A blessed sage ferment, and a preparation method and application thereof

Abstract

The application discloses a kind of blessed herba lepidii ferment and its preparation method and application, wherein the preparation method of blessed herba lepidii ferment includes the following steps: blessed herba lepidii extract is mixed with water, and fermentation substrate is obtained after sterilization;Strain culture solution is inoculated into fermentation substrate, fermentation is carried out, and initial fermentation liquor is obtained;The inoculation amount of strain in strain culture solution is 1~4%, and the strain is one or more of galactosum yeast-like bacteria, saccharomyces cerevisiae or lactobacillus plantarum, and the strain culture solution is 2~8% of fermentation substrate;Initial fermentation liquor is sterilized and separated, and the obtained liquid is blessed herba lepidii ferment.The method of the application can effectively improve the concentration of effective substances in blessed herba lepidii ferment, and blessed herba lepidii ferment as an effective ingredient in cosmetics can improve anti-aging ability and antioxidant capacity.

Description

Technical Field

[0001] This invention relates to a fermented product of thistle, its preparation method, and its application. Background Technology

[0002] Blessed thistle, also known as Blessing thistle, is a plant belonging to the genus *Cirsium* in the family Asteraceae. In recent years, it has been widely cultivated in major botanical gardens and medicinal herb gardens throughout my country. It grows at altitudes of 1,000 to 1,200 meters, often in river valley meadows. The type specimen was collected from Western Europe. The whole plant is used medicinally, possessing stomachic properties. It is a bitter tonic and digestive herb, containing sesquiterpene lactones, triterpenoids, lignans, tannins, essential oils, flavonoids, and polyenes. It treats indigestion and loss of appetite; promotes urination and sweating, purifies the body's fluid system, eliminates toxins, and supports liver repair; it has anti-tumor and anti-cancer properties; it is also a women's supplement, often used to promote lactation, and possesses excellent antioxidant capabilities, showing great potential for development.

[0003] There are currently no studies on the fermentation of thistle. Summary of the Invention

[0004] This invention provides a *Thistle of Fortune* ferment, its preparation method, and its application. The *Thistle of Fortune* ferment of this invention, as an active ingredient in topical skin preparations, exhibits good antioxidant and anti-aging effects.

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

[0006] This invention provides a method for preparing a fermented product of thistle, which includes the following steps:

[0007] (1) Mix the extract of thistle with water and sterilize to obtain the fermentation substrate;

[0008] (2) The inoculum culture is inoculated into the fermentation substrate and fermented to obtain the initial fermentation broth; the inoculation amount of the inoculum culture is 1-4%; the inoculum is one or more of Galactomyces geotrichum, Saccharomyces cerevisiae and Lactobacillus plantarum; the inoculum culture is 2-8% of the fermentation substrate, where % refers to the percentage of the volume of the inoculum culture to the volume of the fermentation substrate;

[0009] (3) The initial fermentation liquid is sterilized and separated, and the resulting liquid is the fermented product of *Cirsium japonicum*.

[0010] In this invention, in step (1), the volume ratio of the herb-blessing thistle extract to the water is preferably 1:(10~50), more preferably 1:(25~50), for example 1:30 or 1:45, and even more preferably 1:30.

[0011] In this invention, the preparation method of the extract of *Thistle arvense* in step (1) can be conventional in the art. Preferably, the extract of *Thistle arvense* is prepared by alcohol extraction.

[0012] More preferably, the preparation method of the Blessed Thistle Extract includes the following steps:

[0013] S1. Crush the Blessed Thistle into powder to obtain Blessed Thistle powder;

[0014] S2. Mix the *Thistle* powder with an alcohol solution, stir and filter at room temperature to obtain a filtrate;

[0015] S3. Remove the alcohol from the filtrate to obtain a concentrated solution. Filter the concentrated solution to obtain a filtrate of thistle extract.

[0016] The *Thistle of Blessing* is generally dried *Thistle of Blessing*. During the drying process, the drying temperature is preferably 55-65°C, for example, 60°C; and the drying time is preferably 3-5 hours.

[0017] In step S1, the pulverization process may further include a sieving step, wherein the particle size of the sieved thistle powder is preferably 70-100 mesh, and more preferably 80 mesh.

[0018] The term "blessed thistle" (Cnicus benedictus L.) is conventional in this field and generally refers to the whole herb of Cnicus benedictus.

[0019] In S2, the ratio of the mass of the *Cirsium japonicum* powder to the volume of the alcohol solution is preferably 1 g: (10~30) mL, more preferably 1 g: 20 mL.

[0020] In step 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 volume ratio of alcohol to water.

[0021] In step S2, the alcohol solution can be conventional in the art, and preferably an ethanol solution.

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

[0023] The room temperature mentioned is conventional in the art and generally refers to 20~30℃, for example 25℃.

[0024] In step S2, the filtering can be conventional in the art, such as vacuum filtration.

[0025] In step S3, the preferred method for removing alcohol from the filtrate is evaporation, such as using a rotary evaporator to evaporate the filtrate to remove alcohol.

[0026] In step S3, the filtration method can be conventional in the art, such as centrifugal filtration.

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

[0028] In this invention, in step (2), the strain is preferably Galactomyces geotrichum, Saccharomyces cerevisiae, or Lactobacillus plantarum, and more preferably Galactomyces geotrichum.

[0029] In this invention, the galactomyces geotrichum, with accession number CGMCCNO.2.3766, is deposited at the China Microbial Culture Collection Center.

[0030] The Saccharomyces cerevisiae strain mentioned, with accession number CGMCC No. 21527, is deposited at the China General Microbiological Culture Collection Center.

[0031] The Lactobacillus plantarum, with accession number CGMCC No. 21528, is deposited at the China General Microbiological Culture Collection Center.

[0032] In this invention, in step (2), the inoculation amount of the bacterial strain in the bacterial culture medium is preferably 2-3%, for example 2%, 2.5%, 3% or 4%, more preferably 2%. The inoculation amount is calculated using conventional methods in the art, namely: inoculation amount = (target colony count / target strain concentration) × (inoculation volume / total culture medium volume).

[0033] In this invention, in step (2), the preparation method of the bacterial culture medium can be conventional in the art, preferably including the following steps: inoculating the bacterial strain into a culture medium, culturing on a shaker, and obtaining the bacterial culture medium.

[0034] The shaking speed of the shaker used for the shaker culture is preferably 100~250 rpm, for example 180 rpm.

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

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

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

[0038] In this invention, in step (2), the microbial culture medium is preferably 2%, 3% or 5% of the fermentation substrate, more preferably 3 to 6%, for example, even more preferably 5%, where % refers to the proportion of the volume of the microbial culture medium to the volume of the fermentation substrate.

[0039] In this invention, in step (2), the fermentation temperature is preferably 15~45℃, and more preferably 30℃.

[0040] In this invention, in step (2), the fermentation time is preferably 24~72h, more preferably 36h.

[0041] In this 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 this invention, the sterilization in step (3) can be conventional in the art. The sterilization temperature is preferably 121°C. The sterilization time is preferably 15~30 min, for example 25 min.

[0043] In this invention, step (3) may further include centrifugation and filtering of the centrifuged liquid after sterilization.

[0044] The filtration method is conventional in the art, generally involving filtering the liquid with 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 method for preparing the aforementioned *Cirsium arvense* ferment, resulting in *Cirsium arvense* ferment.

[0046] The present invention also provides an application of the aforementioned *Thistle* ferment in cosmetics.

[0047] In this invention, preferably, the Blessed Thistle Ferment is used as an anti-aging or antioxidant ingredient in cosmetics.

[0048] In this invention, preferably, the *Thistle* ferment is used in cosmetics as a DPPH scavenger, an elastin synthesis promoter, or a type I procollagen C-terminal peptide expression promoter.

[0049] In this invention, the cosmetic product may be an aqueous solution, emulsion, spray, cream, mask, sunscreen, or foundation, etc.

[0050] The positive and progressive effects of this invention are as follows:

[0051] The method of the present invention can effectively increase the concentration of active substances in the fermented product of *Cirsium affine*; as an active ingredient in cosmetics, *Cirsium affine* fermented product can improve anti-aging and antioxidant capabilities.

[0052] In a further preferred preparation method of the present invention, the active ingredients in the *Thistle sibirica* extract are enhanced by adjusting the pulverization operation, the concentration of the alcohol solution, and the ratio of *Thistle sibirica* powder to the alcohol solution. Attached Figure Description

[0053] Figure 1 The figures show the DPPH free radical scavenging rates of the samples, negative control group, and positive control group in the examples and comparative examples. In the figures, ### indicates P < 0.001, NC indicates the negative control, PC indicates the positive control, IE1~IE11 represent Examples 1~Example 11 respectively, and CE1 represents Comparative Example 1.

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

[0055] Figure 3 The figures show the elastin content of the samples, blank control group, and positive control group for the examples and comparative examples. In the figures, # 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 represent examples 1~11 respectively, and CE1 represents comparative example 1. Detailed Implementation

[0056] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0057] The *Thistle* used in the following examples are all whole plants of *Thistle*, purchased from Mountain Roseherbs, USA.

[0058] Example 1

[0059] S1: Take the whole plant of *Thistle sibirica*, crush it, extract it, filter it, concentrate it, centrifuge it to obtain *Thistle sibirica* extract, add deionized water, sterilize it to obtain the fermentation substrate. The specific steps are as follows:

[0060] The herb *Thistle* was dried at 60℃ for 3 hours, then pulverized and sieved to a particle size of 80 mesh.

[0061] Prepare a 70% ethanol solution. Take the sieved Cirsium japonicum powder, add it to the 70% ethanol solution, with a material-to-liquid ratio of 1:20, stir at room temperature for 2 hours, and then filter to obtain the filtrate.

[0062] The obtained filtrate was removed by rotary evaporation to obtain a concentrated solution. The concentrated solution was centrifuged and filtered a second time to obtain the herb thistle extract.

[0063] Deionized water was added to the obtained *Cirsium japonicum* extract, with a volume ratio of *Cirsium japonicum* extract to deionized water of 1:30. After sterilization (sterilization at 121℃ for 25 min), the fermentation substrate was obtained.

[0064] S2: Galactomyces geotrichum (accession number CGMCC 2.3766, deposited at the China Microbiological Culture Collection Center) was inoculated into YPD medium at an inoculation amount of 2%, and cultured on a shaker at 180 rpm for 30 h at a temperature of 17℃ to obtain the culture solution.

[0065] S3: Inoculate the bacterial culture broth into a fermentation substrate containing *Cirsium japonicum* extract. The inoculation volume of the bacterial culture broth is 5% of the mass of the fermentation substrate. Fermentation is carried out at 30℃ for 36 hours, with a shaking speed of 200 rpm. After constant temperature incubation, the initial fermentation broth is obtained.

[0066] S4: Sterilize the initial fermentation broth (sterilize at 121℃ for 25 min), centrifuge the inactivated fermentation broth, take the supernatant after centrifugation, filter it through a 200nm ceramic membrane, and obtain the fermented product of *Cirsium japonicum*.

[0067] Experimental Example 2

[0068] S1: Take the whole plant of *Thistle sibirica*, crush it, extract it, filter it, concentrate it, centrifuge it to obtain *Thistle sibirica* extract, add deionized water, sterilize it to obtain the fermentation substrate. The specific steps are as follows:

[0069] The thistle was dried at 60℃ for 3 hours, then pulverized and sieved to a particle size of 100 mesh.

[0070] Prepare an 80% ethanol solution by taking sieved Cirsium japonicum powder, adding it to the 80% ethanol solution at a material-to-liquid ratio of 1:30, stirring at room temperature for 2 hours, and then filtering to obtain the filtrate.

[0071] The filtrate was evaporated to remove the ethanol solvent, yielding a concentrated solution. The concentrated solution was centrifuged and filtered twice to obtain thistle extract.

[0072] Deionized water was added to the obtained *Cirsium japonicum* extract, with a volume ratio of *Cirsium japonicum* extract to deionized water of 1:30. After sterilization (sterilization at 121℃ for 25 min), the fermentation substrate was obtained.

[0073] S2: Galactomyces geotrichum (accession number CGMCC 2.3766, deposited at the China Microbiological Culture Collection Center) was inoculated into YPD medium at a concentration of 2.5% of the medium volume. The medium was then cultured on a shaker at 180 rpm for 48 hours at 17°C to obtain the culture broth.

[0074] S3: Inoculate the bacterial culture broth into a fermentation medium containing *Cirsium japonicum* extract. The inoculation amount of the bacterial culture broth is 3% of the mass of the fermentation substrate. Fermentation is carried out at 30℃ for 36 hours, with a shaking speed of 200 rpm. After constant temperature incubation, the initial fermentation broth is obtained.

[0075] S4: Sterilize the initial fermentation broth (sterilize at 121℃ for 25 min), centrifuge the inactivated fermentation broth, take the supernatant after centrifugation, filter it through a 200nm ceramic membrane, and obtain the fermented product of *Cirsium japonicum*.

[0076] Experimental Example 3

[0077] S1: Take the whole plant of *Thistle simonii*, crush it, extract it, filter it, concentrate it, centrifuge it to obtain *Thistle simonii* extract, add deionized water, sterilize it to obtain the fermentation substrate; the specific steps are as follows:

[0078] The herb *Thistle* was dried at 60℃ for 3 hours, then pulverized and sieved to a particle size of 80 mesh.

[0079] Prepare a 60% ethanol solution. Take the sieved Cirsium japonicum powder, add it to the 60% ethanol solution, with a material-to-liquid ratio of 1:30, stir at room temperature for 2 hours, and then filter to obtain the filtrate.

[0080] The filtrate was evaporated to remove the ethanol solvent, yielding a concentrated solution. The concentrated solution was centrifuged and filtered twice to obtain thistle extract.

[0081] Deionized water was added to the obtained *Cirsium japonicum* extract, with a volume ratio of *Cirsium japonicum* extract to deionized water of 1:30. After sterilization (sterilization at 121℃ for 25 min), the fermentation substrate was obtained.

[0082] S2: Galactomyces geotrichum (accession number CGMCC 2.3766, deposited at the China Microbiological Culture Collection Center) was inoculated into YPD medium at a concentration of 3% of the medium volume. The medium was then cultured on a shaker at 180 rpm for 48 hours at 17°C to obtain the culture solution.

[0083] S3: Inoculate the bacterial culture broth into a fermentation medium containing *Cirsium japonicum* extract. The inoculation volume of the bacterial culture broth is 5% of the mass of the fermentation substrate. Fermentation is carried out at 30℃ for 36 hours, with a shaking speed of 200 rpm. After constant temperature incubation, the initial fermentation broth is obtained.

[0084] S4: Sterilize the initial fermentation broth (sterilize at 121℃ for 25 min), centrifuge the inactivated fermentation broth, take the supernatant after centrifugation, filter it through a 200nm ceramic membrane, and obtain the *Cirsium japonicum* fermentation product.

[0085] Experiment Example 4

[0086] This embodiment is basically the same as embodiment 1, except that in this embodiment, the volume ratio of the herb-blessing thistle extract to deionized water in S1 is 1:45.

[0087] Experimental Example 5

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

[0089] Experimental Example 6

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

[0091] Experimental Example 7

[0092] This embodiment is basically the same as Embodiment 1, except that the fermentation time in S3 is changed to 24 hours.

[0093] Experimental Example 8

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

[0095] Experimental Example 9

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

[0097] Example 10

[0098] This embodiment is basically the same as embodiment 1, except that: in this embodiment, the strain in S2 is Saccharomyces cerevisiae (accession number CGMCC No. 21527, deposited at the China General Microbiological Culture Collection Center).

[0099] Example 11

[0100] This embodiment is basically the same as embodiment 1, except that: in this embodiment, the bacterial strain in S2 is Lactobacillus plantarum (its accession number is CGMCC No. 21528, which is deposited at the China General Microbiological Culture Collection Center).

[0101] Comparative Example 1

[0102] This comparative example prepared a *Cirsium japonicum* extract, and the preparation steps were the same as those in step S1 of Example 1.

[0103] Effect Example

[0104] The *Thistle* ferment prepared in the above examples and the *Thistle* extract prepared in the comparative example were added to a polyol preservative system to prepare samples for in vitro cell testing.

[0105] The sample preparation of the embodiment is as follows: 5% butanediol, 3% 1,2-hexanediol, 0.05% ethylhexylglycerin and 91.95% of the *Cirsium japonicum* fermentation product of the above embodiment are mixed to obtain the sample. The percentage refers to the percentage of each substance in the total mass of the sample.

[0106] The comparative sample was prepared by mixing 5% butanediol, 3% 1,2-hexanediol, 0.05% ethylhexylglycerin and 91.95% of the above-mentioned comparative sample of *Cirsium japonicum* extract.

[0107] Test 1: Antioxidant Test

[0108] The antioxidant activity of a test substance is determined by testing its 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 steric hindrance of the three benzene rings through resonance stabilization, preventing the unpaired electrons on the central nitrogen atom from performing their proper electron pairing function. Its anhydrous ethanol solution is purple, with maximum absorption at 517 nm, and absorbance is linearly related to concentration. Adding a free radical scavenger can bind to or replace DPPH, reducing the number of free radicals, decreasing absorbance, and lightening the solution color, thereby evaluating the free radical scavenging ability. This experimental scheme uses DPPH as a substrate and L(+)-ascorbic acid (VC) as a positive agent to determine the in vitro antioxidant activity of cosmetic raw materials.

[0109] Operating steps:

[0110] The experiment was divided into a blank control group, a negative control group, a positive control group, and a test sample group. The samples were added according to Table 1. After adding the samples, the reaction was carried out in the dark for 30 minutes, and the samples were 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, dissolve in 5 mL of anhydrous ethanol, shake well to obtain a DPPH stock solution with a concentration of 1600 μg / mL, and store in a refrigerator. When using, transfer 1 mL of the DPPH stock solution and dilute it 20 times with anhydrous ethanol to obtain an 80 μg / mL DPPH solution.

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

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

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

[0119] Table 2

[0120]

[0121] Results analysis:

[0122] GraphPad was used for plotting, and the results are expressed as Mean ± SD. t-tests were used for comparisons between groups. All statistical analyses were two-tailed.

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

[0124] Figure 1 The DPPH free radical scavenging rate of the samples, negative control group, and positive control group in the examples and comparative examples are shown. Figure 1 In the table, ### indicates P < 0.001, NC indicates negative control, PC indicates positive control, IE1~IE11 represent Examples 1 to 11 respectively, and CE1 represents Comparative Example 1. P < 0.05 is considered statistically significant, and P < 0.01 is considered highly statistically significant.

[0125] Test 2: Anti-aging Test

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

[0127] Principle: Type I collagen is one of the main components of the extracellular matrix of dermal cells. Type I procollagen synthesized intracellularly is secreted extracellularly. Under the action of endopeptidase, the propeptides attached to its N-terminus and C-terminus are cleaved, forming procollagen. Procollagen molecules then polymerize into collagen fibers, constituting the extracellular matrix. The cleaved free propeptides are soluble and can serve as a biochemical indicator reflecting the amount of collagen synthesized in the body. Human dermal fibroblasts can be used as a cell model to study the effect of cosmetics on increasing type I collagen content. By measuring the upregulation of type I procollagen C-terminal peptide content after administration of the test substance to blank controls, positive controls, and the test substance, the efficacy of the test substance in promoting collagen synthesis can be evaluated.

[0128] The content of type I procollagen C-terminal peptide (CTP) was determined using an enzyme-linked immunosorbent assay (ELISA). The principle is as follows: after the type I procollagen CTP specifically binds to the antibody coated on the ELISA plate, it binds to the substrate-labeled anti-type I procollagen CTP antibody. The substrate is catalyzed by the enzyme to generate a colored product. The content of type I procollagen CTP is positively correlated with the intensity of the colored product. The optical density (OD value) was measured at a wavelength of 450 nm using an ELISA reader to calculate the type I procollagen CTP content.

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

[0130] Table 3

[0131]

[0132] Preparation of working fluid:

[0133] 1) Sample solution preparation: The samples were diluted to concentrations of 0.01% and 0.001% using DMEM medium. The samples of each example and comparative example were diluted to these two concentrations. % refers to the volume percentage of the sample in the DMEM medium.

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

[0135] Operating steps:

[0136] 1) Cell seeding: Human dermal fibroblasts (HDF) were seeded into 96-well plates at a density of 8 × 10⁴ / mL, 100 μl per well. The seeded cell culture plates were then placed in an incubator and cultured for 24 h (5% CO₂, 37℃).

[0137] 2) Drug administration: After culturing cells for 24 h, the supernatant was aspirated, and 100 μl of sample solution and 100 μl of TGF-β1 solution were added to the wells respectively. The mixture was then placed in an incubator for 24 h + 1 h.

[0138] 3) Cell viability assay: After 24 hours, collect the supernatant (for ELISA detection), add 100 μl of CCK-8 working solution to each well, and incubate for 1–4 hours. Measure the absorbance at 450 nm. Save the experimental data electronically for subsequent analysis.

[0139] 4) ELISA detection: After collecting the cell culture supernatant, perform ELISA detection.

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

[0141] Table 4

[0142]

[0143] Results analysis:

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

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

[0146] Figure 2The figures show the C-terminal peptide content of type I procollagen in the samples of the examples and comparative examples, the blank control group, and the positive control group. In the figures, # indicates P < 0.05, ## indicates P < 0.01, ### indicates P < 0.001, BC indicates blank control, PC indicates positive control, IE1~IE11 represent Examples 1~11 respectively, and CE1 represents Comparative Example 1. P < 0.05 is considered statistically significant, and P < 0.01 is considered highly statistically significant.

[0147] Test 3: Tightness Test

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

[0149] Principle: Fibroblasts are the main cellular components of the skin, distributed in the dermis. 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 amount of elastin determines the firmness of the skin and thus affects its condition. A decrease in elastin content leads to skin aging. Therefore, the effect of enzyme-linked immunosorbent assay (ELISA) on the elastin content of a sample can be used to evaluate its firming effect.

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

[0152] Table 5

[0153]

[0154] Preparation of working fluid:

[0155] 1) Sample solution preparation: The sample was diluted to a concentration of 0.01% and 0.001% using DMEM medium. The finished raw materials of each example and comparative example were diluted to these two concentrations. % refers to the volume percentage of the sample in the DMEM medium.

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

[0157] Operating steps:

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

[0159] 2) Drug administration: After culturing cells for 24 h, the supernatant was aspirated, and 100 μl of sample solution and 100 μl of TGF-β1 solution were added to the wells respectively. The mixture was then placed in an incubator for 24 h + 1 h.

[0160] 3) Cell viability assay: After 24 hours, collect the supernatant (for ELISA detection), add 100 μl of CCK-8 working solution to each well, and incubate for 1–4 hours. Measure the absorbance at 450 nm. Save the experimental data electronically for subsequent analysis.

[0161] 4) ELISA detection: After collecting the cell culture supernatant, perform ELISA detection.

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

[0163] Results analysis:

[0164] GraphPad Prism was used for plotting, and the results are expressed as Mean±SD. The comparisons between groups were performed using t-test 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, blank control group, and positive control group in the examples and comparative examples are shown. # 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 represent Examples 1~11 respectively, and CE1 represents Comparative Example 1. P < 0.05 is considered statistically significant, and P < 0.01 is considered highly statistically significant.

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

[0168] Table 6

[0169]

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

[0171] Compared with Example 1, Example 4 showed an increase in the volume ratio of deionized water, and a decrease in the antioxidant capacity DPPH scavenging rate, the content of type I procollagen C-terminal peptide, and the content of elastin.

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

[0173] Compared with Example 1, Example 6 showed a reduction in the amount of bacterial culture medium inoculated with, resulting in a decrease in the antioxidant capacity DPPH scavenging rate, the content of type I procollagen C-terminal peptide, and the content of elastin.

[0174] Compared with Example 1, Example 7 showed that the fermentation time of the *Thistle* extract was shorter, and the antioxidant capacity DPPH scavenging rate, the content of type I procollagen C-terminal peptide, and the content of elastin were all reduced.

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

[0176] Compared with Example 1, Example 9 showed an increase in the amount of bacterial inoculum, but a decrease in the antioxidant capacity DPPH scavenging rate, the content of type I procollagen C-terminal peptide, and the content of elastin.

[0177] Compared with Example 1, Example 10 used Saccharomyces cerevisiae as the fermentation strain, and the antioxidant capacity, DPPH scavenging rate, type I procollagen C-terminal peptide content, and elastin content all decreased.

[0178] Compared with Example 1, Example 11 used Lactobacillus plantarum as the fermentation strain, and the antioxidant capacity, DPPH scavenging rate, type I procollagen C-terminal peptide content, and elastin content all decreased.

[0179] Compared with Example 1, Comparative Example 1 did not involve fermentation, but only simple alcohol extraction. The antioxidant capacity DPPH scavenging rate, the content of type I procollagen C-terminal peptide, and the content of elastin all decreased.

Claims

1. A method for preparing a fermented product of *Thistle of Blessing*, characterized in that, It includes the following steps: (1) Mix the extract of thistle with water and sterilize to obtain the fermentation substrate; The volume ratio of the *Thistle* extract to the water is 1:30; The preparation method of the Blessed Thistle Extract includes the following steps: S1. Crush the Blessed Thistle into powder to obtain Blessed Thistle powder; S2. Mix the *Thistle* powder with an alcohol solution, stir and filter at room temperature to obtain a filtrate; S3. Remove the alcohol from the filtrate to obtain a concentrated solution. Filter the concentrated solution to obtain a filtrate of thistle extract. The alcohol concentration in the alcohol solution is 70%; the alcohol solution is an ethanol solution. (2) The bacterial culture medium is inoculated into the fermentation substrate and fermented to obtain the initial fermentation broth; The inoculum size of the bacterial strain in the culture medium is 2%. The bacterial strain is *Galactomyces* (Galactomyces galactosidum). Galactomyces geotrichum The preservation number of the galactosomal yeast-like fungus is CGMCC NO.2.3766; The microbial culture medium is 5% of the fermentation substrate, where % refers to the percentage of the volume of the microbial culture medium to the volume of the fermentation substrate. The fermentation time is 36 hours; (3) The initial fermentation liquid is sterilized and separated, and the resulting liquid is the fermented product of *Cirsium japonicum*.

2. The method for preparing the fermented product of *Thistle of Blessing* as described in claim 1, characterized in that, The sterilization temperature is 121°C; And / or, the sterilization time is 15-30 min.

3. The method for preparing the fermented product of *Thistle of Blessing* as described in claim 2, characterized in that, The sterilization time is 25 minutes.

4. The method for preparing the fermented product of *Thistle of Blessing* as described in claim 1, characterized in that, The preparation method of the *Thistle* extract satisfies one or more of the following conditions: ①The blessed thistle mentioned above is the dried blessed thistle, and the drying temperature is 55~65℃; ②The pulverization process also includes a sieving step, and the particle size of the sieved thistle powder is 70~100 mesh; ③ The mass ratio of the *Thistle* powder to the volume of the alcohol solution is 1 g: (10~30) mL; The stirring time described in section ④ is 2-4 hours.

5. The method for preparing the fermented product of *Thistle of Blessing* as described in claim 1, characterized in that, The preparation method of the *Thistle* extract satisfies one or more of the following conditions: ①The blessed thistle mentioned above is the dried blessed thistle, and the drying temperature is 60℃; ②The blessed thistle mentioned above is the dried blessed thistle, and the drying time is 3~5 hours; ③The pulverization process also includes a sieving step, and the particle size of the sieved thistle powder is 80 mesh; The mass ratio of the *Thistle* powder to the volume of the alcohol solution is 1 g: 20 mL.

6. The method for preparing the fermented product of *Thistle of Blessing* as described in claim 1, characterized in that, Step (2) satisfies one or more of the following conditions: ①The preparation method of the bacterial culture medium includes the following steps: inoculating the bacterial strain into a culture medium, culturing on a shaker, and obtaining the bacterial culture medium; ②The fermentation temperature is 15~45℃; The fermentation described in section ③ is carried out on a shaker with a rotation speed of 100~250 rpm.

7. The method for preparing the fermented product of *Thistle of Blessing* as described in claim 1, characterized in that, Step (2) satisfies one or more of the following conditions: ① The preparation method of the bacterial culture medium includes the following steps: inoculating the bacterial strain into a culture medium, culturing on a shaker to obtain the bacterial culture medium; the shaking speed of the shaker culture is 100~250 rpm; ② The preparation method of the bacterial culture medium includes the following steps: inoculating the bacterial strain into a culture medium, culturing on a shaker to obtain the bacterial culture medium; the temperature of the shaker culture is 15~19℃; ③ The preparation method of the bacterial culture medium includes the following steps: inoculating the bacterial strain into a culture medium, culturing on a shaker to obtain the bacterial culture medium; the shaking culture time is 24~48h; ④ The preparation method of the bacterial culture medium includes the following steps: inoculating the bacterial strain into a culture medium, culturing on a shaker to obtain the bacterial culture medium; the culture medium is YPD medium; ⑤ The fermentation temperature is 30℃; The fermentation described in section ⑥ is carried out on a shaker, and the shaking speed of the shaker is 200 rpm during the fermentation.

8. The method for preparing the fermented product of *Thistle of Blessing* as described in claim 1, characterized in that, Step (2) satisfies one or more of the following conditions: ①The preparation method of the bacterial culture medium includes the following steps: inoculating the bacterial strain into a culture medium, culturing on a shaker to obtain the bacterial culture medium; the shaking speed of the shaker culture is 180 rpm; ② The preparation method of the bacterial culture medium includes the following steps: inoculating the bacterial strain into a culture medium, culturing on a shaker to obtain the bacterial culture medium; the temperature of the shaker culture is 17℃; The preparation method of the bacterial culture medium described in ③ includes the following steps: inoculating the bacterial strain into a culture medium, culturing on a shaker to obtain the bacterial culture medium; the shaking time is 30h or 48h.

9. The method for preparing the fermented product of *Thistle of Blessing* as described in claim 1, characterized in that, Step (3) satisfies one or more of the following conditions: ① The sterilization temperature is 121℃; ② The sterilization time is 15-30 minutes; and, ③The sterilization process also includes centrifugation and filtration of the centrifuged liquid; the filtration is performed using a ceramic membrane.

10. The method for preparing the fermented product of *Thistle of Blessing* as described in claim 1, characterized in that, The sterilization time is 25 minutes; And / or, the sterilization process further includes centrifugation and filtration of the centrifuged liquid; the filtration is performed using a ceramic membrane with a pore size of 150~250 nm.

11. The method for preparing the fermented product of *Thistle of Blessing* as described in claim 1, characterized in that, The sterilization process also includes centrifugation and filtration of the centrifuged liquid; the filtration is performed using a ceramic membrane with a pore size of 200 nm.

12. A fermented herb of *Cirsium japonicum* prepared by a method according to any one of claims 1 to 11.

13. The use of thistle ferment as described in any one of claims 1 to 11 in cosmetics.

14. The application as described in claim 13, characterized in that, The fermented herb *Thistle* is used as an anti-aging or antioxidant ingredient in cosmetics.

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