Preparation method and application of black tea fermented liquid with anti-aging effect

Abstract

The present invention relates to the field of tea fermentation, and in particular to a method for preparing a black tea fermentation liquid with anti-aging effects and its application. The present invention adopts Pichia kudrida and Bacillus to ferment black tea to prepare a black tea fermentation liquid rich in tea polyphenols, thearubigins, theaflavins and theabrownins. The black tea fermentation liquid has the functions of regulating cell metabolism and function, repairing cell oxidative damage, maintaining the integrity of mitochondrial function, promoting the synthesis of high-energy phosphate compounds ATP, enhancing cell activity, and anti-aging, and can be applied to food, cosmetics, and health products. The fermentation process provided by the present invention is green and safe, the method is simple, and has practical application significance.

Description

Technical Field

[0001] The invention relates to the field of tea fermentation, and in particular to a preparation method and application of black tea fermentation liquid with anti-aging effect. Background Art

[0002] Black tea is made from suitable new buds and leaves of tea trees, and is refined through a series of processes such as withering, rolling, fermentation, and drying. It is a fully fermented tea. In the context of Chinese tea, the "fermentation" of black tea is actually completely different from microbial fermentation. It is actually a series of biological enzymatic reactions. That is, after the plant cell wall is damaged by "rolling", the polyphenol oxidase (PPO) and peroxidase (POD) originally present in the cell wall are released. During the "fermentation" period, enzymatic oxidation oxidizes the tea polyphenols in the cell fluid into theaflavins, thearubigins and theabrownins. Finally, it is "baked at high temperature". The high temperature inactivates the enzyme to prevent excessive fermentation and quickly evaporates water.

[0003] Theaflavins (TFs) are one of the important quality components of black tea and are known as the soft gold of tea. Theaflavins are a mixture with a benzophenone structure, mainly composed of catechins and gallocatechins through oxidative condensation. Recent studies have shown that theaflavins have similar functions to tea polyphenols, and are even more effective than tea polyphenols in terms of anti-lipid peroxidation and anti-DNA damage, lowering blood lipids, and anti-cancer. Thearubigins (TRs) are a general term for a class of complex heterogeneous phenolic compounds, with a relative molecular weight of 700~40000 Da, mainly composed of oxidative polymerization and condensation of catechins, theaflavins, etc., accounting for 9-19% of the total dry matter of black tea and about 75% of the polyphenols in black tea. According to modern pharmacological research, thearubigins also show strong antioxidant properties by regulating the activities of various enzymes in the body; and thearubigins can activate some proteins that remove toxins, control the production of some specific enzymes, and improve the body's immune function. Therefore, thearubigins have better anti-mutation, anti-tumor and anti-cancer effects than catechins. Theabrownin (TB) is derived from the further oxidation of polyphenols such as theaflavins and thearubigins, and exists naturally in black tea, green tea and dark tea. Theabrownin is very beneficial to improving the comprehensive metabolic balance of the human body, and has the effects of lowering blood sugar, blood lipids, blood pressure, uric acid, etc. It is also a promising anti-cancer candidate substance. Patent CN111840412A describes the use of theabrownin as a natural substance in drugs to inhibit melanoma. Patent CN114145368A discloses a black tea leaf extract and a preparation method thereof, which uses a special ratio of hard water and hot water to extract a black tea leaf extract with a high content of theabrownin. Although the antioxidant effect of theabrownin is lower than that of EGCG, the main component of tea polyphenols, in the ABTS test, the IC50 value of theabrownin is 13.21 ppm and the IC50 value of EGCG is 1.57 ppm; however, theabrownin has a more excellent anti-aging effect in restoring mitochondrial membrane potential and resisting UVB photoaging.

[0004] Tea polyphenols are one of the main components of tea leaves. They are a class of natural polyphenol compounds with a bitter taste, and have super strong antioxidant and intracellular active oxygen removal functions. So far, there are many patents for the preparation process of black tea fermentation liquid. The rich enzymes and metabolites of microbial metabolism are helpful to extract active ingredients such as amino acids, tea polyphenols, and theaflavins in black tea, and the fermentation liquid is rich in probiotic strains, such as lactic acid bacteria, lactobacillus, yeast, etc., and the metabolites can help the skin anti-aging, whitening and other effects. For example, patent CN115105452A ferments the black tea fermentation liquid with any one or at least two combinations of Saccharomyces cerevisiae, Candida krusei, or Brettanomyces brettanomyces for the first fermentation, and uses lactic acid bacteria, lactobacillus and acetic acid bacteria for the second fermentation, so as to achieve multiple fermentations of multiple probiotics and black tea fermentation liquid, so that the black tea fermentation filtrate is rich in more probiotic metabolic components and has skin care effects. However, the tea polyphenol content in the black tea fermentation filtrate provided by the patent is only 13.5 g / L. The reason why the content of tea polyphenols is not high may be that the process of extracting black tea and then fermenting it is as follows: first, the conventional high-temperature extraction method may make the extraction rate of tea polyphenols in black tea low and easy to be oxidized; second, the active substances produced by multi-strain fermentation cause tea polyphenols to be oxidized to form components such as thearubigins and theabrownins. The patent does not deeply consider the conversion process and efficacy characteristics between tea polyphenols, theaflavins, thearubigins and theabrownins during the fermentation process. Patent CN116334146A discloses a black tea double yeast fermentation product, cosmetics and its preparation method and application, which uses black tea as the fermentation base and is obtained by liquid aerobic fermentation of powdered Pichia pastoris and Saccharomyces cerevisiae. Under the fermentation conditions described in the patent, the release of tea polyphenols from black tea cells can be promoted, the content of tea polyphenols contained in the black tea fermentation filtrate can be increased, and the black tea fermentation product has a significant antioxidant effect. However, the existing research and related patents do not consider the content, conversion degree and efficacy differentiation of tea polyphenols and theaflavins, thearubigins and theabrownins. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for preparing a black tea fermentation liquid with anti-aging effect, by using a specific double bacterial strain for fermentation to prepare a black tea fermentation liquid rich in tea polyphenols, thearubigins, theaflavins and theabrownins.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a method for preparing a black tea fermentation broth, comprising the following steps:

[0008] (1) culturing Pichia kudelni and Bacillus in liquid culture medium to obtain Pichia kudelni seed solution and Bacillus seed solution respectively; the Bacillus is a Bacillus that secretes tannase;

[0009] (2) adding black tea powder to a liquid culture medium to obtain a fermentation medium, inoculating the Pichia kudrida seed liquid and the Bacillus seed liquid obtained in step (1) to ferment and obtain a crude fermentation liquid;

[0010] (3) The crude fermentation liquid obtained in step (2) is inactivated, filtered, and blended to obtain the black tea fermentation liquid.

[0011] The present invention has been found through a large number of experimental studies that a black tea fermentation liquid rich in tea polyphenols, thearubigins, theaflavins and theabrownins can be prepared by fermenting black tea with specific dual strains: Pichia kudrida and Bacillus. Among them, Pichia kudrida is also known as "Oriental Issaffron", which has a rich metabolic enzyme system and can promote the rupture of black tea cells to release active substances such as tea polyphenols (mainly catechins), thearubigins, theaflavins, and theabrownins. The Bacillus strain used in the present invention can secrete tannase during the fermentation process, and tannase can break the ester bond between catechol and gallic acid, so that the ester-type catechins are hydrolyzed and converted into the more rare and difficult-to-obtain thearubigins and theaflavins, and further converted into theabrownins. The present invention selects Pichia kudrida and Bacillus for joint fermentation, which has its own innovation and uniqueness: Pichia kudrida can be used as a "control switch" of tannase: on the one hand, it can quickly reduce organic acids such as lactic acid and acetic acid produced in the fermentation process of Bacillus, maintain the pH balance of the fermentation system, and maintain the activity of tannase; on the other hand, polyols such as ethanol are produced in the fermentation process of Pichia kudrida, which can inhibit the activity of tannase when reaching a certain amount, and "turn off" the conversion, thereby achieving a dynamic balance of tea polyphenols, thearubigins, theaflavins and theabrownins.

[0012] Preferably, the tannase-secreting bacillus includes at least one of Lactobacillus acidophilus, Lactobacillus plantarum, Acetobacter xylinum, Bacillus terequila, and Bacillus subtilis.

[0013] More preferably, the tannase-secreting bacillus is Lactobacillus acidophilus.

[0014] Preferably, in the step (1), the Pichia kudrida and Bacillus are first subjected to plate subculture and recovery culture before inoculation into the liquid culture medium; the culture conditions are activation culture at a temperature of 25-35°C for 2-3 generations, with each generation culture time of 12-24 hours.

[0015] Preferably, in step (1), the concentration of the strains in the Pichia kudrida seed solution and the Bacillus seed solution is not less than 1×10 8 CFU / mL.

[0016] More preferably, the concentration of the strains in the Pichia kudrida seed solution and the Bacillus seed solution is 1×10 8 CFU / mL.

[0017] Preferably, in steps (1) and (2), the liquid culture medium comprises the following components in weight percentage based on a total weight percentage of 1000 parts: 5-15 parts of peptone, 5-15 parts of beef extract, 3-7 parts of yeast extract, 15-25 parts of glucose, 1.5-3.0 parts of citric acid, 2.0-3.5 parts of sodium citrate, 1-3 parts of MgSO4·7H2O, 0.05-0.1 parts of MnSO4·4H2O, and the balance of deionized water; the initial pH of the liquid culture medium is 4.0-5.0.

[0018] The culture medium used in the present invention is a traditional culture medium, which is suitable for industrial production, easy to operate, and has good stability. Adjusting the pH to slightly acidic can make the culture medium more suitable for the growth of microorganisms, thereby affecting the fermentation effect.

[0019] Preferably, the black tea is selected from Fujian Wuyishan Zhengshan Xiaozhong, Huangshan Keemun black tea, Zhaoping black tea, Huohong, Dianhong, Yuehong, Quanchenghong, Suhong, Sichuanhong or Dongjiang Chuyunxian black tea.

[0020] Preferably, in step (2), the black tea powder is obtained by crushing and sterilizing black tea; the particle size of the crushed tea is 100 mesh; and the sterilization conditions are sterilization at a pressure of 0.1 MPa and a temperature of 121° C. for 20 min.

[0021] Preferably, in step (2), the mass percentage of black tea powder in the fermentation medium is 2-5%.

[0022] Preferably, the total inoculation amount of the Pichia kudrida seed solution and the Bacillus seed solution is 4%-10% of the total volume of the liquid culture medium; the volume ratio of the inoculation amount of the Pichia kudrida seed solution to the inoculation amount of the Bacillus seed solution is (2-5): (2-6).

[0023] During the fermentation of Bacillus, a large amount of organic acids such as acetic acid or lactic acid will be produced, and too low pH will inhibit the activity of biological enzymes such as tannase. The present invention has found through a large number of experimental studies that when the volume ratio of Pichia kudrida seed liquid and Bacillus seed liquid is the above-mentioned specific ratio, Pichia kudrida acts as a "control switch" for tannase, regulates the conversion endpoint between tea polyphenols and pigments such as theaflavins, controls the degree of deep conversion, and inhibits excessive conversion of theabrownins. Thus, a black tea fermentation liquid rich in tea polyphenols, theaflavins, thearubigins, and theabrownins is obtained, especially theaflavins and thearubigins therein, so that it has the effects of regulating cell metabolism and function, repairing mitochondrial oxidative damage, enhancing cell vitality, and anti-aging.

[0024] More preferably, the volume ratio of the inoculation amount of the Pichia kuderma seed solution to the inoculation amount of the Bacillus seed solution is 1:1.

[0025] Preferably, in step (2), the fermentation conditions are: during the fermentation process, the ventilation volume is 0.5-1.0 vvm, the fermentation temperature is 25-35°C, the rotation speed is 100-300 r / min, the fermentation time is 24-72 h, and the pH is 3.0-5.0.

[0026] More preferably, in step (2), the fermentation conditions are: fermentation temperature 28°C, rotation speed 150 r / min, and fermentation time 48 h.

[0027] Preferably, in step (3), the inactivation condition is 90-100°C for 15-30 min.

[0028] Preferably, in step (3), the filtration includes coarse filtration and fine filtration; the filtration accuracy of the coarse filtration is 1.2 μm; the filtration accuracy of the fine filtration is 0.45 μm.

[0029] Preferably, the coarse filtration is performed using a plate-and-frame filter or a diatomaceous earth filter, and the filtration area of ​​the plate-and-frame filter is 1 m 2 The filtration accuracy is 1.2μm; the fine filtration is performed by a horizontal centrifuge or a ceramic membrane filter, and the filtration accuracy is 0.45μm.

[0030] Preferably, the preparation method is to add a preservative into the black tea fermentation broth.

[0031] Preferably, the preservative comprises the following components: at least one of butylene glycol, hexylene glycol, pentylene glycol, p-hydroxyacetophenone, ethylhexylglycerin, and caprylhydroxamic acid.

[0032] Preferably, the preservative comprises the following components in parts by weight: 2-5 parts of 1,3-butanediol, 0.3-1 parts of 1,2-hexanediol, and 0.3-1 parts of p-hydroxyacetophenone.

[0033] In a second aspect, the present invention provides a black tea fermented liquid prepared by the above-mentioned method for preparing black tea fermented liquid.

[0034] In a third aspect, the present invention provides the use of the above-mentioned black tea fermentation liquid in food, cosmetics and health products.

[0035] The black tea fermentation liquid obtained by the preparation method of the present invention is rich in active components such as tea polyphenols, thearubigins, theaflavins, theabrownins, etc., and is applied to food, cosmetics, and health care products. It has the effects of inhibiting lipid peroxidation of polyunsaturated fatty acids in mitochondrial membranes to generate malondialdehyde, restoring mitochondrial function, promoting the generation of mitochondrial high-energy phosphate compounds (ATP, the source of cell energy, and a key molecule for regulating cell energy metabolism), providing energy supply for cell activity and regeneration, and promoting cell secretion of collagen and inhibiting human aging.

[0036] The beneficial effects of the present invention are:

[0037] The present invention adopts Pichia kudrida, Bacillus and black tea for symbiotic fermentation to prepare a black tea fermentation liquid; the obtained black tea fermentation liquid is rich in tea polyphenols, thearubigins, theaflavins and theabrownins, has the effects of regulating cell metabolism and function, repairing mitochondrial oxidative damage, enhancing cell vitality, anti-aging and the like, and can be applied to food, cosmetics and health products.

[0038] The strains used in the invention are all beneficial and safe strains, and the prepared black tea fermentation liquid is rich in tea pigments such as tea polyphenols, thearubigins, theaflavins, theabrownins, etc., and has practical application significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 These are pictures of crow's feet of volunteers who tried the product in Example 1 at week 0, week 2, and week 4.

[0040] Figure 2 These are photos of crow's feet of volunteers in comparative example 1 who tried the product in week 0, week 2, and week 4. DETAILED DESCRIPTION

[0041] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0042] The strains used in the examples of the present invention and the comparative examples were all purchased from the market, among which Pichia kudriavzevii, Lactobacillus acidophilus, Bacillus tequilensis, Saccharomyces cerevisiae, and Millerozymafarinosa were all purchased from Beijing Biobo Biotechnology Co., Ltd.; Gluconacetobacterxylinus, Lactobacillus plantarum, and Bacillus subtilis were all purchased from ATCC, USA, and were imported.

[0043] Example 1

[0044] An embodiment of the method for preparing the black tea fermented liquid of the present invention comprises the following steps:

[0045] (1) Preparation of seed solution:

[0046] Take out Pichiakudriavzevii and Lactobacillus acidophilus from -80℃ freezer respectively, carry out plate subculture and resuscitation culture for 2-3 generations, each generation culture for 12 hours, to obtain resuscitated Pichiakudriavzevii and Lactobacillus acidophilus;

[0047] The revived strains were transferred to the culture medium for shaking culture to obtain a concentration of 1×10 8 CFU / mL of Pichia kudrida seed liquid and Lactobacillus acidophilus seed liquid; the culture medium includes the following components in mass: 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of glucose, 2.2 g of citric acid, 2.9 g of sodium citrate, 1 g of MgSO4·7H2O, 0.05 g of MnSO4·4H2O, and the total volume is supplemented with water to 1 L, and the initial pH is 4.6; the culture conditions are: temperature 28°C, culture time 24 h, and rotation speed 150 r / min.

[0048] (2) Fermentation process: The dried Keemun black tea was crushed into 100 mesh, and then sterilized at 0.1 MPa pressure and 121°C temperature for 20 min to obtain black tea powder; 2% of the black tea powder was added into the liquid culture medium and mixed evenly, and 3% of the seed liquid of Pichia kudrida and 3% of the seed liquid of Lactobacillus acidophilus were inoculated for fermentation; the fermentation conditions were: ventilation volume 0.8 vvm, fermentation temperature 28°C, rotation speed 150 r / min, fermentation time 48 h, pH 4.3; and crude fermentation liquid was obtained;

[0049] (3) Inactivation and filtration: After the fermentation is completed, the crude fermentation liquid is inactivated at 90°C for 20 min and filtered to obtain the black tea fermentation filtrate; the filtration condition is to first filter through a coarse filter plate and frame filter with a filtration area of ​​1 m 2 , the filtration accuracy is 1.2μm; then filtered through a precision filtration ceramic membrane filter, the filtration accuracy is 0.45μm.

[0050] (4) Preparation: Add a preservative (3% butylene glycol, 0.8% hexylene glycol, 0.3% p-hydroxyacetophenone) to the black tea fermentation filtrate to obtain the black tea fermentation liquid.

[0051] Example 2

[0052] An embodiment of the method for preparing the black tea fermentation liquid of the present invention, the difference between this embodiment and the preparation method described in Example 1 is that: Lactobacillus acidophilus in Example 1 is replaced with Lactobacillus plantarum, and the black tea powder is fermented by Pichia kudrida and Lactobacillus plantarum, and the other parameters are the same as those in Example 1.

[0053] Embodiment 3:

[0054] An embodiment of the method for preparing the black tea fermentation liquid of the present invention, the difference between this embodiment and the preparation method described in Example 1 is that: Lactobacillus acidophilus in Example 1 is replaced with Gluconacetobacterxylinus, and black tea powder is fermented by Pichia kudrivis and Gluconacetobacterxylinus, and the other parameters are the same as those in Example 1.

[0055] Embodiment 5:

[0056] An embodiment of the method for preparing the black tea fermentation liquid of the present invention, the difference between this embodiment and the preparation method described in Example 1 is that: Lactobacillus acidophilus in Example 1 is replaced with Bacillus tequilensis, and the black tea powder is fermented by Pichia kudrivis and Bacillus tequilensis, and the other parameters are the same as those in Example 1.

[0057] Embodiment 6:

[0058] An embodiment of the method for preparing the black tea fermentation liquid of the present invention, the difference between this embodiment and the preparation method described in Example 1 is that: Lactobacillus acidophilus in Example 1 is replaced with Bacillus subtilis, and black tea powder is fermented by Pichia kudrivis and Bacillus subtilis, and the other parameters are the same as those in Example 1.

[0059] Example 7

[0060] An embodiment of the method for preparing the black tea fermentation broth of the present invention, the difference between this embodiment and the preparation method described in Example 1 is that: the formula of the culture medium is different, and the culture medium used in this embodiment includes the following components by mass: 5 g peptone, 5 g beef extract, 3 g yeast extract, 15 g glucose, 1.5 g citric acid, 3.5 g sodium citrate, 1 g MgSO4·7H2O, 0.05 g MnSO4·4H2O, water is added to a total volume of 1 L, and the initial pH is 5.0; the other parameters are the same as those in Example 1.

[0061] Example 8

[0062] An embodiment of the method for preparing the black tea fermented liquid of the present invention is provided. The difference between this embodiment and the preparation method described in Example 1 is that in step (2) of this embodiment, the mass percentage of black tea powder added is 5%, and the other parameters are the same as those in Example 1.

[0063] Example 9

[0064] An embodiment of the method for preparing the black tea fermentation liquid of the present invention is provided. The difference between this embodiment and the preparation method described in Example 1 is that in step (2) of this embodiment, the inoculation amount of Pichia kudrida seed liquid is 2%, and the inoculation amount of Lactobacillus acidophilus seed liquid is 2%. The other parameters are the same as those in Example 1.

[0065] Example 10

[0066] An embodiment of the method for preparing the black tea fermented liquid of the present invention is provided. The difference between this embodiment and the preparation method described in Example 1 is that in step (2) of this embodiment, the inoculation amount of Pichia kudrida seed liquid is 5%, the inoculation amount of Lactobacillus acidophilus seed liquid is 5%, and the other parameters are the same as those in Example 1.

[0067] Embodiment 11

[0068] An embodiment of the method for preparing the black tea fermentation liquid of the present invention is provided. The difference between this embodiment and the preparation method described in Example 1 is that in step (2) of this embodiment, the inoculation amount of Pichia kudrida seed liquid is 4%, and the inoculation amount of Lactobacillus acidophilus seed liquid is 6%. The other parameters are the same as those in Example 1.

[0069] Example 12

[0070] An embodiment of the method for preparing the black tea fermented liquid of the present invention is provided. The difference between this embodiment and the preparation method described in Example 1 is that the fermentation conditions in step (2) of this embodiment are as follows: the ventilation volume during the fermentation process is 1.0 vvm, the fermentation temperature is 35°C, the rotation speed is 300 r / min, the fermentation time is 72 h, and the pH is 3.0.

[0071] Comparative Example 1

[0072] A comparative example of the method for preparing the black tea fermented liquid of the present invention, the preparation method of this comparative example comprises the following steps:

[0073] (1) Preparation of seed solution:

[0074] Take out the preserved Pichia kudriavzevii from the -80℃ freezer, and perform plate subculture and recovery culture for 2-3 generations, with each generation cultured for 12 hours to obtain the recovered Pichia kudriavzevii;

[0075] The revived strain was transferred to the culture medium for shaking culture to obtain a concentration of 1×10 8 CFU / mL of Pichia kudrida seed liquid; the culture medium includes the following components in mass: 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of glucose, 2.2 g of citric acid, 2.9 g of sodium citrate, 1 g of MgSO4·7H2O, 0.05 g of MnSO4·4H2O, and the total volume is supplemented with water to 1 L, and the initial pH is 4.6; the culture conditions are: temperature 28°C, culture time 24 h, and rotation speed 150 r / min.

[0076] (2) Fermentation process: The dried Keemun black tea was crushed into 100 mesh, and then sterilized at 0.1 MPa pressure and 121°C temperature for 20 min to obtain black tea powder; 2% of the black tea powder was added into the liquid culture medium and mixed evenly, and 6% of the Pichia kudrida seed liquid was inoculated for fermentation; the fermentation conditions were: ventilation volume 0.8 vvm, fermentation temperature 28°C, rotation speed 150 r / min, fermentation time 48 h, pH 4.5; and crude fermentation liquid was obtained;

[0077] (3) Inactivation and filtration: After the fermentation is completed, the crude fermentation liquid is inactivated at 90°C for 20 min and filtered to obtain the black tea fermentation filtrate; the filtration condition is to first filter through a coarse filter plate and frame filter with a filtration area of ​​1 m 2 , the filtration accuracy is 1.2μm; then filtered through a precision filtration ceramic membrane filter, the filtration accuracy is 0.45μm.

[0078] (4) Preparation: Add a preservative (3% butylene glycol, 0.8% hexylene glycol, 0.3% p-hydroxyacetophenone) to the black tea fermentation filtrate to obtain the black tea fermentation liquid.

[0079] Comparative Example 2

[0080] A comparative example of the method for preparing the black tea fermented liquid of the present invention, the difference between this comparative example and the preparation method described in comparative example 1 is that the Pichiakudriavzevii in comparative example 1 is replaced by Lactobacillus acidophilus, and the other parameters are the same as those in comparative example 1.

[0081] Comparative Example 3

[0082] A comparative example of the method for preparing the black tea fermented liquid of the present invention, the difference between this comparative example and the preparation method described in comparative example 1 is that the Pichiakudriavzevii in comparative example 1 is replaced by Lactobacillus plantarum, and the other parameters are the same as those in comparative example 1.

[0083] Comparative Example 4

[0084] A comparative example of the method for preparing the black tea fermented liquid of the present invention, the difference between this comparative example and the preparation method described in comparative example 1 is that the Pichia kudriavzevii in comparative example 1 is replaced by Gluconacetobacter xylinus, and the other parameters are the same as those in comparative example 1.

[0085] Comparative Example 6

[0086] A comparative example of the method for preparing the black tea fermentation liquid of the present invention, the difference between this comparative example and the preparation method described in comparative example 1 is that the Pichia kudriavzevii in comparative example 1 is replaced with Bacillus equilensis, and the other parameters are the same as those in comparative example 1.

[0087] Comparative Example 7

[0088] A comparative example of the method for preparing the black tea fermented liquid of the present invention, the difference between this comparative example and the preparation method described in comparative example 1 is that the Pichia kudriavzevii in comparative example 1 is replaced by Bacillus subtilis, and the other parameters are the same as those in comparative example 1.

[0089] Comparative Example 8

[0090] A comparative example of the method for preparing the black tea fermentation liquid of the present invention, the difference between this comparative example and the preparation method described in Example 1 is that: the Pichiakudriavzevii in Example 1 is replaced by powdered Miller yeast (Millerozymafarinosa), Lactobacillus acidophilus (Lactobacillus acidophilus) is replaced by Saccharomyces cerevisiae (Saccharomyces cerevisiae), and the black tea powder is fermented by powdered Miller yeast and Saccharomyces cerevisiae (the two yeasts cannot produce tannase or produce very little), and the other parameters are the same as those in Example 1.

[0091] Comparative Example 9

[0092] A comparative example of the method for preparing the black tea fermented liquid of the present invention, the difference between the preparation method described in this comparative example 9 and that of Example 1 is that: the Pichiakudriavzevii in Example 1 is replaced by Saccharomyces cerevisiae, and the Lactobacillus acidophilus is replaced by Gluconacetobacterxylinus, and the black tea powder is fermented by using Saccharomyces cerevisiae and Gluconacetobacterxylinus, and the other parameters are the same as those in Example 1.

[0093] Comparative Example 10

[0094] A comparative example of the method for preparing the black tea fermentation broth of the present invention, the difference between this comparative example and the preparation method described in Example 1 is that the culture medium used in this example includes the following components by mass: 10 g peptone, 10 g beef extract, 5 g yeast extract, 20 g glucose, water is added to a total volume of 1 L, and the initial pH is 7.0; the other parameters are the same as those in Example 1.

[0095] Comparative Example 11

[0096] A comparative example of the method for preparing the black tea fermented liquid of the present invention, the difference between this comparative example and the preparation method described in Example 1 is that in step (2) of this comparative example, the inoculation amount of Pichia kudrida seed liquid is 2%, the inoculation amount of Lactobacillus acidophilus seed liquid is 8%, and the other parameters are the same as those in Example 1.

[0097] Comparative Example 12

[0098] A comparative example of the method for preparing the black tea fermented liquid of the present invention, the difference between this comparative example and the preparation method described in Example 1 is that in step (2) of this comparative example, the inoculation amount of Pichia kudrida seed liquid is 6%, the inoculation amount of Lactobacillus acidophilus seed liquid is 2%, and the other parameters are the same as those in Example 1.

[0099] Comparative Example 13

[0100] A comparative example of the method for preparing the black tea fermented liquid of the present invention, the difference between this comparative example and the preparation method of Example 1 is that the fermentation conditions of step (2) of this comparative example are: fermentation is carried out by anaerobic culture method, and oxygen is not passed through the fermentation tank. The other parameters are the same as those of Example 1.

[0101] Comparative Example 14

[0102] This comparative example provides a method for preparing a black tea extract (using a water extraction method), the preparation method comprising the following steps:

[0103] The dried Keemun black tea was crushed to 100 mesh, and then sterilized at 0.1Mpa pressure and 121℃ for 20 min to obtain black tea powder. Black tea powder with a mass percentage of 2% was added to deionized water; the mass ratio of the black tea powder to deionized water was 1:20; the mixture was stirred at 80℃ and a constant speed of 150 r / min, and extracted under condensation reflux conditions for 4 h; and filtered after hot water extraction. The filtering conditions were first filtered through a coarse filter plate and frame filter with a filtration area of ​​1 m 2 The filtration precision is 1.2 μm; and then filtered through a precision filtration ceramic membrane filter with a filtration precision of 0.45 μm. A preservative (3% butanediol, 0.8% hexanediol, 0.3% p-hydroxyacetophenone) is added to the filtrate to obtain the black tea extract.

[0104] Test Example 1:

[0105] This test example detects the contents of tea polyphenols, theaflavins, thearubigins and theabrownins in the black tea fermentation broth / extract obtained in Examples 1-12 and Comparative Examples 1-14.

[0106] The content of tea polyphenols was determined according to the method described in Part 4 of the national standard GB / T 8313-2018: Folin phenol reagent oxidized -OH in tea polyphenols to show blue color, and gallic acid was used as a calibration standard to quantify tea polyphenols at a maximum absorption wavelength of 765 nm. The qualitative and quantitative determination of theaflavins, thearubigins and theabrownins was carried out by solvent extraction colorimetry (reference: Wang, Q., Peng, C., Gong, J. Effects of enzymatic action on the formation of theabrownin during solid state fermentation of Puerhtea. J. Sci. Food Agric. 2011, 91, 2412-2418); in this method, organic solvents such as ethanol, ethyl acetate and n-butanol were used to extract theaflavins, thearubigins and theabrownins, and UV was used to detect the absorbance at 380 nm. The test results are shown in Table 1.

[0107] Table 1 Contents of tea polyphenols, theaflavins, thearubigins and theabrownins in black tea fermentation broth / extract

[0108]

[0109] As can be seen from Table 1, the contents of the four active ingredients of tea polyphenols, theaflavins, thearubigins and theabrownins in the black tea fermentation broth prepared in Examples 1-12 and Comparative Examples 1-14 are compared. Within the conditions specified in the present invention, the black tea fermentation broth prepared in the Examples is measured to have tea polyphenols, theaflavins, thearubigins and theabrownins contents all greater than 1.00 mg / mL, and the contents of theaflavins, thearubigins and theabrownins are not much different, which is relatively balanced; while the contents of the active ingredients in the comparative examples are quite different, and the content of tea polyphenols is significantly higher than that of other active ingredients, indicating that the conversion of active ingredients is not sufficient. The total amount of tea polyphenols, theaflavins, thearubigins and theabrownins contained in the black tea fermentation broth obtained in Examples 1-12 is significantly higher than the total amount of tea polyphenols, theaflavins, thearubigins and theabrownins contained in the black tea fermentation broth obtained in Comparative Examples 1-13 and the black tea extract prepared by water extraction in Comparative Example 14. In particular, the total amount of tea polyphenols, theaflavins, thearubigins and theabrownins contained in the black tea fermentation liquid of Example 8 is the highest.

[0110] The contents of theaflavins and thearubigins in the black tea fermentation broth prepared in the embodiment of the present invention are significantly higher than those in the comparative example, and the content of tea polyphenols is slightly lower than that in some comparative examples. The reason is that the symbiotic fermentation of Pichia kudrida and Bacillus is used in the fermentation process of the embodiment of the present invention to convert the tea polyphenols contained in the black tea fermentation broth into theaflavins, thearubigins and theabrownins. At the same time, Pichia kudrida is used as the conversion endpoint for regulating the conversion between pigments such as tea polyphenols and theaflavins, controls the degree of deep conversion, inhibits the excessive conversion of theabrownins, controls the content of theabrownins to be appropriate, and realizes the dynamic balance of the active ingredients in the fermentation broth, so that it can exert the maximum effect and improve the utilization rate. Among the four active components contained in the black tea fermentation broth in the embodiment of the present invention, the contents of theaflavins and thearubigins, which are known as "soft gold", are significantly higher than those in the comparative example, so that the final black tea fermentation broth has the effect of regulating cell vitality and immunity.

[0111] Compared with Example 1, in the black tea fermentation liquid obtained by fermentation with a single strain of Pichia kudridae in Comparative Example 1 and fermentation with powdered Miller yeast and Saccharomyces cerevisiae in Comparative Example 8, the content of tea polyphenols can reach about 4.0 mg / mL, but due to the lack of tannic acid-producing Bacillus for catalysis, the content of theaflavins and thearubigins in the obtained black tea fermentation liquid is low. In Comparative Example 9, black tea is fermented with Saccharomyces cerevisiae and Acetobacter xylinum, and the content of theaflavins and thearubigins in the black tea fermentation liquid is not high. The reason may be that Saccharomyces cerevisiae cannot regulate the conversion between tea polyphenols and pigments such as theaflavins during the fermentation process. Comparative Examples 2-7 use different tannase-producing Bacillus for single strain fermentation. Compared with Example 1, the theabrownin content is higher, all greater than 2.0 mg / mL. The reason is that during the fermentation process, the excessive catalytic reaction of tannase converts tea polyphenols, theaflavins, and thearubigins into tea brown in depth. In Comparative Example 10, the pH of the culture medium was 7.0, and in Comparative Example 13, anaerobic conditions were maintained during the fermentation process. Compared with Example 1, the metabolic activity of the strains was insufficient, so the contents of theaflavins and thearubigins were not high. In Comparative Example 11, the ratio of Pichia kudrida to Lactobacillus acidophilus was 1:4, and the contents of theaflavins and thearubigins in the fermentation broth were low, while the content of theabrownins was high. The reason for this was that the tannase regulation was unbalanced during the fermentation process, and the over-catalytic reaction converted theaflavins and thearubigins into tea brown. In Comparative Example 12, the ratio of Pichia kudrida to Lactobacillus acidophilus was 3:1, and the contents of theaflavins and thearubigins in the fermentation broth were not high, because the tannase was insufficient, and less tea polyphenols were converted into theaflavins and thearubigins.

[0112] The total content of tea polyphenols, theaflavins, thearubigins and theabrownins in the black tea extract obtained by water extraction in Comparative Example 14 is relatively low, indicating that the extraction effect of the active ingredients in black tea by the microbial fermentation process of the present invention is significantly better than that of the water extraction method.

[0113] Test Example 2:

[0114] This test example detects the changes in cell viability (cytotoxicity) of human skin keratinocytes (HaCaT) after treatment with black tea fermentation broth / extract.

[0115] Test samples: The black tea fermentation broth / extract obtained in Examples 1-12 and Comparative Examples 1-14 was diluted to different concentrations using cell culture medium (DMEM culture medium containing 10% fetal bovine serum FBS): 0.1%, 0.5%, 1.0%, 2.0%, 3.0%, 5.0%, 8.0%, 10.0%.

[0116] Test method:

[0117] (1) Cell culture: DMEM high-glucose culture medium containing 10% FBS was added to human skin keratinocytes (HaCaT) and cultured in a cell culture incubator. The culture conditions were: temperature 37°C, air content 95% (V / V), CO2 content 5% (V / V). The medium was changed every 2 days during the cell culture period.

[0118] (2) Detection of cell viability by MTT colorimetric method: HaCaT cells were cultured for 10 4 Cells were inoculated at a density of 100 μL / well in a 96-well cell culture plate, and 100 μL of different test samples were added to each well. The cells were placed in an incubator and incubated for 24 h, and then the cell morphology and characteristics were observed under a phase contrast inverted microscope. 100 μL of cell culture medium was added to each well of the blank control group; 100 μL of culture medium containing the corresponding concentration of the test sample was added to each well of the sample group; and the zero adjustment group had no cell inoculation in the well, and only 100 μL of cell culture medium was added.

[0119] After 24 h of incubation, discard the culture medium, add 100 μL MTT solution (0.5 mg / mL) to each well, incubate in the incubator for 4 h, remove the supernatant, and add 150 μL / well of DMSO. After shaking on a shaker at a constant temperature (37°C) for 10 min, measure the OD value (OD 570 nm) with an enzyme reader, and calculate the relative cell viability (Viability) of each group according to the following formula:

[0120] Viability(%)=

[0121] In the above formula, OD TA is the test value of the sample group, OD Blank is the test value of the zero adjustment group, OD NC This is the test value of the blank control group.

[0122] The test results are shown in Table 2 and Table 3.

[0123] Table 2 Cytotoxicity results of test samples in Example

[0124]

[0125] Table 3 Cytotoxicity results of comparative test samples

[0126]

[0127] The MTT test results show that the cytotoxicity of the black tea fermented liquid prepared in the embodiment of the present invention is lower than that of the black tea fermented liquid prepared in the comparative example: the cell safety concentration range of the black tea fermented liquid in the embodiment of the present invention is 0.1-10%, that is, the black tea fermented liquid has no obvious cytotoxicity in the concentration range of 0.1-10%, and the irritation is low; while the cell safety concentration range of the black tea fermented liquid in comparative examples 1, 8, 9, 10, 12 and 13 is 0.1-8.0%, that is, at a test concentration higher than 8%, the cell survival rate is lower than 80%, and the cytotoxicity is higher; The cell safety concentration range of the black tea fermentation broth of Examples 2, 3, 4, 6, 7, and 11 is 0.1%-5%, that is, the cell survival rate is lower than 80% at a test concentration higher than 5%, and the cytotoxicity is high. This may be because the use of single bacillus fermentation or mixed bacteria co-fermentation with a large inoculation amount produces excessive organic acids, resulting in a high organic acid content in the black tea fermentation broth, which is more irritating to cells; the black tea extract of Comparative Example 14 is prepared by water extraction method, and has lower irritation. In the test concentration range of 0.1-10%, the black tea extract has no obvious cytotoxicity.

[0128] From the above cell MTT test results, it can be seen that the type of dual-bacteria fermentation strains selected in the present invention and the changes in parameters in the fermentation process will affect the cytotoxicity of the black tea fermentation liquid.

[0129] Test Example 3:

[0130] This test case detects the effect of black tea fermentation liquid / extract on cell damage. Under normal conditions, the body can produce a small amount of ROS to participate in metabolism. At the same time, there is a reaction system in the body that removes free radicals and inhibits free radicals, so that excessive free radicals are removed. If this dynamic imbalance is unbalanced, excessive free radicals can directly act on the body and cause damage to the body. In the body, free radicals will attack polyunsaturated fatty acids in the biological membrane, triggering lipid peroxidation, and the end product of oxidation is malondialdehyde (MDA). MDA can cause cross-linking and polymerization of macromolecules such as proteins and nucleic acids, and is cytotoxic. MDA, the end product of lipid oxidation, affects the mitochondrial respiratory chain complex and the activity of key enzymes in mitochondria in vitro. Its production can also aggravate membrane damage. Therefore, testing the amount of malondialdehyde can reflect the degree of lipid peroxidation in the body and indirectly reflect the degree of cell damage.

[0131] Test principle: One malondialdehyde (MDA) molecule and two thiobarbituric acid (TBA) molecules are heated together under acidic conditions to form a pink complex. With a wavelength of 536 nm as the excitation light, the strongest fluorescence intensity is at 550 nm. The amount of malondialdehyde can be measured in micro amounts using the fluorescence method.

[0132] Test sample: The black tea fermentation liquid / extract obtained in Example 1-12 and Comparative Example 1-14 was diluted to a concentration of 3% and 5% using cell culture medium.

[0133] Test method:

[0134] (1) Cell incubation under UVA-induced model: Human skin keratinocytes (HaCaT) were prepared into a cell suspension and incubated at 1.0 × 10 5 -2.0×10 5 The cells were inoculated in 96-well plates and cultured in DMEM medium containing 10% FBS. After culturing for 24 h, they were divided into three groups for experiments:

[0135] ① Negative control group: UVA light (light dose of 6-10 J / cm 2 );

[0136] ② Experimental group: UVA light (light dose of 6-10 J / cm 2 );

[0137] ③Blank group: normal cells without UVA light induction.

[0138] After the illumination, the cell culture medium was removed and washed with PBS three times. Pure water was added to the negative control group, and 100 μL of 3% and 5% test sample solution (diluent) were added to the experimental group, respectively, and placed in a CO2 incubator for 20 min. After the incubation, 0.5 mL of physiological saline was added and allowed to stand at room temperature for 10 min, centrifuged at 2000 r / min for 10 min, and the supernatant was taken for testing.

[0139] (2) Preparation of standard curve: Dilute 10 nmol / mL tetraethoxypropane with double distilled water to 0.0, 0.25, 0.5, 1.0, 1.5, 2, 3, 5, and 10 nmol / mL, take 0.1 mL of the dilution solution, add 2 mL of acid hydrolysis solution and 0.5 mL of TBA working solution, mix well, protect from light, and place in a boiling water bath for 60 min; cool to room temperature with running water; add 3 mL of n-butanol and shake for 1 min, and centrifuge at 3000 r / min for 5 min. Then, take the supernatant (n-butanol layer) to detect the fluorescence intensity (incident slit 1.5 nm, exit slit 5 nm, excitation wavelength 536 nm, emission wavelength 550 nm). Plot the standard curve with tetraethoxypropane concentration as the horizontal axis and fluorescence intensity as the vertical axis.

[0140] (3) Sample testing process: Take 0.1 mL of the cell supernatant obtained in step (1), add 2 mL of acid hydrolysis solution and 0.5 mL of TBA working solution, mix well, protect from light, place in a boiling water bath for 60 min, cool to room temperature with running water, add 3 mL of n-butanol, shake and extract for 1 min, and centrifuge at 3000 r / min for 5 min. Then, take the supernatant (n-butanol layer) to detect the fluorescence intensity (incident slit 1.5 nm, exit slit 5 nm, excitation wavelength 536 nm, emission wavelength 550 nm)

[0141] The reagents added to each tube during the sample determination process are shown in Table 4:

[0142] Table 4 MDA test method reagent addition amount

[0143]

[0144] The calculation formula of MDA content (nmol / g), the final product of lipid peroxidation, in the cell supernatant is:

[0145] MDA content=(BA) / (FA)×C×K

[0146] Wherein, A represents the fluorescence intensity of the blank tube, B represents the fluorescence intensity of the sample, F represents the fluorescence intensity of tetraethoxypropane, C represents the concentration of tetraethoxypropane (1 nmol / g), and K represents the dilution factor.

[0147] The MDA content of the test samples was tested, and the experimental results are shown in Table 5:

[0148] Inhibition rate = (MDA content in negative control group - MDA content in sample test group) / (MDA content in negative control group - MDA content in blank control group) * 100%

[0149] Table 5

[0150]

[0151] The results of MDA content test showed that the MDA content in normal cells was 2.9 nmol / g. After UVA light exposure, lipid peroxidation occurred in the cells, and the content of malondialdehyde (MDA), the final product of lipid oxidation, increased to 10.2 nmol / g (negative control), which was about three times higher than the MDA content of normal cells. After the test sample was added to the sample test group, the intracellular MDA content decreased, indicating that black tea fermentation liquid / extract can inhibit lipid peroxidation.

[0152] The test results show that the inhibition effect of the black tea fermentation liquid / extract of Examples 1-12 and Comparative Examples 1-14 is significantly higher than that of the comparative sample test group. When the same mass fraction of black tea powder is added for fermentation, the black tea fermentation liquid of Example 1 has the best inhibition effect: when the concentration of the black tea fermentation liquid is 3%, MDA is reduced to 5.21nmol / g, and the inhibition rate is 68.4%, which is significantly higher than that of the comparative group (the inhibition rate of the comparative group is less than 60%); when the concentration of the black tea fermentation liquid is 5%, MDA is reduced to 3.0 nmol / g, which is close to the MDA content level of normal cells (2.9 nmol / g), and the inhibition rate is 98.6%, which is significantly higher than that of the comparative group (the inhibition rate of the comparative group is less than 82%). Further analysis of the inhibitory effect of the fermentation liquid on MDA found that the total content of tea polyphenols and theaflavins in the fermentation liquid was positively correlated with the inhibition rate of MDA.

[0153] From the above results, it can be seen that the type of dual-bacteria fermentation strains selected in the present invention and changes in parameters in the fermentation process will affect the antioxidant damage ability of the obtained black tea fermentation liquid, which is particularly important for removing MDA, inhibiting oxidative damage of cell membrane lipids, and reducing cell damage.

[0154] Test Example 4: Mitochondrial ATP Generation Test

[0155] This test case measures the function of black tea fermented liquid / extract in protecting mitochondria by detecting the generation of mitochondrial ATP. Mitochondria are the key factors affecting human aging. Aging is essentially the aging of cells, and mitochondria are the engine (heart) of cells, providing the energy required for cell activity and regeneration. If mitochondria are damaged, cell function will be impaired, leading to cell apoptosis, and the body will show disease and aging. The main function of mitochondria in cells is to produce high-energy phosphate compounds ATP to provide energy to cells, so the ATP content reflects the function of mitochondria.

[0156] Test sample: Same as Test Example 3.

[0157] Test process: Human skin keratinocytes (HaCaT) cells were cultured at 1.5×10 5The cells were inoculated at a density of 100 μL / well in a 6-well cell culture plate and incubated under the UVA-induced model according to step (1) of test example 3. After UVA treatment, the cells were subjected to sample addition treatment, and DMEM cell culture medium containing 3% and 5% mass fraction of black tea fermentation liquid was added respectively. The control blank group was DMEM basal cell culture medium, and cultured for 48 h. The culture medium was discarded, and the cells were washed twice with pre-cooled PBS. 200 μL of cell lysis solution was added to each well, and the cells were centrifuged at 4°C and 12,000 rpm for 20 min. The supernatant was collected as the sample to be tested, and the operation was strictly carried out according to the operation method described in the ATP (adenosine triphosphate) detection kit (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.). The ATP detection reagent diluent and the ATP detection reagent were diluted in a ratio of 1:4 to prepare the ATP detection working solution for use. 100 μL of ATP detection working solution was added to the detection tube. After standing at room temperature for 5 min, 20 μL of sample (the above-mentioned clear solution to be tested) was added to the detection tube, and the mixture was quickly mixed with a gun. After an interval of 2 s, the RLU value was measured using a chemiluminometer. The results of the mitochondrial ATP content detection and the promotion rate of mitochondrial ATP are shown in Table 6. The promotion rate of mitochondrial ATP is calculated according to the following formula:

[0158] Promotion rate = (ATP content of sample test group - ATP content of negative control group) / (ATP content of blank control group - ATP content of negative control group) × 100%

[0159] In the above formula, the sample test group is the intracellular ATP content after adding black tea fermentation liquid or extract;

[0160] The negative control group was the ATP content in UVA-damaged cells;

[0161] The blank control group was normal intracellular ATP content.

[0162] Table 6

[0163]

[0164] The results of ATP content detection show that the mitochondrial function of cells is severely damaged after UVA irradiation, and the amount of ATP produced is only 18.2% of that of normal cells. After adding the black tea fermentation broth of the embodiment, the ATP content is significantly higher than that of the control example. When the same mass fraction of black tea powder is added for fermentation, the effect of the black tea fermentation broth of Example 1 is the best. When the concentration of the black tea fermentation broth obtained in the embodiment of the present invention is 3%, it can reach more than 52.0% of the ATP production of normal cells, and when the concentration of the black tea fermentation broth is 5%, it can reach more than 93.9% of the ATP production of normal cells. Further analysis of the data on the ATP content and promotion rate of the fermentation broth in the mitochondria of cells shows that the total content of theaflavins, thearubigins and theabrownins in the fermentation broth is positively correlated with the content and promotion rate of ATP.

[0165] The black tea fermentation liquid of the embodiment has a significantly better effect on promoting ATP production than the black tea fermentation liquid / extract of the comparative example, indicating that the black tea fermentation liquid / extract obtained by the dual-species fermentation process of Pichia kudrida and Bacillus kudrida has a stronger mitochondrial protection function than other fermentation processes and extraction processes.

[0166] Test Example 5: Test to promote the production of type I collagen

[0167] Type I collagen is one of the main components of the extracellular matrix of dermis. It is synthesized by dermal fibroblasts in the cells and secreted to the extracellular space. Under the action of terminal procollagen peptidase, the terminal peptides are separated and polymerized to form collagen fibers. Human dermal fibroblasts can be used as a cell model to study the effect of cosmetics on improving the content of type I collagen. By measuring the upregulation rate of type I collagen content after administration of the test substance, the blank control and the test substance, the efficacy of the test substance in promoting collagen synthesis can be evaluated. It is often used as one of the indicators for in vitro evaluation of the firming and anti-wrinkle effects of cosmetics. The specific principle is: after type I collagen specifically binds to the collagen antibody coated on the ELISA plate, it binds to the anti-type I collagen antibody labeled with the substrate. After the substrate is catalyzed by the enzyme, a colored product is generated. The content of type I collagen is positively correlated with the color depth of the colored product. Therefore, the optical density (OD value) is measured at a wavelength of 450 nm using an ELISA instrument to calculate the content of type I collagen.

[0168] Test sample: The black tea fermentation liquid / extract obtained in Example 1-12 and Comparative Example 1-14 was diluted to a concentration of 3% and 5% using cell culture medium.

[0169] Blank sample group: No dose of black tea fermentation liquid or extract was added, and cell culture medium was directly added for testing.

[0170] Test method:

[0171] (1) Cell culture: Prepare human dermal fibroblast suspension 24 h before the test and inoculate the cell suspension into a 96-well cell culture plate, 0.1 mL per well, 5000 cells per well, and add DMEM culture medium containing 10% FBS for 24 h.

[0172] (2) Exposure: Discard the original culture medium in the wells, add 0.15 mL of test sample of different concentrations to each well, and incubate in the incubator for 24 h.

[0173] (3) Sample collection: Centrifuge and obtain the supernatant for fresh use or store at -20°C for later use.

[0174] (4) ELISA test: Follow the instructions of the Collagen I ELISA test kit (purchased from Wuhan Huamei Bioengineering Co., Ltd.) and measure the absorbance at a wavelength of 450 nm on an ELISA reader.

[0175] The relative content of type I collagen (Col) was calculated according to the following formula:

[0176] Relative content of type I collagen (%) = (Col content of sample test group / blank

[0177] Col content of the control group) × 100%

[0178] The relative content of type I collagen is shown in Table 7.

[0179] Table 7

[0180]

[0181] As can be seen from the table above, compared with the content of type I collagen in the blank group without adding the sample, the relative content of type I collagen increased significantly after adding the black tea fermentation liquid. When the same mass fraction of black tea powder was added for fermentation, the black tea fermentation liquid of Example 1 had the best effect: when the concentration of the black tea fermentation liquid was 3%, the relative content of type I collagen in the cells increased by 80%; when the concentration of the black tea fermentation liquid was 5%, the relative content of type I collagen in the cells increased by 130%. The effect of the black tea fermentation liquid of Examples 1-12 on improving the content of type I collagen was significantly better than that of the black tea fermentation liquid / extract of Comparative Examples 1-14. Further analysis of the data on the relative content of type I collagen in cell mitochondria by the fermentation liquid proved that the total content of theaflavins and thearubigins in the fermentation liquid was positively correlated with the relative content of type I collagen. From the above results, it can be seen that the type of double-bacteria fermentation strains selected by the present invention and the changes in parameters in the fermentation process will affect the relative content of type I collagen, thereby affecting the firming and anti-wrinkle effect.

[0182] Test Example 6: Human efficacy evaluation

[0183] The black tea fermented liquor of Example 1 and Comparative Example 1 were added to the skin care products to obtain test cream A (containing 10% black tea fermented liquor of Example 1) and test cream B (containing 10% black tea fermented liquor of Comparative Example 1), respectively. The basic formula is shown in Table 8. 70 healthy volunteers aged 35-60 years old, who had skin problems such as sagging facial skin and fine lines or wrinkles at the corners of the eyes, were selected for human trials to evaluate the anti-aging efficacy. The human trials of the test creams were conducted for 4 consecutive weeks, and the skin firmness and skin surface morphology such as crow's feet on the face of the volunteers were tested before use (initial value) and after 2 and 4 weeks of continuous use to comprehensively evaluate the anti-aging efficacy of the samples.

[0184] Table 8

[0185]

[0186] Skin firmness: Cutometer was used to test 3 times and the average value of skin firmness parameters was taken. The skin firmness parameter values ​​of the tested area decreased and had a significant difference, indicating that the tested sample had the effect of improving skin firmness.

[0187] Crow's feet at the corners of the eyes: PrimosCR was used to photograph crow's feet and analyze wrinkle degree parameters (wrinkle depth, area, length, and number). The values ​​of wrinkle parameters (wrinkle depth, area, wrinkle length, and number) in the test area decreased and showed significant differences, indicating that the test sample has the effect of reducing crow's feet (wrinkle depth, area, wrinkle length, and number).

[0188] Testing instruments: Skin elasticity tester Cutometer MPA580 (Courage & Khazaka, Germany), skin wrinkle analysis system PRIMOS CR (Canfield, USA).

[0189] Test method: 70 subjects were randomly divided into two groups, 35 people in each group, and used cream A and cream B respectively. No facial products were applied to the subjects on the morning of the test day. After cleaning the face with clean water, enter the constant temperature and humidity room with a temperature of 21℃±1.0℃ and a humidity of 50%±10%, and sit quietly for 30 minutes. The subjects were then tested with instruments, screened, and the initial state of the skin was recorded. After the test was completed, the product was distributed, and the method of use each time was as follows: After cleaning the facial area of ​​the subject in the morning and evening every day, take 2 g of the above test sample and apply it evenly on the facial area for 4 consecutive weeks. After using the product for 2 and 4 weeks, the subjects entered the venue for a follow-up visit, and performed the skin elasticity tester and skin wrinkle analysis system instrument tests according to the above steps. A questionnaire was filled out during the last follow-up visit. The test results are shown in Tables 9-10 and Figure 1-2 shown.

[0190] Table 9 Test results of cream A (containing 10% black tea fermented liquid of Example 1)

[0191]

[0192] Table 10 Test results of cream B (containing 10% black tea fermented liquid of comparative example 1)

[0193]

[0194] Note: "ns" indicates no significant difference (P>0.05); "*" indicates statistical difference (0.01≤P≤0.05); "**" indicates significant statistical difference (0.001≤P<0.01); "***" indicates extremely significant statistical difference (P<0.001); "√" indicates improvement with significant difference; "×" indicates no improvement, or improvement but no significant difference.

[0195] The test results of facial cream A (containing 10% of the black tea fermentation liquid of Example 1) are shown in Table 9. Figure 1 Compared with before use, after using the cream A sample for 2 weeks, the skin firmness parameter R0 value of the test area decreased, and there was a significant difference (P=0.002), and the skin firmness parameter R0 improvement rate was 4.17%; after using the test sample for 4 weeks, the skin firmness parameter R0 value of the test area decreased, and the difference was extremely significant (P<0.001), and the skin firmness parameter R0 improvement rate was 8.33%. The test results show that cream A has the effect of improving skin firmness.

[0196] Compared with before use, after using the cream A sample for 2 weeks, the depth, area, length and number of crow's feet in the test area decreased, and the depth had a significant difference (P=0.003), and the area, length and number had extremely significant differences (P<0.001). The improvement rates of the depth, area, length and number of crow's feet were 3.14%, 7.16%, 12.69% and 59.27% ​​respectively; after using the test sample for 4 weeks, the depth, area, length and number of crow's feet in the test area decreased, and the depth had a significant difference (P=0.002), and the area, length and number had extremely significant differences (P<0.001). The improvement rates of the depth, area, length and number of crow's feet were 4.17%, 8.77%, 15.68% and 60.07% respectively. The test results show that cream A has the anti-aging effect of reducing the depth, area, length and number of crow's feet.

[0197] The test results of cream B (containing 10% of the black tea fermented liquid of comparative example 1) are shown in Table 10. Figure 2 Compared with before use, after using the cream B sample for 2 weeks, the skin firmness parameter R0 value of the test area remained unchanged, with no significant difference; after using the test sample for 4 weeks, the skin firmness parameter R0 value of the test area decreased, with a significant difference (P=0.002), and the improvement rate of the skin firmness parameter R0 was 3.33%. The test results show that cream B has the effect of improving skin firmness.

[0198] Compared with before use, after using the cream B sample for 2 weeks, the depth, area, length and number of crow's feet in the test area of ​​this subject population were reduced, but there was no significant difference in wrinkle depth; there were extremely significant differences in wrinkle area and number (P<0.001), and there was a significant difference in wrinkle length (P=0.004), and the improvement rates of crow's feet area, length and number were 3.93%, 4.49% and 17.57% respectively; after using the test sample for 4 weeks, there was still no significant difference in the depth of crow's feet in the test area; the area, length and number of wrinkles were reduced, and there were extremely significant differences in area, length and number (P<0.001), and the improvement rates of crow's feet area, length and number were 4.51%, 5.80% and 22.88% respectively. The test results show that cream B has a certain anti-aging effect, which is reflected in the reduction of crow's feet area, length and number of wrinkles, but the anti-aging effect is limited, and it does not have the anti-aging effect of reducing the depth of crow's feet.

[0199] The above anti-aging effect data show that after the subjects used Cream A, the depth, area, length, number and other aspects of crow's feet were significantly better than those of Cream B. Therefore, the anti-aging effect of the black tea fermented liquid in Example 1 is significantly better than that of Comparative Example 1.

[0200] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing black tea fermentation liquid, characterized in that: The steps include: (1) culturing Pichia kudelni and Bacillus in liquid culture medium to obtain Pichia kudelni seed solution and Bacillus seed solution respectively; the Bacillus is a Bacillus that secretes tannase; the Bacillus that secretes tannase is any one of Lactobacillus acidophilus, Lactobacillus plantarum, Acetobacter xylinum, Bacillus tequila, and Bacillus subtilis; (2) adding black tea powder to a liquid culture medium to obtain a fermentation medium, inoculating the Pichia kudrida yeast seed solution and the Bacillus seed solution obtained in step (1) to ferment and obtain a crude fermentation liquid; the volume ratio of the inoculation amount of the Pichia kudrida yeast seed solution to the inoculation amount of the Bacillus seed solution is (2-5): (2-6); the mass percentage of black tea powder in the fermentation medium is 2-5%; the total inoculation amount of the Pichia kudrida yeast seed solution and the Bacillus seed solution is 4%-10% of the total volume of the liquid culture medium; the fermentation conditions are: the ventilation volume during the fermentation process is 0.5-1.0 vvm, the fermentation temperature is 25-35°C, the rotation speed is 100-300 r / min, the fermentation time is 24-72 h, and the pH is 3.0-5.0; (3) inactivating, filtering and blending the crude fermentation liquid obtained in step (2) to obtain the black tea fermentation liquid; In the steps (1) and (2), the liquid culture medium comprises the following components based on a total weight ratio of 1000 parts: 5-15 parts of peptone, 5-15 parts of beef extract, 3-7 parts of yeast extract, 15-25 parts of glucose, 1.5-3.0 parts of citric acid, 2.0-3.5 parts of sodium citrate, 1-3 parts of MgSO4·7H2O, 0.05-0.1 parts of MnSO4·4H2O, and the balance of deionized water; the initial pH of the liquid culture medium is 4.6-5.

0.

2. The method for preparing black tea fermented liquid according to claim 1, characterized in that: In the step (1), the concentration of the strains in the Pichia kudrida seed solution and the Bacillus seed solution is not less than 1×10 8 CFU / mL.

3. The method for preparing black tea fermented liquid according to claim 1, characterized in that: The volume ratio of the inoculation amount of the Pichia kudrida seed solution to the inoculation amount of the Bacillus seed solution is 1:

1.

4. The black tea fermented liquid prepared by the method for preparing black tea fermented liquid according to any one of claims 1 to 3.

5. Use of the black tea fermented liquid as claimed in claim 4 in cosmetics.

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