A method for solid aerobic fermentation of a chrysosporium complex strain and application thereof

Abstract

The present application relates to the technical field of daily chemical industry, and particularly relates to a black tea extract containing a compound strain of chrysosporium for solid aerobic fermentation. The compound strain contains chrysosporium and yeast. The black tea extract provided by the present application is stable in performance and not prone to discoloration, contains ingredients such as polysaccharides, amino acids and minerals, and can help skin moisturizing. The fermentation process enhances the permeability of these ingredients, helps to lock water in the skin surface, and maintains the water moisture of the skin. The black tea extract has strong antioxidant capacity, can neutralize free radicals, slow down the oxidation process of the skin, delay skin aging, and reduce the generation of wrinkles and fine lines. The black tea extract can have a certain calming effect on the skin prone to redness, itching or slight inflammation.

Description

Technical Field

[0001] This invention relates to the field of daily chemical technology, and in particular to a method for solid-state aerobic fermentation using a compound strain of *Aureobasidium aureum* and its application. Background Technology

[0002] Eurotium cristatum is a fungus belonging to the Ascomycota phylum. It is commonly associated with the fermentation process of fermented teas (such as Fu brick tea from Anhua black tea), and is particularly known for the "golden flowers" produced during fermentation. The yellow or golden spore clusters formed by Eurotium cristatum are a unique marker of fermented products and have been extensively studied due to their significant impact on the quality and flavor of tea.

[0003] During fermentation, *Mycorrhiza uralensis* enhances the aroma, taste, and internal quality of tea through its metabolites, increasing the tea's richness and the content of active ingredients. It can break down large molecules such as polysaccharides and proteins in tea, converting them into easily absorbed small molecules, thereby improving the tea's flavor.

[0004] Yeast is a type of single-celled fungus that is widely found in nature and belongs to the Basidiomycota subphylum of the kingdom Fungi. Yeast can perform both aerobic respiration and anaerobic fermentation (fermentation), and is a key microorganism in many fermentation processes, especially in food, beverage, pharmaceutical, and industrial fermentation.

[0005] Dark tea is a post-fermented tea, one of the six major tea categories in China. It gets its name from its dark brown appearance and is mainly produced in Hunan, Hubei, Sichuan, Yunnan, and Guangxi provinces of China. During its production, dark tea undergoes fermentation with the participation of microorganisms. As the storage time increases, the quality and flavor of the tea gradually change, developing a unique aged aroma and mellow taste.

[0006] This invention provides a black tea extract containing a compound strain of Mycorrhiza uralensis undergoing solid-state aerobic fermentation. Summary of the Invention

[0007] This invention provides a black tea extract containing a compound microbial strain of *Aureobasidium aureum* undergoing solid-state aerobic fermentation, wherein the compound microbial strain contains *Aureobasidium aureum* and yeast.

[0008] As one embodiment of the present invention, the black tea extract is used in the daily chemical industry.

[0009] As one embodiment of the present invention, the extraction steps of the dark tea extract are as follows:

[0010] (1) Activate the bacterial strain in a culture medium;

[0011] (2) Mix the microbial culture with black tea for solid-state aerobic fermentation;

[0012] (3) The solid aerobic fermentation product was extracted with water to obtain black tea extract.

[0013] As one embodiment of the present invention, step (1) is specifically as follows:

[0014] Mix Aureobasidium aureum and yeast at a 1:1 inoculation ratio, and inoculate a total of 1% into liquid culture medium for activation culture.

[0015] The liquid culture medium formula is as follows: 3g yeast powder, 10g peptone, 10g sucrose, 15g soluble starch, 5g sodium acetate, 2g triammonium citrate, 0.15g K2HPO4, 0.5g MnSO4, 0.2g MgSO4, and 1mL Tween 80 dissolved in 1L distilled water, and sterilized at 121℃ under high temperature and pressure.

[0016] Fermentation time: 24 hours, fermentation temperature: 30 degrees Celsius.

[0017] As one embodiment of the present invention, step (2) is specifically as follows:

[0018] After mixing the black tea with the fermentation substrate evenly, place it in a fermentation bucket, add the activated microbial liquid, and carry out solid-state aerobic fermentation. The fermentation temperature is 30℃, the fermentation time is 10 days, and the pile is turned 3 times a day.

[0019] In one embodiment of the present invention, the mass ratio of the black tea mixed with the fermentation substrate to the mass ratio of the added activated microbial liquid is 1:1.

[0020] As one embodiment of the present invention, the specific steps of step (3) are as follows: after the product after fermentation in step (2) is crushed, it is placed at 80°C for 2 hours and then rapidly frozen to 5°C; then the extract is added under stirring and extracted for 2 hours, then filtered after standing, the filtrate is taken and concentrated at low temperature to the same mass as the black tea raw material, and the black tea extract is obtained.

[0021] As one embodiment of the present invention, the black tea extract is used in the daily chemical industry at a dosage of 1-20 wt%.

[0022] By adopting the above technical solution, the present invention has the following beneficial effects:

[0023] The black tea extract provided by this invention is stable and does not easily discolor. It contains polysaccharides, amino acids, and minerals, which help moisturize the skin. The fermentation process enhances the permeability of these components, helping to lock moisture in the skin's surface and maintain skin hydration. It has strong antioxidant capabilities, neutralizing free radicals, slowing down the skin's oxidation process, delaying skin aging, and reducing the formation of wrinkles and fine lines. It can also have a calming effect on skin prone to redness, itching, or mild inflammation. Detailed Implementation

[0024] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] This invention provides a dark tea extract containing a compound strain of *Aureobasidium aureum* undergoing solid-state aerobic fermentation, and its preparation method is as follows:

[0026] (1) Mix the golden flower fungus and yeast at a 1:1 inoculation ratio, and inoculate a total of 1% into the liquid culture medium for activation culture.

[0027] The liquid culture medium formula is as follows: 3g yeast powder, 10g peptone, 10g sucrose, 15g soluble starch, 5g sodium acetate, 2g triammonium citrate, 0.15g K2HPO4, 0.5g MnSO4, 0.2g MgSO4, and 1mL Tween 80 dissolved in 1L distilled water, and sterilized at 121℃ under high temperature and pressure.

[0028] Fermentation time: 24 hours, fermentation temperature: 30 degrees Celsius.

[0029] The *Eurotium cristatum* strain described in this invention is CICC 2099, from the China Industrial Microbial Culture Collection Center. The yeast strains are *Kluyveromyces* and *Hansenula polymorpha*; specifically, *Kluyveromyces*. 1726 and the abnormal variant of Hansenulaanomala var. 1639, with a vaccination ratio of 6:4.

[0030] (2) After mixing the black tea with the fermentation substrate evenly, place it in a fermentation bucket, add the activated bacterial liquid, and carry out solid-state aerobic fermentation. The fermentation temperature is 30℃, the fermentation time is 10 days, and the pile is turned over 3 times a day.

[0031] The mass ratio of the mixed black tea and fermentation substrate to the added activated microbial liquid is 1:1. The height of the black tea and fermentation substrate in the fermentation tank is about 10cm. The fermentation substrate is a mixture of five grains, namely wheat kernels and rice, in a mass ratio of 1:1.

[0032] (4) After the product of fermentation in step (2) is crushed, it is placed at 80°C for 2 hours and then rapidly frozen to 5°C; then the extract is added under stirring and extracted for 2 hours. After standing and filtering, the filtrate is taken and concentrated at low temperature to the same mass as the black tea raw material to obtain the black tea extract.

[0033] The extract consists of water and a phosphate buffer solution with a pH of approximately 7, in a mass ratio of 10:1; the mass ratio of the extract to the fermented product is 10:1.

[0034] As one embodiment of the present invention, the black tea extract is used in the daily chemical industry at a dosage of 1-20 wt%.

[0035] As one embodiment of the present invention, the present invention also provides a cream, the raw materials for which are prepared include:

[0036] The ingredients include 68.483 parts water, 7 parts glycerin, 4 parts cetyl ethylhexanoate, 3 parts cyclopentamethoxysiloxane, 2 parts polydimethylsiloxane, 1.125 parts cetearyl alcohol, 0.375 parts cetearyl glucoside, 1 part betaine, 0.6 parts glyceryl stearate, 0.4 parts PEG-100 stearate, 1 part 1,2-pentanediol, 0.12 parts butylene glycol, 0.015 parts Cynanchum atratum extract, 0.006 parts Sophora flavescens root extract, 0.006 parts Gynostemma pentaphyllum extract, 0.22 parts carbomer, 0.2 parts bisabolol, 0.2 parts arginine, 0.15 parts tocopheryl acetate, 0.03 parts xanthan gum, 0.03 parts (daily use) fragrance, 0.02 parts sodium hyaluronate, 0.02 parts disodium EDTA, and 10 parts of the aforementioned black tea extract.

[0037] The use of dark tea extract in skincare has several drawbacks: firstly, its efficacy is not very obvious; secondly, its color is not easily stabilized. This is because the inventors discovered that half of the components in dark tea extract are extracted with hot water. Furthermore, some polyphenols in dark tea undergo partial oxidation during fermentation, and further oxidation occurs during storage, causing the color to gradually darken. To address this, the inventors found that using the natural microorganisms present in dark tea for further fermentation allows for extraction of better-tasting components under gentler conditions. However, this process resulted in a rapid decrease in amino acid content. Therefore, by adding other microorganisms during the initial fermentation stage and increasing the amount of beneficial components, along with carefully controlling the moisture content, temperature, and fermentation time, the efficacy can be effectively managed.

[0038] The present invention will be further explained below with reference to specific embodiments.

[0039] Example 1

[0040] This embodiment provides a black tea extract containing a compound strain of *Gynostemma pentaphyllum* undergoing solid-state aerobic fermentation. The preparation method is as follows:

[0041] (1) Mix the golden flower fungus and yeast at a 1:1 inoculation ratio, and inoculate a total of 1% into the liquid culture medium for activation culture.

[0042] The liquid culture medium formula is as follows: 3g yeast powder, 10g peptone, 10g sucrose, 15g soluble starch, 5g sodium acetate, 2g triammonium citrate, 0.15g K2HPO4, 0.5g MnSO4, 0.2g MgSO4, and 1mL Tween 80 dissolved in 1L distilled water, and sterilized at 121℃ under high temperature and pressure.

[0043] Fermentation time: 24 hours, fermentation temperature: 30 degrees Celsius.

[0044] The *Eurotium cristatum* strain, strain number CICC 2099, is from the China Industrial Microbial Culture Collection Center. The yeast strains are *Kluyveromyces* and *Hansenula polymorpha*; specifically, *Kluyveromyces*. 1726 and the abnormal variant of Hansenula anomalavar. 1639, with a vaccination ratio of 6:4.

[0045] (2) After mixing the black tea with the fermentation substrate evenly, place it in a fermentation bucket, add the activated inoculum liquid, and carry out solid-state aerobic fermentation. The fermentation temperature is 30℃, the fermentation time is 10 days, and the pile is turned 3 times a day. The mass ratio of black tea to fermentation substrate is 1:1.

[0046] The mass ratio of the mixed black tea and fermentation substrate to the added activated microbial liquid is 1:1. The height of the black tea and fermentation substrate in the fermentation tank is about 10cm. The fermentation substrate is a mixture of five grains, namely wheat kernels and rice, in a mass ratio of 1:1.

[0047] (5) After the product of fermentation in step (2) is crushed, it is placed at 80°C for 2 hours and then cooled to 5°C by liquid nitrogen. Then, the extract is added under stirring and extracted for 2 hours. After standing and filtering, the filtrate is taken and concentrated at low temperature to the same mass as the raw material of black tea to obtain the black tea extract.

[0048] The extract consists of water and a phosphate buffer solution with a pH of approximately 7, in a mass ratio of 10:1; the mass ratio of the extract to the fermented product is 10:1.

[0049] This embodiment also provides a cream, the raw materials for which are prepared include:

[0050] The ingredients include 68.483 parts water, 7 parts glycerin, 4 parts cetyl ethylhexanoate, 3 parts cyclopentamethoxysiloxane, 2 parts polydimethylsiloxane, 1.125 parts cetearyl alcohol, 0.375 parts cetearyl glucoside, 1 part betaine, 0.6 parts glyceryl stearate, 0.4 parts PEG-100 stearate, 1 part 1,2-pentanediol, 0.12 parts butylene glycol, 0.015 parts Cynanchum atratum extract, 0.006 parts Sophora flavescens root extract, 0.006 parts Gynostemma pentaphyllum extract, 0.22 parts carbomer, 0.2 parts bisabolol, 0.2 parts arginine, 0.15 parts tocopheryl acetate, 0.03 parts xanthan gum, 0.03 parts (daily use) fragrance, 0.02 parts sodium hyaluronate, 0.02 parts disodium EDTA, and 10 parts of the aforementioned black tea extract.

[0051] Example 2

[0052] This embodiment provides a black tea extract containing a compound strain of *Gynostemma pentaphyllum* undergoing solid-state aerobic fermentation. The preparation method is as follows:

[0053] (1) Inoculate 1% of the golden flower fungus into the liquid culture medium and mix to activate the culture;

[0054] The liquid culture medium formula is as follows: 3g yeast powder, 10g peptone, 10g sucrose, 15g soluble starch, 5g sodium acetate, 2g triammonium citrate, 0.15g K2HPO4, 0.5g MnSO4, 0.2g MgSO4, and 1mL Tween 80 dissolved in 1L distilled water, and sterilized at 121℃ under high temperature and pressure.

[0055] Fermentation time: 24 hours, fermentation temperature: 30 degrees Celsius.

[0056] The golden flower fungus is Eurotium cristatum, strain number: CICC 2099, China Industrial Microbial Culture Collection Center. (2) After mixing the black tea with the fermentation substrate evenly, place it in a fermentation tank, add the activated strain liquid, and carry out solid-state aerobic fermentation, with a fermentation temperature of 30℃ and a fermentation time of 10 days, and turn the pile 3 times a day.

[0057] The mass ratio of the mixed black tea and fermentation substrate to the added activated microbial liquid is 1:1. The height of the black tea and fermentation substrate in the fermentation tank is about 10cm. The fermentation substrate is a mixture of five grains, namely wheat kernels and rice, with a mass ratio of 1:1. The mass ratio of black tea to fermentation substrate is 1:1.

[0058] (6) After crushing the product after fermentation in step (2), place it at 80°C for 2 hours and then freeze it with liquid nitrogen to 5°C; then add the extract while stirring and continue to extract for 2 hours. After standing and filtering, take the filtrate and concentrate it at low temperature to the same mass as the black tea raw material to obtain the black tea extract.

[0059] The extract consists of water and a phosphate buffer solution with a pH of approximately 7, in a mass ratio of 10:1; the mass ratio of the extract to the fermented product is 10:1.

[0060] This embodiment also provides a cream, the raw materials for which are prepared include:

[0061] The ingredients include 68.483 parts water, 7 parts glycerin, 4 parts cetyl ethylhexanoate, 3 parts cyclopentamethoxysiloxane, 2 parts polydimethylsiloxane, 1.125 parts cetearyl alcohol, 0.375 parts cetearyl glucoside, 1 part betaine, 0.6 parts glyceryl stearate, 0.4 parts PEG-100 stearate, 1 part 1,2-pentanediol, 0.12 parts butylene glycol, 0.015 parts Cynanchum atratum extract, 0.006 parts Sophora flavescens root extract, 0.006 parts Gynostemma pentaphyllum extract, 0.22 parts carbomer, 0.2 parts bisabolol, 0.2 parts arginine, 0.15 parts tocopheryl acetate, 0.03 parts xanthan gum, 0.03 parts (daily use) fragrance, 0.02 parts sodium hyaluronate, 0.02 parts disodium EDTA, and 10 parts of the aforementioned black tea extract.

[0062] Example 3

[0063] This embodiment provides a black tea extract containing a compound strain of *Gynostemma pentaphyllum* undergoing solid-state aerobic fermentation. The preparation method is as follows:

[0064] (1) Mix the golden flower fungus and yeast at a 1:1 inoculation ratio, and inoculate a total of 1% into the liquid culture medium for activation culture.

[0065] The liquid culture medium formula is as follows: 3g yeast powder, 10g peptone, 10g sucrose, 15g soluble starch, 5g sodium acetate, 2g triammonium citrate, 0.15g K2HPO4, 0.5g MnSO4, 0.2g MgSO4, and 1mL Tween 80 dissolved in 1L distilled water, and sterilized at 121℃ under high temperature and pressure.

[0066] Fermentation time: 24 hours, fermentation temperature: 30 degrees Celsius.

[0067] The *Eurotium cristatum* strain, strain number CICC 2099, is from the China Industrial Microbial Culture Collection Center. The yeast strain is *Kluyveromyces*. 1726.

[0068] (2) After mixing the black tea with the fermentation substrate evenly, place it in a fermentation bucket, add the activated microbial liquid, and carry out solid-state aerobic fermentation. The fermentation temperature is 30℃, the fermentation time is 10 days, and the pile is turned over 3 times a day.

[0069] The mass ratio of the mixed black tea and fermentation substrate to the added activated microbial liquid is 1:1. The height of the black tea and fermentation substrate in the fermentation tank is about 10cm. The fermentation substrate is a mixture of five grains, namely wheat kernels and rice, with a mass ratio of 1:1. The mass ratio of black tea to fermentation substrate is 1:1.

[0070] (7) After the product of fermentation in step (2) is crushed, it is placed at 80°C for 2 hours and then cooled to 5°C by liquid nitrogen. Then, the extract is added under stirring and the extraction is continued for 2 hours. After standing and filtering, the filtrate is taken and concentrated at low temperature to the same mass as the black tea raw material to obtain the black tea extract.

[0071] The extract consists of water and a phosphate buffer solution with a pH of approximately 7, in a mass ratio of 10:1; the mass ratio of the extract to the fermented product is 10:1.

[0072] This embodiment also provides a cream, the raw materials for which are prepared include:

[0073] The ingredients include 68.483 parts water, 7 parts glycerin, 4 parts cetyl ethylhexanoate, 3 parts cyclopentamethoxysiloxane, 2 parts polydimethylsiloxane, 1.125 parts cetearyl alcohol, 0.375 parts cetearyl glucoside, 1 part betaine, 0.6 parts glyceryl stearate, 0.4 parts PEG-100 stearate, 1 part 1,2-pentanediol, 0.12 parts butylene glycol, 0.015 parts Cynanchum atratum extract, 0.006 parts Sophora flavescens root extract, 0.006 parts Gynostemma pentaphyllum extract, 0.22 parts carbomer, 0.2 parts bisabolol, 0.2 parts arginine, 0.15 parts tocopheryl acetate, 0.03 parts xanthan gum, 0.03 parts (daily use) fragrance, 0.02 parts sodium hyaluronate, 0.02 parts disodium EDTA, and 10 parts of the aforementioned black tea extract.

[0074] Example 4

[0075] This embodiment provides a black tea extract containing a compound strain of *Gynostemma pentaphyllum* undergoing solid-state aerobic fermentation. The preparation method is as follows:

[0076] (1) Inoculate the golden flower fungus and yeast at a 1:1 ratio, with a total inoculation amount of 1%, into two identical liquid culture media for separate activation culture.

[0077] The liquid culture medium formula is as follows: 3g yeast powder, 10g peptone, 10g sucrose, 15g soluble starch, 5g sodium acetate, 2g triammonium citrate, 0.15g K2HPO4, 0.5g MnSO4, 0.2g MgSO4, and 1mL Tween 80 dissolved in 1L distilled water, and sterilized at 121℃ under high temperature and pressure.

[0078] Fermentation time: 24 hours, fermentation temperature: 30 degrees Celsius.

[0079] The *Eurotium cristatum* strain, strain number CICC 2099, is from the China Industrial Microbial Culture Collection Center. The yeast strains are *Kluyveromyces* and *Hansenula polymorpha*; specifically, *Kluyveromyces*. 1726 and the abnormal variant of Hansenula anomalavar. 1639, with a vaccination ratio of 6:4.

[0080] (2) After mixing the black tea with the fermentation substrate evenly, place it in a fermentation bucket, add the activated inoculum liquid, and carry out solid-state aerobic fermentation. The fermentation temperature is 30℃, the fermentation time is 10 days, and the pile is turned 3 times a day. The mass ratio of black tea to fermentation substrate is 1:1.

[0081] The mass ratio of the mixed black tea and fermentation substrate to the added activated microbial liquid is 1:1. The height of the black tea and fermentation substrate in the fermentation tank is about 10cm. The fermentation substrate is a mixture of five grains, namely wheat kernels and rice, in a mass ratio of 1:1.

[0082] (8) After the product of fermentation in step (2) is crushed, it is placed at 80°C for 2 hours and then cooled to 5°C by liquid nitrogen. Then, the extract is added under stirring and extracted for 2 hours. After standing and filtering, the filtrate is taken and concentrated at low temperature to the same mass as the raw material of black tea to obtain the black tea extract.

[0083] The extract consists of water and a phosphate buffer solution with a pH of approximately 7, in a mass ratio of 10:1; the mass ratio of the extract to the fermented product is 10:1.

[0084] This embodiment also provides a cream, the raw materials for which are prepared include:

[0085] The ingredients include 68.483 parts water, 7 parts glycerin, 4 parts cetyl ethylhexanoate, 3 parts cyclopentamethoxysiloxane, 2 parts polydimethylsiloxane, 1.125 parts cetearyl alcohol, 0.375 parts cetearyl glucoside, 1 part betaine, 0.6 parts glyceryl stearate, 0.4 parts PEG-100 stearate, 1 part 1,2-pentanediol, 0.12 parts butylene glycol, 0.015 parts Cynanchum atratum extract, 0.006 parts Sophora flavescens root extract, 0.006 parts Gynostemma pentaphyllum extract, 0.22 parts carbomer, 0.2 parts bisabolol, 0.2 parts arginine, 0.15 parts tocopheryl acetate, 0.03 parts xanthan gum, 0.03 parts (daily use) fragrance, 0.02 parts sodium hyaluronate, 0.02 parts disodium EDTA, and 10 parts of the aforementioned black tea extract.

[0086] Example 5

[0087] This embodiment provides a black tea extract containing a compound strain of *Gynostemma pentaphyllum* undergoing solid-state aerobic fermentation. The preparation method is as follows:

[0088] (1) Mix the golden flower fungus and yeast at a 1:1 inoculation ratio, and inoculate a total of 1% into the liquid culture medium for activation culture.

[0089] The liquid culture medium formula is as follows: 3g yeast powder, 10g peptone, 10g sucrose, 15g soluble starch, 5g sodium acetate, 2g triammonium citrate, 0.15g K2HPO4, 0.5g MnSO4, 0.2g MgSO4, and 1mL Tween 80 dissolved in 1L distilled water, and sterilized at 121℃ under high temperature and pressure.

[0090] Fermentation time: 24 hours, fermentation temperature: 30 degrees Celsius.

[0091] The *Eurotium cristatum* strain, strain number CICC 2099, is from the China Industrial Microbial Culture Collection Center. The yeast strains are *Kluyveromyces* and *Hansenula polymorpha*; specifically, *Kluyveromyces*. 1726 and the abnormal variant of Hansenula anomalavar. 1639, with a vaccination ratio of 6:4.

[0092] (2) Place the black tea in a fermentation bucket, add the activated bacterial liquid, and carry out solid-state aerobic fermentation. The fermentation temperature is 30℃ and the fermentation time is 10 days. Turn the pile 3 times a day.

[0093] The mass ratio of the black tea to the added activated microbial inoculum is 1:1. The height of the black tea in the fermentation tank is about 5cm.

[0094] (9) After the product of fermentation in step (2) is crushed, it is placed at 80°C for 2 hours and then cooled to 5°C by liquid nitrogen freezing. Then, the extract is added under stirring and extracted for 2 hours. After standing and filtering, the filtrate is taken and concentrated at low temperature to the same mass as the black tea raw material to obtain the black tea extract.

[0095] The extract consists of water and a phosphate buffer solution with a pH of approximately 7, in a mass ratio of 10:1; the mass ratio of the extract to the fermented product is 10:1.

[0096] This embodiment also provides a cream, the raw materials for which are prepared include:

[0097] The ingredients include 68.483 parts water, 7 parts glycerin, 4 parts cetyl ethylhexanoate, 3 parts cyclopentamethoxysiloxane, 2 parts polydimethylsiloxane, 1.125 parts cetearyl alcohol, 0.375 parts cetearyl glucoside, 1 part betaine, 0.6 parts glyceryl stearate, 0.4 parts PEG-100 stearate, 1 part 1,2-pentanediol, 0.12 parts butylene glycol, 0.015 parts Cynanchum atratum extract, 0.006 parts Sophora flavescens root extract, 0.006 parts Gynostemma pentaphyllum extract, 0.22 parts carbomer, 0.2 parts bisabolol, 0.2 parts arginine, 0.15 parts tocopheryl acetate, 0.03 parts xanthan gum, 0.03 parts (daily use) fragrance, 0.02 parts sodium hyaluronate, 0.02 parts disodium EDTA, and 10 parts of the aforementioned black tea extract.

[0098] Example 6

[0099] This embodiment provides a black tea extract containing a compound strain of *Gynostemma pentaphyllum* undergoing solid-state aerobic fermentation. The preparation method is as follows:

[0100] (1) Mix the golden flower fungus and yeast at a 1:1 inoculation ratio, and inoculate a total of 1% into the liquid culture medium for activation culture.

[0101] The liquid culture medium formula is as follows: 3g yeast powder, 10g peptone, 10g sucrose, 15g soluble starch, 5g sodium acetate, 2g triammonium citrate, 0.15g K2HPO4, 0.5g MnSO4, 0.2g MgSO4, and 1mL Tween 80 dissolved in 1L distilled water, and sterilized at 121℃ under high temperature and pressure.

[0102] Fermentation time: 24 hours, fermentation temperature: 30 degrees Celsius.

[0103] The *Eurotium cristatum* strain, strain number CICC 2099, is from the China Industrial Microbial Culture Collection Center. The yeast strains are *Kluyveromyces* and *Hansenula polymorpha*; specifically, *Kluyveromyces*. 1726 and the abnormal variant of Hansenula anomalavar. 1639, with a vaccination ratio of 6:4.

[0104] (2) After mixing the black tea with the fermentation substrate evenly, place it in a fermentation bucket, add the activated bacterial liquid, and carry out solid-state aerobic fermentation. The fermentation temperature is 30℃, the fermentation time is 10 days, and the pile is turned over 3 times a day.

[0105] The mass ratio of the mixed black tea and fermentation substrate to the added activated microbial liquid is 1:1. The height of the black tea and fermentation substrate in the fermentation tank is approximately 10cm. The fermentation substrate consists of five grains, namely wheat kernels and rice, in a 1:1 mass ratio. In this embodiment, the fermentation substrate is ground and pulverized before being mixed with the black tea. The mass ratio of black tea to fermentation substrate is 1:1.

[0106] After the product from step (2) is crushed, it is placed at 80°C for 2 hours and then cooled to 5°C by liquid nitrogen freezing. Then, the extract is added under stirring and extracted for 2 hours. After standing and filtering, the filtrate is taken and concentrated at low temperature to the same mass as the raw material of black tea to obtain the black tea extract.

[0107] The extract consists of water and a phosphate buffer solution with a pH of approximately 7, in a mass ratio of 10:1; the mass ratio of the extract to the fermented product is 10:1.

[0108] This embodiment also provides a cream, the raw materials for which are prepared include:

[0109] The ingredients include 68.483 parts water, 7 parts glycerin, 4 parts cetyl ethylhexanoate, 3 parts cyclopentamethoxysiloxane, 2 parts polydimethylsiloxane, 1.125 parts cetearyl alcohol, 0.375 parts cetearyl glucoside, 1 part betaine, 0.6 parts glyceryl stearate, 0.4 parts PEG-100 stearate, 1 part 1,2-pentanediol, 0.12 parts butylene glycol, 0.015 parts Cynanchum atratum extract, 0.006 parts Sophora flavescens root extract, 0.006 parts Gynostemma pentaphyllum extract, 0.22 parts carbomer, 0.2 parts bisabolol, 0.2 parts arginine, 0.15 parts tocopheryl acetate, 0.03 parts xanthan gum, 0.03 parts (daily use) fragrance, 0.02 parts sodium hyaluronate, 0.02 parts disodium EDTA, and 10 parts of the aforementioned black tea extract.

[0110] Example 7

[0111] This embodiment provides a black tea extract containing a compound strain of *Gynostemma pentaphyllum* undergoing solid-state aerobic fermentation. The preparation method is as follows:

[0112] (1) Mix the golden flower fungus and yeast at a 1:1 inoculation ratio, and inoculate a total of 1% into the liquid culture medium for activation culture.

[0113] The liquid culture medium formula is as follows: 3g yeast powder, 10g peptone, 10g sucrose, 15g soluble starch, 5g sodium acetate, 2g triammonium citrate, 0.15g K2HPO4, 0.5g MnSO4, 0.2g MgSO4, and 1mL Tween 80 dissolved in 1L distilled water, and sterilized at 121℃ under high temperature and pressure.

[0114] Fermentation time: 24 hours, fermentation temperature: 30 degrees Celsius.

[0115] The *Eurotium cristatum* strain, strain number CICC 2099, is from the China Industrial Microbial Culture Collection Center. The yeast strains are *Kluyveromyces* and *Hansenula polymorpha*; specifically, *Kluyveromyces*. 1726 and the abnormal variant of Hansenula anomalavar. 1639, with a vaccination ratio of 6:4.

[0116] (2) Soak the black tea raw material in 10 times the volume of boiling water and boil for 10 minutes. Then cool it to room temperature. After that, mix it evenly with the fermentation substrate and place it in a fermentation bucket. Add the activated bacterial liquid and carry out aerobic fermentation. The fermentation temperature is 30℃ and the fermentation time is 10 days. Stir and mix 3 times a day.

[0117] The mass ratio of the mixed black tea raw material and fermentation substrate to the added activated microbial liquid is 1:1. The fermentation substrate consists of five grains, namely wheat kernels and rice, in a mass ratio of 1:1.

[0118] (10) After the product of fermentation in step (2) is crushed, it is placed at 80°C for 2 hours and then frozen to 5°C. Then, the extract is added under stirring and extracted for 2 hours. After standing and filtering, the filtrate is taken and concentrated at low temperature to the same mass as the black tea raw material to obtain the black tea extract.

[0119] The extract consists of water and a phosphate buffer solution with a pH of approximately 7, in a mass ratio of 10:1; the mass ratio of the extract to the fermented product is 1:1.

[0120] This embodiment also provides a cream, the raw materials for which are prepared include:

[0121] The ingredients include 68.483 parts water, 7 parts glycerin, 4 parts cetyl ethylhexanoate, 3 parts cyclopentamethoxysiloxane, 2 parts polydimethylsiloxane, 1.125 parts cetearyl alcohol, 0.375 parts cetearyl glucoside, 1 part betaine, 0.6 parts glyceryl stearate, 0.4 parts PEG-100 stearate, 1 part 1,2-pentanediol, 0.12 parts butylene glycol, 0.015 parts Cynanchum atratum extract, 0.006 parts Sophora flavescens root extract, 0.006 parts Gynostemma pentaphyllum extract, 0.22 parts carbomer, 0.2 parts bisabolol, 0.2 parts arginine, 0.15 parts tocopheryl acetate, 0.03 parts xanthan gum, 0.03 parts (daily use) fragrance, 0.02 parts sodium hyaluronate, 0.02 parts disodium EDTA, and 10 parts of the aforementioned black tea extract.

[0122] Performance testing

[0123] 1. Hyaluronidase inhibition experiment:

[0124] Hyaluronidase inhibition experiment

[0125] The test was conducted according to the hyaluronidase inhibition rate of HMC-W1-029.

[0126] Examples 1-11: Dilute the sample with pure water to a concentration of 0.2%;

[0127] Positive control (dipotassium glycyrrhizate, purity ≥98%): diluted with water to a positive control concentration of 3%;

[0128] Negative control: pure water.

[0129] Experimental operation steps:

[0130] The design included an example control group, a positive control group, and a negative control group. Each group had three replicates. Different reagent solutions were added to each group, the mixture was shaken, and the mixture was left at room temperature for 30 minutes to develop color. The absorbance value at a wavelength of 528 nm was measured using a UV spectrophotometer.

[0131] Calculation formula

[0132] Hyaluronidase inhibition rate (%) = (1 - (CD) / (AB)) × 100

[0133] Where A is the absorbance of the reaction solution without the sample; B is the absorbance of the reaction solution without the sample and enzyme; C is the absorbance of the reaction solution containing the sample and enzyme; and D is the absorbance of the reaction solution containing the sample and without the enzyme.

[0134] Statistical analysis was performed using SPSS software. Independent sample t-tests were used to compare the hyaluronidase inhibition rates of the test samples, positive controls, and negative controls. All statistical analyses were two-tailed tests, with a significance level of α = 0.05. P > 0.05 indicated no significant difference between the two groups; P < 0.05 indicated a significant difference between the two groups.

[0135] Table 1. Experimental Results

[0136] name Experimental results p-value Example 1 72.783±0.321 ＜0.05 Example 2 60.324±0.153 ＜0.05 Example 3 64.153±0.325 ＜0.05 Example 4 68.231±0.522 ＜0.05 Example 5 56.324±0.231 ＜0.05 Example 6 58.312±0.134 ＜0.05 Example 7 49.382±0.457 ＜0.05 negative control -4.132±0.231 / Positive control 50.982±0.531 ＜0.05

[0137] Hyaluronidase inhibition rate is retained to three decimal places. P < 0.05 indicates that there is a significant difference between the sample and the positive control and the negative control.

[0138] Performance Test 2: Elastase Inhibition Rate

[0139] Fermentation filtrate: Dilute with pure water to a sample concentration of 5%;

[0140] Positive control (epigallocatechin gallate): diluted with water to a positive control concentration of 0.1%;

[0141] Negative control: pure water.

[0142] Experimental operation steps:

[0143] Design sample group, sample background group, solvent group and solvent background group. Each group should be set up in 3 parallels. Add different reagent solutions to 96-well plates, shake gently, incubate at 25°C for 15 min, and then place them in a microplate reader to measure the absorbance at 410 nm.

[0144] Calculation formula: Elastase inhibition rate (%) = (1 - (CD) / (AB)) × 100

[0145] Where A is the absorbance of the reaction solution without the sample; B is the absorbance of the reaction solution without the sample and enzyme; C is the absorbance of the reaction solution containing the sample and enzyme; and D is the absorbance of the reaction solution containing the sample and without the enzyme.

[0146] Statistical analysis was performed using SPSS software. Independent template t-tests were used to compare the elastase inhibition rates of the test samples, positive controls, and negative controls. All statistical analyses were two-tailed tests, with a significance level of α = 0.05. P > 0.05 indicated no significant difference between the two groups, while P < 0.05 indicated a significant difference between the two groups.

[0147] Table 2. Experimental Results

[0148]

[0149]

[0150] The elastase inhibition rate was retained to three decimal places. P < 0.05 indicates that there was a significant difference between the sample and the positive control and the negative control.

[0151] Performance Test 3:

[0152] Difference in transepidermal moisture loss rate

[0153] Healthy subjects aged 25-60 (all subjects already had sensitive skin characteristics, such as allergies, redness, etc.), 12 males and 38 females, with an average age of 37.12 years.

[0154] Part used: hand and forearm

[0155] Instructions for use: When used on the forearm, the test sample concentration is (2.0±0.1) mg / cm². 2 Apply the sample in a single coat using the specified amount of latex, and use a latex finger cot to evenly coat the sample within the test area.

[0156] Test steps

[0157] Subjects did not allow water to come into contact with the inside of their arms before the visit.

[0158] Before the experiment, all participants were required to clean their arms with a dry tissue.

[0159] Two measurement areas are marked on the inside of the subject's left or right arm. Each area is 3cm x 3cm and the distance between the areas is at least 1cm.

[0160] Sample and negative control areas were randomly distributed within the calibration area to ensure statistically balanced locations for each area. Subjects exposed their arms and rested for 30 minutes in an environment with a temperature of 20±1℃ and a relative humidity of 50±10%RH. An inducer (0.1% histamine solution) was applied to the sample and negative control areas for 10 minutes. Before application and sample use (Timm), the transepidermal water loss rate of the calibration area was measured using a Tewameter, and the hemoglobin of the calibration area was measured using a Mexamter.

[0161] Use the sample in the designated sample area according to the sample usage instructions;

[0162] After the sample was used, the subjects rested with their arms exposed in an environment with a temperature of 20±1℃ and a relative humidity of 50±10%RH for 2 hours; 1 hour later (T1), the transepidermal water loss rate of the calibrated area was measured using a Tewameter; 1 hour later (T1), the hemoglobin of the calibrated area was measured using a Mexamter.

[0163] Subjects completed self-assessment questionnaires at 5 minutes (immediately), 30 minutes, and 2 hours after sample administration, and completed satisfaction questionnaires 2 hours later.

[0164] Difference in transepidermal water loss rate

[0165] The Courage+Khazaka Tewameter TM300 transepidermal water loss and evaporative heat loss test probe is used to detect the transepidermal water loss rate of the skin. The transepidermal water loss rate difference refers to the difference in transepidermal water loss rate of the calibrated area before sample application and 1 hour after sample application.

[0166] The calculation formula is:

[0167] Difference in transepidermal water loss rate = Transepidermal water loss rate (Timm) - Transepidermal water loss rate (T1).

[0168] Table 3 Comparison of transepidermal water loss rate before and after.

[0169]

[0170]

[0171] Heme difference.

[0172] The Courage+Khazaka Mexamter MX18 skin melanin and hemoglobin analyzer is used to detect skin hemoglobin. Hemoglobin difference refers to the difference in hemoglobin levels in the calibrated area before and 1 hour after sample application. The calculation formula is as follows:

[0173] Heme difference = Heme (Timm) - Heme (T1).

[0174] Table 4 Comparison of heme levels before and after heme

[0175] Example Similarly, before (mean ± standard deviation) Sample size 1 hour after use (mean ± standard deviation) Example 1 304.42±23.14 156.13±32.12 Example 2 304.21±15.27 171.32±38.52 Example 3 303.94±27.43 167.45±27.42 Example 4 304.32±36.51 163.13±30.12 Example 5 304.73±28.43 183.54±54.12 Example 6 304.48±36.74 180.11±12.54 Example 7 304.18±43.64 201.43±11.42 negative area 304.72±47.64 287.32±65.34

[0176] Performance Test 4:

[0177] The products prepared in Examples 1-7 were placed in the open at 0℃, 25℃, and 40℃ for 90 days, and their properties and color were observed. The test results are shown in Table 5.

[0178] Table 5 shows the test results of performance test 4.

[0179] Example 0℃ 25℃ 40℃ Example 1 No change No change No change Example 2 The color darkened The color darkened The color darkened Example 3 No change No change The color darkened Example 4 No change No change No change Example 5 The color darkened The color darkened The color darkened Example 6 No change No change No change Example 7 The color darkened The color darkened The color darkened

[0180] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cream, characterized in that, Its preparation materials include: Water 68.483 parts, Glycerin 7 parts, Cetyl ethylhexanoate 4 parts, Cyclopentasiloxane 3 parts, Polydimethylsiloxane 2 parts, Cetearyl alcohol 1.125 parts, Cetearyl glucoside 0.375 parts, Betaine 1 part, Glyceryl stearate 0.6 parts, PEG-100 stearate 0.4 parts, 1,2-Pentanediol 1 part, Butylene glycol 0.12 parts, Cynanchum atratum extract 0.015 parts, Sophora flavescens root extract 0.006 parts, Gynostemma pentaphyllum extract 0.006 parts, Carbomer 0.22 parts, Bisabolol 0.2 parts, Arginine 0.2 parts, Tocopheryl acetate 0.15 parts, Xanthan gum 0.03 parts, Daily fragrance 0.03 parts, Sodium hyaluronate 0.02 parts, Disodium EDTA 0.02 parts, Black tea extract 10 parts; The preparation method of the dark tea extract is as follows: (1) Mix the golden flower fungus and yeast at a 1:1 inoculation ratio, and inoculate a total of 1% into the liquid culture medium for activation culture. The liquid culture medium formula is as follows: 3g yeast powder, 10g peptone, 10g sucrose, 15g soluble starch, 5g sodium acetate, 2g triammonium citrate, 0.15g K2HPO4, 0.5g MnSO4, 0.2g MgSO4, and 1mL Tween 80 dissolved in 1L distilled water, and sterilized at 121℃ under high temperature and pressure. Fermentation time: 24 hours, fermentation temperature: 30 degrees Celsius; The *Eurotium cristatum* strain, strain number CICC 2099, is from the China Industrial Microbial Culture Collection Center. The yeast strains are *Kluyveromyces* and *Hansenula polymorpha*, specifically *Kluyveromyces*. 1726 and the aberrant variant of Hansenula anomala var. 1639, vaccination ratio 6:4; (2) After mixing the black tea with the fermentation substrate evenly, place it in a fermentation bucket, add the activated microbial liquid, and carry out solid-state aerobic fermentation. The fermentation temperature is 30℃, the fermentation time is 10 days, and the pile is turned 3 times a day. The mass ratio of black tea to fermentation substrate is 1:

1. The mass ratio of the black tea and fermentation substrate mixture to the added activated microbial liquid is 1:1; the height of the black tea and fermentation substrate in the fermentation tank is about 10cm; the fermentation substrate is five grains, namely wheat kernels and rice, with a mass ratio of 1:

1. After the product from step (2) is crushed, it is placed at 80°C for 2 hours and then cooled to 5°C by liquid nitrogen freezing. Then, the extract is added under stirring and extracted for 2 hours. After standing and filtering, the filtrate is taken and concentrated at low temperature to the same mass as the raw material of black tea to obtain the black tea extract. The extract consists of water and a phosphate buffer solution with a pH of approximately 7, in a mass ratio of 10:1; the mass ratio of the extract to the fermented product is 10:1.

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