Loop thistle fermented product as well as preparation method and application thereof

Abstract

The invention discloses a granary thistle herb fermented product as well as a preparation method and application thereof. The preparation method of the granary thistle herb fermented product comprises the following steps: mixing a granary thistle herb extract with water, and sterilizing to obtain a fermentation substrate; inoculating the strain culture solution into the fermentation substrate, and fermenting to obtain an initial fermentation solution; the inoculum size of a strain in the strain culture solution is 1-4%, the strain is one or more of galactose yeast-like bacteria, saccharomyces cerevisiae or lactobacillus plantarum, and the strain culture solution accounts for 2-8% of the fermentation substrate; and sterilizing and separating the initial fermentation liquid to obtain a liquid which is the fermentation product of the granary thistle herb. According to the method disclosed by the invention, the concentration of effective substances in the granary thistle fermented product can be effectively improved, and the granary thistle fermented product as an effective component in cosmetics can improve the anti-aging capability and the anti-oxidation capability.

Description

Technical Field

[0001] The invention relates to a fermented product of blessed thistle and a preparation method and application thereof. Background Art

[0002] Blessed Thistle, also known as Blessed Thistle, is a plant of the genus Magnolia, Asteraceae, and Magnolia. In recent years, it has been widely cultivated in major botanical gardens and medicinal gardens in my country. It grows in areas with an altitude of 1,000 to 1,200 meters, often in river valley meadows. It is widely cultivated in major botanical gardens and medicinal gardens in my country. The type specimen was collected from Western Europe. The whole herb is used for medicine and has a stomachic effect. It is a bitter tonic and digestive herb, which contains sesquiterpene lactones, triterpenoids, lignans, tannins, essential oils, flavonoids, and polyene compounds. It treats indigestion and loss of appetite; it is a diuretic, perspiration, purifies the body fluid system, eliminates toxins in the body, and supports the liver's self-repair; it is anti-tumor and anti-cancer; it is also a female tonic, often used to promote lactation, has good antioxidant capacity, and has great development potential.

[0003] There is no research on the fermentation of blessed thistle. Summary of the invention

[0004] The present invention provides a fermented product of thistle and its preparation method and application. The fermented product of thistle as an active component in a skin external preparation has good anti-oxidation and anti-aging effects.

[0005] The present invention solves the above technical problems through the following technical solutions:

[0006] The present invention provides a method for preparing a fermented product of blessed thistle, which comprises the following steps:

[0007] (1) mixing the herbaceous thistle extract with water and sterilizing the mixture to obtain a fermentation medium;

[0008] (2) inoculating the bacterial culture solution into the fermentation matrix, fermenting, and obtaining an initial fermentation solution; the inoculation amount of the bacterial species in the bacterial culture solution is 1-4%; the bacterial species is one or more of Galactomyces geotrichum, Saccharomyces cerevisiae, and Lactobacillus plantarum; the bacterial culture solution is 2-8% of the fermentation matrix, and % refers to the ratio of the volume of the bacterial culture solution to the volume of the fermentation matrix;

[0009] (3) The initial fermentation liquid is sterilized and separated, and the obtained liquid is the blessed thistle fermentation product.

[0010] In the present invention, in step (1), the volume ratio of the Herba Thistle extract to the water is preferably 1:(10-50), more preferably 1:(25-50), such as 1:30 or 1:45, and further preferably 1:30.

[0011] In the present invention, in step (1), the preparation method of the extract of the blessed thistle can be conventional in the art. Preferably, the extract of the blessed thistle is prepared by alcohol extraction.

[0012] More preferably, the preparation method of the Herba Thistle extract comprises the following steps:

[0013] S1. crushing the blessed thistle to obtain blessed thistle powder;

[0014] S2, mixing the blessed thistle powder with an alcohol solution, stirring and filtering at room temperature to obtain a filtrate;

[0015] S3, removing the alcohol in the filtrate to obtain a concentrated solution, filtering the concentrated solution, and obtaining a filtrate as the Herba Thistle extract.

[0016] The blessed thistle is generally dried. During the drying process, the drying temperature is preferably 55-65° C., such as 60° C.; and the drying time is preferably 3-5 hours.

[0017] Wherein, in said S1, a sieving step may be further included after said pulverizing, and the particle size of the blessed thistle powder after sieving is preferably 70-100 mesh, more preferably 80 mesh.

[0018] The Cnicus benedictus L. can be conventional in the art, generally referring to the whole herb of Cnicus benedictus L.

[0019] Among them, in S2, the mass ratio of the blessed thistle powder to the volume ratio of the alcohol solution is preferably 1 g: (10-30) mL, more preferably 1 g: 20 mL.

[0020] Wherein, in S2, the alcohol solution can be conventional in the art, generally a mixture of alcohol and water. Preferably, the concentration of alcohol in the alcohol solution is 60-90%, more preferably 70%, where % refers to the ratio of the volume of alcohol to the volume of water.

[0021] Among them, in S2, the alcohol solution can be conventional in the art, preferably an ethanol solution.

[0022] Wherein, in S2, the stirring time is preferably 2 to 4 hours, for example 2 hours.

[0023] The room temperature is conventional in the art, generally 20-30°C, for example 25°C.

[0024] Among them, in S2, the filtration can be conventional in the art, such as suction filtration.

[0025] Among them, in S3, the method for removing the alcohol in the filtrate is preferably an evaporation method, for example, using a rotary evaporator to evaporate the filtrate to achieve the effect of removing the alcohol.

[0026] Among them, in S3, the filtration can be conventional in the art, such as centrifugal filtration.

[0027] In the present invention, in step (1), the sterilization can be conventional in the art. The sterilization temperature is preferably 121° C. The sterilization time is preferably 15 to 30 minutes, for example 25 minutes.

[0028] In the present invention, in step (2), the bacterial species is preferably Galactomyces geotrichum, Saccharomyces cerevisiae or Lactobacillus plantarum, more preferably Galactomyces geotrichum.

[0029] In the present invention, the Galactomyces geotrichum has a deposit number of CGMCCNO.2.3766 and is deposited in the China Center for Microbiological Culture Collection.

[0030] The Saccharomyces cerevisiae has a deposit number of CGMCC No. 21527 and is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration.

[0031] The Lactobacillus plantarum has a preservation number of CGMCC No. 21528 and is preserved in the General Microbiology Center of China National Committee for the Preservation of Microbiological Cultures.

[0032] In the present invention, in step (2), the inoculation amount of the bacterial strain in the bacterial culture solution is preferably 2-3%, such as 2%, 2.5%, 3% or 4%, and more preferably 2%. The inoculation amount is calculated using the conventional method in the art, that is, inoculation amount = (target colony number / bacterial solution concentration of the target strain) × (inoculation volume / total volume of culture medium).

[0033] In the present invention, in step (2), the method for preparing the bacterial culture solution can be conventional in the art, preferably, comprising the following steps: inoculating the bacterial strain into a culture medium, culturing on a shaking table, and obtaining a bacterial culture solution.

[0034] Wherein, the shaking speed of the shaking incubation is preferably 100-250 rpm, for example 180 rpm.

[0035] The shaking culture temperature is preferably 15-19°C, for example 17°C.

[0036] The shaking culture time is preferably 24 to 48 hours, for example 30 hours or 48 hours.

[0037] The culture medium may be conventional in the art, such as YPD medium.

[0038] In the present invention, in step (2), the bacterial culture solution is preferably 2%, 3% or 5% of the fermentation substrate, more preferably 3-6%, for example, further preferably 5%, where % refers to the ratio of the volume of the bacterial culture solution to the volume of the fermentation substrate.

[0039] In the present invention, in step (2), the fermentation temperature is preferably 15-45°C, more preferably 30°C.

[0040] In the present invention, in step (2), the fermentation time is preferably 24 to 72 hours, more preferably 36 hours.

[0041] In the present invention, in step (2), the fermentation is preferably carried out on a shaker, and the rotation speed of the shaker is preferably 100-250 rpm, for example 200 rpm.

[0042] In the present invention, in step (3), the sterilization can be conventional in the art. The sterilization temperature is preferably 121° C. The sterilization time is preferably 15 to 30 minutes, for example 25 minutes.

[0043] In the present invention, in step (3), after the sterilization, the method may further include the steps of centrifugation and filtering the liquid after the centrifugation.

[0044] The filtration may be conventional in the art, and the liquid is generally filtered through a ceramic membrane, wherein the pore size of the ceramic membrane is 150-250 nm, for example, 200 nm.

[0045] The present invention also provides a fermented blessed thistle prepared by the method for preparing the fermented blessed thistle.

[0046] The present invention also provides an application of the aforementioned blessed thistle fermentation product in cosmetics.

[0047] In the present invention, preferably, the fermented product of blessed thistle is used as an anti-aging or antioxidant ingredient in cosmetics.

[0048] In the present invention, preferably, the fermented product of Herba Thistle is used as a DPPH scavenger, an elastin synthesis promoter or a type I procollagen C-terminal peptide expression promoter in cosmetics.

[0049] In the present invention, the cosmetics can be aqueous solutions, emulsions, sprays, creams, facial masks, sunscreens, or liquid foundations.

[0050] The positive and progressive effects of the present invention are:

[0051] The method of the present invention can effectively increase the concentration of effective substances in the fermented product of blessed thistle; the fermented product of blessed thistle as an effective ingredient in cosmetics can improve the anti-aging ability and antioxidant ability.

[0052] In a further preferred preparation method of the present invention, the active ingredients in the Herba Blessed Thistle extract are increased by adjusting the pulverization operation, the concentration of the alcohol solution, and the solid-liquid ratio of the Herba Blessed Thistle powder to the alcohol solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 DPPH free radical scavenging rates of samples of the embodiments and comparative examples, negative control group and positive control group. In the figure, ### indicates P < 0.001, NC indicates negative control, PC indicates positive control, IE1 to IE11 indicate embodiments 1 to 11, respectively, and CE1 indicates comparative example 1.

[0054] Figure 2 It is the C-terminal peptide content of type I procollagen in the samples of the embodiments and comparative examples, the blank control group and the positive control group. In the figure, # indicates P < 0.05, ## indicates P < 0.01, ### indicates P < 0.001, BC indicates blank control, PC indicates positive control, IE1~IE11 indicate embodiments 1 to 11 respectively, and CE1 indicates comparative example 1.

[0055] Figure 3 It is the elastin content of the samples of the embodiments and comparative examples, the blank control group and the positive control group. In the figure, # indicates P < 0.05, ## indicates P < 0.01, ### indicates P < 0.001, ns indicates no significance, BC indicates blank control, PC indicates positive control, IE1~IE11 indicate embodiments 1 to 11 respectively, and CE1 indicates comparative example 1. DETAILED DESCRIPTION

[0056] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0057] The Cnicus benedictus L. used in the following examples are all whole herbs of Cnicus benedictus L. purchased from Mountain Rose Herbs Company, USA.

[0058] Example 1

[0059] S1: Take the whole herb of Blessed Thistle, crush, extract, filter, concentrate, centrifuge, prepare Blessed Thistle extract, add deionized water, sterilize and obtain fermentation matrix. The specific steps are as follows:

[0060] The blessed thistle was dried at 60°C for 3 h, crushed and sieved to a particle size of 80 mesh;

[0061] Prepare a 70% ethanol solution, take the sieved blessed thistle powder, add 70% ethanol solution, the solid-liquid ratio is 1:20, stir at room temperature for 2 hours, and filter to obtain a filtrate;

[0062] The filtrate obtained is subjected to a rotary evaporator to remove the ethanol solvent to obtain a concentrated solution, and the concentrated solution is centrifuged for secondary filtration to obtain a blessed thistle extract;

[0063] Deionized water was added to the obtained blessed thistle extract, wherein the volume ratio of the blessed thistle extract to the deionized water was 1:30, and the fermentation matrix was obtained after sterilization (sterilization at 121° C. for 25 minutes).

[0064] S2: Inoculate the galactose yeast-like bacteria (Galactomyces geotrichum, with the deposit number of CGMCC 2.3766, deposited in the China Microbiological Culture Collection Center) into the YPD medium with an inoculum amount of 2%. Incubate the culture in a shaking table with a shaking speed of 180 rpm, a culture time of 30 h, and a culture temperature of 17°C to obtain a culture solution.

[0065] S3: Inoculate the bacterial culture solution into the fermentation matrix containing the blessed thistle extract, the bacterial culture solution inoculation amount is 5% of the fermentation matrix mass, and fermentation is carried out at a fermentation temperature of 30°C, a fermentation time of 36 hours, and a shaking speed of 200 rpm. Cultivate at a constant temperature, and fermentation is completed to obtain an initial fermentation solution.

[0066] S4: sterilize the initial fermentation broth (sterilize at 121°C for 25 minutes), centrifuge the inactivated fermentation broth, take the supernatant after centrifugation, filter it through a 200nm ceramic membrane, and obtain the blessed thistle fermentation product.

[0067] Experimental Example 2

[0068] S1: Take the whole herb of Blessed Thistle, crush, extract, filter, concentrate, centrifuge, prepare Blessed Thistle extract, add deionized water, sterilize and obtain fermentation matrix. The specific steps are as follows:

[0069] The blessed thistle was dried at 60°C for 3 h, crushed and sieved to a particle size of 100 mesh;

[0070] Prepare 80% ethanol solution, take the sieved blessed thistle powder, add 80% ethanol solution, the solid-liquid ratio is 1:30, stir at room temperature for 2 hours, and obtain filtrate after suction filtration;

[0071] The obtained filtrate is subjected to a rotary evaporator to remove the ethanol solvent to obtain a concentrated solution. The concentrated solution is centrifuged and filtered twice to obtain a blessed thistle extract;

[0072] Deionized water was added to the obtained blessed thistle extract, wherein the volume ratio of the blessed thistle extract to the deionized water was 1:30, and the fermentation matrix was obtained after sterilization (sterilization at 121° C. for 25 minutes).

[0073] S2: Inoculate the galactose yeast-like bacteria (Galactomyces geotrichum, with the deposit number of CGMCC 2.3766, deposited in the China Microorganism Culture Collection Center) into the YPD medium, the inoculation amount of the microorganism strain is 2.5% of the medium, and culture on a shaking table with a shaking speed of 180 rpm, a culture time of 48 h, and a culture temperature of 17°C to obtain a strain culture solution.

[0074] S3: Inoculate the bacterial culture solution into the fermentation medium containing the blessed thistle extract, the bacterial culture solution inoculation amount is 3% of the fermentation substrate mass, and ferment at a temperature of 30°C, a fermentation time of 36 hours, and a shaking speed of 200 rpm. Cultivate at a constant temperature, and the fermentation is completed to obtain the initial fermentation solution.

[0075] S4: sterilize the initial fermentation broth (sterilize at 121°C for 25 minutes), centrifuge the inactivated fermentation broth, take the supernatant after centrifugation, filter it through a 200nm ceramic membrane, and obtain the blessed thistle fermentation product.

[0076] Experimental Example 3

[0077] S1: Take the whole herb of Blessed Thistle, crush, extract, filter, concentrate, centrifuge, prepare Blessed Thistle extract, add deionized water, sterilize and obtain fermentation matrix; the specific steps are as follows:

[0078] The blessed thistle was dried at 60°C for 3 h, crushed and sieved to a particle size of 80 mesh;

[0079] Prepare a 60% ethanol solution, take the sieved blessed thistle powder, add 60% ethanol solution, the solid-liquid ratio is 1:30, stir at room temperature for 2 hours, and obtain a filtrate after suction filtration;

[0080] The obtained filtrate is subjected to a rotary evaporator to remove the ethanol solvent to obtain a concentrated solution. The concentrated solution is centrifuged and filtered twice to obtain a blessed thistle extract;

[0081] Deionized water was added to the obtained blessed thistle extract, wherein the volume ratio of the blessed thistle extract to the deionized water was 1:30, and the fermentation matrix was obtained after sterilization (sterilization at 121° C. for 25 minutes).

[0082] S2: Inoculate the galactose yeast-like bacteria (Galactomyces geotrichum, with the deposit number of CGMCC 2.3766, deposited in the China Microorganism Culture Collection Center) into the YPD medium, the inoculation amount of the microorganism strain is 3% of the medium, and culture is carried out in a shaking table with a shaking speed of 180 rpm, a culture time of 48 h, and a culture temperature of 17°C to obtain a strain culture solution.

[0083] S3: Inoculate the bacterial culture solution into the fermentation medium containing the blessed thistle extract, the bacterial culture solution inoculation amount is 5% of the fermentation substrate mass, and fermentation is carried out at a fermentation temperature of 30°C, a fermentation time of 36 hours, and a shaking speed of 200 rpm. Cultivate at a constant temperature, and fermentation is completed to obtain an initial fermentation solution.

[0084] S4: sterilizing the initial fermentation broth (sterilizing at 121° C. for 25 min), centrifuging the inactivated fermentation broth, taking the supernatant after centrifugation, filtering it through a 200 nm ceramic membrane, and obtaining the blessed thistle fermentation product.

[0085] Experimental Example 4

[0086] This embodiment is substantially the same as Embodiment 1, except that in this embodiment, the volume ratio of the Herba Thistle extract to deionized water in S1 is 1:45.

[0087] Experimental Example 5

[0088] This embodiment is substantially the same as Embodiment 1, except that in this embodiment, the ethanol concentration in S1 is 90%.

[0089] Experimental Example 6

[0090] This embodiment is basically the same as Embodiment 1, except that in this embodiment, the inoculation amount of the bacterial culture solution in S3 is 2% of the fermentation substrate.

[0091] Experimental Example 7

[0092] This embodiment is substantially the same as embodiment 1, except that in this embodiment, the fermentation time in S3 is changed to 24 h.

[0093] Experimental Example 8

[0094] This embodiment is substantially the same as embodiment 1, except that in this embodiment, the fermentation time in S3 is changed to 48 h.

[0095] Experimental Example 9

[0096] This embodiment is basically the same as Embodiment 1, except that in this embodiment, the inoculation amount of the bacterial strain in S2 is 4%.

[0097] Example 10

[0098] This embodiment is basically the same as Embodiment 1, except that: in this embodiment, the bacterial species in S2 is Saccharomyces cerevisiae (with a deposit number of CGMCC No. 21527, deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee).

[0099] Embodiment 11

[0100] This embodiment is basically the same as the embodiment 1, except that: in this embodiment, the bacterial species in S2 is Lactobacillus plantarum (whose deposit number is CGMCC No. 21528, deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee).

[0101] Comparative Example 1

[0102] In this comparative example, a Herba Thistle extract was prepared, and the preparation steps were the same as the preparation steps of the Herba Thistle extract in step S1 of Example 1.

[0103] Effect Example

[0104] The fermented product of Herba Aspergillum prepared in the above examples and the Herba Aspergillum extract prepared in the comparative example were added into a polyol preservative system to prepare samples for in vitro cell testing.

[0105] The sample of the embodiment is prepared by mixing 5% butanediol, 3% 1,2-hexanediol, 0.05% ethylhexylglycerol and 91.95% of the fermented product of the blessed thistle of the above embodiment to obtain , wherein % refers to the percentage of each substance in the total mass of the sample.

[0106] The sample of the comparative example was prepared by mixing 5% butylene glycol, 3% 1,2-hexanediol, 0.05% ethylhexylglycerin and 91.95% of the Herba Thistles Blessed extract of the comparative example.

[0107] Test 1: Antioxidant Test

[0108] The antioxidant effect of the test substance can be determined by testing the sample's ability to scavenge DPPH free radicals. Principle: DPPH, also known as 1,1-diphenyl-2-trinitrophenylhydrazine, is a very stable nitrogen-centered free radical. Its stability mainly comes from the spatial barriers of the three benzene rings of resonance stabilization, which prevents the unpaired electrons on the nitrogen atom in the middle from playing their due electron pairing role. Its anhydrous ethanol solution is purple, with maximum absorption at a wavelength of 517nm, and a linear relationship between absorbance and concentration. When a free radical scavenger is added to it, it can be combined with or replaced by DPPH, reducing the number of free radicals, reducing absorbance, and lightening the color of the solution, thereby evaluating the ability to scavenge free radicals. This experimental scheme uses DPPH as a substrate and L (+) -ascorbic acid (VC) as a positive substance to determine the antioxidant effect of cosmetic raw materials in vitro.

[0109] Steps:

[0110] The experiment was divided into a blank control group, a negative control group, a positive control group and a test sample group. Samples were added according to Table 1. After adding samples, the samples were reacted in the dark for 30 minutes and detected at 517 nm.

[0111] Table 1 shows the test plan.

[0112] Table 1

[0113]

[0114] Solution preparation:

[0115] 1) DPPH solution: Weigh 8 mg of DPPH reagent, add 5 mL of anhydrous ethanol to dissolve, shake well to obtain a 1600 μg / mL DPPH stock solution, and store in a refrigerator. When using, transfer 1 mL of DPPH stock solution and dilute it 20 times with anhydrous ethanol to obtain 80 μg / mL DPPH solution.

[0116] 2) Preparation of VC aqueous solution (5X): Weigh 0.01g VC, add 2ml water to dissolve, and obtain 5mg / ml VC aqueous solution.

[0117] 3) Sample solution: Mix the above sample with water to obtain a sample solution with a sample concentration of 1%.

[0118] The reagents and equipment used in the preparation and detection process are shown in Table 2.

[0119] Table 2

[0120]

[0121] Result analysis:

[0122] GraphPad was used for drawing, and the results were expressed as Mean±SD. The t-test was used for statistical analysis among the groups. All statistical analyses were two-tailed.

[0123] The test results are shown in Table 6 and Figure 1 .

[0124] Figure 1 It is the scavenging rate of DPPH free radicals of the samples of the embodiments and comparative examples, the negative control group and the positive control group. Figure 1 In the table, ### indicates P<0.001, NC indicates negative control, PC indicates positive control, IE1 to IE11 indicate Example 1 to Example 11, respectively, and CE1 indicates Comparative Example 1. P<0.05 is considered to be significantly different, and P<0.01 is considered to be extremely significantly different.

[0125] Test 2: Anti-aging test

[0126] This test uses human dermal fibroblasts HDF to evaluate whether the test substance has the efficacy in promoting collagen synthesis by measuring the upregulation of the content of type I procollagen C-terminal peptide after administration of the test substance.

[0127] Principle: Type I collagen is one of the main components of the extracellular matrix of dermal cells. Type I procollagen synthesized in cells is secreted outside the cells. Under the action of endopeptidase, the propeptides attached to its amino and carboxyl ends are cut off to form tropocollagen. Tropocollagen molecules polymerize into collagen fibers to form the extracellular matrix. The cut free propeptides are soluble and can be used as a biochemical indicator to reflect the amount of collagen synthesis in the body. Human dermal fibroblasts can be used as a cell model to study the effect of cosmetics on improving the content of type I collagen. By measuring the upregulation of the content of type I procollagen C-terminal peptide after administration of blank control, positive control and test substance, the efficacy of the test substance in promoting collagen synthesis can be evaluated.

[0128] The determination of type I procollagen C-terminal peptide content was performed using an enzyme-linked immunosorbent assay (ELISA). The specific principle is that after the type I procollagen C-terminal peptide specifically binds to the antibody coated on the ELISA plate, it binds to the anti-type I procollagen C-terminal peptide antibody labeled with a substrate. After the substrate is catalyzed by the enzyme, a colored product is generated. The type I procollagen C-terminal peptide content is positively correlated with the color depth of the colored product. The optical density (OD value) is measured at a wavelength of 450nm using an ELISA instrument to calculate the type I procollagen C-terminal peptide content.

[0129] The test plan is shown in Table 3.

[0130] Table 3

[0131]

[0132] Preparation of working solution:

[0133] 1) Sample solution preparation: The sample was diluted with DMEM medium to obtain sample solutions with concentrations of 0.01% and 0.001%. The samples of each embodiment and comparative example were diluted to these two concentrations, and % refers to the volume ratio of the sample to the DMEM medium;

[0134] 2) TGF-β1 solution: Dilute 10 μg / mL of TGF-β1 to 100 ng / mL with water to obtain a TGF-β1 solution with a concentration of 100 ng / mL.

[0135] Steps:

[0136] 1) Cell plating: Human dermal fibroblasts (HDF) were seeded into 96-well plates at a cell concentration of 8 × 104 / mL, with 100 μl per well. The seeded cell culture plates were placed in an incubator and cultured for 24 h (5% CO 2 , 37℃).

[0137] 2) Administration: After 24 h of cell culture, remove the supernatant, add 100 μl of sample solution and 100 μl of TGF-β1 solution to each well, mix well, and place in an incubator for 24 h + 1 h.

[0138] 3) Cell viability test: After 24 hours, collect the supernatant (for ELISA test), add 100μl CCK-8 working solution to each well, and place in the incubator for 1~4 hours. Measure the absorbance at 450nm, and save the test data in electronic format for subsequent analysis.

[0139] 4) ELISA test: Collect the cell culture supernatant and perform ELISA test.

[0140] The reagents and equipment used in the preparation and detection process are shown in Table 4.

[0141] Table 4

[0142]

[0143] Result analysis:

[0144] GraphPad Prism was used for graphing, and the results were expressed as Mean ± SD. The groups were compared using t-test for statistical analysis, and all statistical analyses were two-tailed.

[0145] The test results are shown in Table 6 and Figure 2 .

[0146] Figure 2The content of type I procollagen C-terminal peptide in the samples of the examples and comparative examples, the blank control group and the positive control group, in the figure, # indicates P < 0.05, ## indicates P < 0.01, ### indicates P < 0.001, BC indicates blank control, PC indicates positive control, IE1 to IE11 indicate examples 1 to 11, respectively, and CE1 indicates comparative example 1. P < 0.05 is considered to have a significant difference, and P < 0.01 is considered to have a very significant difference.

[0147] Test 3: Compactness test

[0148] This test uses human dermal fibroblasts HDF to evaluate whether the test substance is effective in promoting elastin synthesis by measuring the upregulation of elastin content after administration of the test substance.

[0149] Principle: Fibroblasts are the main cell components of the skin and are distributed in the dermis of the skin. They are the most common cells in loose connective tissue and can produce a large amount of collagen and elastic fibers, playing an important role in maintaining the structural stability and elasticity of the skin.

[0150] The content of elastin determines the firmness of the skin and thus affects the condition of the skin. A decrease in the content of elastin will lead to skin aging. Therefore, the effect of the sample on the content of elastin can be tested by the enzyme-linked immunosorbent assay (ELISA) method to evaluate the firming effect of the sample.

[0151] The test plan is shown in Table 5.

[0152] Table 5

[0153]

[0154] Preparation of working solution:

[0155] 1) Sample solution preparation: The sample was diluted with DMEM medium to obtain sample solutions with concentrations of 0.01% and 0.001%. The finished raw materials of each embodiment and comparative example were diluted to these two concentrations, and % refers to the volume ratio of the sample to the DMEM medium.

[0156] 2) TGF-β1 solution: Dilute 150 μg / mL TGF-β1 stock solution to 200 ng / mL with water to obtain a TGF-β1 solution with a concentration of 200 ng / mL.

[0157] Steps:

[0158] 1) Cell plating: Human dermal fibroblasts (HDF) were seeded into 96-well plates at a cell concentration of 8 × 104 / mL, with 100 μl per well. The seeded cell culture plates were placed in an incubator and cultured for 24 h (5% CO2, 37°C).

[0159] 2) Administration: After 24 h of cell culture, remove the supernatant, add 100 μl of sample solution and 100 μl of TGF-β1 solution to each well, mix well, and place in an incubator for 24 h + 1 h.

[0160] 3) Cell viability test: After 24 hours, collect the supernatant (for ELISA test), add 100μl CCK-8 working solution to each well, and place in the incubator for 1~4 hours. Measure the absorbance at 450nm, and save the test data in electronic format for subsequent analysis.

[0161] 4) ELISA test: Collect the cell culture supernatant and perform ELISA test.

[0162] The reagents and equipment used in the preparation and detection process are shown in Table 4.

[0163] Result analysis:

[0164] GraphPad Prism was used for graphing, and the results were expressed as Mean±SD. The groups were compared using t-test for statistical analysis, and all statistical analyses were two-tailed.

[0165] The test results are shown in Table 6 and Figure 3 .

[0166] Figure 3 The elastin content of the samples of the examples and comparative examples, the blank control group and the positive control group, # indicates P < 0.05, ## indicates P < 0.01, ### indicates P < 0.001, ns indicates no significance, BC indicates blank control, PC indicates positive control, IE1 to IE11 indicate examples 1 to 11, respectively, and CE1 indicates comparative example 1. P < 0.05 is considered to have a significant difference, and P < 0.01 is considered to have a very significant difference.

[0167] The test results of the samples in the above embodiments and comparative examples at different concentrations are shown in Table 6.

[0168] Table 6

[0169]

[0170] It can be seen from the above data that the antioxidant capacity, the content of type I procollagen C-terminal peptide, and the content of elastin in Examples 1, 2, and 3 were all significantly increased, and the efficacy under the conditions of Example 1 was more excellent.

[0171] In Example 4, compared with Example 1, the volume ratio of deionized water increased, and the antioxidant capacity, DPPH clearance rate, type I procollagen C-terminal peptide content, and elastin content all decreased.

[0172] Example 5 Compared with Example 1, the ethanol concentration increased, and the antioxidant capacity DPPH clearance rate, type I procollagen C-terminal peptide content and elastin content all decreased.

[0173] In Example 6, compared with Example 1, the inoculation amount of the bacterial culture solution was reduced, and the antioxidant capacity, DPPH clearance rate, the content of type I procollagen C-terminal peptide and the content of elastin were all reduced.

[0174] Example 7 Compared with Example 1, the fermentation time of the blessed thistle extract was shortened, and the antioxidant capacity, DPPH clearance rate, content of type I procollagen C-terminal peptide and content of elastin were all reduced.

[0175] Example 8 Compared with Example 1, the fermentation time of the blessed thistle extract was prolonged, and the antioxidant capacity, DPPH clearance rate, the content of type I procollagen C-terminal peptide and the content of elastin were all reduced.

[0176] In Example 9, compared with Example 1, the inoculation amount of the bacterial strain was increased, and the antioxidant capacity, DPPH clearance rate, the content of type I procollagen C-terminal peptide and the content of elastin were all decreased.

[0177] In Example 10, compared with Example 1, the fermentation strain was Saccharomyces cerevisiae, and the antioxidant capacity, DPPH clearance rate, content of type I procollagen C-terminal peptide and content of elastin were all decreased.

[0178] Example 11 Compared with Example 1, the fermentation strain is Lactobacillus plantarum, and the antioxidant capacity DPPH clearance rate, the content of type I procollagen C-terminal peptide and the content of elastin are all reduced.

[0179] Compared with Example 1, in Comparative Example 1, no fermentation was performed, and only simple alcohol extraction was performed. The antioxidant capacity, DPPH clearance rate, content of type I procollagen C-terminal peptide and content of elastin were all decreased.

Claims

1. A method for preparing a fermented product of blessed thistle, characterized in that: It includes the following steps: (1) mixing the herbaceous thistle extract with water and sterilizing the mixture to obtain a fermentation medium; (2) inoculating the bacterial culture solution into the fermentation matrix and fermenting to obtain an initial fermentation solution; The inoculation amount of the bacterial strain in the bacterial strain culture solution is 1-4%; The bacterial species is one or more of Galactomyces geotrichum, Saccharomyces cerevisiae and Lactobacillus plantarum; The bacterial culture solution is 2-8% of the fermentation substrate, and % refers to the ratio of the volume of the bacterial culture solution to the volume of the fermentation substrate; (3) The initial fermentation liquid is sterilized and separated, and the obtained liquid is the blessed thistle fermentation product.

2. The method for preparing the fermented product of blessed thistle according to claim 1, characterized in that: Step (1) satisfies one or more of the following conditions: ① The volume ratio of the Herba Thistle extract to the water is 1:(10-50), preferably 1:(25-50), such as 1:30 or 1:45, more preferably 1:30; ② The sterilization temperature is 121°C; and, ③The sterilization time is 15 to 30 minutes, for example 25 minutes.

3. The method for preparing the fermented product of blessed thistle according to claim 1, characterized in that: In step (1), the Blessed Thistle extract is prepared by alcohol extraction; preferably, the preparation method of the Blessed Thistle extract comprises the following steps: S1. crushing the blessed thistle to obtain blessed thistle powder; S2, mixing the blessed thistle powder with an alcohol solution, stirring and filtering at room temperature to obtain a filtrate; S3, removing the alcohol in the filtrate to obtain a concentrated solution, filtering the concentrated solution, and obtaining a filtrate as the Herba Thistle extract.

4. The method for preparing the fermented product of blessed thistle according to claim 3, characterized in that: The preparation method of the blessed thistle extract meets one or more of the following conditions: ① The blessed thistle is a dried blessed thistle, the drying temperature is preferably 55-65° C., for example 60° C.; the drying time is preferably 3-5 hours; ② After the pulverization, the method further comprises a sieving step, wherein the particle size of the blessed thistle powder after sieving is preferably 70-100 meshes, more preferably 80 meshes; ③ The mass ratio of the blessed thistle powder to the volume ratio of the alcohol solution is 1g: (10-30)mL, preferably 1g: 20mL; ④ The alcohol solution is a mixture of alcohol and water, and the concentration of alcohol in the alcohol solution is preferably 60-90%, more preferably 70%, where % refers to the ratio of the volume of alcohol to the volume of water; ⑤ the alcohol solution is an ethanol solution; and, ⑥The stirring time is 2 to 4 hours.

5. The method for preparing the fermented product of blessed thistle according to claim 1, characterized in that: Step (2) satisfies one or more of the following conditions: ① The bacterial species is Galactomyces geotrichum, Saccharomyces cerevisiae or Lactobacillus plantarum. Preferably, the bacterial species is Galactomyces geotrichum; ② The inoculation amount of the bacterial species in the bacterial culture solution is 2-3%, such as 2%, 2.5%, 3% or 4%; and, ③ The bacterial culture solution accounts for 2%, 3% or 5% of the fermentation substrate, preferably 3-6%.

6. The method for preparing the fermented product of blessed thistle according to claim 1, characterized in that: Step (2) satisfies one or more of the following conditions: ① The method for preparing the bacterial culture solution comprises the following steps: inoculating the bacterial species into a culture medium, culturing on a shaking table, and obtaining a bacterial culture solution; The shaking speed of the shaking incubator is preferably 100-250 rpm, such as 180 rpm; the temperature of the shaking incubator is preferably 15-19° C., such as 17° C.; the time of the shaking incubator is preferably 24-48 h, such as 30 h or 48 h; the culture medium is preferably YPD culture medium; ② The fermentation temperature is 15-45°C, preferably 30°C; ③ The fermentation time is 24 to 72 hours, preferably 36 hours; and, ④ The fermentation is carried out on a shaker, and the rotation speed of the shaker is preferably 100-250 rpm, for example 200 rpm.

7. The method for preparing the fermented product of blessed thistle according to claim 1, characterized in that: Step (3) satisfies one or more of the following conditions: ① The sterilization temperature is 121°C; ② The sterilization time is 15 to 30 minutes, for example 25 minutes; and, ③ After the sterilization, the method further includes the steps of centrifugation and filtering the liquid after the centrifugation; the filtration is preferably carried out by ceramic membrane filtration, and the pore size of the ceramic membrane is preferably 150-250 nm, for example 200 nm.

8. A fermented product of blessed thistle prepared by the method for preparing a fermented product of blessed thistle according to any one of claims 1 to 7.

9. Use of the fermented product of blessed thistle according to any one of claims 1 to 7 in cosmetics.

10. The use according to claim 9, characterized in that The fermented product of blessed thistle is used as an anti-aging or antioxidant ingredient in cosmetics; And / or, the Herba Thistle fermented product is used as a DPPH scavenger, an elastin synthesis promoter or a type I procollagen C-terminal peptide expression promoter in cosmetics.

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