CN115887347A

CN115887347A - Traditional Chinese medicine lactobacillus bidirectional fermentation broth, fermentation process and application of fermentation broth

Info

Publication number: CN115887347A
Application number: CN202211584814.5A
Authority: CN
Inventors: 秦立春; 杨欢; 颜少慰; 张廷志
Original assignee: Syoung Cosmetics Manufacturing Co Ltd
Current assignee: Syoung Cosmetics Manufacturing Co Ltd
Priority date: 2022-12-09
Filing date: 2022-12-09
Publication date: 2023-04-04

Abstract

The application provides a traditional Chinese medicine lactobacillus bidirectional fermentation broth, a fermentation process and application of the fermentation broth, and relates to the technical field of skin care products. The fermentation process of the traditional Chinese medicine lactobacillus bidirectional fermentation liquid comprises the steps of inoculating lactobacillus into a fermentation culture medium containing traditional Chinese medicines for fermentation culture, taking the traditional Chinese medicines as bidirectional fermentation substrates, taking the lactobacillus as strains, and fermenting the traditional Chinese medicines and the strains in a combined manner to obtain the bidirectional fermentation liquid with various effects, so that the application of the traditional Chinese medicine lactobacillus fermentation liquid in the field of cosmetics is improved, and the application range of the traditional Chinese medicines is widened. Through bidirectional fermentation of the strawberry tea and the lactic acid bacteria, the strawberry tea can be directly added into a fermentation culture medium after being powdered, and the strawberry tea extract is prepared without methods such as enzymolysis, water extraction or organic reagent extraction, and the like, so that the method is simpler and more convenient, and the energy consumption is low. The total antioxidant capacity, DPPH and ABTS removing capacity of the bidirectional fermentation filtrate of the strawberry tea lactobacillus are superior to those of a strawberry tea fermentation culture medium, and the tyrosinase inhibition rate is obviously superior to that of the strawberry tea fermentation culture medium.

Description

Traditional Chinese medicine lactobacillus bidirectional fermentation broth, fermentation process and application of fermentation broth

Technical Field

The application relates to the technical field of skin care products, in particular to a traditional Chinese medicine lactobacillus bidirectional fermentation liquid, a fermentation process and application of the fermentation liquid.

Background

The strawberry tea is prepared by processing tender stems and leaves of Ampelopsis grossedentata, and is also called as the Ampelopsis grossedentata tea, and is mainly distributed in Hunan, hubei, fujian, jiangxi and other places in China. The plant is rich in natural active ingredient flavone, and flavonoid contained in cells permeates to the surface to form a layer of white cream in the processing process, so that the plant is named as strawberry tea. The raspberry tea is a commonly used national medicine of the Tujia, and is recorded in monographs of traditional Chinese medicine such as Tujia medicine, chinese herbal medicine compilation, chinese medicinal plant in Hunan province (2009 edition), and the like. The strawberry tea has the effects of clearing heat and removing toxicity, promoting blood circulation and removing obstruction in channels, promoting diuresis and reducing edema, calming liver and reducing blood pressure and the like, and is one of the medicine and tea dual-purpose traditional Chinese medicines which are relatively popular in recent research.

At present, the extraction of active ingredients in the raspberry tea mainly comprises the methods of water extraction, enzymolysis, alkali extraction, acid precipitation and the like. For example, patent document CN 111903812A discloses a preparation method and application of an instant raspberry tea extract, and CN 109329524A discloses a preparation method of flavone in ampelopsis grossedentata tea. The extraction time of active ingredients of the raspberry tea in the prior art is long, the operation is complex, and the energy consumption is high.

Disclosure of Invention

The application aims to provide a traditional Chinese medicine lactobacillus bidirectional fermentation broth, a fermentation process and application of the fermentation broth, and aims to solve the problems of long extraction time, complex operation and high energy consumption of active ingredients of the conventional strawberry tea.

In order to achieve the above object, the present application provides a fermentation process of a traditional Chinese medicine lactobacillus bidirectional fermentation broth, comprising: inoculating lactic acid bacteria into a fermentation medium containing traditional Chinese medicines for fermentation culture to obtain traditional Chinese medicine lactic acid bacteria bidirectional fermentation liquid; the addition mass volume ratio of the traditional Chinese medicine in the fermentation culture medium is 0.1-2.0%.

Preferably, the traditional Chinese medicine is selected from: any one of berry tea, glycyrrhrizae radix, camellia oleifera, chia seed and fructus Schisandrae.

Preferably, the traditional Chinese medicine is the raspberry tea, the raspberry tea is the raspberry tea powder, and the mesh number of the raspberry tea powder is 20-50 meshes.

Preferably, the inoculation volume of the lactic acid bacteria is between 3% and 6% of the volume of the fermentation medium.

Preferably, the fermentation temperature of the fermentation culture is 34-38 ℃, and the fermentation time is 1-4 days.

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

Preferably, the lactobacillus is inoculated in 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 and natural pH.

The application also provides a traditional Chinese medicine lactobacillus bidirectional fermentation liquid which is prepared by fermentation of the traditional Chinese medicine lactobacillus bidirectional fermentation liquid.

Preferably, the traditional Chinese medicine lactobacillus bidirectional fermentation liquid is a raspberry tea lactobacillus bidirectional fermentation liquid;

more preferably, the adding mass volume ratio of the raspberry tea powder of the bidirectional fermentation broth of the raspberry tea lactic acid bacteria is 0.5%, and the content of total flavonoids is 400-500 mug/mL; the total phenol content is 800-1000 mug/mL; DPPH clearance rate is 95% -97%; ABTS clearance is 95% -99.9%; the T-AOC is 15-20 mu mol/mL; the tyrosinase inhibition rate is 98-99.9%.

The application also provides application of the traditional Chinese medicine lactobacillus bidirectional fermentation liquid in preparation of cosmetics.

The application also provides a cosmetic, and the raw materials of the cosmetic comprise the traditional Chinese medicine lactobacillus bidirectional fermentation liquid.

Compared with the prior art, the beneficial effect of this application includes:

the fermentation process of the traditional Chinese medicine lactobacillus bidirectional fermentation liquid comprises the steps of inoculating lactobacillus into a fermentation culture medium containing traditional Chinese medicines for fermentation culture, taking the traditional Chinese medicines as bidirectional fermentation substrates, taking the lactobacillus as strains, carrying out combined fermentation on the traditional Chinese medicines and probiotics, and obtaining the bidirectional fermentation liquid with multiple effects through combined fermentation of the traditional Chinese medicines and the probiotics, so that the application of the traditional Chinese medicine lactobacillus fermentation liquid in the field of cosmetics is improved, and the application range of the traditional Chinese medicines is widened. The method has the advantages that the berry tea and the lactic acid bacteria are subjected to bidirectional fermentation through a bidirectional fermentation technology, the berry tea can be directly added into a fermentation culture medium after being powdered, and the method for preparing the berry tea extract does not need enzymolysis, water extraction or organic reagent extraction and the like, so that the method is simpler and more convenient, and the energy consumption is low. The total antioxidant capacity, DPPH and ABTS removing capacity of the bidirectional fermentation filtrate of the raspberry tea lactobacillus are superior to those of a raspberry tea fermentation culture medium, and the tyrosinase inhibition rate is obviously superior to that of the raspberry tea fermentation culture medium.

Detailed Description

The terms as used herein:

"consisting of 8230%" \8230, preparation "and" comprising "are synonymous. The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of 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.

The conjunction "consisting of 823070, 8230composition" excludes any unspecified elements, steps or components. If used in a claim, the phrase is intended to claim as closed, meaning that it does not contain materials other than those described, except for the conventional impurities associated therewith. When the phrase "consisting of (8230) \8230; occurs in a clause of the subject matter of the claims rather than immediately after the subject matter, it only defines the elements described in the clause; other elements are not excluded from the claims as a whole.

When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4," "1 to 3," "1 to 2 and 4 to 5," "1 to 3 and 5," and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.

In these examples, the parts and percentages are by mass unless otherwise indicated.

"parts by mass" means a basic unit of measure indicating a mass ratio of a plurality of components, and 1 part may represent an arbitrary unit mass, for example, 1g or 2.689 g. If the parts by mass of the component A are a parts and the parts by mass of the component B are B parts, the mass ratio of the component A to the component B is expressed as a: b. alternatively, the mass of the A component is aK and the mass of the B component is bK (K is an arbitrary number, and represents a multiple factor). It is not to be misunderstood that the sum of the parts by mass of all the components is not limited to the limit of 100 parts, unlike the parts by mass.

The application provides a fermentation process of traditional Chinese medicine lactobacillus bidirectional fermentation liquor, which comprises the following steps: inoculating lactic acid bacteria into a fermentation medium containing traditional Chinese medicines for fermentation culture to obtain traditional Chinese medicine lactic acid bacteria bidirectional fermentation liquid; the addition mass ratio of the traditional Chinese medicine in the fermentation culture medium is 0.1-2.0%.

Wherein the Chinese medicinal materials are selected from: experiments prove that any one of the traditional Chinese medicines, namely the berry tea, the liquorice, the camellia oleifera, the chia seed and the schisandra chinensis, can be used as a bidirectional fermentation substrate to improve the oxidation resistance and/or whitening capacity of the fermentation liquor. The addition mass volume ratio of the traditional 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%, more specifically, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0%.

Preferably, the traditional Chinese medicine is the strawberry tea, and the oxidation resistance and the whitening capacity of the strawberry tea lactobacillus bidirectional fermentation liquid are improved most obviously.

Among them, lactic Acid Bacteria (LAB) are a general term for a group of bacteria that can produce a large amount of lactic acid using fermentable carbohydrates. The lactobacillus can be any one of Lactobacillus rhamnosus, bifidobacterium lactis, lactobacillus paracasei, lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum, etc.

Further, the lactic acid bacteria may be activated and cultured before being inoculated into the fermentation medium, wherein the components of the medium used in the activation and culture process of the lactic acid bacteria may be, for example: 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 and natural pH. The activated and cultured lactobacillus has better fermentation effect and higher fermentation speed.

Furthermore, when a fermentation tank is required to be used for large-batch fermentation, after the lactobacillus is subjected to activation culture, a fermentation seed solution of the lactobacillus can be obtained through seed culture, and the fermentation seed solution is more beneficial to fermentation in the fermentation culture tank. The formulation of the seed culture medium may be, for example: 10g/L of peptone, 8g/L of beef extract, 5g/L of yeast powder, 15g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.04g/L of manganese sulfate and 80 g/L of Tween, and the pH is natural.

Preferably, the inoculation volume of the lactic acid bacteria is 3% to 6% of the volume of the fermentation medium, which may be 3%, 4%, 5% or 6%, for example.

In the present application, experiments show that the raspberry tea has a certain inhibitory effect on the growth of lactic acid bacteria, and therefore, in the fermentation process of the bidirectional raspberry tea lactic acid bacteria fermentation liquid according to the present application, the addition mass volume ratio of the raspberry tea in the fermentation medium is 0.5% to 2.0%, for example, 0.5% to 1.5%, or 0.5% to 1.0%, and more specifically, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0%.

In a preferred embodiment, the addition mass volume ratio of the raspberry tea in the fermentation medium is 0.5%. The strawberry tea can reduce the inhibition effect on the lactic acid bacteria under the condition of the addition amount, and simultaneously has the synergistic effect of bidirectional fermentation with the lactic acid bacteria.

The addition form of the raspberry tea can be raspberry tea powder, or a raspberry tea water extract, or a raspberry tea enzymolysis extract. Preferably, the berry tea added in the fermentation medium is the berry tea powder, and the berry tea powder is directly added, so that the better fermentation effect can be set, a complex extraction process is not needed, the operation is convenient, and the energy consumption is low.

Preferably, the mesh number of the raspberry tea powder is 20 to 50 meshes, for example, 20 to 30 meshes, or 20 to 40 meshes, or 30 to 50 meshes, and more specifically, for example, 20 meshes, 25 meshes, 30 meshes, 35 meshes, 40 meshes, 45 meshes, or 50 meshes.

Wherein the fermentation medium comprises the following components except the raspberry tea: 10g/L of peptone, 8g/L of beef extract, 5g/L of yeast powder, 20g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.04g/L of manganese sulfate and 80 g/L of Tween, and the pH value is adjusted to 5.7.

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

Preferably, the traditional Chinese medicine lactobacillus bidirectional fermentation liquid is a raspberry tea lactobacillus bidirectional fermentation liquid; more preferably, the adding mass volume ratio of the raspberry tea powder of the bidirectional fermentation broth of the raspberry tea lactic acid bacteria is 0.5%, and the content of total flavonoids is 400-500 mug/mL; the total phenol content is 800-1000 mug/mL; DPPH clearance rate is 95% -97%; ABTS clearance is 95% -99.9%; the T-AOC is 15-20 mu mol/mL; the tyrosinase inhibition rate is 98-99.9%.

The application also provides an application of the traditional Chinese medicine lactobacillus bidirectional fermentation liquid in preparing cosmetics.

The fermentation process of the traditional Chinese medicine lactobacillus bidirectional fermentation liquid provided by the application is characterized in that lactobacillus is inoculated in a fermentation culture medium containing traditional Chinese medicines for fermentation culture, the traditional Chinese medicines are used as bidirectional fermentation substrates, the lactobacillus is used as a strain, and the bidirectional fermentation liquid with various effects is obtained through combined fermentation of the traditional Chinese medicines and the strain, so that the application of the traditional Chinese medicine lactobacillus fermentation liquid in the field of cosmetics is improved, and the application range of the traditional Chinese medicines is widened. The method has the advantages that the berry tea and the lactic acid bacteria are subjected to bidirectional fermentation through a bidirectional fermentation technology, the berry tea can be directly added into a fermentation culture medium after being powdered, and the method for preparing the berry tea extract does not need enzymolysis, water extraction or organic reagent extraction and the like, so that the method is simpler and more convenient, and the energy consumption is low. The total antioxidant capacity, DPPH and ABTS removing capacity of the bidirectional fermentation filtrate of the raspberry tea lactobacillus are superior to those of a raspberry tea fermentation culture medium, and the tyrosinase inhibition rate is obviously superior to that of the raspberry tea fermentation culture medium.

Embodiments of the present application will be described in detail below with reference to specific examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present application and should not be construed as limiting the scope of the present application. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1

The fermentation process of the bidirectional fermentation broth of the berry tea lactobacillus in the embodiment 1 is as follows:

1. strain activation

Inoculating a Lactobacillus paracasei strain into the strain activation culture medium, and performing closed static culture at 34-37 deg.C for 1-2 days to obtain the activated strain.

The formula of the strain activation culture medium is as follows: 12g/L of peptone, 9g/L of beef extract, 5g/L of yeast powder, 15g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.04g/L of manganese sulfate and 80 g/L of Tween, the pH is natural, the preparation volume is 40ml, and the sterilization is carried out for 15min at 118 ℃.

2. Fermentation culture

And (3) inoculating the activated strain in the step (1) into 500mL of shake flask fermentation medium according to the proportion of 3% -6%, fermenting at 34-37 ℃, standing, sealing and fermenting for 1-2 days to obtain the strawberry tea lactobacillus bidirectional fermentation liquid of the example 1.

The formula of the fermentation medium is as follows: taking a proper amount of the strawberry tea, crushing the strawberry tea, sieving the crushed strawberry tea by a sieve of 20-50 meshes, adding the sieved strawberry tea powder into 300ml of prepared culture medium according to the mass volume ratio of 0.5%, wherein the formula of the culture medium is as follows: 10g/L of peptone, 8g/L of beef extract, 5g/L of yeast powder, 20g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.04g/L of manganese sulfate and 80 g/L of Tween, adjusting the pH to 5.7, and sterilizing at 118 ℃ for 15min.

3. Preparation of the filtrate

Centrifuging the fermentation liquor obtained in the step 2 at 8500rpm for 15min, taking the supernatant, and sterilizing the supernatant by a 0.22-micron filter membrane to obtain fermentation filtrate.

Example 2

The difference from example 1 is that: in the fermentation process of embodiment 2, the added berry tea powder in step 2 is replaced by licorice powder, and other steps are the same as those in embodiment 1 and are not described again.

Example 3

The difference from example 1 is that: in the fermentation process of embodiment 3, the added berry tea powder in step 2 is replaced by camellia oleifera seed powder, and other steps are the same as those in embodiment 1 and are not described again.

Example 4

The difference from example 1 is that: in the fermentation process of example 4, the added berry tea powder in step 2 is replaced by chia seed powder, and other steps are the same as those in example 1 and are not described again.

Example 5

The difference from example 1 is that: in the fermentation process of embodiment 5, the added berry tea powder in step 2 is replaced by schisandra chinensis powder, and other steps are the same as those in embodiment 1 and are not described again.

The in vitro efficacy evaluation and detection method for the two-way fermentation liquid of different Chinese medicine substrates obtained in examples 1 to 5 is as follows:

1. DPPH radical scavenging Rate determination

DPPH, the Chinese name 1, 1-diphenyl-2-trinitrophenylhydrazine, is a stable free radical.

(1) Solution preparation

DPPH solution: 0.002g of DPPH is taken each time and dissolved in 50ml of ethanol, and the mixture is stored in dark. Before use, the mixture is diluted by ethanol until the absorbance is about 0.7 and is prepared for use.

Sample treatment: 2ml of fermentation liquor or culture medium is taken, centrifuged for 5min at 8500rpm, and supernatant is taken and diluted to 5%, 10%, 20% and 100% by water.

(2) Procedure of experiment

Under the condition of keeping out of the light, 100 ul of DPPH alcoholic solution and 100 ul of test sample are added into a 96-well plate, incubated for 30min at room temperature in the absence of the light, and the light absorption value at 517nm is measured. Each assay required three sets of sample, blank and control, each set of 3 replicates as follows:

sample: 100. Mu.l sample solution + 100. Mu.l DPPH alcohol solution

Blank: 100. Mu.l of sample solution + 100. Mu.l of absolute ethanol

Control:100 μ l of DPPH alcohol solution +100 μ l of water

And (4) calculating a result: DPPH clearance = (1- (Sample-Blank)/Control) = 100%.

2. ABTS free radical clearance assay

The chinese name of ABTS is 2,2' -diaza-bis-3-ethylbenzothiazoline-6-sulfonic acid, a chemical free radical.

(1) Solution preparation

PBS buffer: 8g of sodium chloride, 0.2g of potassium chloride, 0.24g of potassium dihydrogen phosphate and 3.63g of disodium hydrogen phosphate dodecahydrate, wherein the volume is adjusted to 1000ml by using distilled water, and the pH value is adjusted to 7.4 by using hydrochloric acid or sodium hydroxide.

ABTS stock solution (7 mmol/L) is called ABTS 0.0384g, and is made to 10ml with distilled water.

K ₂ S ₂ O ₈ Stock solution (2.45 mmol/L) is called K ₂ S ₂ O ₈ 0.0066gAnd the volume is adjusted to 10ml by distilled water.

ABTS working solution: stock solutions of ABTS and K ₂ S ₂ O ₈ Stock solution 1:1, uniformly mixing, standing for 12-16h, and preparing the ABTS working solution.

Sample treatment: centrifuging 2ml fermentation liquid or culture medium at 8500rpm for 5min, collecting supernatant, and diluting with buffer solution to 5%, 10%, 20%, 100%.

(2) Procedure of experiment

An appropriate amount of ABTS working solution was taken and diluted with PBS solution until the absorbance of the solution at 734nm was about 0.7. Under the condition of keeping out of the light, 200 μ l of diluted ABTS working solution and 10 μ l of test sample are added into a 96-well plate, incubated for 6min at room temperature in the absence of the light, and the light absorption value at 734nm is measured. Each assay required three sets of sample, blank and control, each set of 3 replicates as follows:

sample:10 μ l sample solution +200 μ l ABTS working solution

Blank: 10. Mu.l sample solution + 200. Mu.l PBS buffer

Control:10 ul distilled water +200 ul ABTS working solution

And (4) calculating a result: ABTS clearance = (1- (Sample-Blank)/Control) × 100%.

3. Tyrosinase inhibition assay

(1) Solution preparation

Phosphate buffer solution: 250mL of 0.2mol/L potassium dihydrogen phosphate solution is taken, 118mL of 0.2mol/L sodium hydroxide solution is added, and deionized water is used for fixing the volume to 1000mL.

L-tyrosine solution: weighing 27.17mg of L-tyrosine, dissolving the L-tyrosine by using a phosphate buffer solution, and then diluting to a volume of 100mL to prepare a 1.5 mmol/L-tyrosine solution for use.

Tyrosinase solution: 18.5mg of tyrosinase (25 KU, solaibao) was weighed, diluted to 2KU/mL with 12.5mL of phosphate buffer, dispensed and frozen. Diluted to 200U/mL with phosphate buffer before use.

(2) Procedure of experiment

And (3) centrifuging the sample for later use, respectively adding phosphate buffer solution, the sample, tyrosine and tyrosinase under the condition of keeping out of the sun, incubating for 10 minutes at the constant temperature of 37 ℃ in an enzyme labeling instrument, and measuring the light absorption value at 475 nm. Each assay required four sets of sample, blank, A0 and A1, each set of 2 replicates as follows:

a0:50 μ l tyrosine solution +100 μ l buffer +50 μ l tyrosinase;

a1: 50. Mu.l tyrosine solution + 150. Mu.l buffer;

sample:50 μ l of the solution of tyrosinase +50 μ l of the sample +50 μ l of the buffer +50 μ l of the tyrosinase;

blank:50 μ l of tyrosine solution +50 μ l of sample +100 μ l of buffer

And (4) calculating a result: tyrosinase inhibition = (1- (Sample-Blank)/(A0-A1)). Times 100%

The fermentation liquids obtained in examples 1 to 5 were tested for antioxidant and whitening activity in vitro, and the results are shown in table 1. The antioxidant capacity of the fermentation liquor is evaluated by DPPH and ABTS free radical clearance, and the whitening capacity is expressed by tyrosinase inhibition rate. In several researched traditional Chinese medicines, the antioxidant and whitening capabilities of the berry tea fermentation liquor are remarkably superior to those of other traditional Chinese medicine fermentation liquor, in addition, the antioxidant capability of the camellia seed fermentation liquor is also excellent, and the whitening capabilities of the schisandra chinensis and licorice fermentation liquor are almost comparable to those of berry tea. Therefore, the strawberry tea is selected as a follow-up research object in consideration of comprehensive antioxidant and whitening capacity.

TABLE 1 evaluation of the efficacy of fermentation broth of different Chinese medicinal substrates in examples 1 to 5

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In order to explore the optimum addition amount of the strawberry tea, the following comparative experiment is designed.

Comparative example 1

Steps 1 and 3 are the same as example 1, and the step of preparing the fermentation medium in step 2 is not added with the berry tea powder.

Comparative example 2

Steps 1 and 3 are the same as example 1, and the addition amount of the berry tea powder in the step of preparing the fermentation medium in the step 2 is 1.0%.

Comparative example 3

Steps 1 and 3 are the same as example 1, and the addition amount of the berry tea powder in the step of preparing the fermentation medium in the step 2 is 1.5%.

Comparative example 4

Steps 1 and 3 are the same as example 1, and the addition amount of the berry tea powder in the step of preparing the fermentation medium in the step 2 is 2.0%.

The growth and pH of the bacteria in example 1 and comparative examples 1-4 were tested, and the statistics of the results are shown in Table 2, and it can be seen from Table 2 that the addition of the berry tea powder has a certain inhibition effect on the growth of lactic acid bacteria, and the antibacterial effect is more and more obvious as the dosage of the berry tea increases. When the addition amount of the raspberry tea powder reaches more than 0.5%, the thalli grow slowly, the pH value is higher after fermentation is finished, and the raspberry tea powder has a certain antibacterial effect. In order to ensure the efficacy of the fermentation liquor and not influence the growth of the lactic acid bacteria, the dosage of the berry tea powder is preferably 0.5%.

TABLE 2 fermentation thallus growth and termination of pH at different berry tea addition levels

	Conditions of the experiment	pH	Growth conditions
				Example 1	Fermentation of 0.5% berry tea powder	3.71	Normal growth of the plant
Comparative example 1	0 fermentation of strawberry tea powder	3.63	Normal growth of the plant
				Comparative example 2	Fermentation of 1.0% berry tea powder	4.22	The amount of the bacteria is small
Comparative example 3	Fermentation of 1.5% strawberry tea powder	4.56	The amount of the bacteria is very small
				Comparative example 4	2.0% fermentation of berry tea powder	4.79	The amount of the bacteria is very small

In order to explore the interaction between the strawberry tea and the lactic acid bacteria in the fermentation process, the following exploration is carried out.

Comparative example 5

The fermentation medium in step 2 of example 1 was used as a blank control without inoculating the medium obtained by direct sterilization without adding berry tea powder.

Comparative example 6

Comparative example 7

The fermentation medium was prepared according to the recipe of step 2 of example 1, but without inoculation of the bacteria.

The fermentation broths obtained in example 1 and comparative examples 5 to 7 were subjected to efficacy tests, which included: DPPH clearance, ABTS clearance, T-AOC, tyrosinase inhibition, total flavonoids and total phenols, the results are shown in Table 3. DPPH clearance, ABTS clearance and tyrosinase inhibition assay methods As described in the previous examples, the in vitro efficacy evaluation assays for T-AOC, total flavonoids and total phenols are as follows:

1. total antioxidant capacity T-AOC assay

The T-AOC detection uses BIOSS kit detection, and the principle is that Fe is reduced in an acidic environment ³⁺ -tripyridotriazine (Fe) ³⁺ TPTZ) produces Fe in blue ²⁺ The ability of TPTZ, reflecting the total antioxidant capacity. And preparing a reaction system according to the kit specification, reacting for 10 minutes, and measuring the absorbance at 593nm in a microplate reader. The measurement calculates the standard curve, which is calculated as y =10.641x +0.0006 ² And =1.x is Fe ²⁺ Concentration, y is the difference in absorbance of the sample and the control.

The sample tube is as follows: 180. Mu.l of detection reagent + 6. Mu.l of sample + 18. Mu.l of distilled water;

the control tube is as follows: mu.l detection reagent + 24. Mu.l distilled water.

And calculating x (mu mol/mL) according to the absorbance difference, and then obtaining the total antioxidant capacity T-AOC = x 34.

2. Total phenol content detection

(1) Determination of the Standard Curve

Weighing pyrogallic acid 4.4mg with pyrogallic acid as standard, and diluting to 10ml with distilled water. Mu.l, 10 mu.l, 20 mu.l, 40 mu.l, 60 mu.l, 80 mu.l, 100 mu.l and 120 mu.l are respectively taken and put into a colorimetric tube, the volume is replenished to 2ml by water, 0.5ml of 2-time diluted forskolin phenol reagent is added, 1.5ml of 26.7% sodium carbonate solution is added, the volume is increased to 10ml by adding water, the mixture is reacted for 2 hours at room temperature after being mixed evenly, and the absorbance is measured at 760 nm. A standard curve was fitted with absorbance versus standard content. The marked curve is y =0.0132x +0.0353 ² ＝0.9986。

(2) Sample assay

Taking a proper amount of sample, adding the reagent according to the steps, reacting for 2 hours, then measuring the absorbance, and calculating the total phenol content.

3. Detection of total flavone content

(1) Determination of the Standard Curve

Taking rutin as standard substance, preparing with 60% ethanol into 1000 μ g/ml,800 μ g/ml,600 μ g/ml,400 μ g/ml,200 μ g/ml,100 μ g/ml,50. Mu.g/ml, 0. Mu.g/ml of the standard solution. Respectively taking 300 mu l of standard solution to an EP tube, adding 100 mu l of 5% sodium nitrite solution, uniformly mixing, standing for reaction for 6min, adding 100 mu l of 10% aluminum nitrate solution, uniformly mixing, standing for reaction for 6min, adding 500 mu l of 4% sodium hydroxide solution, uniformly mixing, standing for reaction for 15min, sucking 200 mu l of reaction solution, and measuring the absorbance at 510 nm. A standard curve was fitted with absorbance versus standard content. The marked curve is y =0.0061x +0.0409 ² ＝0.9994。

(2) Sample assay

Taking a proper amount of samples, adding the reagent according to the steps, measuring absorbance after reaction, and calculating the content of the total flavone.

TABLE 3 detection of efficacy and active substance content of fermentation broth obtained under different fermentation conditions

As can be seen from Table 3, in the basal medium without the strawberry tea, certain antioxidation and stronger tyrosinase inhibition capability can be generated by the fermentation of the lactic acid bacteria, wherein the T-AOC is increased by 1.71 times after the fermentation, the tyrosinase inhibition rate is increased by 5.93 times, and the contents of total flavonoids and total phenols in the basal medium after the fermentation are respectively increased by 29.29 mu g/mL and 35.15 mu g/mL. The lactobacillus fermentation can generate better antioxidant and whitening effects.

After the strawberry tea powder is added, the DPPH and ABTS clearance of the culture medium can reach about 90 percent, the T-AOC reaches 14.36 mu mol/mL, the total phenols and the total flavones are respectively improved by 418.49 mu g/mL and 271.47 mu g/mL compared with the comparative example 5, but the tyrosinase inhibition capability is only 49.25 percent. After the strawberry tea culture medium is fermented by lactic acid bacteria, the free radical scavenging capacity and the T-AOC total reducing power are respectively improved by 6.5-9.4 percent and 1.14 mu mol/mL, the tyrosinase inhibition rate is improved by 101 percent, and the total phenol and the total flavone are respectively improved by 162.12 mu g/mL and 86.45 mu g/mL. Therefore, the strawberry tea has excellent oxidation resistance and certain whitening capacity, the efficacy can be further improved after the fermentation of the lactic acid bacteria, and the two show synergistic effect.

In order to explore the influence of different treatment modes of the raspberry tea on the efficacy, common water extraction methods and enzyme hydrolysis methods are selected to treat the raspberry tea, and the influence of the raspberry tea on the efficacy index of fermentation liquor is researched.

Comparative example 8

Steps 1 and 3 are the same as example 1, and the processing mode of the raspberry tea in step 2 is changed into water extraction, and the method specifically comprises the following steps: weighing the strawberry tea powder with the required dosage, and mixing according to the proportion of 1: adding purified water of 80-90 ℃ in a proportion of 10-20, extracting for 30 minutes in a water bath kettle of 80-90 ℃, filtering the extracting solution while the solution is hot, adding equivalent hot water into filter residues, repeating the extraction step for 2-3 times, combining all filtrates, adding into a fermentation medium, adding water to a constant volume of 3L, and sterilizing for 15min at 118 ℃.

Comparative example 9

Steps 1 and 3 are the same as example 1, and the processing mode of the raspberry tea in step 2 is changed into water extraction after enzymolysis, which specifically comprises the following steps: weighing the berry tea powder with the required dosage, and mixing according to the weight ratio of 1: adding purified water in a proportion of 10-20, adding cellulase in a proportion of 1-2%, carrying out enzymolysis in a water bath kettle at 40-50 ℃ for 2 hours, adding the enzymolysis liquid into a fermentation culture medium, adding water to a constant volume of 3L, and sterilizing at 118 ℃ for 15min.

The results of the detection of the efficacy and the content of the active substances of the fermentation liquid obtained in the example 1 and the comparative examples 8-9 are shown in table 4, and it can be seen from table 4 that the differences of the antioxidant and whitening efficacies of the raspberry tea are not obvious after the water extraction and the enzymolysis treatment, the content of the active substances such as total phenols and total flavonoids is slightly reduced, so that the efficacy of the raspberry tea powder can be well played by directly adding the raspberry tea powder into the culture medium, and the efficacy of the fermentation liquid after the water extraction and the enzymolysis treatment is slightly reduced.

TABLE 4 detection of efficacy and active substance content after fermentation in different berry tea treatment modes

In order to further verify the experimental conclusion, the feasibility in the industrial production field is explored, and the fermentation condition is optimized in a fermentation tank.

Example 2

The fermentation process of the bidirectional fermentation broth of the strawberry and tea lactic acid bacteria in the embodiment 2 is as follows:

1. strain activation

Inoculating a Lactobacillus paracasei strain into the strain activation culture medium, and performing closed standing culture at 34-37 deg.C for 1-2 days to obtain the activated strain.

The formula of the strain activation culture medium is as follows: 12g/L of peptone, 9g/L of beef extract, 5g/L of yeast powder, 15g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.04g/L of manganese sulfate and 80 g/L of Tween, wherein the pH is natural, the preparation volume is 40ml, and the sterilization is carried out for 15min at 118 ℃.

2. Seed culture

Inoculating the activated strain to a seed culture medium according to the volume ratio of 3-6%, and carrying out closed standing culture at 34-37 ℃ for 1 day to obtain a fermented seed solution.

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

3. Fermentation culture

And (3) inoculating the seed liquid obtained in the step (2) into a fermentation medium in a fermentation tank according to the proportion of 3% -6%, wherein the fermentation temperature is 34-38 ℃, the tank pressure is 0.02-0.05MPa, stirring is carried out at 50-80rpm, the pH is natural, and the seed liquid is subjected to closed fermentation for 2 days to obtain the berry tea lactobacillus bidirectional fermentation liquid of the embodiment 2.

The formula of the fermentation medium is as follows: taking a proper amount of berry tea, crushing, sieving with a 20-50 mesh sieve, adding the sieved berry tea powder into 3L of prepared fermentation medium according to the mass volume ratio of 0.5%, wherein the formula comprises 10g/L of peptone, 9g/L of beef extract, 5g/L of yeast powder, 20g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 0.25g/L of magnesium sulfate, 0.04g/L of manganese sulfate and 80 g/L of tween, adjusting the pH to 5.7, and sterilizing at 118 ℃ for 15min.

4. Preparation of the filtrate

Centrifuging the fermentation liquor obtained in the step 3 at 8500rpm for 15min, taking the supernatant, and sterilizing the supernatant by a 0.22-micron filter membrane to obtain fermentation filtrate.

The efficacy test of the fermentation filtrate obtained in example 2 shows that the results are shown in table 5, and the efficacy and the active matter content of the fermentation tank culture process are slightly improved compared with the shake flask culture process in example 1, which indicates that the process has higher industrial amplification potential.

TABLE 5 efficacy test of fermentation filtrates obtained by fermenter culture

Test item	The result of the detection
		DPPH clearance (dilution 20 times)	96.24％
ABTS clearance (Dilute 20 times)	99.35％
		T-AOC(μmol/mL)	15.83
Tyrosinase inhibition rate	99.75％
		Total Flavonoids (mug/mL)	437.53
Total phenols (μ g/mL)	912.35

In conclusion, the strawberry tea has excellent antioxidant capacity and tyrosinase inhibition capacity, and after bidirectional fermentation is carried out by using lactic acid bacteria, the bidirectional fermentation liquid can be endowed with very excellent antioxidant capacity and tyrosinase inhibition capacity, and can be added into a cosmetic formula as an effective component, so that the strawberry tea has very great application potential.

Finally, it should be noted that: the above embodiments are only used for illustrating the technical solutions of the present application, and not for limiting the same; although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present application.

Moreover, those skilled in the art will appreciate that while some embodiments herein include some features included in other embodiments, rather than other features, combinations of features of different embodiments are meant to be within the scope of the application and form different embodiments. For example, in the claims above, any of the claimed embodiments may be used in any combination. The information disclosed in this background section is only for enhancement of understanding of the general background of the application and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Claims

1. A fermentation process of traditional Chinese medicine lactobacillus bidirectional fermentation liquor is characterized by comprising the following steps: inoculating lactobacillus into a fermentation medium containing traditional Chinese medicines for fermentation culture to obtain a traditional Chinese medicine lactobacillus bidirectional fermentation liquid; the addition mass volume ratio of the traditional Chinese medicine in the fermentation medium is 0.1-2.0%.

2. The fermentation process of the bidirectional fermentation broth of lactic acid bacteria of claim 1, wherein the Chinese medicine is selected from the group consisting of: any one of berry tea, glycyrrhrizae radix, camellia oleifera, chia seed and fructus Schisandrae.

3. The fermentation process of the traditional Chinese medicine lactobacillus bidirectional fermentation broth according to claim 2, wherein the traditional Chinese medicine is raspberry tea, the raspberry tea is raspberry tea powder, and the mesh number of the raspberry tea powder is 20-50 meshes.

4. The fermentation process of the traditional Chinese medicine lactic acid bacteria bidirectional fermentation broth according to claim 1, wherein the inoculation volume of the lactic acid bacteria is 3-6% of the volume of the fermentation medium.

5. The fermentation process of the traditional Chinese medicine lactobacillus bidirectional fermentation liquid according to claim 1, wherein the fermentation temperature of the fermentation culture is 34-38 ℃, and the fermentation time is 1-4 days.

6. The fermentation process of the traditional Chinese medicine lactic acid bacteria bidirectional fermentation broth according to claim 1, wherein the fermentation medium further comprises: 10g/L of peptone, 8g/L of beef extract, 5g/L of yeast powder, 20g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.04g/L of manganese sulfate and 80 g/L of Tween, and the pH value is adjusted to 5.7.

7. The fermentation process of the bidirectional fermentation broth of lactic acid bacteria as claimed in claim 1, wherein the lactic acid bacteria are inoculated in the fermentation medium after activation culture, and the culture medium of the activation culture 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 and natural pH.

8. A traditional Chinese medicine lactobacillus bidirectional fermentation liquid, which is characterized in that the traditional Chinese medicine lactobacillus bidirectional fermentation liquid is prepared by fermentation of the fermentation process of the traditional Chinese medicine lactobacillus bidirectional fermentation liquid of any one of claims 1 to 7;

more preferably, the adding mass volume ratio of the raspberry tea powder of the bidirectional fermentation broth of the raspberry tea lactic acid bacteria is 0.5%, and the content of total flavonoids is 400-500 mug/mL; the total phenol content is 800-1000 mug/mL; the DPPH clearance rate is 95-97%; ABTS clearance rate is 95% -99.9%; the T-AOC is 15-20 mu mol/mL; the tyrosinase inhibition rate is 98-99.9%.

9. The use of the bidirectional fermentation broth of lactic acid bacteria of claim 8 for the preparation of cosmetics.

10. A cosmetic characterized in that its raw material comprises the bidirectional fermentation broth of lactic acid bacteria of the Chinese medicine of claim 8.