Method for carrying out solid aerobic fermentation by using eurotium cristatum-containing composite strain and application thereof

Abstract

The invention relates to the technical field of daily chemicals, in particular to a dark tea extract containing eurotium cristatum composite strains for solid aerobic fermentation. The composite strains comprise eurotium cristatum and saccharomycetes. The dark tea extract provided by the invention is stable in performance and not easy to discolor, contains components such as polysaccharide, amino acid and mineral substances, and can help skin moisturize. The permeability of the components is enhanced in the fermentation process, moisture can be locked on the surface layer of the skin, and the moisture degree of the skin is kept. The skin care product has very strong antioxidant capacity, and can neutralize free radicals, slow down the oxidation process of skin, delay skin aging and reduce wrinkles and fine wrinkles. A certain calming effect can be achieved on the skin which is prone to redness and itching or has slight inflammation.

Description

Technical Field

[0001] The invention relates to the technical field of daily chemicals, and in particular to a method for solid-state aerobic fermentation containing a composite strain of golden flower fungi and an application thereof. Background Art

[0002] Eurotium cristatum is a fungus belonging to the phylum Ascomycota. It is commonly associated with the fermentation process of fermented tea (such as Anhua dark tea's "Fuzhuan Tea") and is particularly famous for the "golden flowers" produced during the fermentation process. The yellow or golden spore clusters formed by the golden flower fungus are a unique mark in fermented products and are widely studied because of their significant impact on the quality and flavor of tea.

[0003] During the fermentation process, Jinhua fungus improves the aroma, taste and quality of tea through its metabolites, increasing the mellowness of tea and the content of active ingredients. It can decompose macromolecules such as polysaccharides and proteins in tea and convert them into easily absorbed small molecules, thereby improving the taste of tea.

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

[0005] Dark tea is a post-fermented tea, one of the six major types of tea in China. It is named for its dark brown appearance and is mainly produced in Hunan, Hubei, Sichuan, Yunnan, Guangxi and other places in China. During the production process, dark tea is fermented by microorganisms. As the storage time increases, the quality and flavor of the tea will gradually change, forming a unique aged fragrance and mellow taste.

[0006] The invention provides a dark tea extract obtained by solid-state aerobic fermentation of a composite strain of golden flower fungus. Summary of the invention

[0007] The invention provides a dark tea extract obtained by solid-state aerobic fermentation of a composite strain of golden flower fungi, wherein the composite strain comprises golden flower fungi and yeast.

[0008] As an embodiment of the present invention, the black tea extract is used in the field of daily chemicals.

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

[0010] (1) Activating the bacterial strains in a culture medium;

[0011] (2) mixing the bacterial strain and dark tea for solid-state aerobic fermentation;

[0012] (3) extracting the solid aerobic fermentation product with water to obtain a dark tea extract.

[0013] As an implementation mode of the present invention, the specific steps of step (1) are as follows:

[0014] Mix the golden flower bacteria and yeast at an inoculation ratio of 1:1, and inoculate a total of 1% of the inoculation amount into the liquid culture medium for mixed activation culture;

[0015] The formula of the liquid culture medium 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, 1mL Tween 80 dissolved in 1L distilled water, sterilized at 121℃ with high temperature and high pressure;

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

[0017] As an implementation mode of the present invention, the specific steps of step (2) are as follows:

[0018] After mixing the dark tea and the fermentation substrate evenly, place them in a fermentation barrel, add the activated bacterial liquid, and carry out solid aerobic fermentation. The fermentation temperature is 30°C, the fermentation time is 10 days, and the pile is turned 3 times a day.

[0019] As an embodiment of the present invention, the mass ratio of the mixture of the dark tea and the fermentation substrate to the mass ratio of the activated bacterial liquid added is 1:1.

[0020] As an embodiment of the present invention, the specific steps of step (3) are as follows: the product after fermentation in step (2) is crushed, placed at 80°C for 2 hours, and then rapidly frozen to 5°C; then, the extract is added under stirring, the extraction is continued for 2 hours, the mixture is allowed to stand and filtered, the filtrate is taken, and low-temperature concentrated to the same mass as the dark tea raw material, thereby obtaining the dark tea extract.

[0021] As an embodiment of the present invention, the dark tea extract is used in the field of daily chemicals in an amount of 1 to 20 wt%.

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

[0023] The black tea extract provided by the present invention has stable performance, is not easy to change color, contains ingredients such as polysaccharides, amino acids and minerals, and can help moisturize the skin. The fermentation process enhances the permeability of these ingredients, helps to lock moisture in the surface layer of the skin, and maintains the moisture of the skin. It has a 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. It can have a certain calming effect on skin that is prone to redness, itching or slight inflammation. DETAILED DESCRIPTION

[0024] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] The present invention provides a dark tea extract obtained by solid-state aerobic fermentation of a composite strain of golden flower fungus, and the preparation method thereof is as follows:

[0026] (1) Mixing golden flower bacteria and yeast at an inoculation ratio of 1:1, with a total inoculation amount of 1% inoculated into a liquid culture medium for mixed activation culture;

[0027] The formula of the liquid culture medium 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, 1mL Tween 80 dissolved in 1L distilled water, sterilized at 121℃ with high temperature and high pressure;

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

[0029] The golden flower fungus in the present invention is Eurotium cristatum, strain number: CICC 2099, China Industrial Microbiological Culture Collection Management Center. The yeast is Kluyveromyces and Hansenula; specifically Kluyveromyces marxianus 1726 and Hansenula anomala var. 1639, the inoculation ratio is 6:4.

[0030] (2) After mixing the dark tea and the fermentation substrate evenly, place them in a fermentation barrel, add the activated bacterial liquid, and carry out solid aerobic fermentation. The fermentation temperature is 30°C, the fermentation time is 10 days, and the pile is turned over 3 times a day.

[0031] The mass ratio of the mixed dark tea and fermentation substrate to the activated bacterial liquid added is 1:1. The height of the dark tea and the fermentation substrate in the fermentation barrel is about 10 cm, and the fermentation substrate is grains, namely wheat kernels and rice, with a mass ratio of 1:1.

[0032] (4) The fermented product of step (2) is crushed, placed at 80° C. for 2 h, and then rapidly frozen to 5° C.; then, the extract is added under stirring, and the extraction is continued for 2 h. The filtrate is filtered and concentrated at low temperature to the same mass as the dark tea raw material, thereby obtaining the dark tea extract.

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

[0034] As an embodiment of the present invention, the dark tea extract is used in the field of daily chemicals in an amount of 1 to 20 wt%.

[0035] As an embodiment of the present invention, the present invention also provides a cream, the preparation raw materials of which include:

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

[0037] There are several disadvantages of using black tea extract in the field of skin care: 1. The effect is not obvious enough; 2. The color is not stable. The reason is that the inventor found that half of the ingredients of black tea extract are melted by hot water. In addition, some polyphenols in black tea will be partially oxidized after fermentation, and will be further oxidized during storage, so that the color gradually darkens. In this regard, the inventor found that after further fermentation with the strains of black tea itself, better effective ingredients can be extracted under mild conditions; however, it was found that the amino acid content would drop rapidly during this process. Therefore, other strains were added in the early fermentation, and some low-matter ingredients were added. In addition, the water content, temperature, and fermentation time can be effectively controlled.

[0038] The present invention is further explained below in conjunction with specific implementation modes.

[0039] Example 1

[0040] This embodiment provides a dark tea extract comprising a composite strain of golden flower fungi subjected to solid-state aerobic fermentation, and the preparation method thereof is as follows:

[0041] (1) Mixing golden flower bacteria and yeast at an inoculation ratio of 1:1, with a total inoculation amount of 1% inoculated into a liquid culture medium for mixed activation culture;

[0042] The formula of the liquid culture medium 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, 1mL Tween 80 dissolved in 1L distilled water, sterilized at 121℃ with high temperature and high pressure;

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

[0044] The golden flower fungus is Eurotium cristatum, strain number: CICC 2099, China Industrial Microbiological Culture Collection Management Center. The yeast is Kluyveromyces and Hansenula; specifically Kluyveromyces marxianus 1726 and Hansenula anomalavar. 1639, the inoculation ratio is 6:4.

[0045] (2) After mixing the dark tea and the fermentation substrate evenly, place them in a fermentation barrel, add the activated bacterial liquid, and carry out solid-state aerobic fermentation, wherein the fermentation temperature is 30°C, the fermentation time is 10 days, and the pile is turned over 3 times a day. The mass ratio of dark tea to fermentation substrate is 1:1.

[0046] The mass ratio of the mixed dark tea and fermentation substrate to the activated bacterial liquid added is 1:1. The height of the dark tea and the fermentation substrate in the fermentation barrel is about 10 cm, and the fermentation substrate is grains, namely wheat kernels and rice, with a mass ratio of 1:1.

[0047] (5) The fermented product of step (2) is crushed, placed at 80° C. for 2 h, and then cooled to 5° C. by liquid nitrogen freezing; then, the extract is added under stirring, and the extraction is continued for 2 h. The filtrate is filtered and concentrated at low temperature to the same mass as the dark tea raw material, thereby obtaining the dark tea extract.

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

[0049] This embodiment also provides a cream, the preparation materials of which include:

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

[0051] Example 2

[0052] This embodiment provides a dark tea extract comprising a composite strain of golden flower fungi subjected to solid-state aerobic fermentation, and the preparation method thereof is as follows:

[0053] (1) inoculating 1% of the inoculum of golden flower fungus into a liquid culture medium for mixed activation culture;

[0054] The formula of the liquid culture medium 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, 1mL Tween 80 dissolved in 1L distilled water, sterilized at 121℃ with high temperature and high pressure;

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

[0056] The golden flower fungus is Eurotium cristatum, strain number: CICC 2099, China Industrial Microbiological Culture Collection Management Center. (2) The dark tea and the fermentation substrate are evenly mixed and placed in a fermentation barrel, and the activated strain liquid is added to perform solid aerobic fermentation, wherein the fermentation temperature is 30°C, the fermentation time is 10 days, and the pile is turned 3 times a day.

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

[0058] (6) The product after fermentation in step (2) is crushed, placed at 80° C. for 2 h, and then cooled to 5° C. by liquid nitrogen freezing; then, the extract is added under stirring, and the extraction is continued for 2 h. The filtrate is taken and concentrated at low temperature to the same mass as the dark tea raw material, thereby obtaining the dark tea extract.

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

[0060] This embodiment also provides a cream, the preparation materials of which include:

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

[0062] Example 3

[0063] This embodiment provides a dark tea extract comprising a composite strain of golden flower fungi subjected to solid-state aerobic fermentation, and the preparation method thereof is as follows:

[0064] (1) Mixing golden flower bacteria and yeast at an inoculation ratio of 1:1, with a total inoculation amount of 1% inoculated into a liquid culture medium for mixed activation culture;

[0065] The formula of the liquid culture medium 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, 1mL Tween 80 dissolved in 1L distilled water, sterilized at 121℃ with high temperature and high pressure;

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

[0067] The golden flower fungus is Eurotium cristatum, strain number: CICC 2099, China Industrial Microbiological Culture Collection Management Center. The yeast is Kluyveromyces marxianus 1726.

[0068] (2) After mixing the dark tea and the fermentation substrate evenly, place them in a fermentation barrel, add the activated bacterial liquid, and carry out solid-state aerobic fermentation. The fermentation temperature is 30°C, the fermentation time is 10 days, and the pile is turned over 3 times a day.

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

[0070] (7) The fermented product of step (2) is crushed, placed at 80° C. for 2 h, and then cooled to 5° C. by liquid nitrogen freezing; then, the extract is added under stirring, and the extraction is continued for 2 h. The filtrate is filtered and concentrated at low temperature to the same mass as the dark tea raw material, thereby obtaining the dark tea extract.

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

[0072] This embodiment also provides a cream, the preparation materials of which include:

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

[0074] Example 4

[0075] This embodiment provides a dark tea extract comprising a composite strain of golden flower fungi subjected to solid-state aerobic fermentation, and the preparation method thereof is as follows:

[0076] (1) Inoculating golden flower fungus and yeast in two identical liquid culture media at an inoculation ratio of 1:1, with a total inoculation amount of 1%, respectively, for separate activation culture;

[0077] The formula of the liquid culture medium 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, 1mL Tween 80 dissolved in 1L distilled water, sterilized at 121℃ with high temperature and high pressure;

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

[0079] The golden flower fungus is Eurotium cristatum, strain number: CICC 2099, China Industrial Microbiological Culture Collection Management Center. The yeast is Kluyveromyces and Hansenula; specifically Kluyveromyces marxianus 1726 and Hansenula anomalavar. 1639, the inoculation ratio is 6:4.

[0080] (2) After mixing the dark tea and the fermentation substrate evenly, place them in a fermentation barrel, add the activated bacterial liquid, and carry out solid-state aerobic fermentation, wherein the fermentation temperature is 30°C, the fermentation time is 10 days, and the pile is turned over 3 times a day. The mass ratio of dark tea to fermentation substrate is 1:1.

[0081] The mass ratio of the mixed dark tea and fermentation substrate to the activated bacterial liquid added is 1:1. The height of the dark tea and the fermentation substrate in the fermentation barrel is about 10 cm, and the fermentation substrate is grains, namely wheat kernels and rice, with a mass ratio of 1:1.

[0082] (8) The product after fermentation in step (2) is crushed, placed at 80° C. for 2 h, and then cooled to 5° C. by liquid nitrogen freezing; then, the extract is added under stirring, and the extraction is continued for 2 h. The filtrate is filtered and concentrated at low temperature to the same mass as the dark tea raw material, thereby obtaining the dark tea extract.

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

[0084] This embodiment also provides a cream, the preparation materials of which include:

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

[0086] Example 5

[0087] This embodiment provides a dark tea extract comprising a composite strain of golden flower fungi subjected to solid-state aerobic fermentation, and the preparation method thereof is as follows:

[0088] (1) Mixing golden flower bacteria and yeast at an inoculation ratio of 1:1, with a total inoculation amount of 1% inoculated into a liquid culture medium for mixed activation culture;

[0089] The formula of the liquid culture medium 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, 1mL Tween 80 dissolved in 1L distilled water, sterilized at 121℃ with high temperature and high pressure;

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

[0091] The golden flower fungus is Eurotium cristatum, strain number: CICC 2099, China Industrial Microbiological Culture Collection Management Center. The yeast is Kluyveromyces and Hansenula; specifically Kluyveromyces marxianus 1726 and Hansenula anomalavar. 1639, the inoculation ratio is 6:4.

[0092] (2) Place the dark tea in a fermentation barrel, add the activated bacterial liquid, and carry out solid aerobic fermentation at a temperature of 30°C for 10 days, with the pile turned over three times a day.

[0093] The mass ratio of the dark tea to the activated bacterial liquid is 1:1. The height of the dark tea in the fermentation barrel is about 5 cm.

[0094] (9) The product after fermentation in step (2) is crushed, placed at 80° C. for 2 h, and then cooled to 5° C. by liquid nitrogen freezing; then, the extract is added under stirring, and the extraction is continued for 2 h. The filtrate is filtered and concentrated at low temperature to the same mass as the dark tea raw material, thereby obtaining the dark tea extract.

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

[0096] This embodiment also provides a cream, the preparation materials of which include:

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

[0098] Example 6

[0099] This embodiment provides a dark tea extract comprising a composite strain of golden flower fungi subjected to solid-state aerobic fermentation, and the preparation method thereof is as follows:

[0100] (1) Mixing golden flower bacteria and yeast at an inoculation ratio of 1:1, with a total inoculation amount of 1% inoculated into a liquid culture medium for mixed activation culture;

[0101] The formula of the liquid culture medium 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, 1mL Tween 80 dissolved in 1L distilled water, sterilized at 121℃ with high temperature and high pressure;

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

[0103] The golden flower fungus is Eurotium cristatum, strain number: CICC 2099, China Industrial Microbiological Culture Collection Management Center. The yeast is Kluyveromyces and Hansenula; specifically Kluyveromyces marxianus 1726 and Hansenula anomalavar. 1639, the inoculation ratio is 6:4.

[0104] (2) After mixing the dark tea and the fermentation substrate evenly, place them in a fermentation barrel, add the activated bacterial liquid, and carry out solid aerobic fermentation. The fermentation temperature is 30°C, the fermentation time is 10 days, and the pile is turned over 3 times a day.

[0105] The mass ratio of the mixed dark tea and fermentation substrate to the activated bacterial liquid added is 1:1. The height of the dark tea and the fermentation substrate in the fermentation barrel is about 10 cm, and the fermentation substrate is grains, namely wheat kernels and rice, with a mass ratio of 1:1. In this embodiment, the fermentation substrate is ground and crushed and then mixed with the dark tea. The mass ratio of dark tea to the fermentation substrate is 1:1.

[0106] The product after fermentation in step (2) is crushed, 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 the extraction is continued for 2 hours. The filtrate is taken and concentrated at low temperature to the same mass as the dark tea raw material, thereby obtaining the dark tea extract.

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

[0108] This embodiment also provides a cream, the preparation materials of which include:

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

[0110] Example 7

[0111] This embodiment provides a dark tea extract comprising a composite strain of golden flower fungi subjected to solid-state aerobic fermentation, and the preparation method thereof is as follows:

[0112] (1) Mixing golden flower bacteria and yeast at an inoculation ratio of 1:1, with a total inoculation amount of 1% inoculated into a liquid culture medium for mixed activation culture;

[0113] The formula of the liquid culture medium 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, 1mL Tween 80 dissolved in 1L distilled water, sterilized at 121℃ with high temperature and high pressure;

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

[0115] The golden flower fungus is Eurotium cristatum, strain number: CICC 2099, China Industrial Microbiological Culture Collection Management Center. The yeast is Kluyveromyces and Hansenula; specifically Kluyveromyces marxianus 1726 and Hansenula anomalavar. 1639, the inoculation ratio is 6:4.

[0116] (2) Soak the dark tea raw material in 10 times the volume of boiling water, boil it for 10 minutes, and then cool it to room temperature. Then, mix it evenly with the fermentation substrate and place it in a fermentation barrel. Add the activated strain liquid and perform aerobic fermentation. The fermentation temperature is 30°C and the fermentation time is 10 days. Stir and mix it 3 times a day.

[0117] The mass ratio of the mixed dark tea raw material and the fermentation substrate to the activated bacterial liquid added is 1:1. The fermentation substrate is grains, namely wheat kernels and rice, with a mass ratio of 1:1.

[0118] (10) The fermented product of step (2) is crushed, placed at 80° C. for 2 h, and then frozen to 5° C.; then, the extract is added under stirring, and the extraction is continued for 2 h. The mixture is allowed to stand and filtered, and the filtrate is collected and concentrated at low temperature to the same mass as the dark tea raw material, thereby obtaining the dark tea extract.

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

[0120] This embodiment also provides a cream, the preparation materials of which include:

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

[0122] Performance Testing

[0123] 1. Hyaluronidase inhibition test:

[0124] Hyaluronidase inhibition assay

[0125] The test is based on the HMC-W1-029 hyaluronidase inhibition rate.

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

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

[0128] Negative control: pure water.

[0129] Test operation steps:

[0130] Design the embodiment group, positive control group and negative control group, and set up 3 parallels for each group. Add different reagent solutions respectively, shake well, and place at room temperature for 30 minutes for color development. Use ultraviolet spectrophotometer to measure the absorbance value at a wavelength of 528nm.

[0131] Calculation formula

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

[0133] Wherein, A is the absorbance of the reaction solution without sample; B is the absorbance of the reaction solution without sample and enzyme; C is the absorbance of the reaction solution containing sample and enzyme; D is the absorbance of the reaction solution containing sample and no enzyme.

[0134] The statistical analysis software was SPSS, and the comparison of hyaluronidase inhibition rates among the test samples, positive controls, and negative controls was performed using an independent sample t test. The above statistical analyses were all two-tailed tests, with a significance level of α = 0.05, P > 0.05, indicating no significant difference between the two groups; P < 0.05, indicating a significant difference between the two groups.

[0135] Table 1 Test 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] The hyaluronidase inhibition rate was rounded to three decimal places, and P < 0.05 indicated that there were significant differences 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] Test operation steps:

[0143] Design the sample group, sample background group, solvent group and solvent background group. Set up 3 parallels for each group. Add different reagent solutions into the 96-well plate, shake gently, incubate at 25°C for 15 minutes, place it in the microplate reader, and measure the absorbance at 410nm.

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

[0145] Wherein, A is the absorbance of the reaction solution without sample; B is the absorbance of the reaction solution without sample and enzyme; C is the absorbance of the reaction solution containing sample and enzyme; D is the absorbance of the reaction solution containing sample and no enzyme.

[0146] The statistical analysis software was SPSS, and the comparison of the elastase inhibition rates of the test samples, positive controls and negative controls was performed using an independent sample t test. The above statistical analyses were all two-tailed tests, with a significance level of α = 0.05, P > 0.05, indicating that there was no significant difference between the two groups; P < 0.05, indicating that there was a significant difference between the two groups.

[0147] Table 2 Test results

[0148]

[0149]

[0150] The elastase inhibition rate was rounded to three decimal places, and P < 0.05 indicated that there were significant differences between the sample and the positive control and the negative control.

[0151] Performance Test 3:

[0152] Transepidermal water loss difference

[0153] Healthy subjects aged 25-60 years old (all subjects already have sensitive skin characteristics, such as easy allergies, red blood streaks, etc.), including 12 males and 38 females, with an average age of 37.12 years.

[0154] Part used: hand and forearm

[0155] Usage: When used on the forearm, the test sample is (2.0±0.1) mg / cm 2 Apply a single application of the dosage and use a latex finger cot to evenly spread the sample in the test area.

[0156] Test steps

[0157] The subjects did not touch the inner part of their arms with water before the visit.

[0158] Before the experiment, the subjects were required to uniformly clean their arms with dry facial tissue;

[0159] Mark two measurement areas on the inside of the subject's left or right arm, each area is 3cm*3cm, and the distance between the areas is at least 1cm;

[0160] The sample area and negative control area were randomly distributed in the calibration area to ensure that each area was statistically balanced: the subjects exposed their arms and rested for 30 minutes in an environment with a temperature of 20±1°C and a relative humidity of 50±10%RH; the inducer (0.1% histamine solution) was applied to the sample area and the negative area for 10 minutes, and before the application was completed and before the sample was used (Timm), the transepidermal water loss rate of the calibration area was tested with Tewameter, and the hemoglobin in the calibration area was tested with Mexamter.

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

[0162] After using the sample, 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; after 1 hour (T1), the transepidermal water loss rate of the calibrated area was tested with a Tewameter; after 1 hour (T1), the hemoglobin in the calibrated area was tested with a Mexamter.

[0163] The subjects filled in the self-assessment questionnaire 5 minutes (immediately), 30 minutes, and 2 hours after using the sample, and filled in the satisfaction questionnaire 2 hours later.

[0164] Transepidermal water loss rate difference

[0165] Courage+Khazaka Transepidermal Water Loss and Water Evaporation Heat Loss Test Probe Tewameter TM300 is used to detect the transepidermal water loss rate of the skin. The difference in transepidermal water loss rate refers to the difference in the transepidermal water loss rate of the calibrated area before and 1 hour after the sample is used.

[0166] The calculation formula is:

[0167] Transepidermal water loss rate difference = 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] Courage+Khazaka Skin Melanin and Hemoglobin Tester Mexamter MX18 is used to detect skin hemoglobin. The hemoglobin difference refers to the difference in hemoglobin in the calibration area before and after 1 hour of sample use. The calculation formula is:

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

[0174] Table 4 Comparison of hemoglobin before and after

[0175] Example Same as before (mean ± standard deviation) After 1 hour of 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 to 7 were placed at 0°C, 25°C, and 40°C in an open state for 90 days, and the properties and color of the products were checked. The test results are shown in Table 5.

[0178] Table 5 Test results of performance test 4

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

[0180] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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 method for solid-state aerobic fermentation using a composite strain of golden flower fungi, characterized in that: The composite bacterial strain comprises golden flower fungus and yeast.

2. A dark tea extract, characterized in that: Prepared by the method described in claim 1.

3. The dark tea extract according to claim 2, characterized in that The extraction steps are as follows: (1) Activating the bacteria in a culture medium; (2) mixing the bacterial strain and dark tea for solid-state aerobic fermentation; (3) extracting the solid aerobic fermentation product with water to obtain a dark tea extract.

4. The dark tea extract according to claim 3, characterized in that The specific steps of step (1) are as follows: Mix the golden flower bacteria and yeast at an inoculation ratio of 1:1, and inoculate a total of 1% of the inoculation amount into the liquid culture medium for mixed activation culture; The formula of the liquid culture medium 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, 1mL Tween 80 dissolved in 1L distilled water, sterilized at 121℃ with high temperature and high pressure; Fermentation time: 24h, fermentation temperature 30 degrees Celsius.

5. The dark tea extract according to claim 3, characterized in that The specific steps of step (2) are as follows: After mixing the dark tea and the fermentation substrate evenly, place them in a fermentation barrel, add the activated bacterial liquid, and carry out solid aerobic fermentation. The fermentation temperature is 30°C, the fermentation time is 10 days, and the pile is turned 3 times a day.

6. The dark tea extract according to claim 5, characterized in that The mass ratio of the mixed dark tea and fermentation substrate to the added activated bacterial liquid is 1:

1.

7. The dark tea extract according to claim 3, characterized in that The specific steps of step (3) are as follows: the fermented product of step (2) is crushed, placed at 80°C for 2 hours, and then rapidly frozen to 5°C; then, the extract is added under stirring, the extraction is continued for 2 hours, the mixture is allowed to stand and filtered, the filtrate is taken, and low-temperature concentrated to the same mass as the dark tea raw material, thereby obtaining the dark tea extract.

8. The dark tea extract according to claim 2, characterized in that The dosage is 1-20wt% and is used in the field of daily chemicals.

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