Burdock root fermented liquid, preparation method thereof and application thereof in cosmetics

Abstract

The invention discloses a burdock root fermentation liquid, a preparation method thereof and an application thereof in cosmetics, and belongs to the technical field of cosmetics. The preparation method provided by the invention comprises the following steps: inoculating Pseudoalteromonas or Schizosaccharomyces pombe in a culture medium for activation to obtain a seed liquid; inoculating the seed liquid into a fermentation medium, and adding burdock root powder, wherein the concentration of the burdock root powder added in the fermentation medium is 45-55 g / L, and then fermenting to the end point, and obtaining the burdock root fermentation liquid through post-treatment. The burdock root fermentation liquid prepared by the invention has excellent antioxidant effect, anti-allergic soothing effect and firming effect, and also has certain healing and repairing functions, and is safe and non-toxic, with low irritation, and can be used for the preparation of cosmetic raw materials for antioxidant, anti-allergic and soothing.

Description

Technical Field

[0001] The invention relates to the technical field of cosmetics, and in particular to a burdock root fermented liquid, a preparation method thereof, and application of the burdock root fermented liquid in cosmetics. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance the understanding of the overall background of the invention and should not be necessarily regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Burdock root, also known as "white ginseng", is the root of the burdock plant Arctium lappa L. of the Asteraceae family. Burdock root is rich in polysaccharides, phenolic substances, proteins, amino acids, vitamins, and calcium, zinc, phosphorus, iron and other nutrients and minerals required by the human body. Studies have shown that burdock root has antioxidant, anti-inflammatory, nerve regulation, blood sugar and lipid lowering, immune regulation and other effects, and it is easy to obtain in the market, the raw materials are cheap, and the ingredients are simple and harmless.

[0004] At present, burdock root is mainly used in the field of health food on the market, such as making burdock root tea, but its fermented products are few and its uses are single. There is little research in the cosmetics field. Moreover, when burdock root extract is directly used in cosmetics, its antioxidant effect, anti-allergic soothing effect and firming effect are poor, which limits its wide application in the cosmetics field.

[0005] Therefore, it is of great practical significance to develop a method and product to enhance the antioxidant, anti-allergic, soothing and firming effects of burdock root and expand its application in the field of cosmetics. Summary of the invention

[0006] In view of this, the present invention provides a burdock root fermented liquid, a preparation method thereof and an application thereof in cosmetics. The burdock root fermented liquid prepared by the present invention has better antioxidant effect, anti-allergic soothing effect and firming effect than burdock root water extract, and can be applied to the field of cosmetics.

[0007] In a first aspect, the present invention provides a method for preparing burdock root fermented liquid, comprising the following steps:

[0008] inoculating Pseudoalteromonas or Schizosaccharomyces pombe into a culture medium for activation to obtain a seed solution;

[0009] The seed liquid is inoculated into a fermentation medium, and burdock root powder is added, wherein the concentration of the burdock root powder added to the fermentation medium is 45-55 g / L, and then fermented to an end point, and post-processed to obtain a burdock root fermentation liquid;

[0010] Wherein, the pseudoalteromonas is named as pseudoalteromonas ( Pseudoalteromonas sp.)SE7, which was deposited in China Center for Type Culture Collection on December 13, 2024, with a deposit number of CCTCC NO: M 20242812; the pombe Schizosaccharomyces cerevisiae was named as Pombe Schizosaccharomyces cerevisiae ( Schizosaccharomyces pombe ) JM2-16, which was deposited in the China Center for Type Culture Collection on December 13, 2024, with the deposit number CCTCC NO: M 20242810.

[0011] In a second aspect, the present invention provides burdock root fermented liquid prepared by the above preparation method.

[0012] In a third aspect, the present invention provides an application of the burdock root fermented liquid in cosmetics, wherein the burdock root fermented liquid is used to prepare cosmetics, and the volume fraction of the burdock root fermented liquid in the cosmetics is 1-10%.

[0013] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0014] The invention provides a method for fermenting burdock root based on pseudoalteromonas or Schizosaccharomyces pombe. The burdock root fermented liquid prepared by the method has excellent antioxidant effect, anti-allergic soothing effect and firming effect, and also has certain healing and repairing functions. The biological activity of the burdock root fermented liquid is significantly higher than that of the burdock root water extract, and the burdock root fermented liquid is safe, non-toxic and less irritating, and can be used for the preparation of antioxidant, anti-allergic and soothing cosmetic raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The drawings constituting part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute an improper limitation of the present invention. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 is a bar graph of DPPH free radical scavenging rates of the burdock root fermented liquids of Examples 1 and 2 of the present invention and the burdock root water extract of the comparative example at different concentrations;

[0017] Figure 2 It is a bar graph of superoxide anion free radical scavenging rates of the burdock root fermented liquids of Examples 1 and 2 of the present invention and the burdock root water extract of the comparative example at different concentrations;

[0018] Figure 3 is a bar graph of hydroxyl radical scavenging rates of the burdock root fermented liquids of Examples 1 and 2 of the present invention and the burdock root water extract of the comparative example at different concentrations;

[0019] Figure 4is a bar graph of hyaluronidase inhibition rates of the burdock root fermented liquids of Examples 1 and 2 of the present invention and the burdock root water extract of the comparative example at different concentrations;

[0020] Figure 5 is a bar graph of the elastase inhibition rate of the burdock root fermented liquid of Examples 1 and 2 of the present invention and the burdock root water extract of the comparative example at different concentrations;

[0021] Figure 6 It is a bar graph of cell survival rates of the burdock root fermented liquid of Examples 1 and 2 of the present invention and the burdock root water extract of the comparative example at different concentrations;

[0022] Figure 7 The burdock root fermented liquid of Example 1 and Example 2 of the present invention and the burdock root water extract of the comparative example are scratch healing test pictures at a concentration of 1%, wherein a1 is a cell scratch micrograph of the burdock root fermented liquid of Example 1 at 0h; a2 is a cell scratch micrograph of the burdock root fermented liquid of Example 1 at 24h; b1 is a cell scratch micrograph of the burdock root fermented liquid of Example 2 at 0h; b2 is a cell scratch micrograph of the burdock root fermented liquid of Example 2 at 24h; c1 is a cell scratch micrograph of the burdock root water extract at 0h; c2 is a cell scratch micrograph of the burdock root water extract at 24h;

[0023] Figure 8 The present invention is a picture of the burdock root fermented liquid of Example 1 and Example 2 and the chicken embryo stimulation experiment of the positive control group; wherein, a1 is a microscopic picture of the chorioallantoic membrane of the chicken embryo before stimulation by the burdock root fermented liquid of Example 1; a2 is a microscopic picture of the chorioallantoic membrane of the chicken embryo after stimulation by the burdock root fermented liquid of Example 1; b1 is a microscopic picture of the chorioallantoic membrane of the chicken embryo before stimulation by the burdock root fermented liquid of Example 2; b2 is a microscopic picture of the chorioallantoic membrane of the chicken embryo after stimulation by the burdock root fermented liquid of Example 2; c1 is a microscopic picture of the chorioallantoic membrane of the chicken embryo before stimulation by the positive control group; c2 is a microscopic picture of the chorioallantoic membrane of the chicken embryo after stimulation by the positive control group. DETAILED DESCRIPTION

[0024] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0025] The present invention provides a method for preparing burdock root fermented liquid, comprising the following steps:

[0026] inoculating Pseudoalteromonas or Schizosaccharomyces pombe into a culture medium for activation to obtain a seed solution;

[0027] The seed liquid is inoculated into a fermentation medium, and burdock root powder is added, wherein the concentration of the burdock root powder added to the fermentation medium is 45-55 g / L, and then fermented to an end point, and post-processed to obtain a burdock root fermentation liquid;

[0028] Wherein, the pseudoalteromonas is named as pseudoalteromonas ( Pseudoalteromonas sp. )SE7, which was deposited in China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province) on December 13, 2024, with a deposit number of CCTCC NO: M 20242812; the pombe Schizosaccharomyces cerevisiae was named as Pombe Schizosaccharomyces cerevisiae ( Schizosaccharomyces pombe ) JM2-16, which was deposited in the China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province) on December 13, 2024, with the deposit number of CCTCC NO: M 20242810.

[0029] The pseudoalteromonas provided by the present invention can promote the decomposition and transformation of plant polysaccharides, release more antioxidant active ingredients, and can grow stably in an extremely cold environment. It has excellent antioxidant synthesis ability, which is beneficial to improving the antioxidant properties of burdock roots. The pombe schizosaccharomyces provided by the present invention can produce many beneficial metabolites during fermentation, which is helpful to improve the antioxidant properties of burdock roots.

[0030] In the present invention, the activation method of the Pseudoalteromonas is specifically as follows: inoculating the Pseudoalteromonas into a sea salt culture medium at an inoculum amount of 0.8-1.2 vol%, and culturing for 10-15 h at 13-17° C. and 150-200 rpm;

[0031] The activation method of the Schizosaccharomyces pombe is specifically as follows: the Schizosaccharomyces pombe is inoculated into a yeast extract peptone glucose medium at an inoculation rate of 0.8-1.2 vol%, and cultured at 28-32° C. and 150-200 rpm for 10-15 hours.

[0032] In the present invention, the sea salt culture medium comprises: 2-4 g / L yeast powder, 4-6 g / L peptone, 4-6 g / L sea salt, the solvent is water, and the pH is 7.5-8.0;

[0033] The yeast extract peptone glucose culture medium comprises: 2wt% glucose, 1-2wt% peptone, 0.5-1wt% yeast extract, water as solvent, and pH value of 6.0-6.5. The yeast extract peptone glucose culture medium can be obtained through commercial channels.

[0034] In the present invention, the inoculation amount of the seed liquid in the fermentation medium is 0.8-1.2 vol%, more preferably 1 vol%.

[0035] In the present invention, when the seed liquid is obtained by activating Pseudoalteromonas, the fermentation medium includes 2-4 g / L yeast powder, 4-6 g / L peptone, 4-6 g / L sea salt, the solvent is water, and the pH is 7.5-8.0; the process of fermenting to the end point is specifically: fermenting for 18-48 hours at 13-17°C and 150-200 rpm.

[0036] In the present invention, when the seed liquid is obtained by activating Schizosaccharomyces pombe, the fermentation medium includes 2wt% glucose, 1-2wt% peptone, 0.5-1wt% yeast extract, the solvent is water, and the pH is 6.0-6.5; the fermentation process to the end point is specifically: fermentation at 28-32°C and 150-200rpm for 18-48h.

[0037] In the present invention, the post-treatment includes centrifugation, sterilization, decolorization and filtration. The above-mentioned treatment steps are to obtain a clear and transparent fermentation broth. Specifically, the centrifugation step is: centrifugation at 10000-12000 r / min for 15-25 min; the sterilization step is: sterilization at 120-125°C for 20-40 min; the decolorization step uses activated carbon for decolorization; the filtration step uses a filter membrane with a pore size of 0.1-0.4 μm, more preferably 0.22 μm.

[0038] The present invention also provides burdock root fermented liquid prepared by the above preparation method. The burdock root fermented liquid prepared by the above preparation method has excellent antioxidant effect, anti-allergic soothing effect and firming effect, and also has certain healing and repairing functions, and is safe, non-toxic and less irritating.

[0039] The present invention also provides the use of the burdock root fermented liquid in cosmetics, wherein the volume fraction of the burdock root fermented liquid in the cosmetics is 1-10%, more preferably 5-10%. The cosmetics have antioxidant, anti-allergic, soothing and firming effects.

[0040] The technical solution of the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified, the present invention has no special restrictions on the sources of the reagents used in the following embodiments, and commercially available products known to those skilled in the art can be used.

[0041] In the following examples, Pseudoalteromonas is named as Pseudoalteromonas ( Pseudoalteromonas sp. ) SE7, which was deposited in China Center for Type Culture Collection on December 13, 2024, with a deposit number of CCTCC NO: M20242812; the pombe Schizosaccharomyces cerevisiae was named as Pombe Schizosaccharomyces cerevisiae ( Schizosaccharomyces pombe) JM2-16, which was deposited in the China Center for Type Culture Collection on December 13, 2024, with the deposit number CCTCC NO: M20242810.

[0042] Sea salt culture medium: yeast powder 3g / L, peptone 5g / L, sea salt 5g / L, solvent is deionized water, pH is 7.5~8.0.

[0043] Yeast extract peptone glucose medium (YPD medium): glucose 2wt%, peptone 2wt%, yeast extract 1wt%, solvent is deionized water, pH is 6.0~6.5.

[0044] Example 1

[0045] This embodiment provides a method for preparing burdock root fermentation broth based on Pseudoalteromonas.

[0046] (1) Activation:

[0047] Take Pseudoalteromonas ( Pseudoalteromonas sp. )SE7 was inoculated into sea salt medium at a rate of 1 vol% and cultured at 15 °C and 180 rpm for 12 h to obtain seed solution.

[0048] (2) Fermentation culture:

[0049] The seed liquid of step (1) was inoculated into the sea salt culture medium at an inoculum amount of 1 vol%, and then burdock root powder was added to control the concentration of burdock root powder to be 50 g / L. The culture medium was sealed with gauze and fermented in a shaking flask at 15° C. and 180 rpm for 24 h to obtain a fermentation product.

[0050] (3) Post-processing:

[0051] The fermentation product of step (2) was centrifuged at 11000 r / min for 20 min to obtain a clarified fermentation product, which was then sterilized in a high-temperature autoclave at 121° C. for 30 min, and then decolorized with activated carbon and filtered with a 0.22 μm needle filter to obtain a burdock root fermentation liquid.

[0052] Example 2

[0053] This embodiment provides a method for preparing burdock root fermentation broth based on Schizosaccharomyces pombe.

[0054] (1) Activation:

[0055] Take Schizosaccharomyces pombe ( Schizosaccharomyces pombe ) JM2-16 was inoculated into sea salt medium at 1 vol% and cultured at 30 °C and 180 rpm for 12 h to obtain seed solution.

[0056] (2) Fermentation culture:

[0057] The seed liquid of step (1) was inoculated into a yeast extract peptone glucose medium at an inoculation rate of 1 vol%, and then burdock root powder was added to control the concentration of burdock root powder to be 50 g / L. The medium was sealed with gauze and fermented in a shaking flask at 30° C. and 180 rpm for 24 h to obtain a fermentation product.

[0058] (3) Post-processing:

[0059] The fermentation product of step (2) was centrifuged at 11000 r / min for 20 min to obtain a clarified fermentation product, which was then sterilized in a high-temperature autoclave at 121° C. for 30 min, and then decolorized with activated carbon and filtered with a 0.22 μm needle filter to obtain a burdock root fermentation liquid.

[0060] Comparative Example

[0061] This comparative example provides a method for preparing a burdock root water extract.

[0062] The burdock root water extract was prepared according to the same ratio as in Example 1 and Example 2 (solid-liquid ratio of 1:20), centrifuged at 11000 r / min for 20 min to obtain a clarified supernatant, and then sterilized in a high-temperature autoclave at 121° C. for 30 min, and then decolorized with activated carbon and filtered with a 0.22 μm needle filter in sequence to obtain the burdock root water extract.

[0063] Test example

[0064] Sample solution: The burdock root fermented liquid of Example 1 and Example 2 and the burdock root water extract of the comparative example were respectively prepared into solutions with volume fractions of 1%, 5% and 10% with deionized water. The sample solutions were recorded as Example 1-1%, Example 1-5%, Example 1-10%, Example 2-1%, Example 2-5%, Example 2-10%, Comparative Example -1%, Comparative Example -5% and Comparative Example -10%.

[0065] 1. Analysis of Antioxidant Efficacy

[0066] (1) DPPH free radical scavenging experiment

[0067] Prepare 0.1 mM DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) solution. Take 0.002 g DPPH each time and dissolve it in 50 mL ethanol to prepare a 0.1 mM DPPH solution. Keep it away from light. The experiment was carried out in a 96-well plate. Three replicates were set for each group to reduce experimental errors. Anhydrous ethanol was used to adjust the zero. Sample group: 180 μL of the above sample solution was taken and added to 90 μL DPPH solution; control group: 180 μL of distilled water was added to 90 μL DPPH solution; blank group: 90 μL of anhydrous ethanol was added to 180 μL sample solution. Shake in the dark for 10 min and test the absorbance. The calculation formula for DPPH free radical scavenging rate is as follows:

[0068] DPPH clearance rate = [A 对照组 -(A 样品组 -A 空白组 )] / A 对照组 ×100%;

[0069] In the formula, A 样品组 is the absorbance of the sample group, A 对照组 is the absorbance of the control group, A 空白组 is the absorbance of the blank group. Figure 1 shown.

[0070] from Figure 1 It can be seen that with the increase in the concentration of the burdock root fermentation liquid fermented by Pseudoalteromonas and Schizosaccharomyces pombe, the DPPH free radical scavenging rate is also gradually increasing. When the concentration of the burdock root fermentation liquid fermented by Pseudoalteromonas and Schizosaccharomyces pombe reaches 5%, the DPPH free radical scavenging rate is higher than 70%; when the fermentation liquid concentration reaches 10%, the scavenging rates reach 95.81% and 91.18%, respectively, which are much higher than the DPPH free radical scavenging rate of the burdock root water extract at the same concentration. It can be seen that the burdock root fermentation liquid prepared in Example 1 and Example 2 has a good scavenging effect on DPPH free radicals.

[0071] (2) Superoxide anion free radical scavenging experiment

[0072] Take 4.5mL of 0.05mol / L Tris-HCl buffer with a pH of 8.2, place it in a 25℃ water bath for 20min, add 1mL of the above sample solution and 0.4mL of 25mmol / L pyrogallol solution respectively, mix well and react in a 25℃ water bath for 5min, add 1.0mL of 8mol / L HCl solution to terminate the reaction, use Tris-HCl buffer as a reference, measure the absorbance at 299nm, and calculate the superoxide anion radical scavenging rate. The blank control group replaced the sample solution with 1mL of 0.05mol / L Tris-HCl buffer with a pH of 8.2, and each treatment was repeated three times. The calculation formula for the superoxide anion radical scavenging rate of the sample is as follows:

[0073] Superoxide anion radical scavenging rate = (A1-A2) / A1×100%;

[0074] In the formula, A1 is the average absorbance of the blank control group; A2 is the average absorbance of the experimental group containing the sample solution. Figure 2 shown.

[0075] from Figure 2 It can be seen that the burdock root fermentation liquid fermented by Pseudoalteromonas and Schizosaccharomyces pombe has a certain ability to scavenge superoxide anion free radicals. When the concentration of the burdock root fermentation liquid fermented by Pseudoalteromonas and Schizosaccharomyces pombe is 5%, the scavenging rate can be higher than 70%; when the fermentation liquid concentration reaches 10%, the scavenging rates reach 90.64% and 81.59%, respectively, which are much higher than the superoxide anion free radical scavenging rate of the burdock root water extract at the same concentration. This shows that the burdock root fermentation liquid fermented by Pseudoalteromonas and Schizosaccharomyces pombe has a good scavenging effect on superoxide anion free radicals, and the effect is better than that of the burdock root water extract.

[0076] (3) Hydroxyl radical scavenging experiment

[0077] Hydroxyl radicals are produced by the Fenton reaction. ·OH oxidizes salicylic acid to produce 2,3-dihydroxybenzoic acid with characteristic absorption at 510nm. The scavenging rate of ·OH is determined by measuring the product obtained by salicylic acid capturing ·OH. Add 3mL each of 2mmol / L FeSO4 and 1mmol / L H2O2 to a 25mL colorimetric tube, shake well, then add 3mL of 6mmol / L salicylic acid, shake well, heat in a 37℃ water bath for 15min, take out, and measure its absorbance A1. Then add 1mL of sample solution of different concentrations, shake well, continue to heat in a water bath for 15min, take out and measure its absorbance A2. The calculation formula for the scavenging rate of hydroxyl radicals by the sample is as follows:

[0078] Hydroxyl radical scavenging rate = (A1-A2) / A1×100%.

[0079] Test results such as Figure 3 As shown, from Figure 3 It can be seen that with the increase of the concentration of burdock root fermentation liquid fermented by Pseudoalteromonas and Schizosaccharomyces pombe, the scavenging effect on hydroxyl free radicals increases; when the concentration of burdock root fermentation liquid fermented by Pseudoalteromonas and Schizosaccharomyces pombe is 5%, the hydroxyl free radical scavenging rate can be higher than 70%; when the concentration of burdock root fermentation liquid fermented by Pseudoalteromonas and Schizosaccharomyces pombe is 10%, the scavenging rates reach 90.72% and 94.80%, respectively, which are much higher than the hydroxyl free radical scavenging rate of burdock root water extract.

[0080] 2. Analysis of anti-allergic and soothing effects

[0081] Hyaluronic acid (HA) is the component with the highest content and the largest proportion in the extracellular matrix (ECM). It can maintain the volume of the extracellular matrix, regulate the secretion of cell growth factors and cytokines, and affect the adhesion, growth, proliferation and differentiation of cells. Therefore, it plays an important role in maintaining skin moisture and elasticity, wound healing and angiogenesis. Hyaluronidase (HAase) is a specific cleavage enzyme of HA. Inhibiting the activity of HAase can ensure the normal content and function of HA. Therefore, the anti-allergic activity (anti-allergic soothing activity) of a substance can be evaluated by the HAase inhibition rate. The greater the HAase inhibition rate, the stronger the anti-allergic activity.

[0082] Hyaluronidase inhibition test: Take 0.02 mL of 0.25mmol / L CaCl2 solution and 0.1mL of hyaluronidase solution, incubate at 37℃ for 20 min; add sample solutions of different concentrations, continue to incubate at 37℃ for 20 min; add 0.1mL of sodium hyaluronate solution, incubate at 37℃ for 30 min, and place at room temperature for 5 min; add 0.02 mL of 0.4mol / L NaOH solution and 0.1 mL of acetylacetone solution, heat in a boiling water bath for 15 min, immediately cool with ice water for 5 min, add 0.2 mL of Ehrlich reagent, dilute with 0.6 mL of anhydrous ethanol, place for color development for 20 min, and measure the absorbance at 510 nm with a multifunctional microplate reader. The hyaluronidase inhibition rate is calculated according to the following formula:

[0083] Hyaluronidase inhibition rate = [(A1-A2)-(B1-B2)] / (A1-A2);

[0084] Wherein, A1 is the absorbance value of the reference solution (using acetate buffer solution instead of the sample solution); A2 is the absorbance value of the reference blank solution (using acetate buffer solution instead of hyaluronidase and sample solution); B1 is the absorbance value of the sample solution; B2 is the absorbance value of the sample blank solution (using acetate buffer solution instead of hyaluronidase and sodium hyaluronate solution).

[0085] Test results such as Figure 4 As shown in the figure, it can be seen that with the increase of the concentration of burdock root fermented by Pseudoalteromonas and Schizosaccharomyces pombe, the inhibition rate of hyaluronidase is also gradually increasing. When the concentration reaches 5%, the inhibition rates of hyaluronidase are 71.32% and 76.56%, respectively, both higher than 70%; the inhibition rates of burdock root fermented by Pseudoalteromonas and Schizosaccharomyces pombe at a concentration of 10% are 80.23% and 82.33%, respectively, and the inhibition rates are higher than those of burdock root water extract. The experimental results show that burdock root fermented by Pseudoalteromonas and Schizosaccharomyces pombe have a good inhibitory effect on hyaluronidase activity, and the effect is significantly better than that of burdock root water extract.

[0086] 3. Analysis of firming effect

[0087] Elastase has the ability to degrade collagen, elastin and other proteins. The change in elastin structure during skin aging is one of the important causes of wrinkles and skin sagging, and inhibiting elastase from decomposing elastin can play an anti-wrinkle and firming role. Elastase is mainly synthesized and secreted by fibroblasts, and can degrade elastin in the skin, causing skin aging. The firming effect can be characterized by evaluating the inhibition rate of the test sample on elastase. The higher the elastase inhibition rate, the better the firming effect of the sample.

[0088] Elastase inhibition test: 0.05 mL of sample solution of the same concentration was added to the sample group and the sample background group, 0.1 mL of 50 mM Tris-HCl buffer at pH 8.0 and 0.025 mL of 2.5 U / mL elastase solution were added to the sample group, 0.125 mL of 50 mM Tris-HCl buffer at pH 8.0 were added to the sample background group, 0.125 mL of 50 mM Tris-HCl buffer at pH 8.0 and 0.025 mL of elastase solution were added to the control group, and 0.150 mL of 50 mM Tris-HCl buffer at pH 8.0 was added to the control blank group. The mixture was fully mixed and reacted at a constant temperature of 25°C for 15 minutes. Add 0.025mL 1mM AAPAN (N-acetyl-L-alanyl-L-alanyl-prolyl-phenylnitroaniline) solution to the sample group, sample background group, control group, and control blank group, and react at 25°C for 15 minutes. Transfer each reaction solution into a 96-well plate and measure the absorbance at 410nm. The calculation formula for elastase inhibition rate is as follows:

[0089] Elastase inhibition rate: 1-(A1-A2) / (B1-B2)×100%;

[0090] In the formula, A1 is the absorbance value of the sample group; A2 is the absorbance value of the sample background group; B1 is the absorbance value of the control group; B2 is the absorbance value of the control blank group. The test results are as follows Figure 5 shown.

[0091] from Figure 5 It can be seen that when the burdock root fermented liquid fermented by Pseudoalteromonas and Schizosaccharomyces pombe and the burdock root water extract are at the same concentration gradient, the inhibition rate of elastase is much higher than that of the burdock root water extract. This shows that the burdock root fermented liquid fermented by Pseudoalteromonas and Schizosaccharomyces pombe can better inhibit the synthesis and secretion of elastase by fibroblasts, alleviate skin aging caused by the reduction of elastin in the skin, and has a better firming effect.

[0092] 4. Cytotoxicity assay

[0093] HaCaT cell model establishment: Keratinocytes (HaCaT) were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS). The cells were incubated in an incubator containing 5% CO2 at 37°C and used for experiments when they were passed to the fifth generation.

[0094] HaCaT cell toxicity assay: 0.8×10 4Keratinocytes (HaCaT) were inoculated in a 96-well plate at a seeding density of cells / well and incubated overnight in an incubator (37°C, 5% CO2). The drug was administered when the cell plating rate in the 96-well plate reached about 50%. The 96-well plate was washed before administration. Administration: There was no cell inoculation in the blank group, and only 200μL of cell culture medium was added; 200μL of culture medium was added to each well of the control group; 200μL of culture medium containing samples of corresponding concentrations was added to each well of the sample group. After the administration was completed, the 96-well plate was placed in an incubator (37°C, 5% CO2) and cultured for 24 hours. After the cells were incubated for 24 hours, 10μL of CCK-8 was added and incubated at 37°C in the dark for 3 hours. After the incubation was completed, the OD value was read at 490nm. The formula for calculating the cell viability is shown as follows:

[0095] Cell viability = (OD 样品组 -OD 空白组 / OD 对照组 -OD 空白组 )×100%;

[0096] Among them, OD 样品组 is the OD value of the sample group, OD 对照组 is the OD value of the control group, OD 空白组 is the OD value of the blank group.

[0097] Test results such as Figure 6 As shown, from Figure 6 It can be seen that when the burdock root fermented liquid fermented by Pseudoalteromonas and Schizosaccharomyces pombe is applied at a low concentration (1%), the survival rate of keratinocytes remains at about 98%, indicating that low concentrations of burdock root fermented liquid have almost no toxic effects on cells and will not cause irritation or damage; when the concentration reaches 10%, the cell survival rate can still be maintained above 80%, indicating that burdock root fermented liquid has good safety.

[0098] 5. Cell scratch test

[0099] Before the experiment, use a marker pen to mark three horizontal lines on the back of the 6-well plate. Take logarithmic phase HaCaT cells at 0.8×10 4 The cells were inoculated into a 6-well plate at a density of 100 / well and incubated overnight in an incubator (37°C, 5% CO2). The cells were cultured until a monolayer of adherent cells was formed before the experiment. Before administration, a 200μL sterile pipette tip was used to evenly draw a line perpendicular to the marked line in the center of the cell plate, washed 3 times with PBS, and fresh serum-free culture medium was added. Pictures were taken under a microscope (0h), and the drug treatment (the sample group was treated with 1% sample solution, and the control group was not treated with drug treatment) was continued for 24h. After the experiment, pictures were taken under a microscope, and the scratch grayscale value and scratch healing rate were calculated using ImageJ. The calculation formula for the scratch healing rate is as follows:

[0100] Scratch healing rate = (S 样品 -S 对照 ) / S 对照 ×100%;

[0101] Among them, S 对照 The area of ​​the scratch reduction in the control group after 24 hours of culture; S 样品 The area of ​​the scratches reduced after the sample group was treated with drugs and cultured for 24 hours. Figure 7 shown.

[0102] from Figure 7 It can be seen that compared with 1% burdock root water extract (scratch healing rate of 17.2%), 1% burdock root fermented liquid fermented by Pseudoalteromonas and Schizosaccharomyces pombe showed significantly improved repair ability, with scratch healing rates of 36.3% and 32.5%, respectively. This shows that burdock root fermented liquid has a significant healing and repair function for HaCaT cells, further confirming its excellent potential as a natural skin care raw material in the field of cell proliferation and skin damage repair.

[0103] 6. Chicken embryo stimulation experiment

[0104] This test uses the characteristics of intact, clear and transparent vascular system of the chorioallantoic membrane of mid-stage incubated chicken embryos. A certain amount of the test substance is directly exposed to the chorioallantoic membrane of the chicken embryo. After a period of exposure, the changes in the toxic effect indicators of the chorioallantoic membrane (such as bleeding, vascular melting and coagulation) are observed, and the degree of toxic effect is scored. The higher the score, the stronger the irritation.

[0105] Reaction time method: Take 0.3 mL of the burdock root fermentation liquid of Example 1 and Example 2 with a volume fraction of 1% and directly drip it on the surface of the chicken embryo membrane, observe the reaction, and record the time when the toxic effect appears within 5 minutes. A 1wt% sodium dodecyl sulfate (SDS) aqueous solution is used as the positive control group. The following formula is used to calculate the irritation score (IS), and the result is rounded to two decimal places:

[0106] ;

[0107] Wherein, SecH is the bleeding time, in seconds (s); SecL is the vascular melting time, in seconds (s); SecC is the coagulation time, in seconds (s).

[0108] The irritation rating standard is: IS <1, no irritation; 1≤IS <5, mild irritation; 5≤IS <9, moderate irritation; IS≥10, strong irritation / corrosiveness.

[0109] The experimental results are shown in Table 1 and Figure 8As shown, it can be seen that in the chicken embryo stimulation experiment with burdock root fermented liquid as the test substance, the chicken embryos in each experimental group showed a good growth state and did not show any signs of abnormal stimulation reaction, which strongly proved that burdock root fermented liquid is safe and non-toxic.

[0110] Table 1 Test results of stimulation scoring method

[0111]

[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of burdock root fermented liquid in cosmetics, characterized in that: The volume fraction of the burdock root fermented liquid in the cosmetic is 1-10%, and the cosmetic has antioxidant, anti-allergic, soothing and firming effects; The preparation method of the burdock root fermented liquid comprises: inoculating Pseudoalteromonas or Schizosaccharomyces pombe into a culture medium for activation to obtain a seed solution; The seed liquid is inoculated into a fermentation medium, and burdock root powder is added, wherein the concentration of the burdock root powder added to the fermentation medium is 45-55 g / L, and then fermented to an end point, and post-processed to obtain a burdock root fermentation liquid; The inoculation amount of the seed liquid in the fermentation medium is 0.8-1.2 vol%; When the seed solution is obtained by activating Pseudoalteromonas, the fermentation to the end point is specifically as follows: fermenting at 13-17° C. and 150-200 rpm for 18-48 hours; When the seed liquid is obtained by activating Schizosaccharomyces pombe, the fermentation to the end point is specifically as follows: fermenting at 28-32° C. and 150-200 rpm for 18-48 hours; Wherein, the pseudoalteromonas is named as pseudoalteromonas ( Pseudoalteromonas sp. )SE7, which was deposited in China Center for Type Culture Collection on December 13, 2024, with a deposit number of CCTCC NO: M 20242812; the pombe Schizosaccharomyces cerevisiae was named as Pombe Schizosaccharomyces cerevisiae ( Schizosaccharomyces pombe ) JM2-16, which was deposited in the China Center for Type Culture Collection on December 13, 2024, with the deposit number CCTCC NO: M 20242810.

2. The use according to claim 1, characterized in that The activation method of the Pseudoalteromonas is specifically as follows: inoculating the Pseudoalteromonas into a sea salt culture medium at an inoculum amount of 0.8-1.2 vol%, and culturing for 10-15 hours at 13-17° C. and 150-200 rpm; The activation method of the Schizosaccharomyces pombe is specifically as follows: the Schizosaccharomyces pombe is inoculated into a yeast extract peptone glucose medium at an inoculation rate of 0.8-1.2 vol%, and cultured at 28-32° C. and 150-200 rpm for 10-15 hours.

3. The use according to claim 2, characterized in that The sea salt culture medium comprises: 2-4 g / L yeast powder, 4-6 g / L peptone, 4-6 g / L sea salt, the solvent is water, and the pH is 7.5-8.0; The yeast extract peptone glucose culture medium comprises: 2wt% glucose, 1-2wt% peptone, 0.5-1wt% yeast extract, water as solvent, and pH value of 6.0-6.

5.

4. The use according to claim 1, characterized in that When the seed solution is obtained by activating Pseudoalteromonas, the fermentation medium includes 2-4 g / L yeast powder, 4-6 g / L peptone, 4-6 g / L sea salt, the solvent is water, and the pH is 7.5-8.0; When the seed liquid is obtained by activating Schizosaccharomyces pombe, the fermentation medium includes 2wt% glucose, 1-2wt% peptone, 0.5-1wt% yeast extract, the solvent is water, and the pH is 6.0-6.

5.

5. The use according to claim 1, characterized in that The post-treatment includes centrifugation, sterilization, decolorization and filtration.

6. The use according to claim 5, characterized in that The centrifugation step is specifically: centrifugation at 10000-12000 r / min for 15-25 min; the sterilization step is specifically: sterilization at 120-125°C for 20-40 min; the decolorization step uses activated carbon for decolorization; the filtration step uses a filter membrane with a pore size of 0.1-0.4 μm.

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