A fermentation method for improving skin care efficacy of black tea fermented liquid

Abstract

The present invention provides a fermentation method for improving the skin care effect of black tea fermented liquid, belonging to the fields of microbial technology and black tea fermentation technology. The method comprises the following steps: (1) the seed liquid of Clostridium butyricum is inoculated into the fermentation medium with an inoculum amount of 1-3%, and 1-3% black tea dry powder is added; (2) after the above-mentioned mixed solution is cultured under anaerobic conditions for 3-7 hours, the seed liquid of Bifidobacterium longum is then inoculated into the fermentation medium with an inoculum amount of 3-6%, and the fermentation is stopped when the pH drops to 4.5; (3) the mixed solution obtained after the fermentation is sterilized, cooled to room temperature, centrifuged or filtered by suction and then passed through a membrane to obtain a clear black tea fermented liquid. The present invention improves the skin care effect of black tea fermented liquid by optimizing the fermentation medium, and the raw materials used in the fermentation medium are not traditional chemicals, which can save costs and make it have certain quality and price advantages compared with the products currently on the market.

Description

Technical Field

[0001] The invention relates to the field of microbial technology and black tea fermentation technology, in particular to a fermentation method for improving the skin care effect of black tea fermentation liquid. Background Art

[0002] Black tea is a fully fermented tea, which is a tea refined after a series of processes. The processing and fermentation process causes chemical reactions among the ingredients in the tea, making its functional ingredients richer than green tea, including theanine, multiple amino acids, tea polyphenols, tea polysaccharides and theaflavins, etc., which can be used in health care, medicine and skin care industries.

[0003] At present, black tea fermentation liquid has received a lot of attention. Some studies have fermented black tea to obtain fermentation liquid with certain efficacy. However, due to the differences between black tea varieties and processing methods, different conditions will also produce different efficacy. Therefore, it is necessary to explore a stable black tea fermentation liquid with high efficacy.

[0004] In the preparation process of black tea fermentation liquid, the choice of strains can determine the fermentation results to a certain extent. Bifidobacterium, also known as Bifidobacterium, is a strict anaerobic bacteria and is widely used in the fermentation series of the skin care industry. Clostridium butyricum, Bacillus subtilis, yeast, and lactic acid bacteria are all common probiotics and are also considered for use in the cosmetics industry.

[0005] The optimal conditions for fermentation of two different strains are different, and joint fermentation has high requirements for fermentation conditions. The selection of fermentation medium is particularly important. It not only provides the necessary nutrients for the strain, but also affects the metabolites of the strain. The appropriate culture medium and fermentation matrix have a strong influence on the fermentation of the strain. Therefore, optimizing the culture medium and fermentation matrix can allow the two to ferment symbiotically, which is also a powerful means to change the efficacy of the fermentation liquid.

[0006] In order to improve the biological activity of black tea fermentation liquid, adding a certain amount of Chinese herbal medicine to the fermentation medium can help improve the functional components, and Chinese herbal medicine residue, as a kind of residue, can be reused as a kind of recycling resource. In recent years, there have been many studies on adding Ganoderma lucidum and other substances into the formula of fermentation medium, and there are also very few methods of adding mixed Chinese herbal medicine residue, which can significantly improve the biological activity of fermentation products, but there has been no single variety of Chinese herbal medicine added to black tea liquid. Summary of the invention

[0007] In view of the above problems existing in the prior art, the present invention provides a fermentation method for improving the skin care effect of black tea fermented liquid. The present invention improves the skin care effect of black tea fermented liquid by optimizing the fermentation medium, and the raw materials used in the fermentation medium are not traditional chemicals, which can save costs and make it have certain quality and price advantages compared with the products currently on the market.

[0008] The technical solution of the present invention is as follows:

[0009] A fermentation method for improving the skin care effect of black tea fermented liquid, the method comprising the following steps:

[0010] (1) Inoculating the seed liquid of Clostridium butyricum into the fermentation medium at an inoculum amount of 1-3%, and adding 1-3% of black tea dry powder; preferably, after adding the black tea dry powder in step (1), the initial pH value is adjusted to 7.0.

[0011] (2) After culturing the mixed solution at 35-37° C., 120-180 rpm and anaerobic conditions for 3-7 hours, the seed solution of Bifidobacterium longum is inoculated into the fermentation medium at an inoculum amount of 3-6%, and the fermentation is stopped when the pH drops to 4.5; preferably, the pH is measured every 2 hours in step (2).

[0012] (3) Sterilize the mixed solution obtained after the fermentation, cool it to room temperature, centrifuge or filter it, and then pass it through a membrane to obtain a clear black tea fermentation solution. Preferably, the sterilization condition of step (3) is sterilization at 70-80°C for 10-45 minutes.

[0013] Preferably, the formula of the fermentation medium in step (1) is: 0.5-1.5% of astragalus residue, 1-3% of glutinous rice flour, 1-2% of the sum of soybean powder and corn steep liquor powder, and the balance is water, and is sterilized at 105° C. for 10-45 minutes; wherein the mass ratio of soybean powder to corn steep liquor powder is 1:0.5-1:2.

[0014] Preferably, the black tea dry powder in step (1) is crushed to 80-100 mesh.

[0015] Preferably, the method for culturing the seed liquid of Clostridium butyricum in step (1) is as follows: the strain of Clostridium butyricum is inoculated into a liquid culture medium and cultured in an anaerobic environment for 48-72 hours at a culture temperature of 30-40°C, a pH of 4-6, and a shaking speed of 180-200 rpm, so that the bacterial concentration of Clostridium butyricum reaches 1*10 7 -1*10 9 CFU / mL.

[0016] Furthermore, the formula of the liquid culture medium for culturing Clostridium butyricum is: 60g corn steep liquor, 20g glucose, 1g Tween-80, 2g dipotassium hydrogen phosphate, 5g anhydrous sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate heptahydrate, 0.05g manganese sulfate monohydrate, and 1L deionized water.

[0017] Preferably, the method for culturing the seed solution of Bifidobacterium longum in step (2) is as follows: the Bifidobacterium longum strain is inoculated into MRS liquid culture medium for anaerobic culture for 24-72 hours at a culture temperature of 30-40°C, a pH of 6.5-7.0, and a shaking speed of 120-180 rpm, so that the bacterial concentration of Bifidobacterium longum reaches 1*10 7 -1*10 9 CFU / mL.

[0018] Furthermore, the formula of the MRS liquid culture medium for culturing Bifidobacterium longum is: 5 g peptone, 10 g beef extract powder, 4 g yeast powder, 5 g glucose, 1 g soluble starch, 5 g NaCl, 3 g NaAc, 0.5 g L-cysteine ​​hydrochloride, 0.2 g MgSO4, 0.05 g MnSO4, and 1 L deionized water.

[0019] Furthermore, the percentages described in the present invention are all mass percentages.

[0020] The beneficial technical effects of the present invention are:

[0021] 1. The present invention obtains the fermentation method for improving the skin care effect of black tea fermented liquid by screening and optimization. First, the best mixed strains are screened, then the amount and order of adding the strains are selected, and finally the fermentation medium formula is optimized so that the two strains of Bifidobacterium longum and Clostridium butyricum can co-ferment with some strains of black tea itself, and the fermentation medium is optimized, and the obtained black tea liquid has a strong skin care effect.

[0022] 2. The present invention obtains a mixed strain with good moisturizing effect by screening strains and optimizing the order and amount of addition of strains, and realizes the combined fermentation of Bifidobacterium longum and Clostridium butyricum by optimizing the fermentation medium.

[0023] 3. The present invention improves the skin care effect of black tea fermentation liquid by optimizing the fermentation medium, and screens the Chinese medicine residue, carbon source and nitrogen source in the fermentation medium to obtain a black tea fermentation liquid with strong antioxidant capacity, high tyrosinase inhibition rate and high moisturizing and skin repair barrier capacity.

[0024] 4. The present invention increases the amount of fermentation products and enhances the efficacy of black tea fermentation liquid through the combined fermentation of Bifidobacterium longum and Clostridium butyricum, the addition of Chinese medicine residues, and the optimization of the fermentation medium, thereby obtaining a method for producing black tea fermentation liquid with strong antioxidant, whitening and skin barrier repair capabilities.

[0025] 5. The present invention is easy to operate, cost-saving, safe and pollution-free, and can ensure the efficacy of black tea fermentation liquid. DETAILED DESCRIPTION

[0026] The present invention is described in detail below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] The strains and sources used in the present invention are as follows: Bifidobacterium longum, Lactobacillus rhamnosus, Clostridium butyricum, Bacillus subtilis, Lactococcus lactis, and Saccharomyces cerevisiae are purchased from Ningbo Testo Biotechnology Co., Ltd.; Lactobacillus plantarum-JJL-1 is from China Microorganism Culture Collection Center CGMCC 24962.

[0028] Example 1: Antioxidant capacity determination method (DPPH free radical scavenging rate)

[0029] DPPH was prepared into 0.2mM DPPH-anhydrous ethanol solution using anhydrous ethanol. 200μL of each 1% black tea fermentation broth sample was placed in a 0.5mL centrifuge tube with the DPPH-anhydrous ethanol solution, mixed evenly, and reacted in a dark water bath at 37°C for 30 minutes. The absorbance (Ap) of the sample group was measured at 517nm on a 96-well ELISA plate. Water was used to replace the sample solution to measure the absorbance (Aq) of the sample control group, and anhydrous ethanol was used to replace the DPPH-anhydrous ethanol solution to measure the absorbance (Aw) of the blank group. Vc was used as a positive control to compare the scavenging rate of each black tea fermentation broth on DPPH free radicals. The calculation formula is as follows:

[0030] DPPH free radical scavenging rate (%) = [Aq-(Ap-Aw)] / Aq*100%

[0031] Wherein, Ap is the absorbance value of the sample group; Aq is the absorbance value of the sample control group; Aw is the absorbance value of the blank group.

[0032] Example 2: Whitening effect determination method (anti-tyrosinase activity)

[0033] The monophenolase activity of tyrosinase was determined in an activity test system with 1 mg / mL DOPA solution as substrate and 0.05 mol / L PBS (phosphate buffer, pH 6.8). First, add 0.5 mL 100 U / mL tyrosinase solution, add 1 mL 1% of each black tea fermentation solution sample, react at 37°C for 10 minutes, then add 2 mL of substrate solution that was previously kept warm in a 37°C constant temperature water bath, mix immediately, and measure the absorbance value Aa at a wavelength of 475 nm under 37°C constant temperature conditions. Use PBS solution instead of enzyme solution as sample control Ab, use PBS solution instead of sample solution as blank Ac, and use PBS instead of enzyme solution and sample as blank control Ad. The calculation formula is as follows:

[0034] Tyrosinase inhibition rate (%) = [1-(Aa-Ab) / (Ac-Ad)]*100%

[0035] Example 3: Moisturizing ability determination method (determination of transepidermal water loss TEWL)

[0036] A skin tester (CK Tewameter from Germany) was used, and 250 healthy volunteers aged 18-45 were selected as subjects and divided into 25 groups, with 10 people in each group. After cleaning the facial area of ​​the subjects, 1-2 mL of black tea fermentation liquid sample was randomly applied to half of the face area, and the other half of the face area was used as a blank control group and deionized water was applied; it was used once in the morning and evening every day, and TEWL was measured by the skin tester before using the sample, 10 days after using the sample, and 20 days after using the sample, and the average value was taken after repeating 3 times.

[0037] Example 4: Bacteria screening

[0038] 1. Conventional fermentation medium: 2% glucose, 1% peptone and beef extract (1:1) and the balance water, sterilized at 105°C for 30 minutes.

[0039] 2. Inoculate the seed liquid of different strains (1:1) (see Table 1 for strain selection) into the above fermentation medium at an inoculum rate of 3%, and add 2% dry black tea powder (crushed black tea to 80 mesh).

[0040] 3. The mixed solution in step 2 was cultured at 36°C, 180 rpm, and oxygen conditions as shown in Table 1. The pH was measured every 4 hours, and the fermentation was stopped when the pH dropped to 4.5.

[0041] 4. After the fermentation is completed, the mixed liquid is sterilized at 80°C for 30 minutes, cooled to room temperature, centrifuged and then filtered through a membrane to obtain a clear black tea fermentation liquid.

[0042] 5. The moisturizing effect of the obtained black tea fermentation liquid was measured using the test method as shown in Example 3.

[0043] Table 1 Screening of mixed strains

[0044]

[0045] Moisturizing effect is the most basic requirement of the skin care industry for lotions. Transepidermal water loss (TEWL) is one of the main indicators for evaluating skin barrier function. It measures the water vapor pressure gradient on the skin surface to indicate water loss, thereby reflecting the water permeability barrier state of the skin. The smaller the TEWL value, the less water loss and the better the skin barrier. As can be seen from the results in Table 1, the black tea liquid obtained by the combined use of Bifidobacterium longum and Butyricum acidum has the lowest TEWL value after 20 days of use, proving that it produces a more obvious moisturizing effect.

[0046] Example 5: Selection of the order and amount of bacteria added

[0047] 1. Conventional fermentation medium: 2% glucose, 1% peptone and beef extract (1:1) and the balance water, sterilized at 105°C for 30 minutes.

[0048] Bifidobacterium longum seed culture: The strain was inoculated into MRS liquid culture medium and cultured in an anaerobic environment for 48 hours at a culture temperature of 35°C, pH 6.5-7.0, and a shaking speed of 180 rpm. The bacterial concentration of Bifidobacterium longum reached 1*10 7 -1*10 9 CFU / mL. (MRS liquid medium: peptone 5g, beef extract powder 10g, yeast powder 4g, glucose 5g, soluble starch 1g, NaCl 5g, NaAc 3g, L-cysteine ​​hydrochloride 0.5g, MgSO4 0.2g, MnSO4 0.05g, 1L deionized water, sterilized at 105℃ for 40 minutes)

[0049] Seed liquid culture of Clostridium butyricum: The strain was inoculated into liquid culture medium and cultured in an anaerobic environment for 48 hours at a temperature of 35°C, pH 4-6, and a shaking speed of 180 rpm. The concentration of Clostridium butyricum reached 1*10 7 -1*10 9 CFU / mL. (Liquid culture medium for Clostridium butyricum: 60 g corn syrup, 20 g glucose, 1 g Tween-80, 2 g potassium dihydrogen phosphate, 5 g sodium acetate anhydrous, 2 g triammonium citrate, 0.2 g magnesium sulfate heptahydrate, 0.05 g manganese sulfate monohydrate, 1 L deionized water, sterilized at 105°C for 40 minutes)

[0050] 2. Inoculate one of the bacterial species (see Table 2 for inoculation order and ratio) into the above fermentation medium, and add 2% dry black tea powder (crushed black tea to 80 mesh).

[0051] 3. After culturing the mixture in step 2 at 36°C, 180rpm and anaerobic conditions for 4 hours, add seed solution of another strain, measure the pH every 2 hours, and stop fermentation when the pH drops to 4.5.

[0052] 4. After the fermentation is completed, the mixed liquid is sterilized at 80°C for 30 minutes, cooled to room temperature, centrifuged or filtered, and then passed through a membrane to obtain a clear black tea fermentation liquid.

[0053] 5. The moisturizing effect of the obtained black tea fermentation liquid was measured using the test method as shown in Example 3.

[0054] Table 2 Selection of order and proportion of bacterial addition

[0055]

[0056]

[0057] From the results in Table 2, it can be seen that different addition orders and proportions will also affect the effect of the black tea liquid. First, 2% of the Butyric Acid Bacterium seed liquid is added and cultured for 4 hours, and then 4% of the Bifidobacterium longum seed liquid is added and cultured until the pH drops to 4.5. This can obtain a black tea liquid with a better moisturizing effect than other addition orders and addition amounts.

[0058] Example 6: Optimization of selection of Chinese medicine residues

[0059] 1. The fermentation medium to be optimized: 1% Chinese medicinal residue (types of Chinese medicinal residue: peony bark, polygonatum, rhubarb, astragalus), 2% glucose, 1% peptone and beef extract (1:1) and the rest water, sterilized at 105°C for 30 minutes.

[0060] 2. Inoculate the seed liquid of Clostridium butyricum into the above fermentation medium at an inoculation rate of 2%, and add 2% dry black tea powder (crushed black tea to 80 mesh).

[0061] 3. After culturing the mixed solution in step 2 at 36°C, 180 rpm and anaerobic conditions for 4 hours, Bifidobacterium longum was inoculated into the fermentation medium at an inoculum size of 4%, and the pH was measured every 2 hours. The fermentation was stopped when the pH dropped to 4.5.

[0062] 4. After the fermentation is completed, the mixed liquid is sterilized at 80°C for 30 minutes, cooled to room temperature, centrifuged or filtered, and then passed through a membrane to obtain a clear black tea fermentation liquid.

[0063] 5. The skin care efficacy of the obtained black tea fermented liquid was measured. The test method is as shown in Example 1 to Example 3, and the test results are shown in Table 3 and Table 4.

[0064] Table 3 Determination results of antioxidant and whitening ability of different Chinese medicinal residues on black tea fermentation liquid

[0065] Chinese medicine residue No drug residue Peony bark Polygonatum rhubarb Astragalus DPPH free radical scavenging rate (%) 61.8 77.3 67.4 68.5 80.8 Tyrosinase inhibition rate (%) 50.7 55.5 54 51.2 56.3

[0066] Table 4 Effects of different Chinese medicinal residues on the moisturizing effect of black tea fermentation liquid

[0067]

[0068] Antioxidant activity is a commonly used evaluation index for skin care, and whitening effect is an advantageous index of the black tea fermented liquid of the present invention, which can broaden the application scope of the fermented liquid. The smaller the TEWL value, the less water loss and the better the skin barrier. Therefore, it is very reasonable to use antioxidant, anti-tyrosinase activity and TEWL value to evaluate the skin care effect of the fermented liquid.

[0069] From the results in Tables 3 and 4, it can be seen that the skin care effect of adding Astragalus residue is significantly higher than that of other types of Chinese medicine residues. The antioxidant capacity of the black tea fermentation liquid obtained by adding Astragalus residue at a concentration of 1% can reach 80.8%.

[0070] Example 7: Optimization of carbon source selection

[0071] 1. The fermentation medium to be optimized: 1% astragalus residue, 2% carbon source (carbon source: one of glycerol, bagasse, soybean residue, rice bran residue, glutinous rice flour), 1% peptone and beef extract (1:1) and the rest water, sterilized at 105°C for 30 minutes.

[0072] 2. Inoculate the seed liquid of Clostridium butyricum into the above fermentation medium at an inoculation rate of 2%, and add 2% dry black tea powder (crushed black tea to 80 mesh).

[0073] 3. After culturing the mixed solution in step 2 at 36°C, 180 rpm and anaerobic conditions for 4 hours, Bifidobacterium longum was inoculated into the fermentation medium at an inoculum size of 4%, and the pH was measured every 2 hours. The fermentation was stopped when the pH dropped to 4.5.

[0074] 4. After the fermentation is completed, the mixed liquid is sterilized at 80°C for 30 minutes, cooled to room temperature, centrifuged or filtered, and then passed through a membrane to obtain a clear black tea fermentation liquid.

[0075] 5. The activity and efficacy of the obtained black tea fermentation liquid were measured. The test methods were as shown in Examples 1 to 3. The test results are shown in Tables 5 and 6.

[0076] Table 5 The results of the antioxidant and whitening ability test of different carbon sources on 1% black tea fermentation broth

[0077] Carbon source glycerin Bagasse Soybean residue Glutinous rice flour Rice bran residue DPPH free radical scavenging rate (%) 74.2 78.4 77.4 81.5 77.9 Tyrosinase inhibition rate (%) 53 52.7 55.4 57.1 54.8

[0078] Table 6 Effect of different carbon sources on the moisturizing effect of black tea fermentation liquid

[0079]

[0080] From the results in Tables 5 and 6, it can be seen that selecting glutinous rice flour as the carbon source can achieve the best skin care effect. The antioxidant capacity of the black tea fermentation liquid obtained by adding glutinous rice flour to the culture medium is significantly higher than that of others, and the TEWL value is reduced by 9.3 after using the product for 20 days.

[0081] Example 8: Optimization of nitrogen source selection

[0082] 1. The fermentation medium to be optimized: 1% astragalus residue, 2% glutinous rice flour, 1% nitrogen source (nitrogen source: two of soybean powder, corn steep liquor powder, bran and cottonseed hull powder) and the balance water, sterilized at 105°C for 30 minutes.

[0083] 2. Inoculate the seed liquid of Clostridium butyricum into the above fermentation medium at an inoculation rate of 2%, and add 2% dry black tea powder (crushed black tea to 80 mesh).

[0084] 3. After culturing the mixed solution in step 2 at 36°C, 180 rpm and anaerobic conditions for 4 hours, Bifidobacterium longum was inoculated into the fermentation medium at an inoculum size of 4%, and the pH was measured every 2 hours. The fermentation was stopped when the pH dropped to 4.5.

[0085] 4. After the fermentation is completed, the mixed liquid is sterilized at 80°C for 30 minutes, cooled to room temperature, centrifuged or filtered, and then passed through a membrane to obtain a clear black tea fermentation liquid.

[0086] 5. The activity and efficacy of the obtained black tea fermentation liquid were measured. The test methods were as shown in Examples 1 to 3. The test results are shown in Tables 7 and 8.

[0087] Table 7 Determination results of antioxidant and whitening ability of black tea fermentation broth with different nitrogen sources

[0088]

[0089] Table 8 Effect of different nitrogen sources on the moisturizing effect of black tea fermentation liquid

[0090]

[0091] From the results in Tables 7 and 8, it can be seen that the best choice of nitrogen source is the combination of soybean flour and corn steep liquor powder (1:1).

[0092] Example 9: Optimization of initial pH selection

[0093] 1. Fermentation medium: 1% astragalus residue, 2% glutinous rice flour, 1% soybean flour and corn steep liquor powder (1:1) and the balance water, sterilize at 105°C for 30 minutes.

[0094] 2. Inoculate the seed liquid of Clostridium butyricum into the above fermentation medium at an inoculum rate of 2%, add 2% dry black tea powder (crushed black tea to 80 mesh), and adjust the initial pH value to 8.0, 7.0 or 6.0 respectively.

[0095] 3. After culturing the mixed solution in step 2 at 36°C, 180 rpm and anaerobic conditions for 4 hours, Bifidobacterium longum was inoculated into the fermentation medium at an inoculum size of 4%, and the pH was measured every 2 hours. The fermentation was stopped when the pH dropped to 4.5.

[0096] 4. After the fermentation is completed, the mixed liquid is sterilized at 80°C for 30 minutes, cooled to room temperature, centrifuged or filtered, and then passed through a membrane to obtain a clear black tea fermentation liquid.

[0097] 5. The skin care efficacy of the obtained black tea fermented liquid was measured. The test method was as shown in Example 1 to Example 3. The test results are shown in Table 9 and Table 10.

[0098] Table 9 Determination results of antioxidant and whitening ability of black tea fermentation broth at different initial pH values

[0099] pH 8.0 7.0 6.0 DPPH free radical scavenging rate (%) 70.7 81.9 74.4 Tyrosinase inhibition rate (%) 48.5 58.3 52.7

[0100] Table 10 Effect of different initial pH values ​​on the moisturizing effect of black tea fermentation liquid

[0101]

[0102]

[0103] From the results of Tables 9 and 10, it can be concluded that the selection of the initial pH also has a certain influence on the effect of the fermentation liquid. When the initial pH value is 7.0, the various functions and moisturizing effects of the obtained black tea fermentation liquid are better.

[0104] From the results of Example 4 to Example 9, it can be seen that by selecting Bifidobacterium longum and Clostridium butyricum as bacterial strains, first adding 2% butyric acid Bacillus seed solution and culturing for 4 hours, and then adding 4% Bifidobacterium longum seed solution and culturing until the pH drops to 4.5, the highest moisturizing effect can be obtained; when the formula of the fermentation medium selected by the bacterial strain is 1% astragalus residue, 2% glutinous rice flour, 1% soybean flour and corn steep liquor powder (1:1) and the balance is water, the best fermentation medium formula is obtained; and when the initial pH is 7.0, the fermentation effect is best.

[0105] Under this optimal formula, the DPPH free radical scavenging rate of the black tea fermented liquid reached 81.9% at its 1% concentration, and the tyrosine inhibition rate was 58.3%. The TEWL reduction value of the black tea fermented liquid obtained under this condition exceeded 10 after 20 days of use, indicating that it has strong moisturizing and skin barrier repair capabilities.

[0106] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and implementation modes, and can be fully applicable to various fields suitable for the present invention. For those familiar with the art, for those of ordinary skill in the art, various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principle and spirit of the present invention. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A fermentation method for improving the skin care effect of black tea fermented liquid, characterized in that: The method comprises the following steps: (1) inoculating the seed liquid of Clostridium butyricum into the fermentation medium at an inoculum amount of 1-3%, adding 1-3% of black tea dry powder, and adjusting the initial pH value to 7.0; (2) culturing the mixed solution at 35-37° C., 120-180 rpm and anaerobic conditions for 3-7 hours, then inoculating the seed solution of Bifidobacterium longum into the fermentation medium at an inoculum rate of 3-6%, and stopping the fermentation when the pH drops to 4.5; (3) sterilizing the mixed solution obtained after the fermentation, cooling it to room temperature, centrifuging or filtering it with suction, and then passing it through a membrane to obtain a clear black tea fermentation solution; The formula of the fermentation medium in step (1) is: 0.5-1.5% of astragalus residue, 1-3% of glutinous rice flour, 1-2% of the sum of soybean powder and corn steep liquor powder, and the balance is water, and the medium is sterilized at 105° C. for 10-45 minutes; wherein the mass ratio of soybean powder to corn steep liquor powder is 1:1; The formula of the liquid culture medium for culturing Clostridium butyricum is: 60 g corn steep liquor, 20 g glucose, 1 g Tween-80, 2 g dipotassium hydrogen phosphate, 5 g anhydrous sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate heptahydrate, 0.05 g manganese sulfate monohydrate, and 1 L deionized water; The formula of MRS liquid culture medium for culturing Bifidobacterium longum is: 5g peptone, 10g beef extract powder, 4g yeast powder, 5g glucose, 1g soluble starch, 5g NaCl, 3g NaAc, 0.5g L-cysteine ​​hydrochloride, 0.2g MgSO4, 0.05g MnSO4, and 1L deionized water.

2. The fermentation method according to claim 1, characterized in that The black tea dry powder in step (1) is crushed to 80-100 mesh.

3. The fermentation method according to claim 1, characterized in that The method for culturing the seed liquid of Clostridium butyricum in step (1) is as follows: the strain of Clostridium butyricum is inoculated into a liquid culture medium and cultured in an anaerobic environment for 48-72 hours at a culture temperature of 30-40°C, a pH of 4-6, and a shaking speed of 180-200 rpm, so that the bacterial concentration of Clostridium butyricum reaches 1*10 7 -1*10 9 CFU / mL.

4. The fermentation method according to claim 1, characterized in that Step (2) The method for culturing the seed solution of Bifidobacterium longum is as follows: the Bifidobacterium longum strain is inoculated into MRS liquid culture medium for anaerobic culture for 24-72 hours at a culture temperature of 30-40°C, a pH of 6.5-7.0, and a shaking speed of 120-180 rpm, so that the bacterial concentration of Bifidobacterium longum reaches 1*10 7 -1*10 9 CFU / mL.

5. The fermentation method according to claim 1, characterized in that The sterilization conditions of step (3) are sterilization at 70-80°C for 10-45 minutes.

Images