Preparation method and application of a composition for repairing sensitive skin

Abstract

This invention belongs to the field of skincare technology, specifically relating to a method for preparing and applying a composition for sensitive skin repair. The invention employs a segmented fermentation method using Bletilla striata root extract as the carbon source. The composition undergoes sequential low-temperature fermentation with Bifidobacterium and high-temperature fermentation with Thermophilic Bacillus, followed by compounding with excipients to obtain a sensitive skin repair composition. This overcomes the shortcomings of Bifida Ferment Lysate, such as poor stability, low permeability, and strong irritation. The dual fermentation achieves a stepwise degradation of macromolecular polysaccharides, improving product permeability and stability; high-temperature fermentation inactivates unwanted bacteria and consumes residual organic acids, reducing irritation. This composition, used in water-based and serum-type skincare products, exhibits excellent stability, is gentle and non-irritating, and provides long-lasting moisturizing and antioxidant properties, offering an effective solution for sensitive skin repair.

Description

Technical Field

[0001] This invention belongs to the field of skincare technology, specifically relating to a method for preparing a composition for repairing sensitive skin and its application in the preparation of skincare products for repairing sensitive skin. Background Technology

[0002] Sensitive skin, also known as "sensitive skin," is a condition characterized by high skin intolerance. Subjective symptoms include burning, stinging, itching, and tightness when exposed to physical, chemical, or psychological stimuli, often accompanied by objective signs such as erythema, peeling, and telangiectasia. The root cause lies in impaired skin barrier function, leading to a decreased ability of the skin to defend against external stimuli, while the skin's internal neuroimmune system is in a state of irritation. With the accelerated pace of modern life, increased environmental pollution, and the prevalence of improper skincare habits, the proportion of people with sensitive skin worldwide is rising annually. Therefore, developing safe and effective sensitive skin repair products has become a crucial demand in the skincare market.

[0003] Currently, there are many types of products on the market for repairing sensitive skin. Their mechanisms of action mainly focus on the following aspects: (1) Replenishing intercellular lipids and repairing the physical barrier: such as adding ingredients such as ceramides, cholesterol, and free fatty acids. (2) Increasing skin hydration and reducing transepidermal water loss (TEWL): such as using moisturizers such as hyaluronic acid, glycerin, and butylene glycol. (3) Soothing and anti-inflammatory, inhibiting inflammatory reactions: such as using bisabolol, dipotassium glycyrrhizate, and various plant extracts (such as purslane extract and centella asiatica extract). Well-known commercial products such as Winona Soothing and Moisturizing Special Care Cream utilize the soothing and repairing effects of plant extracts such as purslane and sea buckthorn oil as their core ingredients.

[0004] However, existing technologies for sensitive skin repair still have the following technical problems: (1) Single function: Many products focus only on moisturizing or single anti-inflammatory effects, failing to address the complex issues of skin barrier damage, microecological imbalance, and chronic inflammation at the root. Sensitive skin is caused by multiple factors, and single-pathway repair has limited effect and is prone to recurrence. (2) Difficulty in achieving both immediate relief and long-term repair: Some products can quickly relieve symptoms such as redness and itching, but they are insufficient for deep repair and reconstruction of the skin barrier; while other products that focus on long-term repair do not provide satisfactory relief during acute sensitive periods. (3) Insufficient synergy of ingredients: Simple ingredient layering may not produce a 1+1>2 effect, and may even increase the burden on the skin or cause new irritation due to improper formulation. For example, some products containing fermented ingredients may not be suitable for severely sensitive skin. (4) Insufficient protection against environmental damage: Sensitive skin has a damaged barrier and is more vulnerable to environmental damage such as ultraviolet and infrared rays, while most repair products on the market fail to provide effective protection against these environmental pressures, thus hindering the repair process.

[0005] Bifida ferment lysate, with its comprehensive effects in repairing the skin barrier, anti-inflammatory and soothing, and promoting DNA repair, has proven its significant application value and advantages in the field of sensitive skin care. However, as a complex biologically derived ingredient, its inherent risk of allergies and individual intolerance are unavoidable drawbacks in its application to sensitive skin. For example, its poor stability makes it susceptible to inactivation due to temperature fluctuations and extreme pH values, leading to a decrease in efficacy within the product's shelf life. Trace amounts of organic acids remaining during fermentation may cause adverse reactions such as itching, redness, stinging, swelling, and even breakouts (papules, pustules). Furthermore, its high water solubility results in a short residence time on the skin surface, making it difficult to effectively penetrate the stratum corneum, leading to low permeability and thus affecting the full extent of its repair effects. Therefore, there is an urgent need to develop a Bifida ferment lysate composition suitable for sensitive skin repair with a gentle formula, optimal performance temperature, and excellent skin feel. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the existing technology, such as poor stability of Bifida ferment lysate in sensitive skin repair, low stratum corneum permeability, and adverse reactions caused by residual organic acids from fermentation, and to provide a method for preparing and applying a composition for sensitive skin repair.

[0007] This invention relates to a method for preparing a composition for repairing sensitive skin, comprising the following steps:

[0008] S1. Mix Bletilla striata root extract, nitrogen source and inorganic salt to obtain fermentation medium;

[0009] S2, First step of fermentation: Sterilize the fermentation medium, adjust the pH to 6.5-7.0, inoculate 3-10% of Bifidobacterium inoculum for fermentation, and obtain the primary fermentation broth of Bifida Ferment Lysate;

[0010] S3. Second fermentation: Heat the primary fermentation broth to 60-80℃, adjust the pH to 7.0-7.5, and inoculate with 0.5-2% of thermophilic bacteria to ferment and obtain the secondary fermentation broth of Bifida ferment lysate.

[0011] S4. Post-processing: The secondary fermentation broth is homogenized and crushed under high pressure at 80-100MPa, filtered, sterilized, and the filtrate is collected to obtain the final fermentation broth of Bifida ferment lysate.

[0012] S5. Mix the final fermentation broth of Bifida Ferment Lysate, 1,2-pentanediol, octanoyl hydroxamic acid, 1,3-propanediol, penetration enhancer, and surfactant to obtain the sensitive skin repair composition.

[0013] Preferably, the preparation of Bletilla striata root extract in step S1 includes the following steps: take dried Bletilla striata root, crush it through a 30-80 mesh sieve, add 10-20 times the mass of deionized water, reflux extract at 80-90℃ for 2-5 hours, filter and concentrate the filtrate to a solid content of 5-15wt% to obtain Bletilla striata root extract.

[0014] Preferably, the culture medium contains 97-99.4 wt% Bletilla striata root extract, 0.5-2 wt% nitrogen source and 0.1-1 wt% inorganic salts.

[0015] Preferably, the nitrogen source in step S1 includes at least one of soybean peptone and tryptone; the inorganic salt includes at least one of potassium dihydrogen phosphate, magnesium sulfate, and zinc sulfate.

[0016] Preferably, the solid content of the Bletilla striata root extract in step S1 is 5-10 wt%.

[0017] Preferably, the fermentation temperature in step S2 is 30-40℃, and the fermentation time is 24-72 hours; the concentration of Bifidobacterium is 1×10⁻⁶. 8 ~1×10 10 CFU / mL.

[0018] Preferably, the fermentation temperature in step S3 is 50-60℃, and the fermentation time is 24-48 hours; the concentration of the thermophilic bacteria strain is 1×10⁻⁶. 7 ~1×10 8 CFU / mL.

[0019] Preferably, the filtration is performed using a polypropylene microfiltration membrane with a diameter of 1.5 μm to 3.5 μm.

[0020] Preferably, the sterilization in step S4 is low-temperature pasteurization, with a sterilization temperature of 80-100°C and a time of 30-60 minutes.

[0021] Preferably, the sensitive skin repair composition in step S5 includes 80-100 parts of final fermentation broth, 3-10 parts of 1,2-pentanediol, 0.01-0.2 parts of capryloyl hydroxamic acid, 3-7 parts of 1,3-propanediol, 0-10 parts of penetration enhancer, and 1-5 parts of surfactant.

[0022] Preferably, the penetration enhancer is at least one of squalane and ceramide, and more preferably, the penetration enhancer is a mixture of squalane and ceramide in a mass ratio of 1-5:1.

[0023] Preferably, according to some embodiments of the present invention, the surfactant is at least one selected from PEG-40 hydrogenated castor oil, Tween-20, Tween-60, and PEG-100 stearate.

[0024] The present invention also provides a composition for repairing sensitive skin, prepared according to the method described above.

[0025] Preferably, the present invention also provides the use of the composition for sensitive skin repair in the preparation of skin care products.

[0026] Preferably, the present invention also provides a skin care product containing the composition for repairing sensitive skin as described above.

[0027] Preferably, according to some embodiments of the present invention, the skin care product further includes excipients acceptable for skin care products.

[0028] Preferably, according to some embodiments of the present invention, the excipients include at least one of chelating agents, humectants, thickeners, emulsifiers, and flavor modifiers.

[0029] Preferably, according to some embodiments of the present invention, the excipients, by weight percentage, include at least one of the following: 0.05-0.5% chelating agent, 1-10% humectant, 0.5-2% thickener, 1-5% emulsifier, and 0.1-0.5% flavor modifier.

[0030] Preferably, the chelating agent is selected from at least one of disodium ethylenediaminetetraacetate, trisodium ethylenediaminetetraacetate, tetrasodium ethylenediaminetetraacetate, trisodium ethylenediaminedisuccinate, and sodium citrate.

[0031] Preferably, the moisturizer includes at least one of glycerin, sodium hyaluronate, niacinamide, butylene glycol, and sorbitol.

[0032] Preferably, the thickener is at least one of sodium polyacrylate, carboxymethyl cellulose, hydroxyethyl cellulose, and xanthan gum.

[0033] Preferably, the emulsifier is at least one of cocamidopropyl betaine, polysorbate-20, polysorbate-60, polyglycerol-10 stearate, and potassium lauryl phosphate.

[0034] Preferably, the fragrance modifier is selected from one or a combination of menthol, rose essential oil, jasmine essential oil, carnation essential oil, gardenia essential oil, and violet essential oil.

[0035] Preferably, according to some embodiments of the present invention, the amount of the composition for repairing sensitive skin added to the skin care product is 0.01 to 2 wt%.

[0036] Preferably, according to some embodiments of the present invention, the skin care product is one of the following: aqueous solution, serum, essence lotion, facial mask liquid, and cream.

[0037] The core component of Bletilla striata root extract is Bletilla striata polysaccharide. This invention uses Bletilla striata root extract as a unique carbon source and utilizes Bifidobacterium for the first stage of low-temperature fermentation; subsequently, it conducts the second stage of high-temperature fermentation by raising the temperature and introducing Thermophilic bacteria. Compared with the traditional Bifidobacterium fermentation product lysate (Bifida ferment lysate), this invention solves the key problems of poor stability, low permeability and strong irritation of Bifida ferment lysate: (1) The dual fermentation gradually decomposes the large molecular polysaccharide in Bletilla striata root extract into small molecular active substances that are easier to penetrate the skin; (2) The high-temperature fermentation stage of Thermophilic bacteria can not only inactivate the Bifidobacterium and harmful microorganisms in the first stage and terminate the continuous generation of organic acids, but also utilize Thermophilic bacteria to metabolize some residual lactic acid, reducing the risk of irritation; (3) Thermophilic bacteria introduced in the second stage can produce heat-stable superoxide dismutase SOD, which can effectively remove free radicals, resist the damage to the skin caused by ultraviolet rays and environmental stress, and give the sensitive skin repair composition stronger antioxidant and repair functions.

[0038] Furthermore, for individuals with sensitive skin, the ceramide content in their skin is often significantly lower than that of healthy skin, leading to impaired barrier function. Supplementing with ceramides can repair the damaged barrier and provide a stable penetration environment for Bifida Ferment Lysate products. The added squalane penetration enhancer is a lipophilic component, similar in structure to the lipids (such as cholesterol and fatty acids) of the stratum corneum. It can quickly integrate into the lipid bilayer of the stratum corneum, reducing the density of lipid molecules and decreasing the physical resistance to the penetration of Bifida Ferment Lysate filtrate (containing water-soluble and lipid-soluble active molecules). This invention uses a combination of squalane and ceramides in a mass ratio of 1-5:1, synergistically promoting the efficiency of various small-molecule active ingredients in Bifida Ferment Lysate penetrating deep into the skin along the concentration gradient, providing an important guarantee for repairing sensitive skin.

[0039] Furthermore, the capryloyl hydroxamic acid added in this invention not only buffers the pH of the system and chelates harmful metal ions to inhibit chemical degradation, but also has antibacterial effects. Together with 1,2-pentanediol, it forms a broad-spectrum microbial defense network, which together maintains the structural integrity and activity of the sensitive skin repair composition under complex environments (such as temperature and pH fluctuations), thereby delaying its inactivation and improving the stability of skin care products.

[0040] The advantages or beneficial effects of the composition for sensitive skin repair of the present invention include at least the following:

[0041] This invention utilizes Bletilla striata root extract as a unique carbon source, employing a dual fermentation process: a first-stage low-temperature fermentation using Bifidobacterium and a second-stage high-temperature fermentation using Thermophilic Bacillus. This dual fermentation process not only progressively degrades the large-molecule polysaccharides in the Bletilla striata root extract into smaller-molecule active substances but also inactivates unwanted bacteria and metabolizes residual organic acids. It reduces LPS-induced pro-inflammatory factor mRNA expression for anti-inflammatory and soothing effects, promotes HaCaT cell migration, and repairs the skin barrier. The product exhibits excellent gentleness, permeability, and stability. In skincare products, only 0.01-2 wt% of this sensitive skin repair composition is needed to achieve long-lasting hydration with a 4-hour moisturizing rate ≥61.8% and a maximum DPPH free radical scavenging rate of 79.3%, effectively overcoming the shortcomings of traditional Bifida ferment lysate fermentation products, such as poor stability, low permeability, and strong irritation. Attached Figure Description

[0042] Figure 1 Example 1 describes the composition for sensitive skin repair and its ability to migrate HaCaT cells.

[0043] Figure 2 This is a microscopic image showing the ability of the composition for sensitive skin repair in Example 1 to migrate HaCaT cells.

[0044] Figure 3 The effect of the composition for sensitive skin repair in Example 1 on the activity of RAW264.7 cells.

[0045] Figure 4 The effect of the composition for sensitive skin repair in Example 1 on the expression of inflammatory factor mRNA in LPS-induced RAW264.7 cells. Detailed Implementation

[0046] To more clearly illustrate the purpose, technical solution, and advantages of this invention, the technical solution of this invention will be described in detail below through specific embodiments. It should be noted that these embodiments are only for illustrating this invention and not for limiting its scope of protection; the actual scope of protection of this invention should be determined by the claims.

[0047] Unless otherwise specified, the materials and reagents used in the following examples and comparative examples are commercially available. Unless otherwise specified, the amount of each component in the following examples is 1 g per part by weight.

[0048] 1. Experimental materials:

[0049] Bifidobacterium, accession number: CGMCC 1.5014, purchased from China General Microbiological Culture Collection Center.

[0050] Thermostats, with accession number CGMCC 1.15099, were purchased from the China General Microbiological Culture Collection Center.

[0051] PEG-40 hydrogenated castor oil: CO40, CAS No. 61788-85-0, purchased from Prui Xing Chemical.

[0052] Squalane: Bestway Biotechnology (Zhejiang) Co., Ltd.

[0053] Ceramides: Shenzhen Dickman Biotechnology Co., Ltd.

[0054] 1,2-Pentanediol; purchased from Symrise Fragrance & Flavor (Nantong) Co., Ltd.

[0055] Capryloyl hydroxamic acid: purchased from Guangdong Hainuo Kewei Biomedical Co., Ltd.

[0056] 1,3-Propanediol: purchased from Shandong Xinheng Chemical Co., Ltd.

[0057] Preparation of Bletilla striata root extract: Take dried Bletilla striata root, crush it through a 40-mesh sieve, weigh 100g, add 10 times the mass of deionized water, reflux at 85℃ for 3h, extract 3 times, filter and concentrate to a solid content of 8wt% to obtain Bletilla striata root extract for later use.

[0058] 2. Examples and Comparative Examples

[0059] Example 1

[0060] S1. Preparation of fermentation medium: Mix 98wt% Bletilla striata root extract, 1.2wt% soybean peptone, and 0.8wt% potassium dihydrogen phosphate by weight percentage, and stir well.

[0061] S2, First step fermentation: Sterilize the culture medium at 121℃ for 20 minutes, cool it, adjust the pH to 6.8, and inoculate with 5% volume of Bifidobacterium inoculum (concentration 5×10⁻⁶). 9 The primary fermentation broth was obtained by fermenting at 37°C for 48 hours (CFU / mL) at 37°C.

[0062] S3, Second Fermentation: Heat the primary fermentation broth to 65℃, adjust the pH to 7.2, and inoculate with 1% volume of thermophilic bacteria (concentration 5×10⁻⁶). 7 The secondary fermentation broth was obtained by fermenting at 50°C for 36 hours (CFU / mL) at 50°C.

[0063] S4. Post-processing: The secondary fermentation broth was homogenized and crushed under high pressure at 90 MPa, filtered through a 2.0 μm polypropylene microfiltration membrane, pasteurized at 90℃ for 40 min, and the filtrate was collected to obtain the final fermentation broth.

[0064] S5. Composition formulation: By weight, take 90 parts of final fermentation broth, 5 parts of 1,2-pentanediol, 0.1 parts of capryloyl hydroxamic acid, 5 parts of 1,3-propanediol, and 3 parts of Tween-60, stir and mix evenly to obtain the sensitive skin repair composition.

[0065] Example 2

[0066] S1. Preparation of fermentation medium: Mix 98.5wt% Bletilla striata root extract, 1wt% tryptone and 0.5wt% magnesium sulfate by weight percentage and stir well.

[0067] S2, First step fermentation: Sterilize the culture medium at 120℃ for 30 minutes, cool it, adjust the pH to 6.5, and inoculate with 3% volume of Bifidobacterium inoculum (concentration 1×10⁻⁶). 8 The primary fermentation broth was obtained by fermenting at 35°C for 72 hours (CFU / mL).

[0068] S3, Second Fermentation: Heat the primary fermentation broth to 75℃, adjust the pH to 7.0, and inoculate with 0.5% volume of thermophilic bacteria (concentration 1×10⁻⁶). 7 The secondary fermentation broth was obtained by fermenting at 60℃ for 48 hours (CFU / mL) with the fermentation concentration of CFU / mL.

[0069] S4. Post-processing: The secondary fermentation broth was homogenized and crushed under high pressure at 80 MPa, filtered through a 1.5 μm polypropylene microfiltration membrane, pasteurized at 85℃ for 60 min, and the filtrate was collected to obtain the final fermentation broth.

[0070] S5. Composition formulation: By weight, take 85 parts of final fermentation broth, 3 parts of 1,2-pentanediol, 0.05 parts of capryloyl hydroxamic acid, 3 parts of 1,3-propanediol, and 2 parts of PEG-40 hydrogenated castor oil, stir and mix evenly to obtain a sensitive skin repair composition.

[0071] Example 3

[0072] S1. Preparation of fermentation medium: Mix 97.5wt% Bletilla striata root extract, 2wt% soybean peptone, and 0.5wt% zinc sulfate by weight percentage, and stir well.

[0073] S2, First Fermentation Step: Sterilize the culture medium at 121℃ for 25 minutes, cool it, adjust the pH to 7.0, and inoculate with 10% volume of Bifidobacterium inoculum (concentration 1×10⁻⁶). 10 The primary fermentation broth was obtained by fermenting at 40℃ for 24 hours (CFU / mL) at 40℃.

[0074] S3. Second fermentation step: Heat the primary fermentation broth to 70℃, adjust the pH to 7.5, and inoculate with 2% volume of thermophilic bacteria (concentration 1×10⁻⁶). 8The secondary fermentation broth was obtained by fermenting at 55℃ for 24 hours (CFU / mL) to produce a secondary fermentation broth.

[0075] S4. Post-processing: The secondary fermentation broth was homogenized and crushed under high pressure at 100 MPa, filtered through a 3.5 μm polypropylene microfiltration membrane, and pasteurized at 100℃ for 30 min. The filtrate was collected to obtain the final fermentation broth.

[0076] S5. Composition formulation: By weight, take 100 parts of final fermentation broth, 10 parts of 1,2-pentanediol, 0.2 parts of capryloyl hydroxamic acid, 7 parts of 1,3-propanediol, and 5 parts of PEG-100 stearate, stir and mix evenly to obtain a sensitive skin repair composition.

[0077] Example 4

[0078] The difference from Example 1 is that the amount of penetration enhancer (squalane: ceramide = 1:1) added is 3 parts, while the rest of the steps and parameters are the same as in Example 1.

[0079] Example 5

[0080] The difference from Example 1 is that the amount of penetration enhancer (squalane:ceramide = 3:1) added is 5 parts, while the rest of the steps and parameters are the same as in Example 1.

[0081] Example 6

[0082] The difference from Example 5 is that the penetration enhancer is 5 parts of squalane, while the remaining steps and parameters are the same as in Example 1.

[0083] Example 7

[0084] The difference from Example 5 is that the penetration enhancer is 5 parts of ceramide, while the remaining steps and parameters are the same as in Example 1.

[0085] Comparative Example 1

[0086] Compared with Example 1, Comparative Example 1 omits the second fermentation of thermophilic bacteria, and the other conditions are the same as in Example 1.

[0087] Comparative Example 2

[0088] Compared with Example 1, Comparative Example 2 used a sucrose solution with a solid content of 8 wt% instead of Bletilla striata root extract, and the other conditions were the same as in Example 1.

[0089] Comparative Example 3

[0090] Compared with Example 1, Comparative Example 3 used an equal amount of 1,2-pentanediol instead of octanoyl hydroxamic acid, and other conditions were the same as in Example 1.

[0091] Comparative Example 4

[0092] Compared with Example 1, Comparative Example 4 used an equal amount of octanoyl hydroxamic acid instead of 1,2-pentanediol, and other conditions were the same as in Example 1.

[0093] 3. Performance characterization of the sensitive skin repair composition.

[0094] (1) Migration ability of HaCaT cells

[0095] (I) Experimental conditions:

[0096] Cell line: HaCaT cells.

[0097] Density: 3*10 5 / ml.

[0098] Tested sample: Raw material for sensitive skin repair in Example 1.

[0099] Positive control: 8% FBS.

[0100] (II) Experimental method: HaCaT cells in the logarithmic growth phase were seeded into 12-well plates. After 24 hours, when the cells had filled the bottom, the bottom was gently cut with a 200 μL pipette tip to simulate human skin injury. The culture medium was aspirated and the detached cells were gently rinsed with PBS. Then, culture medium containing different concentrations of the test sample was added. A blank control group and a positive control group were set up. Microscopic images were taken at 0 h, 12 h and 24 h. The scar area was then statistically analyzed and the healing rate was calculated using ImageJ software.

[0101] Test results are as follows Figure 1-2 As shown: *: P < 0.05 compared to the control group; **: P < 0.01 compared to the control group; ***: P < 0.001 compared to the control group; ***: P < 0.0001 compared to the control group. Figure 1 From left to right: blank group, 0.0625wt% repair material, 0.125wt% repair material, and positive control.

[0102] according to Figure 1-2 Test results showed that the sensitive skin repair material significantly increased the cell scratch healing area at 12 h and 24 h after scratching, which was significantly different from the control group.

[0103] In summary, based on the test results of HaCaT migration ability, the sensitive skin repair composition of the present invention has comprehensive effects such as repairing the skin barrier and promoting skin renewal and healing.

[0104] (2) Effects on the viability of RAW264.7 cells and on the expression of inflammatory factors mRNA in RAW264.7 cells induced by LPS.

[0105] A: Effects on the viability of RAW264.7 cells.

[0106] (I) Experimental conditions

[0107] Tested sample: Sensitive skin repair raw material from Example 1.

[0108] Duration of action: 24 hours.

[0109] Detection method: CCK 8.

[0110] (II) Experimental method: RAW264.7 cells were collected by centrifugation and the cell density was adjusted to 1×10⁻⁶. 5 The drug was seeded in 96-well plates at a concentration of 100 ng / mL and cultured overnight in a cell culture incubator. After RAW264.7 cells adhered, the drug was diluted with serum-free basal medium, and the stimulant LPS was added to a final concentration of 100 ng / mL. After culturing for 24 h, the cell viability was detected by adding CCK8 reagent.

[0111] Test results are as follows Figure 3 As shown, the results indicate that when the concentration of the sensitive skin repair ingredient is below 2.0%, the RAW264.7 cell viability is greater than 80%, and when the concentration is 2.0%, the RAW264.7 cell viability is also close to 80%, both of which can be used as non-toxic concentrations for sensitive skin repair ingredients.

[0112] B: Effects on LPS-induced expression of inflammatory cytokines mRNA in RAW264.7 cells.

[0113] (I) Experimental conditions

[0114] Tested sample: Raw material for sensitive skin repair in Example 1.

[0115] Positive control: 10 μM dexamethasone.

[0116] Cell density: 2.5 x 10 5 / mL; 12-well plate.

[0117] (II) Experimental method: RAW264.7 cells were collected by centrifugation and the cell density was adjusted to 2.5 × 10⁻⁶. 5 The drug was seeded in 12-well plates at a concentration of 100 ng / mL and cultured overnight in a cell culture incubator. After RAW264.7 cells adhered, the drug was diluted with serum-free basal medium and the stimulant LPS was added to a final concentration of 100 ng / mL. After culturing for 6 h, RNA was extracted and the expression level of inflammatory factor mRNA in RAW264.7 cells was detected by qPCR.

[0118] Test results are as follows Figure 4As shown, concentrations of 0.125, 0.25, and 1.0 mg / mL of the sensitive skin repair ingredient effectively reduced the expression levels of IL-6, IL-1β, and TNF-α mRNA in LPS-induced RAW264.7 cells, with the effect increasing with increasing concentration. This indicates that the sensitive skin repair ingredient of the present invention possesses anti-inflammatory and soothing effects and can be used in skincare products for sensitive skin repair.

[0119] In summary, based on the test results of RAW264.7 cell activity and LPS-induced expression of inflammatory factor mRNA in RAW264.7 cells, it can be seen that the sensitive skin repair raw material of the present invention has important anti-inflammatory and soothing effects, and is very suitable for sensitive skin care for sensitive skin with inflammation such as sensitivity, redness, and acne.

[0120] 4. Application Examples

[0121] The application of the composition of the present invention for sensitive skin repair in a serum, the mass percentage of each component in the serum is shown in Table 1, and the preparation method includes the following steps:

[0122] (1) Accurately weigh each raw material component according to the formula in Table 1;

[0123] (2) Glycerin, nicotinamide, xanthan gum and deionized water were stirred evenly at 50°C to obtain phase A;

[0124] (3) Sodium citrate and polysorbate-20 were stirred evenly at 80°C to obtain phase B;

[0125] (4) Add phase B to phase A, emulsify and homogenize, add the sensitive skin repair composition and jasmine essential oil and stir evenly to obtain the essence emulsion.

[0126] Table 1: The mass percentages of each component in Application Examples 1-5 are as follows:

[0127]

[0128] In Examples 1-5, the sensitive skin repair compositions described in Examples 1-5 were used respectively.

[0129] Application Examples 6-7: The sensitive skin repair composition of Examples 6-7 was used instead of the sensitive skin repair composition in Application Example 5, and everything else was the same as in Application Example 5.

[0130] Compare and contrast examples 1-4

[0131] Compared with Application Example 1, in Comparative Application Examples 1-4, the sensitive skin repair composition of Example 1 was replaced with the sensitive skin repair composition provided in Comparative Examples 1-4, and the composition, amount and preparation method of other raw materials were the same as in Application Example 1.

[0132] 5. Performance evaluation of the serum containing the sensitive skin repair composition of the present invention.

[0133] (1) Stability evaluation

[0134] Cold and heat stability: 100mL of essence emulsion was placed in a 250mL transparent glass bottle and placed at 50℃ for 24h, then placed at -10℃ for 24h. This cycle was repeated 15 times for a total of 30 days. The separation of layers and oil and water was observed.

[0135] Room temperature stability: 100mL of essence lotion was placed in a 250mL transparent glass bottle and placed at 25℃ for 30 days. The separation of layers and oil and water was observed.

[0136] The grades are as follows: A (no stratification and no oil-water separation); B (minor stratification or oil-water separation); and C (extensive stratification or oil-water separation). The test results are shown in Table 2 below.

[0137] Table 2 Stability Test Results

[0138]

[0139] Based on the above test results, the serum of the present invention exhibits good stability at room temperature and during thermal cycling, with no stratification or oil-water separation. In contrast, Application Examples 3-4, which used only one of capryloyl hydroxamic acid or 1,2-pentanediol, showed a significant decrease in stability at room temperature and after thermal cycling, with obvious stratification. This demonstrates that the combination of capryloyl hydroxamic acid and 1,2-pentanediol can synergistically improve the stability of the serum.

[0140] (2) Transdermal permeation test of the sample

[0141] Fermentation products are complex mixtures. The permeability of the entire fermentation product is indirectly characterized by measuring the transdermal absorption of its key functional components. Small molecule peptides (<1000 Da) are one of the core functional components for repair and anti-photoaging. High-performance liquid chromatography (HPLC) was used to test small molecule peptides below 1000 Da to characterize the permeability of Bifida ferment lysate fermentation products.

[0142] Using pig skin to simulate human epidermal skin, a Franz diffusion cell was used as the static diffusion cell. The tested essence emulsion was used as the transdermal release solution, and transdermal absorption experiments were conducted under constant temperature (37℃) and constant flow rate (600 r / min). High-performance liquid chromatography (HPLC) was used to determine small molecule peptides below 1000 Da in the samples.

[0143] The permeability of small molecule peptides below 1000 Da = (amount of small molecule peptides below 1000 Da in the receiving pool / amount of small molecule peptides below 1000 Da in the serum) × 100%.

[0144] Table 3: Results of in vitro transdermal absorption test

[0145]

[0146] The test results in Table 3 show that the serum prepared in the embodiments of the present invention exhibits significantly higher in vitro transdermal permeability than the comparative examples. Furthermore, in Application Examples 4-5, which use a combination of squalane and ceramides, the serum's skin penetration is significantly higher than in Example 1, indicating that the present invention uses a combination of ceramides and squalane in a specific ratio to synergistically promote the penetration of active ingredients in the Bifida ferment lysate, which helps improve skin repair performance. Compared to Application Example 1, which did not use Thermophilic bacteria fermentation, and Application Example 2, which used ordinary sucrose as a carbon source, the permeability of small molecule peptides decreased.

[0147] (3) Skin patch test

[0148] Healthy volunteers aged 35-45 years were selected and randomly divided into 12 groups of 20 people each. Using a patch tester, 0.02g of the serum was placed in the patch tester and applied to the back of the subject. After 24 hours, the patch tester was removed, and the appearance of the volunteer's skin was assessed after another 60 minutes.

[0149] Evaluation criteria: Grade 0: Negative reaction; Grade 1: Suspicious reaction, only slight erythema; Grade 2: Weak positive reaction, erythema, infiltration, edema, and possible papules; Grade 3: Strong positive reaction, erythema, infiltration, edema, and possible papules, reaction may extend beyond the test area; Grade 4: Very strong positive reaction, obvious erythema, severe infiltration, edema, confluent herpes, reaction extends beyond the test area.

[0150] Table 4 Results of Skin Patch Test

[0151]

[0152] According to the skin patch test results in Table 4, the essence provided in this embodiment of the invention is gentle and non-irritating to the skin. According to the comparison between Application Example 1 and Comparative Application Example 1, the high-temperature fermentation stage of Thermophilic Bacteria can not only inactivate Bifidobacteria and harmful microorganisms in the first stage and terminate the continuous generation of organic acids, but also utilize Thermophilic Bacteria to metabolize some residual lactic acid, reduce the risk of irritation, and reduce symptoms such as erythema.

[0153] (4) Determination of long-lasting moisturizing performance

[0154] Healthy female volunteers aged 35–55 years were selected and used the serum provided in both the application case and the control case. Six participants were in each group. Facial skin hydration was quantitatively measured using a Corneometer CM825 at 0 h, 1 h, and 4 h to assess the serum's moisturizing ability. The hydration rate and moisturizing rate are average values ​​for each group. Moisturizing rate after 1 h / % = (1 h hydration rate / 0 h hydration rate) * 100; Moisturizing rate after 4 h / % = (4 h hydration rate / 0 h hydration rate) * 100.

[0155] Table 5: Moisturizing Performance Test

[0156]

[0157] According to the test data in Table 5, the skin hydration rate of the serums in the application examples and the control examples was similar after 1 hour of use. However, after 4 hours of use, the serum in the application examples was able to maintain the skin hydration level at a stable level of over 61.8%. Furthermore, application examples 5-6 used a combination of squalane and ceramide as a penetration enhancer, which facilitated the penetration of active ingredients into the skin and improved the long-lasting moisturizing performance of the serums. In contrast, the moisturizing performance of the serums in the control examples decreased slightly, especially the moisturizing rate of control example 1, which decreased significantly. This may be because the double fermentation process gradually breaks down the large polysaccharides in the Bletilla striata root extract into smaller active molecules that are easier for the skin to penetrate, thereby improving the long-lasting moisturizing rate.

[0158] (5) DPPH free radical scavenging activity assay

[0159] Prepare a 0.05 mg / mL DPPH solution (1,1-diphenyl-2-trinitrophenylhydrazine) using anhydrous ethanol. Place 2 g of the essence emulsion and 2 mL of the DPPH solution in a test tube and incubate at 37°C for 3 hours. Use anhydrous ethanol as a blank control. Measure the absorbance A1 of each test sample at 517 nm and calculate the free radical scavenging rate using the following formula: Free radical scavenging rate = [1-(A1-A2) / A3]×100%. Wherein, A3: absorbance of 2 g anhydrous ethanol plus 2 mL DPPH solution; A1: absorbance of 2 g test reagent plus 2 mL DPPH solution; A2: absorbance of 2 g test reagent plus 2 g anhydrous ethanol solution. Measure three parallel samples for each test sample and calculate the average value.

[0160] Table 6 Antioxidant Performance Test

[0161]

[0162] According to the test results in Table 6, the DPPH scavenging rate of Comparative Application Example 1 was lower than that of Application Example 1. Comparative Application Example 1 did not use thermophilic bacteria for fermentation and failed to produce heat-stable superoxide dismutase (SOD), which reduced its ability to scavenge free radicals and resist oxidation, resulting in a lower DPPH scavenging rate of Comparative Application Example 1 compared to Application Example 1.

[0163] The above embodiments are merely examples to illustrate the present invention and are not intended to limit the possible implementations of the invention. Based on the disclosure of this invention, those skilled in the art can make various modifications and adjustments. It is neither possible nor necessary to list all possible implementations. Any modifications, equivalent substitutions, or improvements made within the basic principles and scope of this invention should be considered to fall within the protection scope of this invention.

Claims

1. A method for preparing a composition for repairing sensitive skin, characterized in that, Includes the following steps: S1. Mix the Bletilla striata root extract, nitrogen source and inorganic salt to obtain fermentation culture medium; The preparation of Bletilla striata root extract includes the following steps: Take dried Bletilla striata root, crush it through a 30-80 mesh sieve, add 10-20 times the mass of deionized water, reflux extract at 80-90℃ for 2-5 hours, filter and concentrate the filtrate to a solid content of 5-15wt% to obtain Bletilla striata root extract. S2, First step of fermentation: Sterilize the fermentation medium, adjust the pH to 6.5-7.0, inoculate 3-10% of Bifidobacterium inoculum for fermentation, and obtain the primary fermentation broth of Bifida Ferment Lysate; S3. Second fermentation: Heat the primary fermentation broth to 60-80℃, adjust the pH to 7.0-7.5, and inoculate with 0.5-2% of thermophilic bacteria to ferment and obtain the secondary fermentation broth of Bifida ferment lysate. S4. Post-processing: The secondary fermentation broth is homogenized and crushed under high pressure at 80-100MPa, filtered, sterilized, and the filtrate is collected to obtain the final fermentation broth of Bifida ferment lysate. S5. Mix the final fermentation broth of Bifida Ferment Lysate, 1,2-pentanediol, octanoyl hydroxamic acid, 1,3-propanediol, penetration enhancer, and surfactant to obtain the sensitive skin repair composition. The culture medium in step S1 contains 97-99.4 wt% Bletilla striata root extract, 0.5-2 wt% nitrogen source, and 0.1-1 wt% inorganic salts; the nitrogen source in step S1 includes at least one of soybean peptone and tryptone; the inorganic salts include at least one of potassium dihydrogen phosphate, magnesium sulfate, and zinc sulfate. The fermentation temperature in step S2 is 30-40℃, and the fermentation time is 24-72 hours; the concentration of Bifidobacterium is 1×10⁻⁶. 8 ~1×10 10 CFU / mL; the fermentation temperature in step S3 is 50-60℃, and the fermentation time is 24-48h; the concentration of the thermophilic bacteria is 1×10⁻⁶. 7 ~1×10 8 CFU / mL; The sensitive skin repair composition in step S5 includes 80-100 parts of final fermentation broth, 3-10 parts of 1,2-pentanediol, 0.01-0.2 parts of capryloyl hydroxamic acid, 3-7 parts of 1,3-propanediol, 0-10 parts of penetration enhancer, and 1-5 parts of surfactant.

2. The method for preparing the sensitive skin repair composition as described in claim 1, characterized in that, The filtration in step S4 is performed using a 1.5μm to 3.5μm polypropylene microfiltration membrane; the sterilization in step S4 is low-temperature pasteurization, with a sterilization temperature of 80 to 100℃ and a time of 30 to 60 minutes.

3. The method for preparing the sensitive skin repair composition as described in claim 1 or 2, characterized in that, The penetration enhancer is at least one of squalane and ceramide.

4. The method for preparing the sensitive skin repair composition as described in claim 1 or 2, characterized in that, The surfactant is at least one of PEG-40 hydrogenated castor oil, Tween-20, Tween-60, and PEG-100 stearate.

5. A composition for repairing sensitive skin, characterized in that, Prepared by the method according to any one of claims 1-4.

6. A skincare product, characterized in that, The skincare product contains the composition of claim 5 for repairing sensitive skin.

7. The skincare product as described in claim 6, characterized in that, The composition for repairing sensitive skin is added to skincare products in an amount of 0.01–2 wt%.

8. The skincare product as described in claim 7, characterized in that, The skin care product also includes acceptable excipients; the excipients include at least one of chelating agents, moisturizers, thickeners, emulsifiers, and fragrance modifiers.

9. The skincare product as described in claim 8, characterized in that, The excipients, by weight percentage, include at least one of the following: chelating agent 0.05-0.5%, humectant 1-10%, thickener 0.5-2%, emulsifier 1-5%, and flavor modifier 0.1-0.5%.

10. The skincare product as described in claim 9, characterized in that, The chelating agent is at least one of disodium EDTA, tetrasodium EDTA, trisodium EDTA-disuccinate, and sodium citrate; the moisturizer is at least one of glycerin, sodium hyaluronate, niacinamide, butylene glycol, and sorbitol; the thickener is at least one of sodium polyacrylate, carboxymethyl cellulose, hydroxyethyl cellulose, and xanthan gum; the emulsifier is at least one of cocamidopropyl betaine, polysorbate-20, polysorbate-60, polyglycerol-10 stearate, and potassium lauryl phosphate; and the fragrance modifier is one or a combination of menthol, rose essential oil, jasmine essential oil, carnation essential oil, gardenia essential oil, and violet essential oil.

11. The skincare product according to any one of claims 6-10, characterized in that, The skincare product is at least one of the following: water-based, serum, essence lotion, facial mask liquid, and cream.

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