Black rose fermentation liquor for repairing skin barrier as well as preparation method and application of black rose fermentation liquor

Abstract

The invention belongs to the technical field of cosmetics, particularly relates to the field of A61K8 / 9789, and more particularly relates to black rose fermentation liquor for repairing a skin barrier as well as a preparation method and application of the black rose fermentation liquor. The method comprises the following steps: S1, taking freshly picked roses, putting into pure water, heating and extracting, filtering and sterilizing after the solid content reaches the standard, and sterilizing to obtain a fermentation substrate; s2, inoculating bacillus into an MRS liquid culture medium, and performing shake cultivation to obtain a seed culture solution; s3, adding the seed culture solution into the black rose fermentation substrate according to the inoculum size of 1-5wt%, and fermenting to obtain an initial fermentation solution; s4, sequentially performing sterilization, wall-breaking treatment and filtration on the initial fermentation liquor, and collecting filtrate to obtain the feed additive. A fermentation substrate containing black roses is used, bacillus is used as a fermentation strain for fermentation, and the obtained fermentation liquor can improve the skin moisture retention degree, strengthen the skin barrier function and inhibit damage of inflammation to the skin barrier, and can be applied to cosmetics as a functional raw material.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, in particular to the field of A61K8 / 9789, and more specifically relates to a black rose fermentation broth for repairing skin barrier, its preparation method and application. Background Art

[0002] The "Guidelines for the Diagnosis and Treatment of Sensitive Skin in China" defines sensitive skin (SS) as: specifically a hyper-responsive state that occurs under physiological or pathological conditions of the skin, mainly occurring on the face, and manifested as subjective symptoms such as burning, stinging, itching and tightness when the skin is stimulated by physical, chemical, mental and other factors, with or without objective signs such as erythema, scales, telangiectasia, etc.

[0003] Sensitive skin has a relatively high incidence rate in various countries around the world. Due to different investigation methods, the reported incidence rates vary greatly in different regions, ranging from 25.4% to 89.9% in Europe and 50% in Australia. Females are generally higher than males. The incidence rate of American females is 22.3% - 50.9%, that of Asian females is 40% - 55.98%, and that of Chinese females is 36.1%. In recent years, the research on the repair of skin barrier has attracted more and more attention.

[0004] In China, the history of rose cultivation can be traced back to more than 2,000 years ago in the Han Dynasty. It is said in "Miscellaneous Records of the Western Capital" that there were "rose trees" planted in the Leyou Garden of Emperor Wu of the Han Dynasty. In Pingyin, the hometown of roses in China, there has been a legend that monks in Cuiping Mountain planted roses as early as the Tang Dynasty. At the same time, roses have high medicinal value and economic value. Research shows that the nutritional components contained in rose flowers are: flavonoids, organic acids, phenols, tannins, alkaloids, sugars, amino acids, proteins and various mineral elements. According to analysis and determination, roses contain 1.2% soluble sugar and 68% carbohydrates. The black rose in the Netherlands, also known as "Zhu Mo Shuang Hui", is one of the edible rose varieties with a long history in Yunnan Province. Its flower shape is dark red with black, and it has a high content of melanin itself, with a velvet-like sense of hierarchy and a strong and authentic fragrance. It is expected that its active substances can be used as active efficacy components of cosmetics after further extraction and fermentation.

[0005] The prior art CN111904878A discloses a preparation method and application of a liposome containing rose fermentation broth: the preparation steps of the rose fermentation broth are as follows: dry rose flower buds are crushed and sieved, and then mixed with water to form a mixed solution, sterilized, then the strain is activated, mixed fermentation is carried out, and post-treatment is carried out to obtain the rose fermentation broth. However, the selection of raw materials above is not detailed enough, only dry rose flower buds are mentioned, and there is no further limitation. Flower buds in different growth cycles may have a greater impact on the active efficacy of the rose fermentation broth. In addition, the prior art does not explain in detail the specific micro-mechanism of the beauty and skin care effect of the rose fermentation broth. Summary of the Invention

[0006] The object of the present invention is to provide a black rose fermentation broth for repairing skin barrier, its preparation method and application. The method of the present invention uses a fermentation matrix containing matcha powder and ferments with lactic acid bacteria as the fermentation strain. The obtained fermentation broth can synergistically enhance the effect from three dimensions, giving all-round prevention and repair to the skin barrier, and can be added to cosmetics as an effective ingredient.

[0007] The present application is achieved through the following technical solutions:

[0008] In order to achieve the above object, the present application provides a preparation method of a black rose fermentation broth for repairing skin barrier in the first aspect, including the following steps:

[0009] S1. Take freshly picked black roses, put them into pure water for heating extraction, filter and sterilize after the solid content reaches the standard, and obtain a rose fermentation matrix after sterilization;

[0010] S2. Inoculate Bacillus in MRS liquid medium and culture it in a shaking table to obtain a seed culture solution;

[0011] S3. Add the seed culture solution to the rose fermentation matrix with an inoculation amount of 1-5 wt%, and ferment to obtain an initial fermentation broth;

[0012] S4. Sterilize the initial fermentation broth in sequence, perform cell wall breaking treatment, filter, and collect the filtrate to obtain a black rose fermentation broth for repairing skin barrier.

[0013] Preferably, the black roses in step S1 are fresh rose buds with the calyx open and the outermost petals of the flower just turning outwards.

[0014] Preferably, in step S1, the heating temperature is 60-100 °C.

[0015] Preferably, in step S1, the solid content requirement is 1.5-2%.

[0016] Preferably, in step S1, the sterilization cycle used is 20-60 min, and the temperature is 80-125 °C.

[0017] Preferably, in step S2, the inoculation amount of Bacillus is 1-5 wt%, the shaking table rotation speed is 100-200 rpm, the culture temperature is 35-45 °C, and the culture time is 24-72 h.

[0018] Preferably, in step S3, the fermentation temperature is 35-45 °C, and the fermentation time is 48-96 h.

[0019] Preferably, in the step S4, the sterilization cycle is 20 - 60 min, and the sterilization temperature is 80 - 125 °C.

[0020] Preferably, in the step S4, the cell wall breaking treatment is carried out by using a high-pressure homogenizer at a temperature of 70 - 90 °C, a pressure of 400 - 600 bar, for 4 - 6 min of cell wall breaking and 4 - 6 min of gap, and repeated 3 - 5 times.

[0021] The second aspect of the present invention provides a black rose fermentation broth prepared by the above preparation method.

[0022] The third aspect of the present invention provides a cosmetic comprising the black rose fermentation broth, and the cosmetic includes but is not limited to aqueous solutions, emulsions, sprays, creams, masks, liquid foundations, and the mass percentage concentration of the black rose fermentation broth in the cosmetic is 0.1 - 5 wt%. Description of the Drawings

[0023] Figure 1 It is a schematic diagram of the test result of the promoting effect on aquaporin AQP3 in Test 2 of the present invention.

[0024] Figure 2 It is a schematic diagram of the test result of the promoting effect on filaggrin gene expression in Test 3 of the present invention.

[0025] Figure 3 It is a schematic diagram of the test result of the promoting effect on transglutaminase TGM-5 in Test 4 of the present invention.

[0026] Figure 4 It is a schematic diagram of the test result of the cell migration rate in Test 5 of the present invention.

[0027] Figure 5 It is a schematic diagram of the test result of the expression of NO in Test 6 of the present invention.

[0028] Figure 6 It is a schematic diagram of the measurement result of the trans-epidermal water loss Tewl value in Test 8 of the present invention.

[0029] Beneficial Effects

[0030] (1) The inventors of the present invention found that the growth cycle stage of the raw materials of the black rose fermentation broth has a great influence on the performance of the product. When the black rose is the fresh black rose bud with the outermost petals just turning outwards, the obtained fermentation broth can effectively improve its performance of repairing the skin barrier on the premise of maintaining high safety. If the fresh black rose bud with the calyx not yet opened and the outermost petals not yet turned outwards is selected as the raw material of the fermentation broth, the content of active ingredients such as polysaccharides, flavonoids, and aromatic oils in its system is relatively low, which will affect the performance of the black rose fermentation broth in repairing the skin barrier in cosmetics.

[0031] (2) In addition to strictly controlling the growth cycle stage of the raw material black rose buds in the present invention, the inventor also found that process parameters such as water extraction temperature, sterilization temperature, fermentation temperature, etc. during the extraction and fermentation process will have a certain impact on the performance of the black rose fermentation broth. If not within the protection scope described in the present invention, some active substances in the black rose may be partially inactivated or denatured, affecting the efficacy of the product in repairing the skin barrier.

[0032] (3) The black rose fermentation broth provided by the present invention improves skin moisture retention by promoting the expression of aquaporin AQP3 and filaggrin FLG.

[0033] (4) The black rose fermentation broth can also strengthen the skin barrier function by promoting the expression of transglutaminase TGM-5 and promoting the migration of damaged human fibroblasts, and inhibit the damage of inflammation to the skin barrier by inhibiting the expression of NO.

[0034] (5) The black rose fermentation broth for repairing the skin barrier obtained in the present invention synergistically enhances from the above three dimensions. When applied in cosmetics, it can provide all-round prevention and repair of the skin barrier for users after use. Specific Embodiments

[0035] Example 1

[0036] In the first aspect of this embodiment, a preparation method of a black rose fermentation broth for repairing the skin barrier is provided, and the following process is specifically adopted for preparation:

[0037] S1. Take the flower buds of fresh black roses with the calyx open and the outermost petals just turning outwards, put them into pure water and heat-extract at 90 °C. After the solid content reaches 1.8%, filter, sterilize at 121 °C for 30 min, and then obtain the fermentation substrate;

[0038] S2. Inoculate Bacillus at an inoculation amount of 2 wt% into MRS liquid medium, and culture it on a shaker at a temperature of 38 °C, a rotation speed of 180 rpm, and a culture time of 48 h to obtain the seed culture solution;

[0039] S3. Add the seed culture solution to the rose fermentation substrate at an inoculation amount of 5 wt%, ferment at a temperature of 40 °C, a shaker rotation speed of 200 rpm, and a fermentation time of 72 h to obtain the initial fermentation broth;

[0040] S4. Sterilize the initial fermentation broth at 121 °C for 40 min, perform cell wall breaking treatment with a high-pressure homogenizer at a temperature of 80 °C, a pressure of 500 bar, break the wall for 5 min, with an interval of 5 min, repeat 4 times, filter, and collect the filtrate to obtain a black rose fermentation broth for repairing the skin barrier.

[0041] In the second aspect of this embodiment, a black rose fermentation broth prepared by a method for preparing a black rose fermentation broth for repairing the skin barrier is provided.

[0042] In the third aspect of this embodiment, a cosmetic product including the black rose fermentation broth is provided. The cosmetic product includes, but is not limited to, aqueous solutions, emulsions, sprays, creams, facial masks, and liquid foundations. The mass percentage concentration of the black rose fermentation broth in the cosmetic product is 0.1 - 5 wt%.

[0043] Comparative Example 1

[0044] In the first aspect of this comparative example, a method for preparing a black rose fermentation broth for repairing the skin barrier is provided. The specific preparation process is as follows:

[0045] S1. Take the flower buds of fresh black roses whose sepals have not opened and the outermost petals of the flowers have not turned outwards, put them into pure water and heat-extract at 90 °C. After the solid content reaches 1.8%, filter, sterilize at 121 °C for 30 min, and then obtain the fermentation substrate.

[0046] S2. Inoculate Bacillus with an inoculation amount of 2 wt% into MRS liquid medium, and culture it on a shaker at a temperature of 38 °C, a rotation speed of 180 rpm, and a culture time of 48 h to obtain the seed culture solution.

[0047] S3. Add the seed culture solution to the rose fermentation substrate with an inoculation amount of 5 wt%, ferment at a temperature of 40 °C, a shaker rotation speed of 200 rpm, and a fermentation time of 72 h to obtain the initial fermentation broth.

[0048] S4. Sterilize the initial fermentation broth at 121 °C for 40 min, perform cell wall breaking treatment using a high-pressure homogenizer at a temperature of 80 °C, a pressure of 500 bar, break the cells for 5 min, with an interval of 5 min, repeat 4 times, filter, and collect the filtrate to obtain a black rose fermentation broth for repairing the skin barrier.

[0049] Comparative Example 2

[0050] In the first aspect of this comparative example, a method for preparing a black rose fermentation broth for repairing the skin barrier is provided. The specific preparation process is as follows:

[0051] S1. Take the flower buds of fresh black roses whose sepals have opened and the outermost petals of the flowers have just turned outwards, put them into pure water and heat-extract at 100 °C. After the solid content reaches 1.6%, filter, sterilize at 121 °C for 30 min, and then obtain the fermentation substrate.

[0052] S2. Inoculate Bacillus with an inoculation amount of 1 wt% into MRS liquid medium, and culture it on a shaker at a temperature of 38 °C, a rotation speed of 180 rpm, and a culture time of 48 h to obtain the seed culture solution.

[0053] S3. Add the seed culture solution to the rose fermentation substrate at an inoculation amount of 0.5 wt%, ferment at a temperature of 40 °C, a shaker speed of 200 rpm, and a fermentation time of 72 h to obtain the initial fermentation broth.

[0054] S4. Sterilize the initial fermentation broth at 121 °C for 40 min, perform cell wall breaking treatment using a high-pressure homogenizer at a temperature of 100 °C, a pressure of 600 bar, break the wall for 5 min, with an interval of 5 min, repeat 5 times, filter, and collect the filtrate to obtain a black rose fermentation broth for repairing the skin barrier.

[0055] Comparative Example 3

[0056] This comparative example provides a method for preparing a black rose fermentation broth for repairing the skin barrier, which is specifically prepared by the following process:

[0057] S1. Take the flower buds of fresh black roses with the calyx not yet opened and the outermost petals of the flower not yet turned outwards, put them into pure water and heat-extract at 70 °C. After the solid content reaches 1.6%, filter, sterilize at 121 °C for 30 min, and then obtain the fermentation substrate.

[0058] S2. Inoculate Bacillus spores into MRS liquid medium at an inoculation amount of 2 wt%, culture on a shaker at a temperature of 38 °C, a rotation speed of 180 rpm, and a culture time of 48 h to obtain the seed culture solution.

[0059] S3. Add the seed culture solution to the rose fermentation substrate at an inoculation amount of 6 wt%, ferment at a temperature of 40 °C, a shaker speed of 200 rpm, and a fermentation time of 72 h to obtain the initial fermentation broth.

[0060] S4. Sterilize the initial fermentation broth at 121 °C for 40 min, perform cell wall breaking treatment using a high-pressure homogenizer at a temperature of 90 °C, a pressure of 600 bar, break the wall for 5 min, with an interval of 5 min, repeat 5 times, filter, and collect the filtrate to obtain a black rose fermentation broth for repairing the skin barrier.

[0061] Performance Test

[0062] Test 1. Cytotoxicity Evaluation Test

[0063] MTT is a yellow powdery chemical reagent widely used in the detection of cytotoxicity or cell proliferation. The detection principle is that succinate dehydrogenase in the mitochondria of living cells can reduce MTT to water-insoluble blue-violet crystalline formazan and deposit in the cells, while dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve formazan in the cells. The number of living cells is judged according to the measured absorbance value. The specific experimental method is as follows:

[0064] In a 96-well plate, 100 μL each of DMEM medium containing 10% bovine serum and human immortalized epidermal cells (HaCaT) were inoculated at a density of 1×10 4 cells per well, and after culturing for 24 hours, the medium was replaced with serum-free medium; the moisturizing anti-aging compositions prepared in Example 1 and Comparative Examples 1-3 above were added to the serum-free medium respectively, and after treatment, the cells were cultured for 24 hours; the medium was removed, and the cells were treated with 20 μL of MTT solution (the solvent was phosphate buffer solution, with a concentration of 5 mg / mL), and cultured for 4 hours; 150 μL of DMSO was added to the cells from which the MTT solution had been removed, and the reaction was carried out at 37 °C in a water bath for 4 h until the crystalline formazan was completely dissolved; the absorbance was measured at 570 nm, and the cell survival rate was calculated according to the following formula.

[0065] Cell survival rate (%) = absorbance value of the sample / average absorbance of the control group × 100%

[0066] For the control group, the test was carried out without adding the sample. The results related to cytotoxicity are shown in Table 1:

[0067] Table 1. Cell survival rate

[0068]

[0069] The data results in Table 2 show that Example 1 and Comparative Examples 1-3 have no obvious cytotoxicity.

[0070] Test 2. Determination of promoting aquaporin AQP3

[0071] Cells were inoculated into a 96-well plate at a density of 3×10 5 / mL, 100 μL per well, and the inoculated cell culture plate was placed in an incubator and cultured for another 24 h (5% CO2, 37 °C). After culturing the cells for 24 h, 100 μl of test substances and retinoic acid solutions with different concentrations were added to each well respectively, mixed evenly, and placed in the incubator for treatment for 24 h ± 1 h. RNA was extracted using a cell-to-reverse transcription one-step reagent. cDNA was synthesized using a cDNA synthesis kit. BeyoFast TM SYBR Green qPCR Mix (2X, Low ROX) was used for RT-PCR detection. GraphPad Prism was used for plotting, and the results were expressed as Mean ± SD. The results are as Figure 1 shown.

[0072] It can be Figure 1 seen that: the promoting effect of the fermentation broth obtained in Example 1 on aquaporin AQP3 is significantly higher than that of other comparative examples, and the order is Example 1 > Comparative Example 1 > Comparative Example 2 > Comparative Example 3.

[0073] Test 3: Determination of promoting filaggrin gene expression

[0074] For HEKa cells of normal human skin, use Epilife containing 1% HKGS (human keratinocyte growth supplement) TM medium (containing PSA solution: penicillin, streptomycin and amphotericin B), and culture routinely in an incubator at 37°C and 5% CO2. When the cells grow to 80% confluence, digest them with Trypsin / EDTA (trypsin), and passage them at a density of about 2.5×10 3 cells / cm 2 to 25 cm 2 culture flasks, and passage them once every 3 - 5 days. Take cells in the logarithmic growth phase and digest them routinely, adjust the concentration of HEKa cells to 5×10 4 cells / mL, take 100 μL and inoculate it into a 96-well plate. When it grows to near confluence, change the medium in the 96-well plate to Epilife TM medium, and continue to culture for 24 h. Then add 100 μL of the prepared EpilifeTM medium mixed solution that meets the requirements in Table 1 to each well. Set up a negative control group (cell and medium group), with 5 replicates for each sample, place it in the incubator for 24 h, and collect the supernatant of HEKa cells treated with different concentrations of the composition respectively for ELISA determination.

[0075] Use the ELISA method to determine the synthesis of HA in HEKa cells, and operate according to the instructions. The steps are as follows:

[0076] S1. Dilute the prepared cell supernatant 10-fold with the buffer (reaction buffer) in the kit;

[0077] S2. Add 100 μL of the diluted sample solution to the appropriate wells, and incubate at room temperature for 60 min;

[0078] S3. Discard the liquid in the wells, wash the plate 4 times with PBS working solution, and suck off the residual liquid with a filter paper;

[0079] S4. Add 100 μL of horseradish peroxidase-conjugated hyaluronic acid binding protein (HRP-conjugated HABP) solution to each well, and incubate at room temperature for 30 min;

[0080] S5. Discard the liquid in the wells, wash the plate 4 times with PBS working solution, and suck off the residual liquid with a filter paper;

[0081] S6. Add 100 μL of substrate solution (containing 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide) to each well, and incubate at room temperature for 30 min;

[0082] S7. Add 100 μL of stop solution (0.36 N sulfuric acid) to terminate the enzyme reaction. The order and rate of adding the stop solution are the same as those of the substrate solution;

[0083] S8. Measure the OD value of each well at 450 nm.

[0084] The results are shown in Figure 2 , Figure 2 indicating the effect of the composition on hyaluronic acid synthesis in HEKa cells, where n = 5, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

[0085] It can be seen from Figure 2 that the fermentation broth obtained in Example 1 has a significantly higher promoting effect on filaggrin gene expression than other comparative examples, and the order is Example 1 > PC > Comparative Example 1 > Comparative Example 2 > Comparative Example 3.

[0086] Test Four. Determination of promoting transglutaminase TGM-5 gene expression

[0087] Inoculate cells into a 96-well plate at a density of 3×10 5 / mL, 100 μl per well, and place the inoculated cell culture plate in an incubator for continued culture for 24 h (5% CO2, 37 °C). After 24 h of cell culture, add 100 μl of test substances and salicylic acid solution with different concentrations to each well, mix well, and place in the incubator for treatment for 24 h ± 1 h. Use a cell-to-reverse transcription one-step reagent to extract RNA. Use a cDNA synthesis kit to synthesize cDNA. Use BeyoFast TM SYBR Green qPCRMix (2X, Low ROX) for RT-PCR detection. Use GraphPad Prism to plot the graph, and the results are expressed as Mean ± SD. The results are as shown in Figure 3 shown.

[0088] It can be seen from Figure 3 that the fermentation broth obtained in Example 1 has a significantly higher promoting effect on transglutaminase TGM-5 than other comparative examples, and the order is Example 1 > Comparative Example 1 > Comparative Example 2 > Comparative Example 3.

[0089] Test Five. Determination of promoting cell migration rate

[0090] (1) Sample concentration selection: At a cell concentration of 8×10 4Cells were inoculated into 96-well plates at a density of

[0091] (2) Plating: Select P7 human fibroblasts with good growth status and inoculate the cells into 60 mm culture dishes at a density of 1×10 6 / 3 mL, 3 mL per well. Place the inoculated cell culture dishes in the incubator and continue culturing for 24 h (5% CO2, 37 °C).

[0092] (3) Sample addition: After the plating rate reaches 90%, sample addition can be carried out. Aspirate the original culture medium and add the prepared test substances according to the grouping. After sample addition, place the culture dishes in the incubator and continue culturing for 24 h (5% CO2, 37 °C).

[0093] (4) Scratching: When the cell plating rate reaches about 90%, use a 200 μL pipette tip to damage the cells and make scratches. During the scratching process, the pipette tip is slightly inclined to the plane and evenly scrape across the bottom of the plate with force.

[0094] (5) Photographing: Immediately take a 0 h photograph after scratching to record the size of the original scratch and record the observation point numbers (numbered from top to bottom). Take 4 or more photos for each well. Mark the observation points. After photographing, aspirate the PBS and add serum-free medium to each well, then place it back in the incubator and continue incubating for about 24 h. After incubation, take a 24 h photograph at the same position using the same magnification objective lens as the 0 h photographing to record the scratch repair situation.

[0095] (6) Calculation of scratch area: Use ImageJ software to calculate the scratch area. Process the pictures according to the fixed command and calculate the scratch area. The results are as Figure 4 shown.

[0096] It can be Figure 4 seen that the promoting effect of the fermentation broth obtained in Example 1 on cell migration rate is significantly higher than that of other comparative examples, and the order is Example 1 > Comparative Example 1 > Comparative Example 2 > Comparative Example 3.

[0097] Test Six: Determination of NO Expression

[0098] Macrophages (Raw 264.7) were inoculated and cultured on 24-well plates. When performing the anti-inflammatory experiment, lipopolysaccharide (LPS) and the test sample were treated in fresh medium and then cultured in a microbial incubator at 37 °C and 5% CO2. After the culture ended, the culture medium of the cells was recovered and transferred to a 96-well plate. Griess' reagent was added for reaction, and the absorbance was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The percentage of absorbance was reflected after comparison with the lipopolysaccharide that inhibited NO production. On the well plate from which the culture medium was removed, a culture medium containing MTT solution was placed for reaction. After removing the supernatant, dimethyl sulfoxide (DMSO) was added to dissolve the formed MTT formazan crystals, and then the absorbance was measured using an ELISA reader. When performing the immune enhancement experiment, the test sample was directly treated alone without lipopolysaccharide treatment, and then the same experimental procedure was carried out. The results are as Figure 5 shown.

[0099] It can be Figure 5 seen that the promoting effect of the fermentation broth obtained in Example 1 on cell migration rate was significantly higher than that of other comparative examples, and the order was Example 1 > Comparative Example 1 > Comparative Example 2 > Comparative Example 3.

[0100] Test Seven: Safety Test

[0101] 20 μL of the test solution containing the composition was added dropwise to the patch test device, and the control well was a blank control (pure water); the patch test device with the test substance added was attached to the flexor side of the forearm of the test subject, and gently pressed with the palm to make it evenly adhere to the skin for 24 hours; the skin irritation and sensitization were observed according to Table 4 at 30 min, 24 hours, and 48 hours after removing the patch test device with the test substance, and the observation results were recorded. The skin reaction grading criteria for the skin closed patch test are shown in Table 2.

[0102] Table 2 Skin Reaction Grading Criteria for Skin Closed Patch Test

[0103]

[0104] Experimental results: The compositions obtained in Example 1 and Comparative Examples 1, 2, and 3 were subjected to human skin patch tests, and the results are shown in Table 3. The results of the human skin patch test showed that among 30 people, there were no skin adverse reactions, and Example 1 and Comparative Examples 1, 2, and 3 were all safe and non-irritating.

[0105] Table 3

[0106]

[0107]

[0108] Test Eight: Human Test (Results of Transdermal Water Loss Measurement)

[0109] Thirty-three healthy volunteers aged 18 - 45 years were selected. An emulsion containing 2% (by mass) of the fermentation broth obtained in Example 1 was used and applied to the entire face every day, once in the morning and once in the evening, about the size of 3 - 4 soybeans each time, and used continuously for 4 weeks. The transdermal water loss value of the face was measured using a Tewameter, and the results are as Figure 6 shown.

Claims

1. A preparation method of a black rose fermentation liquid for repairing skin barrier, characterized in that, The preparation method described above includes the following steps: S1. Take freshly picked black roses, put them into pure water for heating extraction, filter and sterilize after the solid content reaches the standard, and obtain a rose fermentation matrix after sterilization; S2. Inoculate Bacillus in MRS liquid medium and culture it on a shaker to obtain a seed culture solution; S3. Add the seed culture solution to the rose fermentation matrix at an inoculation amount of 1-5 wt%, and ferment to obtain an initial fermentation broth; S4. Sterilize the initial fermentation broth successively, perform cell wall breaking treatment, filter, and collect the filtrate to obtain a black rose fermentation broth for repairing the skin barrier.

2. The preparation method of the black rose fermentation liquid for repairing skin barrier according to claim 1, wherein: The black roses in step S1 are fresh rose buds with the calyx open and the outermost petals of the flower just turning outwards.

3. The preparation method of the black rose fermentation liquid for repairing the skin barrier according to claim 2, characterized in that: In step S1, the heating temperature is 60-100 °C, the required solid content is 1.5-2 wt%, the sterilization cycle is 20-60 min, and the temperature is 80-125 °C.

4. The preparation method of the black rose fermentation liquid for repairing the skin barrier according to claim 1, wherein: In step S2, the inoculation amount of Bacillus is 1-5 wt%, the shaker rotation speed is 100-200 rpm, the culture temperature is 35-45 °C, and the culture time is 24-72 h.

5. The preparation method of the black rose fermentation liquid for repairing the skin barrier according to claim 1, characterized in that: In step S3, the fermentation temperature is 35-45 °C, and the fermentation time is 48-96 h.

6. The preparation method of the black rose fermentation broth for repairing the skin barrier according to claim 1, wherein: In step S4, the cell wall breaking treatment is carried out using a high-pressure homogenizer, the temperature is 70-90 °C, the pressure is 400-600 bar, the cell wall breaking time is 4-6 min, the gap time is 4-6 min, repeated 3-5 times, the sterilization cycle is 20-60 min, and the temperature is 80-125 °C.

7. The preparation method of the black rose fermentation broth for repairing the skin barrier according to claim 6, wherein: In step S4, the sterilization cycle is 20-60 min, and the temperature is 80-125 °C.

8. A black rose fermentation broth for repairing the skin barrier prepared by the preparation method of the black rose fermentation broth for repairing the skin barrier according to any one of claims 1-7.

9. Use of the black rose fermentation broth for repairing skin barrier according to claim 8, characterized in that, The black rose fermentation broth for repairing the skin barrier is applied in cosmetics, and the cosmetics include but are not limited to aqueous solutions, emulsions, sprays, creams, masks, and liquid foundations.

10. Use of the black rose fermentation liquid for repairing skin barrier according to claim 9, characterized in that, The mass percentage concentration of the black rose fermentation broth for repairing the skin barrier in the cosmetics is 0.1-5 wt%.

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