full reaction for 15 min, and use a microplate reader to detect the absorbance values at the excitation wavelength of 485 nm and the absorption wavelength of 565 nm for the fluorescence intensity of nile red in each well;

[0208] d. Calculation: The results are determined by the fluorescence intensity of nile red. The percentage of intracellular neutral lipids (%) = fluorescence intensity of the control group or sample group / fluorescence intensity of the model group * 100%.

[0209] 2. Experimental results

[0210] Measure the oil secretion inhibition rate of the samples of Example 4 and Comparative Example 2 at a concentration of 1% on human immortalized sebaceous gland cells SZ95. The measured results are shown in Table 5 below. It can be seen from the results in Table 5 that the fermented Ampelopsis grossedentata (Example 4) at a concentration of 1% can inhibit 39.66% of the oil content. Compared with the inhibition rate of 17.33% of the unfermented Ampelopsis grossedentata (Comparative Example 2) at the same concentration, the Ampelopsis grossedentata fermentation broth obtained by the fermentation method of the present invention also has a significant improvement in the efficacy of inhibiting oil control.

[0211] Oil secretion inhibition rate = (1 - percentage of intracellular neutral lipids in the sample group / percentage of intracellular neutral lipids in the negative control group) * 100%.

[0212] Table 5

[0213]

[0214] Example 7: Evaluation of Bacteriostatic Efficacy

[0215] 1. Experimental scheme

[0216] Inoculate Propionibacterium acnes into FT liquid medium and anaerobically culture it at 37 °C for 24 h, and then dilute it to 2×10 ^ 6Reserve at CFU / mL; Take a sterile 96-well plate, and first add 100 μL of the diluted Propionibacterium acnes suspension to each well. Then add 100 μL of gradient sample solutions respectively: Use the fermented Ampelopsis grossedentata and unfermented Ampelopsis grossedentata with 100% Aspergillus niger MicF_An3127 content as the stock solutions, and perform gradient dilution with FT liquid medium; At the same time, set up a positive model group (only add Propionibacterium acnes suspension), a positive control group (Propionibacterium acnes suspension + erythromycin), and a negative control group (only FT liquid medium).

[0217] After placing the sterile 96-well plate in an anaerobic workstation and culturing at 37 °C for 24 h, observe the growth of Propionibacterium acnes in the wells. Select the lowest concentration of the sample in the wells that can keep the culture medium still clear and transparent as the minimum inhibitory concentration (MIC) of this sample against this bacterium. Respectively aspirate 100 μL of the liquid in the wells with a final concentration greater than or equal to MIC, spread it on the RCM agar culture dish plate, and culture anaerobically at 37 °C for 48 h. The lowest drug concentration without colony growth is the minimum bactericidal concentration (MBC).

[0218] 2. Experimental results

[0219] Measure the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the samples in Example 4 and Comparative Example 2 against Propionibacterium acnes. The measured results are shown in Table 6 below. It can be seen from the results in Table 6 that both the fermented Ampelopsis grossedentata (Example 4) and the unfermented Ampelopsis grossedentata (Comparative Example 2) showed good antibacterial effects, and their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were 0.78% and 1.56% respectively.

[0220] Table 6

[0221]

[0222] Example 8: Evaluation of Anti-Inflammatory Efficacy

[0223] 1. Experimental scheme

[0224] Prepare a THP-1 cell suspension of 5×10 5 cells / mL with RPMI-1640 complete medium, and add phorbol ester (PMA, dissolved in DMSO) to make its concentration 50 ng / mL. Add 100 μL of the THP-1 cell suspension to each well in a 96-well plate, incubate in an incubator at 37 °C and 5% CO2 for 24 h, and the cells adhere to the wall after 24 h. Wash off the culture medium, add 90 μL of RPMI-1640 complete medium to each well, add 5 μL of the sample (prepared with PBS) and 5 μL of the Propionibacterium acnes suspension (2.5×108 CFU / mL, inactivated at 85 °C for 1 h), 5 μL of PBS and 5 μL of Propionibacterium acnes were added to the control group, and 10 μL of PBS was added to the blank group. The cells were cultured in an incubator for 24 h. The cell culture medium was collected, and the relevant inflammatory factors were detected using a TNF-α ELISA kit.

[0225] TNF-α inhibition rate = (1 - TNF-α content in the sample group / TNF-α content in the negative control group) * 100%

[0226] 2. Experimental results

[0227] The TNF-α inhibition rates of the samples of Example 4 and Comparative Example 2 at a concentration of 1% on the cell inflammation model caused by Propionibacterium acnes were measured, and the measured results are shown in Table 7 below. From the results in Table 7, it can be seen that both the fermented Ampelopsis grossedentata (Example 4) and the unfermented Ampelopsis grossedentata (Comparative Example 2) at a concentration of 1% showed good anti-inflammatory effects, with TNF-α inhibition rates of 70.60% and 69.1% respectively.

[0228] Table 7

[0229]

[0230] Example 9: Evaluation of Antioxidant Efficacy

[0231] 1. Experimental protocol

[0232] 100 μL of HDF cells with a density of (1 - 1.5) × 10⁴ cells / well were added to a 96-well plate and incubated in an incubator at 37 °C and 5% CO₂ for 24 h. After 24 h, the culture medium was removed. 100 μL of complete medium DMEM was added to each well of the blank group and the control group, and 100 μL of the sample solution diluted with complete medium DMEM was added to the sample group. The plate was placed in the incubator for 18 h. After 18 h, the cells were rinsed 1 - 2 times with PBS. 100 μL of DMEM containing 20 μM DCFH-DA and 5 μg / mL Hoechst was added to each well of all groups, and the plate was placed in the incubator for 20 min. The cells were rinsed 1 - 2 times with PBS. 100 μL of PBS-prepared H₂O₂ (200 μM) was added to each well of the blank group, and 100 μL of PBS-prepared H₂O₂ (200 μM) was added to each well of the control group and the sample group. The plate was placed in the incubator for 1 h. The detection wavelengths of DCFH-DA are 485 nm (excitation wavelength) and 525 nm (emission wavelength), and the detection wavelengths of Hoechst are 350 nm (excitation wavelength) and 461 nm (emission wavelength).

[0233] ROS inhibition rate = (1 - fluorescence intensity in the sample group / fluorescence intensity in the negative control group) * 100%

[0234] 2. Experimental results

[0235] The inhibition rates of reactive oxygen species (ROS) of the samples of Example 4 and Comparative Example 2 at a concentration of 1% were measured, and the measured results are shown in Table 8 below. It can be seen from the results in Table 8 that at a concentration of 1%, fermented Ampelopsis grossedentata (Example 4) has an inhibitory effect of 36.72% on the increase in ROS level caused by H2O2, while unfermented Ampelopsis grossedentata (Comparative Example 2) has an inhibitory effect of 31.6% on the ROS level caused by H2O2. The inhibitory effect of unfermented Ampelopsis grossedentata on ROS is lower than that of fermented Ampelopsis grossedentata. Therefore, after being fermented by the technology of this patent, Ampelopsis grossedentata has further improvement in antioxidant efficacy.

[0236] Table 8

[0237]

[0238] Example 10: Evaluation of Whitening Efficacy

[0239] 1. Experimental protocol

[0240] Add 0.5 mL of B16 cells with a density of 200,000 cells / mL (RPMI 1640 complete medium, RPMI 1640 containing 10% FBS and 1% double antibody) into a 24-well plate, and incubate overnight in an incubator at 37°C and 5% CO2. Aspirate the culture medium, wash once with PBS, add 450 μL of DMEM complete medium (DMEM containing 10% FBS and 1% double antibody, also used for preparing samples). Add 50 μL of the sample to each well in the sample group, add 50 μL of DMEM complete medium to the blank control group, and add 50 μL of 0.1 mg / mL kojic acid to the positive control. Place in the incubator for 48 h. Collect the culture medium and cells, and detect the absorbance of the supernatant at 405 nm.

[0241] Melanin production inhibition rate = (1 - melanin content in the sample group / melanin content in the negative control group) * 100%

[0242] 2. Experimental results

[0243] The inhibition rates of the samples of Example 4 and Comparative Example 2 at a concentration of 1% on melanin production of B16 cells were measured, and the measured results are shown in Table 9 below. It can be seen from the results in Table 9 that fermented Ampelopsis grossedentata showed extremely significant improvement in the inhibitory ability of melanin production in B16 cells. The originally unfermented Ampelopsis grossedentata (Comparative Example 2) did not show inhibitory effect in the melanin inhibition experiment, that is, it does not have the function of whitening. However, after being fermented by the technology of this patent (Example 4), it has a melanin inhibition effect of 74.46% at a concentration of 1%. Therefore, after being fermented by the technology of this patent, Ampelopsis grossedentata that does not have the whitening effect can have a significant whitening effect.

[0244] Table 9

[0245]

[0246] Example 11: Evaluation of Anti-Wrinkle Efficacy

[0247] 1. Experimental protocol

[0248] 1×10 5 HFF-1 cells at a density of / mL were seeded in 24-well plates. After the cells adhered to the wall the next day, they were irradiated with 4 + 4 J / cm 2 UVA (4 J / cm of light irradiation in the morning and evening each) 2 . Samples were prepared with DMEM to incubate the cells. The control group and the model group (UVA group) were not treated with samples. The positive group was TGF-β1 at 50 ng / mL. After culturing the cells continuously for 16 h, the cells were lysed and total cellular RNA was collected. RNA was extracted and purified using the SteadyPure Universal RNA Extraction Kit, and reverse transcription and probe modification were performed using III All-in-one RT SuperMix Perfect for qPCR and qPCR SYBR Green Master Mix (Vazyme).

[0249] 2. Experimental results

[0250] The transcriptional expression ability of type I procollagen in fibroblasts by the samples of Example 4 and Comparative Example 2 at a concentration of 0.1% was measured, and the results obtained are shown in Table 10 below. From the results in Table 10, it can be seen that before and after fermentation of Ampelopsis grossedentata, there are also differences in the transcriptional expression ability of type I procollagen in fibroblasts. The relative mRNA expression level of the fermented Ampelopsis grossedentata at 0.1% (Example 4) increased to 1.34 compared with the model group, while that of the unfermented Ampelopsis grossedentata at the same concentration (Comparative Example 2) was only 1.15. Therefore, after fermentation by the technology of this patent, Ampelopsis grossedentata has a significant improvement in the anti-wrinkle effect.

[0251] Table 10

[0252]

[0253] Example 12: Evaluation of Barrier Repair Efficacy

[0254] 1. Experimental protocol

[0255] 2×10 5 HaCaT cells at a density of / mL were seeded in 24-well plates. After the cells adhered to the wall the next day, they were treated with 0.2 J / cm 2The cells were incubated with the sample prepared with DMEM after being irradiated with UVB for 1 minute. The control group and the model group (UVB group) were not treated with the sample and were cultured for 24 hours. After continuous culture for 24 hours, the cells were lysed and total cellular RNA was collected. RNA was extracted and purified using the SteadyPure Universal RNA Extraction Kit, and reverse transcription and probe modification were performed using III All-in-one RT SuperMix Perfect for qPCR and qPCR SYBR Green Master Mix (Vazyme).

[0256] 2. Experimental results

[0257] Filaggrin (FLG), Claudin-1 (CLDN1), and Occludin (OCLN) play key roles in the barrier and repair capabilities of the skin. The transcriptional expression capabilities of the samples of Example 4 and Comparative Example 2 at a concentration of 0.1% on the above proteins were measured, and the results obtained are shown in Table 11 below. From the results in Table 11, it can be seen that after the vine tea was fermented, the transcriptional expression capabilities of the 0.1% fermented vine tea (Example 4) on the above proteins were all improved to varying degrees, especially the improvement on CLDN1 and OCLN was the most significant, with the relative mRNA expression levels being 1.81 and 1.74 respectively, and the relative mRNA expression level of FLG was 1.29. The relative mRNA expression levels of the corresponding unfermented vine tea were 1.37, 1.35, and 1.12 respectively. Therefore, after the vine tea was fermented by the technology of this patent, there was a significant improvement in the barrier repair efficacy.

[0258] Table 11

[0259]

Claims

1. A method for preparing a fermented liquor of rattan tea, the method comprising the following steps: (a) culturing an Aspergillus fungus on a solid culture medium to obtain spore powder of the fungus; (b) inoculating the spore powder of the fungus obtained in step (a) into a liquid culture medium to obtain a seed solution of the fungus; (c) inoculating the seed solution obtained in step (b) into a fermentation matrix of rattan tea for fermentation, The liquid culture medium in step (b) comprises glucose, corn cob powder, rattan tea powder, yeast extract powder, (NH4)2SO4, KH2PO4, CaCl2, KCl, MgSO4, MnSO4 and vitamin B12, The rattan tea fermentation matrix is ​​a solution of rattan tea in water.

2. The method according to claim 1, wherein the liquid culture medium in step (b) comprises 2-10 g / L glucose, 20-30 g / L corn cob powder, 5-20 g / L rattan tea powder, 2-10 g / L yeast extract, 1-5 g / L (NH4)2SO4, 1-5 g / L KH2PO4, 0.1-2 g / L CaCl2, 0.01-0.5 g / L KCl, 0.1-1 g / L MgSO4, 0.01-0.5 g / L MnSO4 and 0.1-2 mg / mL vitamin B12, and the pH of the liquid culture medium is 4.5-6.

3. The method according to claim 1 or 2, wherein the Aspergillus fungus is selected from Aspergillus niger, Aspergillus candidus, Aspergillus oryzae or one or more combinations thereof, preferably Aspergillus niger, most preferably Aspergillus niger MicF_An3127.

4. The method according to any one of the preceding claims, wherein the solid culture medium in step (a) is potato dextrose agar medium, and the solid culture in step (a) is cultured at a temperature of 25-35° C. for 60-90 h.

5. The method according to any one of the preceding claims, wherein the ratio between the spore powder inoculated in step (b) and the liquid culture medium is (0.5-5)*10 5 CFU / mL, the liquid culture in step (b) is cultured at 25-35°C for 60-90h.

6. The method according to any one of the preceding claims, wherein the fermentation substrate of the rattan tea in step (c) is a solution of 5-20 g / L of rattan tea in water.

7. The method according to any one of the preceding claims, wherein the amount of seed liquid introduced in step (c) is 5%-15% by volume of the volume of the rattan tea fermentation substrate.

8. The method according to any one of the preceding claims, wherein the fermentation time in step (c) is 15-36 h.

9. The method according to any one of the preceding claims, wherein the fermentation temperature in step (c) is 25-40°C; the rotation speed during fermentation is 150-250rpm; the pressure during fermentation is 0.01-0.05MPa; and the ventilation volume during fermentation is 0.1-5L / min.

10. The method according to any one of the preceding claims, wherein the method further comprises step (d): filtering and using a membrane to separate the rattan tea fermentation liquid obtained in step (c) to obtain a rattan tea fermentation filtrate.

11. A rattan tea fermentation liquid, which is prepared by the method according to any one of claims 1 to 10.

12. Use of the rattan tea fermentation liquid according to claim 11 in the preparation of cosmetics / medicines.

13. The use according to claim 12, wherein the cosmetic / drug has oil control, antibacterial, anti-inflammatory, acne-removing, antioxidant, whitening, anti-wrinkle, anti-aging and / or barrier repair effects.

14. A cosmetic or medicine comprising the rattan tea fermentation liquid according to claim 11, wherein the rattan tea fermentation liquid accounts for 0.1%-99% of the weight of the cosmetic or medicine.

15. An organism, which is Aspergillus niger deposited in China Center for Type Culture Collection (CCTCC) under the accession number M20242388.

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