A black tea fermented liquid with antioxidant and brightening effects, and preparation method and application thereof

Abstract

The invention discloses a black tea fermentation liquid with antioxidant and brightening effects, a preparation method and application thereof, and belongs to the technical field of cosmetics. The black tea fermentation liquid with antioxidant and brightening effects of the invention is obtained by fermenting black tea powder with composite bacteria, wherein the composite bacteria are animal Bifidobacterium lactis subspecies and Lactobacillus plantarum; the end mark of the fermentation is: the pH of the black tea fermentation liquid drops to 3.5-4.5. The invention adopts specific composite bacteria to ferment black tea powder, and optimizes the conditions during the fermentation process, so that the prepared black tea fermentation liquid has excellent antioxidant, brightening, firming and soothing effects.

Description

Technical Field

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

[0002] Black tea is a fully fermented tea. It is made from new buds and leaves of suitable tea trees through a series of processes such as withering, rolling, fermentation, and drying. It has the effects of warming the stomach, refreshing the mind, eliminating fatigue, eliminating edema, stopping diarrhea, antibacterial, and enhancing immunity. At present, adding black tea extract to cosmetics to achieve the beauty and skin care effects of black tea extract in cosmetics has been widely studied.

[0003] However, due to the chemical reaction centered on the enzymatic oxidation of tea polyphenols during the processing of black tea, the active substances such as fresh leaf components change greatly during the extraction process, especially tea polyphenols with antioxidant effects will be reduced by more than 90%, resulting in a significant reduction in the antioxidant effect of black tea extract, making the skin care effect of black tea extract in cosmetics poor. CN114586865A uses fermentation to extract black tea extract, which reduces the waste of active substances in black tea, but the fermentation strains used in this method are relatively single, and the obtained black tea extract still cannot meet the skin care needs of current cosmetics consumers.

[0004] In view of the above-mentioned defects in the extraction process of black tea extract, developing a black tea extraction method that can reduce the loss of active substances in the black tea extraction process and enable the black tea extract to fully exert its efficacy is the current research focus. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a black tea fermented liquid with antioxidant and brightening effects, and a preparation method and application thereof, wherein the black tea fermented liquid has excellent antioxidant, brightening, firming and soothing effects.

[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 fermented liquid having antioxidant and brightening effects, the method comprising the following steps:

[0008] S1, adding the activated composite bacteria seed liquid into the fermentation matrix containing black tea powder and fermenting until the pH drops to 3.5-4.5, stopping the fermentation to obtain a crude fermentation liquid;

[0009] S2, sterilizing and filtering the crude fermentation liquid described in step S1 to obtain the black tea fermentation liquid with antioxidant and brightening effects;

[0010] Wherein, in step S1, the composite bacteria are Bifidobacterium animalis subspecies lactis and Lactobacillus plantarum.

[0011] The invention adopts animal bifidobacterium lactis subspecies and plant lactobacillus to ferment black tea powder, so that the prepared black tea fermented liquid has excellent anti-oxidation, brightening, firming and soothing effects.

[0012] Adding the black tea fermented liquid with antioxidant and brightening effects as an active ingredient into cosmetics can greatly improve the antioxidant, brightening, firming and soothing effects of the cosmetics, and the black tea fermented liquid with antioxidant and brightening effects can also show excellent stability in the products to which it is applied.

[0013] As a preferred embodiment of the method for preparing the black tea fermented liquid with antioxidant and brightening effects of the present invention, in the step S1, the inoculation amounts of animal Bifidobacterium lactis subspecies and Lactobacillus plantarum are both 2%-5%, and the inoculation amount ratio of animal Bifidobacterium lactis subspecies and plantarum Lactobacillus plantarum is animal Bifidobacterium lactis subspecies: plantarum Lactobacillus = 1-2:1-2; the inventors found through a large number of experiments that when the inoculation amount and inoculation amount ratio of animal Bifidobacterium lactis subspecies and plantarum Lactobacillus plantarum in the composite bacterial seed liquid are within the above range, the antioxidant effect, brightening effect, firming and soothing effect of the final black tea fermented liquid can be made more obvious.

[0014] As a more preferred embodiment of the method for preparing the black tea fermented liquid with antioxidant and brightening effects of the present invention, the inoculation amounts of animal Bifidobacterium lactis subspecies and Lactobacillus plantarum are both 3%, and the inoculation ratio of animal Bifidobacterium lactis subspecies and plantarum Lactobacillus plantarum is animal Bifidobacterium lactis subspecies: plantarum Lactobacillus = 1:1; the inventors have found through a large number of experiments that when the inoculation amounts of animal Bifidobacterium lactis subspecies and plantarum Lactobacillus in the composite bacterial seed liquid are the above values, the antioxidant effect, brightening effect, firming and soothing effect of the final black tea fermented liquid can be best.

[0015] As a preferred embodiment of the method for preparing the black tea fermentation broth with antioxidant and brightening effects of the present invention, in the step S1, the fineness of the black tea powder is 60-100 mesh; the inventors have found through a large number of experiments that the fineness of the black tea powder in the black tea fermentation broth with antioxidant and brightening effects has a significant impact on the fermentation effect of the final black tea fermentation broth, and the active substances of black tea are usually distributed in cells. After the black tea powder is crushed, the cell walls are broken and the effective ingredients in the cells are exposed. Therefore, the finer the black tea powder is ground, the more conducive it is to the dissolution of the active substances in the black tea powder. However, due to static electricity and adsorption, the finer the black tea powder is ground, the more difficult it is to disperse evenly in the fermentation matrix, and the black tea powder is prone to agglomeration and caking, resulting in poor fermentation effect; therefore, in the present invention, only when the fineness of the black tea powder is within the above range can the black tea fermentation effect be better.

[0016] As a more preferred embodiment of the method for preparing the black tea fermented liquid with antioxidant and brightening effects of the present invention, the fineness of the black tea powder is 80 mesh; the inventors found through a large number of experiments that when the fineness of the black tea powder is 80 mesh, the fermentation effect of the fermented liquid can be best.

[0017] As a preferred embodiment of the method for preparing the black tea fermentation broth with antioxidant and brightening effects of the present invention, the composite bacteria are purchased from the China Industrial Microbiological Culture Collection Management Center.

[0018] As a preferred embodiment of the method for preparing the black tea fermented liquid with antioxidant and brightening effects of the present invention, the method for preparing the composite bacteria seed liquid comprises the following steps: adding animal Bifidobacterium lactis subspecies colonies or plant Lactobacillus colonies to a liquid culture medium for culturing, thereby obtaining the animal Bifidobacterium lactis subspecies seed liquid or plant Lactobacillus seed liquid.

[0019] As a more preferred embodiment of the method for preparing the black tea fermented liquid with antioxidant and brightening effects of the present invention, the parameters of the composite bacteria seed liquid culture process are: the culture temperature is 35-40°C, the culture time is 24-72h, the rotation speed during culture is 100-300rpm, and the bacterial concentrations of animal Bifidobacterium lactis subspecies and plantarum Lactobacillus in the composite bacteria are both 1x10 7 -1x10 9 cfu / ml.

[0020] As the most preferred embodiment of the method for preparing the black tea fermented liquid with antioxidant and brightening effects of the present invention, the parameters of the composite bacteria seed liquid culture process are: the culture temperature is 37°C, the culture time is 48h, the rotation speed during the culture is 150rpm, and the bacterial concentrations of animal Bifidobacterium lactis subspecies and plantarum Lactobacillus in the composite bacteria are both 1x10 8 cfu / ml.

[0021] As a preferred embodiment of the method for preparing the black tea fermentation liquid with antioxidant and brightening effects of the present invention, the culture medium in the process of preparing the composite bacteria seed liquid is a culture medium commonly used in the art, and the present invention adopts MRS liquid culture medium.

[0022] As a more preferred embodiment of the method for preparing the black tea fermented liquid with antioxidant and brightening effects of the present invention, the method for preparing the MRS liquid culture medium is: after mixing the raw materials, adding distilled water, sterilizing at 120° C. for 20 minutes, and then storing for later use.

[0023] As the most preferred embodiment of the method for preparing the black tea fermented liquid with antioxidant and brightening effects of the present invention, the MRS liquid culture medium includes the following raw materials per liter of culture medium: 10g of peptone, 5g of beef powder, 4g of yeast powder, 2g of glucose, 1mL of Tween 80, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 2g of triammonium citrate, 0.2g of magnesium sulfate, and 0.05g of manganese sulfate.

[0024] As a preferred embodiment of the method for preparing the black tea fermented liquid with antioxidant and brightening effects of the present invention, the fermentation matrix includes black tea powder, a carbon source, a nitrogen source and water.

[0025] As a more preferred embodiment of the method for preparing the black tea fermented liquid with antioxidant and brightening effects of the present invention, the fermentation matrix includes the following components in weight percentage: 1%-5% black tea powder, 1%-4% carbon source, 1%-4% nitrogen source and the remainder water.

[0026] As the most preferred embodiment of the method for preparing the black tea fermented liquid with antioxidant and brightening effects of the present invention, the fermentation matrix includes the following components in weight percentage: 2% black tea powder, 2% carbon source, 2% nitrogen source and the balance water.

[0027] As a preferred embodiment of the method for preparing the black tea fermented liquid with antioxidant and brightening effects of the present invention, at least one of the following (1)-(4) is adopted:

[0028] (1) The carbon source comprises at least one of corn starch, potato starch, cyclodextrin, fructose syrup, and maltose;

[0029] (2) The nitrogen source includes at least two of ammonium salt, peptone, and yeast extract;

[0030] (3) The fermentation conditions are: temperature of 35-40°C and time of 50-80h;

[0031] (4) The sterilization conditions are: temperature of 85-115°C and time of 15-30 min.

[0032] As a more preferred embodiment of the method for preparing the black tea fermented liquid with antioxidant and brightening effects of the present invention, at least one of the following (5)-(8) is adopted:

[0033] (5) The carbon source is corn starch;

[0034] (6) The nitrogen source comprises ammonium chloride and soy peptone, and the weight ratio of ammonium chloride to soy peptone is 1:1-3;

[0035] (7) The fermentation conditions are: temperature of 37°C;

[0036] (8) The sterilization conditions are: temperature of 90°C and time of 20 min.

[0037] As the most preferred embodiment of the method for preparing the black tea fermented liquid with antioxidant and brightening effects of the present invention, the nitrogen source comprises the following components in parts by weight: the weight ratio of ammonium chloride to soy peptone is 1:1.

[0038] It should be noted that the sign of the end of fermentation in the present invention is that the pH of the fermentation liquid drops to 3.5-4.5.

[0039] In a second aspect, the present invention provides a black tea fermented liquid with antioxidant and brightening effects, wherein the black tea fermented liquid with antioxidant and brightening effects is prepared by the method described in the first aspect.

[0040] In a third aspect, the present invention provides a cosmetic comprising the black tea fermented liquid with antioxidant and brightening effects as described in the second aspect.

[0041] As a preferred embodiment of the cosmetic of the present invention, the added amount of the black tea fermentation liquid is 1-30% of the total mass of the cosmetic.

[0042] As a preferred embodiment of the cosmetics of the present invention, the cosmetics can be skin softeners, lotions, creams, facial masks, gels, powders, sprays, essences, cleansing products and makeup products.

[0043] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention uses animal Bifidobacterium lactis subspecies and Lactobacillus plantarum to ferment black tea leaf powder of a specific fineness, and optimizes the fermentation conditions in the preparation process of the black tea fermentation liquid, and the prepared black tea fermentation liquid has excellent antioxidant, brightening, firming and soothing effects. DETAILED DESCRIPTION

[0044] 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.

[0045] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0046] The strains used in the present invention are all purchased from the China Industrial Microbiological Culture Collection Center. The animal Bifidobacterium lactis subspecies is CICC 21710, CICC 21718 or CICC 21712, and the plant lactobacillus is JYLP-326, CICC 22219 or CICC 23132.

[0047] Example 1

[0048] In the method for preparing the black tea fermentation liquid with antioxidant and brightening effects described in this embodiment, the method for preparing black tea powder is: select dry black tea leaves, grind them into powder with a grinder, pass them through an 80-mesh sieve, remove impurities in the upper layer, and take the tea powder in the lower layer as a fermentation raw material for later use.

[0049] The animal Bifidobacterium lactis subspecies used in this embodiment is CICC 21710, and the plant Lactobacillus is JYLP-326; the preparation method of the composite bacterial seed liquid is: using an inoculation loop to pick animal Bifidobacterium lactis subspecies colonies and plant Lactobacillus colonies into MRS liquid culture medium, respectively, and culture them at 37°C and 150rpm for 48h to obtain animal Bifidobacterium lactis subspecies seed liquid and plant Lactobacillus seed liquid; wherein the bacterial concentrations of animal Bifidobacterium lactis subspecies seed liquid and plant Lactobacillus seed liquid are both about 1×10 8 cfu / ml.

[0050] The fermentation matrix includes the following components in weight percentage: 2% black tea powder, 2% corn starch, 1% soy peptone, 1% ammonium chloride and balance water; the preparation method of the fermentation matrix is: the components are mixed evenly in a fermentation tank, the fermentation tank is sealed, the temperature is increased to 121° C., maintained for 30 minutes, and then sterilized to obtain the fermentation matrix.

[0051] The method for preparing the black tea fermented liquid with antioxidant and brightening effects described in this embodiment comprises the following steps:

[0052] S1, adding the activated composite bacteria seed liquid to the fermentation matrix, standing and fermenting at 37°C, and stopping the fermentation when the pH value reaches 4.2 to obtain a crude black tea fermentation liquid;

[0053] S2, sterilizing the crude black tea fermentation liquid in step S1 at 95° C. for 15 minutes by pasteurization, and then filtering the sterilized crude black tea fermentation liquid through a diatomaceous earth filter to remove impurities, thereby obtaining the black tea fermentation liquid with antioxidant and brightening effects;

[0054] Wherein, in step S1, the inoculation amount of Bifidobacterium animalis subspecies lactis seed solution and Lactobacillus plantarum seed solution are both 3%.

[0055] Example 2-11

[0056] The differences between the method for preparing the black tea fermented liquid with antioxidant and brightening effects described in Examples 2-11 and the method for preparing the black tea fermented liquid with antioxidant and brightening effects described in Example 1 are shown in Table 1 below. Except for the differences in Table 1, the rest of the method for preparing the black tea fermented liquid with antioxidant and brightening effects described in Examples 2-11 are consistent with Example 1.

[0057] Table 1

[0058]

[0059]

[0060]

[0061]

[0062] Example 12

[0063] In the method for preparing the black tea fermentation liquid with antioxidant and brightening effects described in this embodiment, the method for preparing black tea powder is: select dried black tea leaves, grind them into powder with a grinder, pass them through a 70-mesh sieve, remove impurities in the upper layer, and take the tea powder in the lower layer as a fermentation raw material for later use.

[0064] The animal Bifidobacterium lactis subspecies used in this embodiment is CICC 21718, and the plant Lactobacillus is CICC 22219; the preparation method of the composite bacterial seed liquid is: using an inoculation loop to pick animal Bifidobacterium lactis subspecies colonies and plant Lactobacillus colonies into MRS liquid culture medium, respectively, and culture them at 40°C and 100rpm for 24h to obtain animal Bifidobacterium lactis subspecies seed liquid and plant Lactobacillus seed liquid; wherein the bacterial concentrations of animal Bifidobacterium lactis subspecies seed liquid and plant Lactobacillus seed liquid are both about 1×10 7 cfu / ml.

[0065] The fermentation matrix includes the following components in weight percentage: 1% black tea powder, 4% corn starch, 2% soy peptone, 2% ammonium chloride and balance water; the preparation method of the fermentation matrix is: the components are mixed evenly in a fermentation tank, the fermentation tank is sealed, the temperature is increased to 121° C., maintained for 30 minutes, and then sterilized to obtain the fermentation matrix.

[0066] The method for preparing the black tea fermented liquid with antioxidant and brightening effects described in this embodiment comprises the following steps:

[0067] S1, adding the activated composite bacteria seed liquid to the fermentation matrix, standing and fermenting at 35°C, and stopping the fermentation when the pH value reaches 4.3 to obtain a crude black tea fermentation liquid;

[0068] S2, sterilizing the crude black tea fermentation liquid in step S1 at 115° C. for 15 minutes by pasteurization, and then filtering the sterilized crude black tea fermentation liquid through a diatomaceous earth filter to remove impurities, thereby obtaining the black tea fermentation liquid with antioxidant and brightening effects;

[0069] Wherein, in step S1, the inoculation amount of Bifidobacterium animalis subspecies lactis seed solution and Lactobacillus plantarum seed solution are both 5%.

[0070] Embodiment 13

[0071] In the method for preparing the black tea fermentation liquid with antioxidant and brightening effects described in this embodiment, the method for preparing black tea powder is: select dry black tea leaves, grind them into powder with a grinder, pass them through a 90-mesh sieve, remove impurities in the upper layer, and take the tea powder in the lower layer as a fermentation raw material for later use.

[0072] The animal Bifidobacterium lactis subspecies CICC 21712 and the plant Lactobacillus plantarum subspecies CICC23132 used in this embodiment are prepared by using an inoculation loop to pick animal Bifidobacterium lactis subspecies colonies and plant Lactobacillus plantarum colonies into MRS liquid culture medium, and culture them at 35°C and 300rpm for 72h to obtain animal Bifidobacterium lactis subspecies seed liquid and plant Lactobacillus plantarum seed liquid; wherein the bacterial concentrations of animal Bifidobacterium lactis subspecies seed liquid and plant Lactobacillus plantarum seed liquid are both about 1×10 9 cfu / ml.

[0073] The fermentation matrix comprises the following components in percentage by weight: 5% black tea powder, 1% corn starch, 0.5% soy peptone, 2% ammonium sulfate and the balance water; the preparation method of the fermentation matrix is: the components are mixed evenly in a fermentation tank, the fermentation tank is sealed, the temperature is raised to 121° C., maintained for 30 minutes, and then sterilized to obtain the fermentation matrix.

[0074] The method for preparing the black tea fermented liquid with antioxidant and brightening effects described in this embodiment comprises the following steps:

[0075] S1, adding the activated composite bacteria seed liquid to the fermentation matrix, standing and fermenting at 40°C, and stopping the fermentation when the pH value reaches 4.0 to obtain a crude black tea fermentation liquid;

[0076] S2, sterilizing the crude black tea fermentation liquid in step S1 at 85° C. for 30 minutes by pasteurization, and then filtering the sterilized crude black tea fermentation liquid through a diatomaceous earth filter to remove impurities, thereby obtaining the black tea fermentation liquid with antioxidant and brightening effects;

[0077] Wherein, in step S1, the inoculation amount of Bifidobacterium animalis subspecies lactis seed solution and Lactobacillus plantarum seed solution are both 2%.

[0078] Comparative Examples 1-11

[0079] The differences between the method for preparing the black tea fermented liquid with antioxidant and brightening effects described in Comparative Examples 1-11 and the method for preparing the black tea fermented liquid with antioxidant and brightening effects described in Example 1 are shown in Table 2 below. Except for the differences in Table 2, the rest of the method for preparing the black tea fermented liquid with antioxidant and brightening effects described in Comparative Examples 1-10 is consistent with Example 1. The difference between Comparative Example 11 and Example 1 is that the step S1 is fermented at a rotation speed of 150 rpm.

[0080] Table 2

[0081]

[0082]

[0083]

[0084] Test Case

[0085] 1. Antioxidant test - Cellular reactive oxygen species (ROS) inhibition test

[0086] Test method: After making human skin keratinocytes (Hacat) into a cell suspension, inoculate it in a 96-well plate, with 10,000-20,000 cells per well, and culture it with DMEM medium. After culturing for 24 hours, change the medium, add the black tea fermented liquid with antioxidant and brightening effects described in Examples 1-13 and Comparative Examples 1-11 (the amount of black tea fermented liquid added is 2% of the amount of cell culture medium), continue to culture for 24 hours, and then give UVA light to the control group and the experimental group, and the light dose is 6-10J / cm 2After the illumination, the cell culture medium was removed, washed with PBS for 3 times, 100 μL 10 μmol / L DCFH-DA was added to each well, and placed in a CO2 incubator for 20 min. After the incubation, PBS was used to wash 3 times, and serum-free DMEM culture medium was added again. The ROS content was detected using a fluorescence microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 525 nm; the relative level of cell reactive oxygen species (ROS) = OD value of the experimental group or OD value of the control group / OD value of the blank group. Blank group: PBS buffer was added without illumination; control group: PBS buffer was added. The results are shown in Table 3 below.

[0087] Table 3

[0088]

[0089]

[0090] According to the data in Table 3, the relative levels (%) of cellular active oxygen in the black tea fermented liquors with antioxidant and brightening effects described in Examples 1-13 of the present invention are all lower than that in the control group, indicating that the black tea fermented liquors prepared in the present invention have antioxidant properties.

[0091] By comparing the examples with the comparative examples, it is found that the relative level (%) of cellular active oxygen in the black tea fermentation broth of Examples 1-3 is lower than that of Comparative Example 1. It can be seen that in the present invention, the choice of carbon source in the fermentation matrix will affect the fermentation effect, thereby affecting the antioxidant properties of the final black tea fermentation broth; the present invention is suitable for using at least one of corn starch, potato starch, cyclodextrin, fructose syrup, and maltose as a carbon source; at the same time, the relative level (%) of cellular active oxygen in Example 1 is lower than that in Examples 2 and 3, indicating that the present invention is most suitable for using corn starch as a carbon source.

[0092] The relative level (%) of cellular active oxygen in the black tea fermentation broth of Examples 1, 4 and 5 is lower than that of Comparative Examples 2 and 3. It can be seen that in the present invention, the selection of nitrogen source in the fermentation matrix will affect the fermentation effect, thereby affecting the antioxidant properties of the final black tea fermentation broth; the present invention is suitable for using at least two of ammonium salt, peptone and yeast extract as nitrogen sources; at the same time, the relative level (%) of cellular active oxygen in Example 1 is lower than that of Examples 4 and 5, indicating that the present invention is most suitable for using ammonium chloride and soy peptone as nitrogen sources, and the relative level (%) of cellular active oxygen in Example 1 is lower than that of Example 6, which also indicates that when the mass ratio of ammonium chloride and soy peptone in the nitrogen source of the present invention is 1:1, the antioxidant properties of the final black tea fermentation broth are the best.

[0093] The relative level (%) of cellular active oxygen in the black tea fermentation broth described in Examples 1 and 9 is lower than that in Comparative Examples 4-6. It can be seen that in the present invention, the type of fermentation bacteria selected will affect the antioxidant properties of the final black tea fermentation broth. The present invention is suitable for symbiotic fermentation using animal Bifidobacterium lactis subspecies and plant Lactobacillus; at the same time, the relative level (%) of cellular active oxygen in Example 1 is lower than that in Example 9, indicating that when the inoculation amount of animal Bifidobacterium lactis subspecies and plant Lactobacillus seed liquid is 1:1, the antioxidant property of the black tea fermentation broth is the best.

[0094] The relative level (%) of cellular active oxygen in the black tea fermentation broth of Examples 1, 7 and 8 is lower than that of Comparative Examples 7 and 8. It can be seen that in the present invention, the pH at the end of fermentation affects the antioxidant properties of the final black tea fermentation broth. When the pH at the end of fermentation of the present invention is 3.5-4.5, the antioxidant properties of the final black tea fermentation broth are better; at the same time, the relative level (%) of cellular active oxygen in Example 1 is lower than that of Examples 7 and 8, indicating that when the pH at the end of fermentation of the present invention is 4.2, the antioxidant properties of the final black tea fermentation broth are the best.

[0095] The relative level (%) of cellular active oxygen in the black tea fermentation broth of Examples 1, 10 and 11 is lower than that of Comparative Examples 10 and 11. It can be seen that in the present invention, the fineness of black tea powder affects the antioxidant properties of the final black tea fermentation broth. The present invention is suitable for using black tea powder with a fineness of 60-100 mesh as a raw material; at the same time, the relative level (%) of cellular active oxygen in Example 1 is lower than that of Examples 10 and 11, indicating that when the present invention uses black tea powder with a fineness of 80 mesh as a raw material, the antioxidant properties of the final black tea fermentation broth can be the best.

[0096] The relative level (%) of cellular active oxygen in the black tea fermentation liquid of Example 1 is lower than that of Comparative Example 11. It can be seen that in the present invention, the fermentation method affects the antioxidant properties of the final black tea fermentation liquid; the antioxidant properties of the black tea fermentation liquid finally prepared by fermentation under static conditions and fermentation under stirring conditions are also different. The present invention is suitable for fermentation by static fermentation.

[0097] 2. Oxidation test - hydroxyl radical-Fenton colorimetry

[0098] Test method: Step 1, prepare salicylic acid-ethanol solution (9.1mmol / L): accurately weigh 0.13g of salicylic acid, dissolve it in 95% ethanol and dilute the solution to 100mL, shake well to obtain 9.1mmol / L salicylic acid-ethanol solution, and place it in a brown bottle for later use;

[0099] Step 2: Prepare Fe 2+ Solution (9mmol / L): Accurately weigh 0.35g of ammonium ferrous sulfate, dilute to 100mL with boiled deionized water, and shake to obtain 9mmol / L Fe-containing2+ The solution was placed in a brown bottle for later use;

[0100] Step 3, prepare H2O2 solution (88mmol / L): accurately weigh 2g of 30% H2O2, dilute to 200mL with deionized water, shake well to obtain 88mmol / L H2O2 solution, and place in a brown bottle for later use;

[0101] Step 4, diluting the black tea fermented liquor with antioxidant and brightening effects described in Examples 1-13 and Comparative Examples 1-11 of the present invention with deionized water to a concentration of 0.5% and 1%, respectively, to obtain a sample solution;

[0102] According to Table 4 below, add the above samples to the sample tubes, shake well and let stand for 5 minutes, then add H2O2 solution, and after Fenton reaction, leave it in dark for 10 minutes. Measure the absorbance of each sample solution at a wavelength of 510nm, and calculate the scavenging rate of hydroxyl radicals. The recorded data are shown in Table 5 below:

[0103] The calculation formula of hydroxyl radical scavenging rate is:

[0104]

[0105] P—clearance rate;

[0106] A1—Salicylic acid-ethanol solution, Fe added to the sample 2+ Solution absorbance of the test solution;

[0107] A2—the absorbance value of the salicylic acid-ethanol solution to be tested after adding the sample;

[0108] A3—Salicylic acid-ethanol solution without sample addition, Fe 2+ Solution The absorbance value of the test solution.

[0109] Table 4

[0110]

[0111] Table 5

[0112]

[0113]

[0114] According to the data in Table 5, the hydroxyl radical scavenging rates of the black tea fermented liquors with antioxidant and brightening effects described in Examples 1-13 of the present invention are all higher than those of the comparative example, indicating that the black tea fermented liquors prepared in the present invention have antioxidant properties.

[0115] By comparing the examples with the comparative examples, it is found that the hydroxyl radical scavenging rate of the black tea fermentation broth described in Examples 1-3 is higher than that in Comparative Example 1. It can be seen that in the present invention, the selection of the carbon source in the fermentation matrix will affect the fermentation effect, thereby affecting the antioxidant properties of the final black tea fermentation broth; the present invention is suitable for using at least one of corn starch, potato starch, cyclodextrin, fructose syrup, and maltose as a carbon source; at the same time, the absorbance of Example 1 is higher than that of Examples 2 and 3, indicating that the present invention is most suitable for using corn starch as a carbon source.

[0116] The hydroxyl radical scavenging rate of the black tea fermentation broth in Examples 1, 4 and 5 is higher than that in Comparative Examples 2 and 3. It can be seen that in the present invention, the selection of the nitrogen source in the fermentation matrix will affect the fermentation effect, thereby affecting the antioxidant properties of the final black tea fermentation broth; the present invention is suitable for using at least two of ammonium salts, peptones and yeast extract as nitrogen sources; at the same time, the hydroxyl radical scavenging rate of Example 1 is higher than that of Examples 4 and 5, indicating that the present invention is most suitable for using ammonium chloride and soy peptone as nitrogen sources. The hydroxyl radical scavenging rate of Example 1 is higher than that of Example 6, which also indicates that when the mass ratio of ammonium chloride to soy peptone in the nitrogen source of the present invention is 1:1, the antioxidant properties of the final black tea fermentation broth are the best.

[0117] The hydroxyl radical scavenging rate of the black tea fermented liquor in Examples 1 and 9 is higher than that in Comparative Examples 4-6. It can be seen that in the present invention, the type of fermented bacteria will affect the antioxidant properties of the final black tea fermented liquor. The present invention is suitable for symbiotic fermentation of animal Bifidobacterium lactis subspecies and plant Lactobacillus; at the same time, the hydroxyl radical scavenging rate of Example 1 is higher than that of Example 9, indicating that when the inoculation amount of animal Bifidobacterium lactis subspecies and plant Lactobacillus seed liquid is 1:1, the antioxidant property of the black tea fermented liquor is the best.

[0118] The hydroxyl radical scavenging rate of the black tea fermentation broth in Examples 1, 7 and 8 is higher than that in Comparative Examples 7 and 8. It can be seen that in the present invention, the pH at the end of fermentation affects the antioxidant properties of the final black tea fermentation broth. When the pH at the end of fermentation of the present invention is 3.5-4.5, the antioxidant properties of the final black tea fermentation broth can be better; at the same time, the hydroxyl radical scavenging rate of Example 1 is higher than that of Examples 7 and 8, indicating that when the pH at the end of fermentation of the present invention is 4.2, the antioxidant properties of the final black tea fermentation broth can be the best.

[0119] The hydroxyl radical scavenging rate of the black tea fermentation liquor in Examples 1, 10 and 11 is higher than that in Comparative Examples 10 and 11. It can be seen that in the present invention, the fineness of the black tea powder affects the antioxidant properties of the final black tea fermentation liquor. The present invention is suitable for using black tea powder with a fineness of 60-100 mesh as a raw material; at the same time, the hydroxyl radical scavenging rate of Example 1 is higher than that in Examples 10 and 11, indicating that when the present invention uses black tea powder with a fineness of 80 mesh as a raw material, the antioxidant properties of the final black tea fermentation liquor can be the best.

[0120] The hydroxyl radical scavenging rate of the black tea fermented liquid in Example 1 is higher than that in Comparative Example 11. It can be seen that in the present invention, the fermentation method affects the antioxidant properties of the final black tea fermented liquid; the antioxidant properties of the black tea fermented liquid finally prepared by fermentation under static conditions and fermentation under stirring conditions are also different. The present invention is suitable for fermentation by static fermentation.

[0121] 3. Brightening test

[0122] Test sample: The black tea fermented liquor with antioxidant and brightening effects described in Examples 1-13 and Comparative Examples 1-11 of the present invention was prepared into a 10 wt % aqueous solution, and deionized water was used as a blank control group.

[0123] Testing instrument: Glossymeter (GL200, Courage and Khazaka, Germany).

[0124] Subject selection: 250 healthy volunteers aged 18-45 years were divided into 25 groups, with 10 people in each group.

[0125] Test method: After cleaning the facial area of ​​the subject, take 1-2mL of the above sample and apply it randomly on half of the face area, and the other half of the face area is used as a blank control group and applied with deionized water; use it once in the morning and evening every day, and test the skin gloss of the marked parts of the subject through the active surface analysis software SELS before using the sample, 7 days after using the sample, and 14 days after using the sample. The results are averaged and the skin gloss improvement rate is calculated according to the following formula. Table 6 below shows the skin gloss improvement rate after use by the subjects.

[0126] Skin glossiness improvement rate (%) = (average value after use - average value before use) / average value before use × 100%.

[0127] Table 6

[0128]

[0129]

[0130] According to the data in Table 6, the improvement rate of skin glossiness of the black tea fermented liquor with antioxidant and brightening effects described in Examples 1-13 of the present invention on the subjects after 7 days and 14 days of use was higher than that of the control example, indicating that the black tea fermented liquor prepared by the present invention has a brightening effect.

[0131] By comparing the examples and the comparative examples, it was found that the improvement rates of the skin glossiness of the black tea fermented liquids of Examples 1-3 after being used for 7 days and 14 days on the subjects were higher than those of Comparative Example 1. It can be seen that in the present invention, the selection of the carbon source in the fermentation matrix will affect the fermentation effect, thereby affecting the brightening effect of the final black tea fermented liquid; the present invention is suitable for using at least one of corn starch, potato starch, cyclodextrin, fructose syrup, and maltose as a carbon source; at the same time, the improvement rates of the skin glossiness of Example 1 after being used for 7 days and 14 days on the subjects were higher than those of Examples 2 and 3, indicating that the present invention is most suitable for using corn starch as a carbon source.

[0132] The improvement rate of skin glossiness of the black tea fermented liquid in Examples 1, 4 and 5 after being used for 7 and 14 days on the subjects was higher than that in Comparative Examples 2 and 3. It can be seen that in the present invention, the selection of the nitrogen source in the fermentation matrix will affect the fermentation effect, thereby affecting the brightening effect of the final black tea fermented liquid; the present invention is suitable for using at least two of ammonium salts, peptones and yeast extracts as nitrogen sources; at the same time, the improvement rate of skin glossiness of Example 1 after being used for 7 and 14 days on the subjects was higher than that in Examples 4 and 5, indicating that the present invention is most suitable for using ammonium chloride and soy peptone as nitrogen sources, and the improvement rate of skin glossiness of Example 1 after being used for 7 and 14 days on the subjects was higher than that in Example 6, which also indicates that when the mass ratio of ammonium chloride and soy peptone in the nitrogen source of the present invention is 1:1, the final black tea fermented liquid has the best brightening effect.

[0133] The improvement rate of skin glossiness of the black tea fermented liquid in Examples 1 and 9 after being used for 7 days and 14 days on the subjects was higher than that in Comparative Examples 4-6. It can be seen that in the present invention, the selection of the type of fermented bacteria will affect the brightening effect of the final black tea fermented liquid. The present invention is suitable for symbiotic fermentation of animal Bifidobacterium lactis subspecies and plant Lactobacillus; at the same time, the improvement rate of skin glossiness of Example 1 after being used for 7 days and 14 days on the subjects was higher than that in Example 9, indicating that when the inoculation amount of animal Bifidobacterium lactis subspecies seed liquid and plant Lactobacillus seed liquid is 1:1, the black tea fermented liquid has the best brightening effect.

[0134] The improvement rate of skin glossiness of the black tea fermented liquid in Examples 1, 7 and 8 after being used for 7 and 14 days on the subjects was higher than that in Comparative Examples 7 and 8. It can be seen that in the present invention, the pH at the end of fermentation affects the brightening effect of the final black tea fermented liquid. When the pH at the end of fermentation of the present invention is 3.5-4.5, the brightening effect of the final black tea fermented liquid can be better; at the same time, the improvement rate of skin glossiness of Example 1 after being used for 7 and 14 days on the subjects was higher than that in Examples 7 and 8, indicating that when the pH at the end of fermentation of the present invention is 4.2, the brightening effect of the final black tea fermented liquid can be the best.

[0135] The improvement rate of skin glossiness of the black tea fermented liquid in Examples 1, 10 and 11 after being used for 7 days and 14 days on the subjects was higher than that in Comparative Examples 10 and 11. It can be seen that in the present invention, the fineness of the black tea powder affects the brightening effect of the final black tea fermented liquid. The present invention is suitable for using black tea powder with a fineness of 60-100 mesh as a raw material; at the same time, the improvement rate of skin glossiness of Example 1 after being used for 7 days and 14 days on the subjects was higher than that in Examples 10 and 11, indicating that when the present invention uses black tea powder with a fineness of 80 mesh as a raw material, the final black tea fermented liquid can have the best brightening effect.

[0136] The improvement rate of skin glossiness of the black tea fermented liquid in Example 1 after being used for 7 days and 14 days on the subjects was higher than that in Comparative Example 11. It can be seen that in the present invention, the fermentation method affects the brightening effect of the final black tea fermented liquid. The brightening effect of the black tea fermented liquid finally prepared by fermentation under static conditions and fermentation under stirring conditions is also different. The present invention is suitable for fermentation by static fermentation.

[0137] 4. Anti-wrinkle and firming test - zebrafish type I collagen gene expression promotion test

[0138] Test principle: Collagen is the most abundant extracellular matrix protein in the human body, among which type I collagen is the most abundant protein in the skin. The distribution of type I collagen in zebrafish is the same as that in humans, and it shows a high degree of conservation with humans. Their innate expression decreases significantly at 6 days after fertilization and becomes very low between 10-12 days after fertilization. The expression of zebrafish type I collagen gene (collala) is tested, and the relative expression changes of zebrafish type I collagen gene in the experimental group and the blank control group are compared. By calculating the promotion rate of type I collagen gene expression, the anti-wrinkle and firming effects of the product can be evaluated.

[0139] Test method: The blank control group randomly selected 36 healthy zebrafish 6 days after fertilization and transferred them to a 24-well plate, with each well containing 12 zebrafish and 2.5 mL of fish embryo culture medium; the experimental group dissolved the black tea fermented liquid of Examples 1-13 and Comparative Examples 1-11 in fish embryo culture medium to prepare a sample solution with a mass percentage of 5%, randomly selected 36 healthy zebrafish 6 days after fertilization, and transferred them to a 24-well plate, with each well containing 12 zebrafish and 2.5 mL of 5wt% sample solution.

[0140] Cultivation process: The blank control group and experimental group were placed in a 28°C incubator for 24 h, and 12 zebrafish in each well were collected into a 1.5 mL test tube. The solution was removed, and 0.5 mL RNAlater solution (Invitrogen; AM7020) was added and stored frozen.

[0141] RNA extraction process: remove RNAlater solution from the test tube, rinse with PBS solution three times, place in ice bath, remove PBS solution, add 500 μL TRIzol reagent (Invitrogen; 15596018), homogenize embryos with a pellet pestle, place in ice bath for 10 min, add 100 μL chloroform, vortex for 1 min, place in ice bath for 5 min, centrifuge sample for 20 min, transfer upper layer solution to 1.5 mL centrifuge tube, add 250 μL isopropanol, vortex for 1 min, place in ice bath for 10 min, centrifuge sample for 20 min, remove all supernatant, add 500 μL ethanol, vortex for 1 min, centrifuge sample for 5 min, place in ice bath for 10 min; repeat this step 3 times. Remove ethanol, centrifuge sample for 5 min, open the lid in the fume hood to air dry the sample for 10 min, add 10 μL DNase / RNase-free ultrapure distilled water, and heat at 55°C for 15 min.

[0142] cDNA synthesis process: The RNA sample was diluted to 1000 ng / 7 μL with DNase / RNase-free ultrapure distilled water, and the RNA was synthesized into cDNA using the PrimerScript RT kit containing gDNA Eraser (Takara; Cat no.RR047A), and stored frozen.

[0143] Real-time RT-PCR process: prepare primer mixture and real-time PCR master mixture for each primer, dilute cDNA sample 10 times with DNase / RNase-free ultrapure distilled water, add 9 μL PCR master mixture and 1 μL diluted cDNA sample to each well of 96-well plate for PCR, seal the 96-well plate for PCR with optical film, centrifuge the plate for 5 minutes, and perform real-time PCR amplification. Commercially available PCR kit uses SYBR Premix Ex Taq (Takara; Cat no.RR420A).

[0144] Data and result calculation: Real-time PCR data were collected, and the relative expression level of each gene (collala) was calculated as the test result. The collagen gene expression promotion rate was calculated by the following formula. The test results of the promotion of zebrafish type I collagen gene expression by Examples 1-13 and Comparative Examples 1-11 are shown in Table 7 below.

[0145]

[0146] Among them, T represents the relative expression level of zebrafish collagen gene in the experimental group; C represents the relative expression level of zebrafish collagen gene in the blank control group.

[0147] Table 7

[0148] Zebrafish type I collagen gene expression promotion rate (%) Example 1 64±2 Example 2 60±3 Example 3 62±2 Example 4 62±1 Example 5 61±4 Example 6 60±2 Example 7 59±3 Example 8 61±2 Example 9 61±3 Example 10 59±1 Embodiment 11 62±2 Example 12 58±2 Embodiment 13 63±1 Comparative Example 1 39±4 Comparative Example 2 43±3 Comparative Example 3 48±2 Comparative Example 4 47±3 Comparative Example 5 43±3 Comparative Example 6 27±2 Comparative Example 7 48±1 Comparative Example 8 50±1 Comparative Example 9 46±2 Comparative Example 10 48±2 Comparative Example 11 42±4

[0149] According to the data in Table 7, the black tea fermented liquor with antioxidant and brightening effects described in Examples 1-13 of the present invention has a higher promotion rate (%) on the expression of type I collagen gene in zebrafish than that in the control example, indicating that the black tea fermented liquor prepared by the present invention has anti-wrinkle and firming effects.

[0150] By comparing the examples with the comparative examples, it was found that the black tea fermented liquid of Examples 1-3 had a higher promotion rate (%) on the expression of type I collagen gene in zebrafish than that of comparative example 1. It can be seen that in the present invention, the choice of carbon source in the fermentation matrix will affect the fermentation effect, thereby affecting the anti-wrinkle and firming effects of the final black tea fermented liquid; the present invention is suitable for using at least one of corn starch, potato starch, cyclodextrin, fructose syrup, and maltose as a carbon source; at the same time, the promotion rate (%) of type I collagen gene expression in zebrafish in Example 1 is higher than that in Examples 2 and 3, indicating that the present invention is most suitable for using corn starch as a carbon source.

[0151] The black tea fermentation broth described in Examples 1, 4, and 5 has a higher promotion rate (%) on zebrafish type I collagen gene expression than that in Comparative Examples 2 and 3. It can be seen that in the present invention, the choice of nitrogen source in the fermentation matrix will affect the fermentation effect, thereby affecting the anti-wrinkle and firming effects of the final black tea fermentation broth; the present invention is suitable for using at least two of ammonium salts, peptones, and yeast extracts as nitrogen sources; at the same time, the zebrafish type I collagen gene expression promotion rate (%) in Example 1 is higher than that in Examples 4 and 5, indicating that the present invention is most suitable for using ammonium chloride and soy peptone as nitrogen sources. The zebrafish type I collagen gene expression promotion rate (%) in Example 1 is higher than that in Example 6, which also indicates that when the mass ratio of ammonium chloride and soy peptone in the nitrogen source described in the present invention is 1:1, the final black tea fermentation broth has the best anti-wrinkle and firming effects.

[0152] The promotion rate (%) of the black tea fermented liquid on the expression of type I collagen gene in zebrafish in Examples 1 and 9 is higher than that in Comparative Examples 4-6. It can be seen that in the present invention, the selection of the type of fermented bacteria will affect the anti-wrinkle and firming effects of the final black tea fermented liquid. The present invention is suitable for the symbiotic fermentation of animal Bifidobacterium lactis subspecies and plant Lactobacillus; at the same time, the promotion rate (%) of zebrafish type I collagen gene expression in Example 1 is higher than that in Example 9, indicating that when the inoculation amount of animal Bifidobacterium lactis subspecies seed liquid and plant Lactobacillus seed liquid is 1:1, the black tea fermented liquid has the best anti-wrinkle and firming effects.

[0153] The promotion rate (%) of the black tea fermentation liquid on the expression of type I collagen gene in zebrafish is higher than that in comparative examples 7 and 8. It can be seen that in the present invention, the pH at the end of fermentation affects the anti-wrinkle and firming effects of the final black tea fermentation liquid. When the pH at the end of fermentation of the present invention is 3.5-4.5, the anti-wrinkle and firming effects of the final black tea fermentation liquid can be better; at the same time, the promotion rate (%) of the expression of type I collagen gene in zebrafish in Example 1 is higher than that in Examples 7 and 8, indicating that when the pH at the end of fermentation of the present invention is 4.2, the anti-wrinkle and firming effects of the final black tea fermentation liquid can be the best.

[0154] The promotion rate (%) of the black tea fermentation liquid on the expression of type I collagen protein in zebrafish is higher than that in comparative examples 10 and 11. It can be seen that in the present invention, the fineness of the black tea powder affects the anti-wrinkle and firming effects of the final black tea fermentation liquid. The present invention is suitable for using black tea powder with a fineness of 60-100 mesh as a raw material; at the same time, the promotion rate (%) of the expression of type I collagen protein in zebrafish in Example 1 is higher than that in Examples 10 and 11, indicating that when the present invention uses black tea powder with a fineness of 80 mesh as a raw material, the final black tea fermentation liquid can have the best anti-wrinkle and firming effects.

[0155] The promotion rate (%) of the black tea fermented liquid in Example 1 on the expression of type I collagen gene in zebrafish is higher than that in Comparative Example 11. It can be seen that in the present invention, the fermentation method affects the anti-wrinkle and firming effects of the final black tea fermented liquid. The present invention is suitable for fermentation by static fermentation.

[0156] 5. Relief test-neutrophil aggregation inhibition test

[0157] Test principle: The neutrophils of zebrafish embryos are highly similar to those of human bodies in morphology, biochemistry and physiological functions. Neutrophils are the first white blood cells to appear at the site of injury or pathogen invasion, and they act to clear infection or harmful substances. The copper sulfate-induced damage to the neuromast cells in the lateral line region of zebrafish embryos was used for testing, and the changes in the number of neutrophils in the lateral line region of the fish embryos between the test substance treatment group and the model control group were compared. The neutrophil inhibition rate was calculated to evaluate the soothing effect of the sample.

[0158] Blank control group: 24 healthy zebrafish embryos 3 days after fertilization were randomly selected, transferred to a 3 cm culture dish, and 5 mL of fish embryo culture medium containing 0.16 mg / L copper sulfate pentahydrate (CuSO4·5H2O) was added; positive control group: 24 healthy zebrafish embryos 3 days after fertilization were randomly selected, transferred to a 3 cm culture dish, and 5 mL of fish embryo culture medium containing 0.16 mg / L copper sulfate pentahydrate (CuSO4·5H2O) and 0.0036 mg / L indomethacin was added (indomethacin has a soothing effect and can inhibit neutrophil aggregation. The positive control is used to judge the effectiveness of the experiment.).

[0159] Experimental group: The black tea fermented liquor of Examples 1-13 and Comparative Examples 1-11 was dissolved in fish embryo culture medium to prepare a sample solution with a mass percentage of 5wt%. 24 healthy zebrafish embryos 3 days after fertilization were randomly selected and transferred to a 3 cm culture dish, and 5 mL of fish embryo culture medium containing 0.16 mg / L copper sulfate pentahydrate (CuSO4·5H2O) and the sample to be tested was added.

[0160] Test method: The blank control group, positive control group and experimental group were placed in a 28°C incubator and cultured for 40 min. After the fish embryos of each test group were fixed in a paraformaldehyde solution (4%) for at least 1 h, the fish embryos were treated with PBST (a PBS solution containing 0.04% Triton X-100 by mass) for 3 times, each for 5 min, and then treated with 50% ethanol for 3 min. After the fish embryos were stained with Sudan black (0.4%) staining solution at room temperature for 1 h, they were rinsed with 70% ethanol for 4 times, each for 5 min, and then treated with PBST for 2 times, each for 5 min. The fish embryos were treated with a bleaching solution (a mixed aqueous solution containing 3% and 1% H2O2 and KOH by mass, respectively) for 10 min, and then treated with 70% ethanol solution for 5 min, PBST for 1 min, and clear solution 1 (sugar solution) for 10 min. The fish embryos were placed sideways on a stereomicroscope, and the number of neutrophils in the lateral line area of ​​the tail of each fish embryo at three-quarters of the way from the hilum of the liver was counted. The neutrophil aggregation inhibition rate was calculated by the following formula. The results of the inhibition of neutrophil aggregation of zebrafish embryos by the samples of Examples 1-13 and Comparative Examples 1-11 are shown in Table 8 below.

[0161]

[0162] Among them, T represents the average number of neutrophils in zebrafish embryos in the experimental group; C represents the average number of neutrophils in zebrafish embryos in the blank control group.

[0163] Table 8

[0164]

[0165]

[0166] According to the data in Table 8, the number of neutrophils in the black tea fermented liquor with antioxidant and brightening effects described in Examples 1-13 of the present invention is less than that in the control example, and the inhibition rate of neutrophil aggregation on zebrafish embryos is higher than that in the control example, indicating that the black tea fermented liquor prepared by the present invention has a soothing effect.

[0167] By comparing the examples with the comparative examples, it was found that the number of neutrophils in the black tea fermented broth of Examples 1-3 was less than that in Comparative Example 1, and the inhibition rate of neutrophil aggregation on zebrafish embryos was higher than that in Comparative Example 1. It can be seen that in the present invention, the choice of carbon source in the fermentation matrix will affect the fermentation effect, thereby affecting the soothing effect of the final black tea fermented broth; the present invention is suitable for using at least one of corn starch, potato starch, cyclodextrin, fructose syrup, and maltose as a carbon source; at the same time, compared with Examples 1-3, Example 1 has the least number of neutrophils and the highest inhibition rate of neutrophil aggregation on zebrafish embryos, indicating that the present invention is most suitable for using corn starch as a carbon source.

[0168] The number of neutrophils in the black tea fermented liquid of Examples 1, 4, and 5 is less than that in Comparative Examples 2 and 3, and the inhibition rate of neutrophil aggregation on zebrafish embryos is higher than that in Comparative Examples 2 and 3. It can be seen that in the present invention, the choice of nitrogen source in the fermentation matrix will affect the fermentation effect, thereby affecting the soothing effect of the final black tea fermented liquid; the present invention is suitable for using at least two of ammonium salts, peptones, and yeast extracts as nitrogen sources; at the same time, compared with Examples 1, 4, 5, and 6, Example 1 has the least number of neutrophils and the highest inhibition rate of neutrophil aggregation on zebrafish embryos, indicating that the present invention is most suitable for using ammonium chloride and soy peptone as nitrogen sources, and when the mass ratio of ammonium chloride and soy peptone in the nitrogen source is 1:1, the final black tea fermented liquid has the best soothing effect.

[0169] The number of neutrophils in the black tea fermented liquid described in Examples 1 and 9 is less than that in Comparative Examples 4-6, and the inhibition rate of neutrophil aggregation on zebrafish embryos is higher than that in Comparative Examples 4-6. It can be seen that in the present invention, the selection of the type of fermented bacteria will affect the soothing effect of the final black tea fermented liquid. The present invention is suitable for symbiotic fermentation of animal Bifidobacterium lactis subspecies and plant Lactobacillus; at the same time, compared with Examples 1 and 9, Example 1 has the least number of neutrophils and the highest inhibition rate of neutrophil aggregation on zebrafish embryos, indicating that when the inoculation amount of animal Bifidobacterium lactis subspecies seed liquid and plant Lactobacillus seed liquid is 1:1, the black tea fermented liquid has the best soothing effect.

[0170] The number of neutrophils in the black tea fermented liquor described in Examples 1, 7, and 8 is less than that in Comparative Examples 7 and 8, and the inhibition rate of neutrophil aggregation on zebrafish embryos is higher than that in Comparative Examples 7 and 8. It can be seen that in the present invention, the pH at the end of fermentation affects the soothing effect of the final black tea fermented liquor. When the pH at the end of fermentation of the present invention is 3.5-4.5, the soothing effect of the final black tea fermented liquor can be better; at the same time, compared with Examples 1, 7, and 8, Example 1 has the least number of neutrophils and the highest inhibition rate of neutrophil aggregation on zebrafish embryos, indicating that when the pH at the end of fermentation of the present invention is 4.2, the soothing effect of the final black tea fermented liquor can be the best.

[0171] The number of neutrophils in the black tea fermented liquor described in Examples 1, 10 and 11 is less than that in Comparative Examples 10 and 11, and the inhibition rate of neutrophil aggregation on zebrafish embryos is higher than that in Comparative Examples 10 and 11. It can be seen that in the present invention, the fineness of black tea powder affects the soothing effect of the final black tea fermented liquor, and the present invention is suitable for using black tea powder with a fineness of 60-100 mesh as a raw material; at the same time, compared with Examples 1, 10 and 11, Example 1 has the least number of neutrophils and the highest inhibition rate of neutrophil aggregation on zebrafish embryos, indicating that when the present invention uses black tea powder with a fineness of 80 mesh as a raw material, the soothing effect of the final black tea fermented liquor can be the best.

[0172] The number of neutrophils in the black tea fermented liquid in Example 1 is less than that in Comparative Example 11, and the inhibition rate of neutrophil aggregation on zebrafish embryos is higher than that in Comparative Example 11. It can be seen that in the present invention, the fermentation method will also affect the soothing effect of the final black tea fermented liquid. The present invention is suitable for fermentation by static fermentation.

[0173] In summary, the selection of the type of carbon source, the selection of the type and content of the nitrogen source, the fermentation strain and the inoculation amount, the pH at the end of fermentation, the fineness of the black tea powder and the fermentation method in the fermentation matrix during the preparation of the black tea fermentation liquid of the present invention have a significant effect on the antioxidant effect, brightening effect, anti-wrinkle firming effect and soothing effect of the final black tea fermentation liquid; when the fineness of the black tea powder of the present invention is 80 mesh, the carbon source is 2% corn starch, the nitrogen source is soy peptone and ammonium chloride in a mass ratio of 1:1, the pH at the end of fermentation is 4.2, the composite bacteria are inoculated with animal Bifidobacterium lactis subspecies seed liquid and plant Lactobacillus seed liquid in an inoculation amount of 1:1, and fermentation is carried out in a static fermentation manner, the antioxidant effect, brightening effect, firming effect and soothing effect of the final black tea fermentation liquid with antioxidant and brightening effects are the best.

[0174] 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 a black tea fermented liquid having antioxidant and brightening effects, characterized in that: The following steps are involved: S1, adding the activated composite bacteria seed liquid to the fermentation matrix containing black tea powder and fermenting until the pH drops to 3.5-4.5, and then stopping the fermentation to obtain a crude fermentation liquid; S2, sterilizing and filtering the crude fermentation liquid described in step S1 to obtain the black tea fermentation liquid with antioxidant and brightening effects; Wherein, in step S1, the composite bacteria are Bifidobacterium animalis subspecies lactis and Lactobacillus plantarum; The fineness of the black tea powder is 60-100 mesh; The fermentation matrix comprises the following components in weight percentage: 1%-5% black tea powder, 1%-4% carbon source, 1%-4% nitrogen source and the balance water; The carbon source is at least one of corn starch, potato starch and cyclodextrin; the nitrogen source is ammonium salt and peptone, or ammonium salt and yeast extract.

2. The method for preparing the black tea fermented liquid with antioxidant and brightening effects according to claim 1, characterized in that: In the step S1, the inoculation amounts of Bifidobacterium animalis subsp. lactis and Lactobacillus plantarum are both 2%-5%, and the ratio of the inoculation amounts of Bifidobacterium animalis subsp. lactis to Lactobacillus plantarum is Bifidobacterium animalis subsp. lactis: Lactobacillus plantarum = 1-2:1-2.

3. The method for preparing the black tea fermented liquid with antioxidant and brightening effects as claimed in claim 1, characterized in that: The fermentation matrix comprises the following components in weight percentage: 2% black tea powder, 2% carbon source, 2% nitrogen source and the balance water.

4. The method for preparing the black tea fermented liquid with antioxidant and brightening effects according to claim 1, characterized in that: At least one of the following (1)-(2) is adopted: (1) The fermentation conditions are: temperature of 35-40°C and time of 50-80h; (2) The sterilization conditions are: temperature of 85-115°C and time of 15-30 minutes.

5. The method for preparing the black tea fermented liquid with antioxidant and brightening effects as claimed in claim 1, characterized in that: At least one of the following (3)-(6) is adopted: (3) The carbon source is corn starch; (4) The nitrogen source is ammonium chloride and soy peptone, and the weight ratio of ammonium chloride to soy peptone is 1:1-3; (5) The fermentation conditions are: temperature is 37°C; (6) The sterilization conditions are: temperature 90°C and time 20 min.

6. A black tea fermented liquid with antioxidant and brightening effects, characterized in that: The black tea fermented liquid with antioxidant and brightening effects is prepared by the method according to any one of claims 1 to 5.

7. A cosmetic, characterized in that: The cosmetic comprises the black tea fermented liquid with antioxidant and brightening effects as claimed in claim 6.

8. The cosmetic according to claim 7, characterized in that The added amount of the black tea fermented liquid with antioxidant and brightening effects is 1-30% of the total mass of the cosmetics.

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