A Chinese medicine lactic acid bacteria bidirectional fermentation liquid, fermentation process and application of fermentation liquid

Abstract

The present application provides a Chinese medicine lactic acid bacteria bidirectional fermentation liquid, a fermentation process and an application of the fermentation liquid, and relates to the technical field of skin care products. The fermentation process of the Chinese medicine lactic acid bacteria bidirectional fermentation liquid is to inoculate lactic acid bacteria in a fermentation medium containing Chinese medicine for fermentation and culture, with Chinese medicine as a bidirectional fermentation substrate, and lactic acid bacteria as a strain, and a bidirectional fermentation liquid with multiple functions is obtained by a combination of the two. The application of Chinese medicine lactic acid bacteria fermentation liquid in the field of cosmetics is improved, and the application range of Chinese medicine is broadened. By bidirectional fermentation of berry tea and lactic acid bacteria, the berry tea powder can be directly added to the fermentation medium after treatment, and the berry tea extract is obtained without enzymatic hydrolysis, water extraction or organic reagent extraction. The method is simpler and has low energy consumption. The total antioxidant capacity, DPPH and ABTS scavenging ability of the obtained berry tea lactic acid bacteria bidirectional fermentation filtrate are better than those of berry tea fermentation medium, and the tyrosinase inhibition rate is significantly better than that of berry tea fermentation medium.

Description

Technical Field

[0001] The present application relates to the technical field of skin care products, and in particular to a Chinese medicine lactic acid bacteria bidirectional fermentation liquid, a fermentation process and an application of the fermentation liquid. Background Art

[0002] Berry tea is made from the tender stems and leaves of Agave sphenanthera, also known as small-leaf vine tea. It is mainly distributed in Hunan, Hubei, Fujian, Jiangxi and other places in my country. The plant is rich in natural active ingredients such as flavonoids. During the processing, the flavonoids contained in the cells penetrate to the surface and form a layer of white frost, hence the name berry tea. Berry tea was originally a common ethnic medicine used by the Tujia ethnic group, and is included in traditional Chinese medicine monographs such as "Tujia Medicine", "National Compilation of Chinese Herbal Medicines", and "Hunan Chinese Medicinal Materials (2009 Edition)". Berry tea has the effects of clearing away heat and detoxifying, promoting blood circulation and unblocking collaterals, removing dampness and swelling, and calming the liver and lowering blood pressure. It is one of the more popular Chinese medicines for both medicinal and tea use in recent years.

[0003] At present, the extraction of active ingredients in berry tea mainly includes water extraction, enzymatic hydrolysis, alkali extraction and acid precipitation. For example, patent document CN111903812 A discloses a preparation method and application of instant berry tea extract, and CN109329524 A discloses a preparation method of flavonoids in berry tea. The extraction time of berry tea active ingredients in the prior art is long, the operation is complicated, and the energy consumption is high. Summary of the invention

[0004] The purpose of the present application is to provide a Chinese medicine lactic acid bacteria bidirectional fermentation liquid, a fermentation process and an application of the fermentation liquid, aiming to solve the problems of long extraction time, complex operation and high energy consumption of the existing berry tea active ingredients.

[0005] To achieve the above objectives, the present application provides a fermentation process for a bidirectional fermentation broth of lactic acid bacteria containing traditional Chinese medicine, comprising: inoculating lactic acid bacteria in a fermentation medium containing traditional Chinese medicine for fermentation and culturing to obtain the bidirectional fermentation broth of lactic acid bacteria containing traditional Chinese medicine; the added mass volume ratio of the traditional Chinese medicine in the fermentation medium is 0.1%-2.0%.

[0006] Preferably, the traditional Chinese medicine is selected from any one of berry tea, liquorice, oil tea, chia seeds and schisandra chinensis.

[0007] Preferably, the traditional Chinese medicine is berry tea, the berry tea is berry tea powder, and the mesh number of the berry tea powder is 20-50 meshes.

[0008] Preferably, the inoculation volume of the lactic acid bacteria is 3%-6% of the volume of the fermentation medium.

[0009] Preferably, the fermentation temperature of the fermentation culture is 34-38° C., and the fermentation time is 1-4 days.

[0010] Preferably, the fermentation medium further comprises: 10 g / L peptone, 8 g / L beef extract, 5 g / L yeast powder, 20 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium hydrogen citrate, 5 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, 1 g / L Tween 80, and the pH is adjusted to 5.7.

[0011] Preferably, the lactic acid bacteria are inoculated into the fermentation medium after activation culture, and the activation culture medium is: peptone 12g / L, beef extract 9g / L, yeast powder 5g / L, glucose 15g / L, dipotassium hydrogen phosphate 2g / L, diammonium hydrogen citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.04g / L, Tween 80 1g / L, natural pH.

[0012] The present application also provides a Chinese medicine lactic acid bacteria bidirectional fermentation liquid, which is prepared by fermentation using the above-mentioned Chinese medicine lactic acid bacteria bidirectional fermentation liquid fermentation process.

[0013] Preferably, the Chinese medicine lactic acid bacteria bidirectional fermentation liquid is berry tea lactic acid bacteria bidirectional fermentation liquid;

[0014] More preferably, the added mass volume ratio of berry tea powder in the berry tea lactic acid bacteria bidirectional fermentation broth is 0.5%, and its total flavonoids content is 400-500 μg / mL; the total phenol content is 800-1000 μg / mL; the DPPH clearance rate is 95%-97%; the ABTS clearance rate is 95%-99.9%; T-AOC is 15-20 μmol / mL; and the tyrosinase inhibition rate is 98%-99.9%.

[0015] The present application also provides the use of the above-mentioned Chinese medicine lactic acid bacteria bidirectional fermentation liquid in the preparation of cosmetics.

[0016] The present application also provides a cosmetic, the raw materials of which include the above-mentioned Chinese medicine lactic acid bacteria bidirectional fermentation liquid.

[0017] Compared with the prior art, the beneficial effects of this application include:

[0018] The fermentation process of the Chinese medicine lactic acid bacteria bidirectional fermentation liquid provided in the present application is to inoculate lactic acid bacteria in a fermentation medium containing Chinese medicine for fermentation culture, use Chinese medicine as a bidirectional fermentation substrate, use lactic acid bacteria as a strain, and ferment Chinese medicine and probiotics in combination. The two-way fermentation liquid with multiple functions is obtained by the combined fermentation of the two, and the application of Chinese medicine lactic acid bacteria fermentation liquid in the field of cosmetics is improved, and the application range of Chinese medicine is broadened. Through the bidirectional fermentation technology, berry tea and lactic acid bacteria are bidirectionally fermented, and the berry tea powder can be directly added to the fermentation medium after treatment, and the berry tea extract is obtained without enzymolysis, water extraction or organic reagent extraction. The method is simpler and has low energy consumption. The total antioxidant capacity, DPPH and ABTS scavenging ability of the obtained berry tea lactic acid bacteria bidirectional fermentation filtrate are better than those of berry tea-fermentation medium, and the tyrosinase inhibition rate is significantly better than that of berry tea-fermentation medium. DETAILED DESCRIPTION

[0019] As used herein:

[0020] "Prepared from" is synonymous with "comprising." As used herein, the terms "comprising," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0021] The conjunction "consisting of excludes any unspecified element, step, or component. If used in a claim, this phrase renders the claim closed-ended so that it does not include materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of" appears in a clause of the body of a claim rather than immediately following the subject matter, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0022] When an amount, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described in this article, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

[0023] In these examples, parts and percentages are by mass unless otherwise indicated.

[0024] "Parts by mass" refers to the basic unit of measurement for expressing the mass ratio of multiple components. 1 part can represent any unit mass, such as 1g or 2.689g. If we say that the mass of component A is a parts and the mass of component B is b parts, it means the ratio of the mass of component A to the mass of component B is a:b. Alternatively, it means that the mass of component A is aK and the mass of component B is bK (K is an arbitrary number, indicating a multiple factor). It should not be misunderstood that, unlike the mass parts, the sum of the mass of all components is not limited to 100 parts.

[0025] The present application provides a fermentation process for a bidirectional fermentation liquid of lactic acid bacteria containing traditional Chinese medicine, comprising: inoculating lactic acid bacteria in a fermentation medium containing traditional Chinese medicine for fermentation culture to obtain the bidirectional fermentation liquid of lactic acid bacteria containing traditional Chinese medicine; the mass ratio of the traditional Chinese medicine added in the fermentation medium is 0.1%-2.0%.

[0026] The Chinese medicine is selected from any one of berry tea, liquorice, tea oil, chia seeds and schisandra chinensis. Experiments have shown that any of these Chinese medicines can improve the antioxidant and / or whitening ability of the fermentation liquid as a bidirectional fermentation substrate. The added mass volume ratio of the Chinese medicine in the fermentation medium is 0.1%-2.0%, for example, 0.1%-0.5%, 0.5%-1.5%, or 0.5%-1.0%, and more specifically, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2.0%.

[0027] Preferably, the Chinese medicine is berry tea, and the antioxidant and whitening abilities of the berry tea lactic acid bacteria bidirectional fermentation liquid are most significantly improved.

[0028] Lactic acid bacteria (LAB) is a general term for bacteria that can produce a large amount of lactic acid using fermentable carbohydrates. Lactic acid bacteria can be any species such as Lactobacillus rhamnosus, Bifidobacterium lactis, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum, etc.

[0029] Furthermore, the lactic acid bacteria can be activated before being inoculated into the fermentation medium, wherein the medium components used in the lactic acid bacteria activation culture process can be, for example, 12 g / L peptone, 9 g / L beef extract, 5 g / L yeast powder, 15 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium hydrogen citrate, 5 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, 1 g / L Tween 80, and natural pH. The lactic acid bacteria that have been activated have better fermentation effects and faster fermentation speeds.

[0030] Furthermore, when a fermentation tank is needed for large-scale fermentation, the lactic acid bacteria can be cultured after activation culture to obtain the fermentation seed liquid of the lactic acid bacteria, and the fermentation seed liquid is more conducive to fermentation in the fermentation culture tank. The formula of the seed culture medium can be, for example: peptone 10g / L, beef extract 8g / L, yeast powder 5g / L, glucose 15g / L, dipotassium hydrogen phosphate 2g / L, diammonium hydrogen citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.04g / L, Tween 80 1g / L, pH natural.

[0031] Preferably, the inoculation volume of the lactic acid bacteria is 3%-6% of the volume of the fermentation medium, for example, 3%, 4%, 5% or 6%.

[0032] Among them, the present application has found through experiments that berry tea has a certain inhibitory effect on the growth of lactic acid bacteria. Therefore, in the fermentation process of the berry tea lactic acid bacteria bidirectional fermentation liquid of the present application scheme, the added mass volume ratio of berry tea in the fermentation medium is 0.5%-2.0%, for example, it can be 0.5%-1.5%, or 0.5%-1.0%, and more specifically, for example, it can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2.0%.

[0033] In a preferred embodiment, the mass volume ratio of berry tea added to the fermentation medium is 0.5%. At this addition amount, berry tea can reduce the inhibitory effect on lactic acid bacteria and play a synergistic role in bidirectional fermentation with lactic acid bacteria.

[0034] The berry tea can be added in the form of berry tea powder, berry tea water extract, or berry tea enzymatic extract. Preferably, the berry tea added to the fermentation medium is berry tea powder. Directly adding berry tea powder can achieve the same better fermentation effect without the need for a complicated extraction process, is easy to operate, and has low energy consumption.

[0035] Preferably, the mesh size of the berry tea powder is 20-50 mesh, for example, 20-30 mesh, or 20-40 mesh, or 30-50 mesh, more specifically, for example, 20 mesh, 25 mesh, 30 mesh, 35 mesh, 40 mesh, 45 mesh or 50 mesh.

[0036] Among them, the components of the fermentation medium besides berry tea also include: peptone 10g / L, beef extract 8g / L, yeast powder 5g / L, glucose 20g / L, dipotassium hydrogen phosphate 2g / L, diammonium hydrogen citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.04g / L, Tween 80 1g / L, and the pH is adjusted to 5.7.

[0037] Preferably, the fermentation temperature of the fermentation culture is 34-38°C, for example, 34°C, 35°C, 36°C, 37°C or 38°C, and the fermentation time is 1-4 days, for example, 1 day, 2 days, 3 days or 4 days.

[0038] The present application also provides a Chinese medicine lactic acid bacteria bidirectional fermentation liquid, which is prepared by fermentation using the above-mentioned Chinese medicine lactic acid bacteria bidirectional fermentation liquid fermentation process.

[0039] Preferably, the Chinese medicinal lactic acid bacteria bidirectional fermentation broth is berry tea lactic acid bacteria bidirectional fermentation broth; more preferably, the added mass volume ratio of berry tea powder in the berry tea lactic acid bacteria bidirectional fermentation broth is 0.5%, and its total flavonoids content is 400-500 μg / mL; the total phenol content is 800-1000 μg / mL; the DPPH clearance rate is 95%-97%; the ABTS clearance rate is 95%-99.9%; T-AOC is 15-20 μmol / mL; and the tyrosinase inhibition rate is 98%-99.9%.

[0040] The present application also provides the use of the above-mentioned Chinese medicine lactic acid bacteria bidirectional fermentation liquid in the preparation of cosmetics.

[0041] The present application also provides a cosmetic, the raw materials of which include the above-mentioned Chinese medicine lactic acid bacteria bidirectional fermentation liquid.

[0042] The fermentation process of the Chinese medicine lactic acid bacteria bidirectional fermentation liquid provided in the present application is to inoculate lactic acid bacteria in a fermentation medium containing Chinese medicine for fermentation culture, using Chinese medicine as a bidirectional fermentation substrate, and lactic acid bacteria as a strain, and obtaining a bidirectional fermentation liquid with multiple functions through the combined fermentation of the two, thereby improving the application of Chinese medicine lactic acid bacteria fermentation liquid in the field of cosmetics and broadening the scope of application of Chinese medicine. Through the bidirectional fermentation technology, berry tea and lactic acid bacteria are bidirectionally fermented, and the berry tea powder can be directly added to the fermentation medium after being treated, without the need for enzymatic hydrolysis, water extraction or organic reagent extraction to obtain berry tea extract, and the method is simpler and has low energy consumption. The total antioxidant capacity, DPPH and ABTS scavenging ability of the obtained berry tea lactic acid bacteria bidirectional fermentation filtrate are better than those of berry tea-fermentation medium, and the tyrosinase inhibition rate is significantly better than that of berry tea-fermentation medium.

[0043] The embodiments of the present application will be described in detail below in conjunction with specific examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If specific conditions are not specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0044] Example 1

[0045] The fermentation process of the berry tea lactic acid bacteria bidirectional fermentation liquid of Example 1 is as follows:

[0046] 1. Bacteria activation

[0047] Take a strain of Lactobacillus paracasei and inoculate it into a strain activation culture medium, and culture it in a closed environment at 34-37° C. for 1-2 days to obtain an activated strain.

[0048] The formula of the strain activation culture medium is: peptone 12g / L, beef extract 9g / L, yeast powder 5g / L, glucose 15g / L, dipotassium hydrogen phosphate 2g / L, diammonium hydrogen citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.04g / L, Tween 801g / L, natural pH, preparation volume 40ml, sterilization at 118℃ for 15min.

[0049] 2. Fermentation culture

[0050] The activated bacteria in step 1 are inoculated into 500 mL shake flask fermentation medium at a ratio of 3%-6%, the fermentation temperature is 34-37° C., and the fermentation is allowed to stand in a closed state for 1-2 days to obtain the berry tea lactic acid bacteria bidirectional fermentation liquid of Example 1.

[0051] The formula of the fermentation medium is as follows: take an appropriate amount of berry tea, crush it and pass it through a 20-50 mesh sieve, and add the sieved berry tea powder into 300 ml of the prepared culture medium at a mass volume ratio of 0.5%. The culture medium formula is as follows: 10 g / L peptone, 8 g / L beef extract, 5 g / L yeast powder, 20 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium hydrogen citrate, 5 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, 1 g / L Tween 80, adjust the pH to 5.7, and sterilize at 118°C for 15 minutes.

[0052] 3. Preparation of filtrate

[0053] The fermentation liquid obtained in step 2 was centrifuged at 8500 rpm for 15 min, the supernatant was taken, and the fermentation filtrate was obtained after sterilization through a 0.22 micron filter membrane.

[0054] Example 2

[0055] The difference from Example 1 is that in the fermentation process of Example 2, the berry tea powder added in step 2 is replaced by licorice powder, and the other steps are the same as those in Example 1 and will not be repeated here.

[0056] Example 3

[0057] The difference from Example 1 is that in the fermentation process of Example 3, the berry tea powder added in step 2 is replaced by camellia seed powder, and the other steps are the same as those in Example 1 and will not be repeated here.

[0058] Example 4

[0059] The difference from Example 1 is that in the fermentation process of Example 4, the berry tea powder added in step 2 is replaced by chia seed powder, and the other steps are the same as those in Example 1 and will not be repeated here.

[0060] Example 5

[0061] The difference from Example 1 is that in the fermentation process of Example 5, the berry tea powder added in step 2 is replaced by Schisandra chinensis powder, and the other steps are the same as those in Example 1, which will not be repeated here.

[0062] The efficacy of the two-way fermentation broths of different Chinese medicine substrates obtained in Examples 1-5 was tested, and the in vitro efficacy evaluation test method was as follows:

[0063] 1. Determination of DPPH free radical scavenging rate

[0064] The Chinese name of DPPH is 1,1-diphenyl-2-trinitrophenylhydrazine, which is a stable free radical.

[0065] (1) Solution preparation

[0066] DPPH solution: Dissolve 0.002g DPPH in 50ml ethanol each time and store in dark place. Dilute with ethanol to an absorbance of about 0.7 before use and use immediately after preparation.

[0067] Sample treatment: Take 2 ml of fermentation liquid or culture medium, centrifuge at 8500 rpm for 5 min, take the supernatant, and dilute with water to 5%, 10%, 20%, or 100%.

[0068] (2) Experimental process

[0069] Under light-proof conditions, add 100 μl of DPPH alcohol solution and 100 μl of test sample to a 96-well plate, incubate at room temperature in the dark for 30 minutes, and measure the absorbance at 517 nm. Each measurement requires three groups of samples, blanks, and controls, and each group has three parallels, as follows:

[0070] Sample: 100μl sample solution + 100μl DPPH alcohol solution

[0071] Blank: 100 μl sample solution + 100 μl anhydrous ethanol

[0072] Control: 100 μl DPPH alcohol solution + 100 μl water

[0073] Result calculation: DPPH clearance rate = (1-(Sample-Blank) / Control)*100%.

[0074] 2. ABTS free radical scavenging rate determination

[0075] The Chinese name of ABTS is 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid, which is a chemical free radical.

[0076] (1) Solution preparation

[0077] PBS buffer: 8 g sodium chloride, 0.2 g potassium chloride, 0.24 g potassium dihydrogen phosphate, 3.63 g disodium hydrogen phosphate dodecahydrate, make up to 1000 ml with distilled water, and adjust the pH to 7.4 with hydrochloric acid or sodium hydroxide.

[0078] ABTS stock solution (7mmol / L): Weigh 0.0384g of ABTS and make up to 10ml with distilled water.

[0079] K2S2O8 stock solution (2.45mmol / L): Weigh 0.0066g of K2S2O8 and make up to 10ml with distilled water.

[0080] ABTS working solution: Mix ABTS stock solution and K2S2O8 stock solution in a ratio of 1:1, let stand for 12-16 hours, and prepare ABTS working solution.

[0081] Sample treatment: Take 2 ml of fermentation liquid or culture medium, centrifuge at 8500 rpm for 5 min, take the supernatant, and dilute it with buffer to 5%, 10%, 20%, or 100%.

[0082] (2) Experimental process

[0083] Take an appropriate amount of ABTS working solution and dilute it with PBS solution until the absorbance of the solution at 734nm is about 0.7. Under light-proof conditions, take 200μl of the diluted ABTS working solution and 10μl of the test sample and add them to a 96-well plate. Incubate at room temperature in the dark for 6 minutes and measure the absorbance at 734nm. Each measurement requires three groups of samples, blanks and controls, and each group has 3 parallels, as follows:

[0084] Sample: 10μl sample solution + 200μl ABTS working solution

[0085] Blank: 10μl sample solution + 200μl PBS buffer

[0086] Control: 10 μl distilled water + 200 μl ABTS working solution

[0087] Result calculation: ABTS clearance rate = (1-(Sample-Blank) / Control)*100%.

[0088] 3. Tyrosinase inhibition rate detection

[0089] (1) Solution preparation

[0090] Phosphate buffer: Take 250 mL of 0.2 mol / L potassium dihydrogen phosphate solution, add 118 mL of 0.2 mol / L sodium hydroxide solution, and make up to 1000 mL with deionized water.

[0091] L-tyrosine solution: Weigh 27.17 mg of L-tyrosine, dissolve it in phosphate buffer and dilute to 100 mL volumetric flask to prepare 1.5 mmol / L L-tyrosine solution, which should be used immediately after preparation.

[0092] Tyrosinase solution: weigh 18.5 mg tyrosinase (25 KU, Solebol), add 12.5 mL phosphate buffer to dilute to 2 KU / mL, aliquot and freeze. Dilute to 200 U / mL with phosphate buffer before use.

[0093] (2) Experimental process

[0094] After the sample is centrifuged, add phosphate buffer, sample, tyrosine and tyrosinase respectively under light-proof conditions, incubate at 37°C for 10 minutes in an ELISA reader, and measure the absorbance at 475nm. Each measurement requires four groups: sample, blank, A0 and A1, with 2 parallels in each group, as follows:

[0095] A0: 50 μl tyrosine solution + 100 μl buffer + 50 μl tyrosinase;

[0096] A1: 50 μl tyrosine solution + 150 μl buffer;

[0097] Sample: 50μl tyrosine solution + 50μl sample + 50μl buffer + 50μl tyrosinase;

[0098] Blank: 50μl tyrosine solution + 50μl sample + 100μl buffer

[0099] Result calculation: Tyrosinase inhibition rate = (1-(Sample-Blank) / (A0-A1))*100%

[0100] The fermentation broth obtained in Examples 1-5 was tested for in vitro antioxidant and whitening activity, and the results are shown in Table 1. The antioxidant capacity of the fermentation broth was evaluated by DPPH and ABTS free radical scavenging rates, and the whitening ability was expressed by tyrosinase inhibition rate. Among the several Chinese medicines studied, the antioxidant and whitening abilities of berry tea fermentation broth were significantly better than those of other Chinese medicine fermentation broths. In addition, the antioxidant capacity of camellia seed fermentation broth was also excellent, and the whitening abilities of Schisandra chinensis and licorice fermentation broth were almost comparable to those of berry tea. Therefore, considering the comprehensive antioxidant and whitening abilities, berry tea was selected as the subject of subsequent research.

[0101] Table 1 Evaluation of efficacy of fermentation broths of different Chinese medicine substrates in Examples 1-5

[0102]

[0103] In order to explore the optimal amount of berry tea added, the following comparative experiment was designed.

[0104] Comparative Example 1

[0105] Steps 1 and 3 are the same as those in Example 1, except that berry tea powder is not added in the fermentation medium preparation step in step 2.

[0106] Comparative Example 2

[0107] Steps 1 and 3 are the same as those in Example 1, and in step 2, the amount of berry tea powder added in the fermentation medium preparation step is 1.0%.

[0108] Comparative Example 3

[0109] Steps 1 and 3 are the same as those in Example 1, and the amount of berry tea powder added in the fermentation medium preparation step in step 2 is 1.5%.

[0110] Comparative Example 4

[0111] Steps 1 and 3 are the same as those in Example 1, and the amount of berry tea powder added in the fermentation medium preparation step in step 2 is 2.0%.

[0112] The bacterial growth and pH of Example 1 and Comparative Examples 1-4 were tested, and the statistical results are shown in Table 2. According to Table 2, the addition of berry tea powder has a certain inhibitory effect on the growth of lactic acid bacteria, and the antibacterial effect becomes more and more obvious as the amount of berry tea increases. When the amount of berry tea powder added reaches more than 0.5%, the bacterial growth is slow and the fermentation termination pH is high, indicating that berry tea has a certain antibacterial effect. In order to ensure the efficacy of the fermentation liquid without affecting the growth of lactic acid bacteria, the amount of berry tea powder is preferably 0.5%.

[0113] Table 2 Fermentation bacterial growth and termination pH at different berry tea addition amounts

[0114] Experimental conditions pH Growth Example 1 0.5% berry tea powder fermentation 3.71 Normal growth Comparative Example 1 0 Berry tea powder fermentation 3.63 Normal growth Comparative Example 2 1.0% Fermented berry tea powder 4.22 Small amount of bacteria Comparative Example 3 1.5% berry tea powder fermentation 4.56 Very little bacteria Comparative Example 4 2.0% berry tea powder fermentation 4.79 Very little bacteria

[0115] In order to explore the interaction between berry tea and lactic acid bacteria during fermentation, the following investigation was conducted.

[0116] Comparative Example 5

[0117] The fermentation medium in step 2 of Example 1 was directly sterilized without adding berry tea powder and was not inoculated, serving as a blank control.

[0118] Comparative Example 6

[0119] Steps 1 and 3 are the same as those in Example 1, except that berry tea powder is not added in the fermentation medium preparation step in step 2.

[0120] Comparative Example 7

[0121] The fermentation medium was prepared according to the formula of step 2 of Example 1, but without inoculating bacteria.

[0122] The fermentation broth obtained in Example 1 and Comparative Examples 5-7 was subjected to efficacy tests, including: DPPH clearance rate, ABTS clearance rate, T-AOC, tyrosinase inhibition rate, total flavonoids and total phenols, and the results are shown in Table 3. The test methods for DPPH clearance rate, ABTS clearance rate and tyrosinase inhibition rate are as described in the above examples, and the in vitro efficacy evaluation detection methods for T-AOC, total flavonoids and total phenols are as follows:

[0123] 1. Total antioxidant capacity T-AOC determination

[0124] T-AOC is detected using the BIOSS kit. Its principle is to reduce Fe 3+ -Tripyridine triazine (Fe 3+ -TPTZ) produces blue Fe 2+ -TPTZ capacity reflects the total antioxidant capacity. Prepare the reaction system according to the kit instructions. After 10 minutes of reaction, measure the absorbance at 593nm in an ELISA reader. Determine and calculate the standard curve. After calculation, the standard curve is y=10.641x+0.0006, R 2 = 1. x is Fe 2+ concentration, and y is the absorbance difference between the sample and the control.

[0125] The sample tube is: 180μl detection reagent + 6μl sample + 18μl distilled water;

[0126] The control tube is: 180μl detection reagent + 24μl distilled water.

[0127] According to the absorbance difference, x (μmol / mL) is calculated, and the total antioxidant capacity T-AOC = x*34.

[0128] 2. Total phenol content detection

[0129] (1) Determination of standard curve

[0130] Take pyrogallic acid as the standard, weigh 4.4 mg pyrogallic acid, and dilute to 10 ml with distilled water. Take 0μl, 10μl, 20μl, 40μl, 60μl, 80μl, 100μl, 120μl into colorimetric tubes respectively, make up to 2 ml with water, add 0.5 ml of 2-fold diluted Folin phenol reagent, then add 1.5 ml of 26.7% sodium carbonate solution, add water to make up to 10 ml, mix well, react at room temperature for 2 hours, and measure the absorbance at 760 nm. Fit the standard curve with the absorbance to the standard content. The obtained standard curve is y=0.0132x+0.0353, R 2 =0.9986.

[0131] (2) Sample measurement

[0132] Take an appropriate amount of sample, add reagents according to the above steps, measure the absorbance after 2 hours of reaction, and calculate the total phenol content.

[0133] 3. Total flavonoids content detection

[0134] (1) Determination of standard curve

[0135] Take rutin as the standard, and use 60% ethanol to prepare standard solutions of 1000μg / ml, 800μg / ml, 600μg / ml, 400μg / ml, 200μg / ml, 100μg / ml, 50μg / ml, and 0μg / ml. Take 300μl of the standard solution into the EP tube, add 100μl of 5% sodium nitrite solution, mix well and let it stand for 6 minutes, then add 100μl of 10% aluminum nitrate solution, mix well and let it stand for 6 minutes, then add 500μl of 4% sodium hydroxide solution, mix well and let it stand for 15 minutes, take 200μl of the reaction solution, and measure the absorbance at 510nm. Fit the standard curve with the absorbance to the standard content. The obtained standard curve is y=0.0061x+0.0409, R 2 =0.9994.

[0136] (2) Sample measurement

[0137] Take an appropriate amount of sample, add reagents according to the above steps, measure the absorbance after the reaction, and calculate the total flavonoid content.

[0138] Table 3 Detection of efficacy and active substance content of fermentation broth obtained under different fermentation conditions

[0139]

[0140]

[0141] As can be seen from Table 3, in the basic medium without berry tea, lactic acid bacteria fermentation can produce certain antioxidant and strong tyrosinase inhibition ability, among which T-AOC increased by 1.71 times after fermentation, tyrosinase inhibition rate increased by 5.93 times, and the total flavonoids and total phenols content of the basic medium increased by 29.29μg / mL and 35.15μg / mL respectively after fermentation. This shows that lactic acid bacteria fermentation itself can produce good antioxidant and whitening effects.

[0142] After adding berry tea powder, the DPPH and ABTS clearance rates of the culture medium can reach about 90%, T-AOC reaches 14.36μmol / mL, and the total phenols and total flavonoids are increased by 418.49μg / mL and 271.47μg / mL respectively compared with comparative example 5, but the tyrosinase inhibition ability is only 49.25%. After the berry tea culture medium is fermented by lactic acid bacteria, the free radical scavenging ability and T-AOC total reducing power are increased by 6.5%-9.4% and 1.14μmol / mL respectively, the tyrosinase inhibition rate is increased by 101%, and the total phenols and total flavonoids are increased by 162.12μg / mL and 86.45μg / mL respectively. It can be seen that berry tea has excellent antioxidant and certain whitening ability, and the efficacy can be further improved after lactic acid bacteria fermentation, and the two show a synergistic effect.

[0143] In order to explore the effects of different processing methods on the efficacy of berry tea, common water extraction and enzymatic hydrolysis methods were selected to process berry tea, and their effects on the efficacy indicators of fermentation liquid were studied.

[0144] Comparative Example 8

[0145] Steps 1 and 3 are the same as those in Example 1. In step 2, the berry tea processing method is changed to water extraction, which is as follows: weigh the required amount of berry tea powder, add purified water at 80-90°C in a ratio of 1:10-20, extract in a water bath at 80-90°C for 30 minutes, filter out the extract while hot, add an equal amount of hot water to the residue, repeat the extraction step 2-3 times, combine all the filtrates, add to the fermentation medium, dilute to 3L with water, and sterilize at 118°C for 15 minutes.

[0146] Comparative Example 9

[0147] Steps 1 and 3 are the same as those in Example 1. In step 2, the berry tea treatment method is changed to enzymatic hydrolysis followed by water extraction, as follows: weigh the required amount of berry tea powder, add purified water at a ratio of 1:10-20, add cellulase at a ratio of 1% to 2%, and perform enzymatic hydrolysis in a 40-50°C water bath for 2 hours. After adding the enzymatic hydrolyzate to the fermentation medium, dilute to 3 L with water, and sterilize at 118°C for 15 min.

[0148] The efficacy and active ingredient content of the fermentation broth obtained in Example 1 and Comparative Examples 8-9 were tested, and the results are shown in Table 4. It can be seen from Table 4 that after the berry tea was subjected to water extraction and enzymatic hydrolysis, the antioxidant and whitening effects were not significantly different, and the contents of active ingredients such as total phenols and total flavonoids were slightly reduced. It can be seen that the berry tea powder can be directly added to the culture medium to exert its efficacy well, and the efficacy of the fermentation broth after water extraction and enzymatic hydrolysis is slightly reduced.

[0149] Table 4 Detection of efficacy and active substance content of different berry tea processing methods after fermentation

[0150]

[0151] In order to further verify the above experimental conclusions and explore its feasibility in the field of industrial production, the fermentation conditions were optimized in the fermentation tank.

[0152] Example 2

[0153] The fermentation process of the berry tea lactic acid bacteria bidirectional fermentation liquid of Example 2 is as follows:

[0154] 1. Bacteria activation

[0155] Take a strain of Lactobacillus paracasei and inoculate it into a strain activation culture medium, and culture it in a closed environment at 34-37° C. for 1-2 days to obtain an activated strain.

[0156] The formula of the strain activation culture medium is: peptone 12g / L, beef extract 9g / L, yeast powder 5g / L, glucose 15g / L, dipotassium hydrogen phosphate 2g / L, diammonium hydrogen citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.04g / L, Tween 801g / L, natural pH, preparation volume 40ml, sterilization at 118℃ for 15min.

[0157] 2. Seed cultivation

[0158] The activated bacteria were inoculated into the seed culture medium at a volume ratio of 3%-6%, and cultured in a sealed manner at 34-37° C. for 1 day to obtain a fermentation seed liquid.

[0159] The seed culture medium formula is: peptone 10g / L, beef extract 8g / L, yeast powder 5g / L, glucose 15g / L, dipotassium hydrogen phosphate 2g / L, diammonium hydrogen citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.04g / L, Tween 80 1g / L, pH natural, preparation volume 150ml, sterilization at 118℃ for 15min.

[0160] 3. Fermentation culture

[0161] The seed liquid in step 2 is added to the fermentation medium in the fermentation tank at a ratio of 3%-6%, the fermentation temperature is 34-38°C, the tank pressure is 0.02-0.05MPa, the stirring is 50-80rpm, the pH is natural, and the closed fermentation is carried out for 2 days to obtain the berry tea lactic acid bacteria bidirectional fermentation liquid of Example 2.

[0162] The formula of the fermentation medium is as follows: take an appropriate amount of berry tea, grind it and pass it through a 20-50 mesh sieve, add the sieved berry tea powder into 3L of the prepared fermentation medium at a mass volume ratio of 0.5%, the formula is 10g / L peptone, 9g / L beef extract, 5g / L yeast powder, 20g / L glucose, 2g / L dipotassium hydrogen phosphate, 2g / L diammonium hydrogen citrate, 5g / L sodium acetate, 0.25g / L magnesium sulfate, 0.04g / L manganese sulfate, 1g / L Tween 80, adjust the pH to 5.7, and sterilize at 118°C for 15min.

[0163] 4. Preparation of filtrate

[0164] The fermentation liquid obtained in step 3 was centrifuged at 8500 rpm for 15 min, the supernatant was taken, and the fermentation filtrate was obtained after sterilization through a 0.22 micron filter membrane.

[0165] The fermentation filtrate obtained in Example 2 was tested for efficacy, and the results are shown in Table 5. Compared with the shake flask culture process in Example 1, the efficacy and active ingredient content of the fermenter culture process were slightly improved, indicating that the process has a high potential for industrial scale-up.

[0166] Table 5 Efficacy test of fermentation filtrate obtained by fermentation tank culture

[0167] Detection items Test results DPPH clearance rate (20 times dilution) 96.24％ ABTS clearance rate (20 times dilution) 99.35％ T-AOC (μmol / mL) 15.83 Tyrosinase inhibition rate 99.75％ Total flavonoids (μg / mL) 437.53 Total phenols (μg / mL) 912.35

[0168] In summary, berry tea has excellent antioxidant and tyrosinase inhibitory abilities. After bidirectional fermentation using lactic acid bacteria, the bidirectional fermentation liquid can be endowed with excellent antioxidant and tyrosinase inhibitory abilities. It can be added to cosmetic formulas as an effective ingredient and has enormous application potential.

[0169] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

[0170] In addition, those skilled in the art will appreciate that, although some embodiments herein include certain features included in other embodiments but not other features, the combination of features of different embodiments is meant to be within the scope of the present application and form different embodiments. For example, in the above claims, any one of the claimed embodiments may be used in any combination. The information disclosed in this background technology section is intended only to deepen the understanding of the overall background technology of the present application and should not be regarded as an admission or in any form of implication that the information constitutes prior art known to those skilled in the art.

Claims

1. A fermentation process for a two-way fermentation broth of Chinese medicinal lactic acid bacteria, characterized in that: include: Inoculating lactic acid bacteria in a fermentation medium containing traditional Chinese medicine for fermentation culture to obtain the traditional Chinese medicine lactic acid bacteria bidirectional fermentation liquid; The added mass volume ratio of the Chinese medicine in the fermentation medium is 0.5%; the Chinese medicine is berry tea, the berry tea is berry tea powder, and the mesh size of the berry tea powder is 20-50 meshes; the inoculation volume of the lactic acid bacteria is 3%-6% of the volume of the fermentation medium; the lactic acid bacteria is Lactobacillus paracasei; The pH of the fermentation medium is adjusted to 5.7; The fermentation temperature of the fermentation culture is 34-38° C., and the fermentation time is 1-2 days.

2. The fermentation process of the Chinese medicine lactic acid bacteria bidirectional fermentation liquid according to claim 1, characterized in that: The fermentation medium also includes: 10 g / L peptone, 8 g / L beef extract, 5 g / L yeast powder, 20 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium hydrogen citrate, 5 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, and 1 g / L Tween 80.

3. The fermentation process of the Chinese medicine lactic acid bacteria bidirectional fermentation liquid according to claim 1, characterized in that: The lactic acid bacteria are inoculated into the fermentation medium after activation culture. The activation culture medium comprises: 12 g / L peptone, 9 g / L beef extract, 5 g / L yeast powder, 15 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium hydrogen citrate, 5 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, 1 g / L Tween 80, and natural pH.

4. A Chinese medicine lactic acid bacteria bidirectional fermentation liquid, characterized in that: Prepared by fermentation of the Chinese medicine lactic acid bacteria bidirectional fermentation broth according to any one of claims 1 to 3; The Chinese medicine lactic acid bacteria bidirectional fermentation liquid is berry tea lactic acid bacteria bidirectional fermentation liquid.

5. The Chinese medicinal lactic acid bacteria bidirectional fermentation liquid according to claim 4, characterized in that: The total flavonoid content of the berry tea lactic acid bacteria bidirectional fermentation liquid is 400-500 μg / mL; the total phenol content is 800-1000 μg / mL; the DPPH clearance rate is 95%-97%; the ABTS clearance rate is 95%-99.9%; the T-AOC is 15-20 μmol / mL; and the tyrosinase inhibition rate is 98%-99.9%.

6. Use of the Chinese medicinal lactic acid bacteria bidirectional fermentation liquid according to claim 4 or 5 in the preparation of cosmetics.

7. A cosmetic, characterized in that: The raw materials include the bidirectional fermentation liquid of Chinese medicinal lactic acid bacteria as claimed in claim 4 or 5.

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