Composite probiotic fermented camellia oil sensitive skin repair agent and preparation method thereof

Abstract

This product relates to the technical field of medical, dental, or cosmetic formulations, and discloses a compound probiotic fermented camellia oil sensitive skin repair agent and its preparation method. The repair agent contains, by weight percentage, 45%-55% compound probiotic fermented camellia oil, 8%-12% soothing agents, 4%-6% emulsifiers, 0.8%-1.5% thickeners, 12%-18% moisturizers, 0.8%-1.5% antioxidants, and the balance deionized water. The compound probiotic fermented camellia oil is obtained by fermenting camellia oil with *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, and *Bifidobacterium adolescentis*. The soothing agents include bisabolol, allantoin, and *Centella asiatica* extract. This product utilizes microbial enzymes to dissociate large-molecule triglycerides into smaller molecules, improving transdermal absorption. Metabolites synergistically promote ceramide synthesis and inhibit peroxide formation, reducing irritation and soothing redness in sensitive skin.

Description

Technical Field

[0001] This product relates to the technical field of medical, dental, or cosmetic formulations, and discloses a compound probiotic fermented camellia oil sensitive skin repair agent and its preparation method. Background Technology

[0002] Existing repair compositions for sensitive skin often use camellia oil as a base oil. The conventional preparation process involves using pressed camellia oil directly as the oil phase, combined with conventional moisturizers such as glycerin and sodium hyaluronate, as well as synthetic ester emulsifiers. This mixture is physically mixed and emulsified through heating and stirring to produce a repair cream or skincare oil. In this physically mixed system, camellia oil acts only as an occlusive oil, covering the skin surface to reduce moisture loss.

[0003] Directly added camellia oil triglycerides have large molecules, making it difficult for them to penetrate the stratum corneum and be absorbed by the skin. Their repairing effect remains at the level of physical occlusion. The unsaturated fatty acids in camellia oil are easily oxidized during conventional heating, emulsification, and storage, producing peroxides that can irritate sensitive skin. Current conventional methods do not modify camellia oil, resulting in poor transdermal absorption and easy oxidation that irritates sensitive skin. Summary of the Invention

[0004] In response to the shortcomings of existing technologies where camellia oil molecules are difficult to absorb and easily oxidized, thus irritating sensitive skin, this product provides a compound probiotic fermented camellia oil sensitive skin repair agent and its preparation method.

[0005] To address the aforementioned technical issues, this product provides a compound probiotic fermented camellia oil sensitive skin repair agent, comprising the following technical features: by mass percentage, it consists of 45%-55% compound probiotic fermented camellia oil, 8%-12% soothing agent, 4%-6% emulsifier, 0.8%-1.5% thickener, 12%-18% moisturizer, 0.8%-1.5% antioxidant, and the remainder being deionized water; the compound probiotic fermented camellia oil is prepared by fermenting camellia oil and compound probiotic liquid at a mass ratio of 1:0.8-1.2, and the compound probiotic liquid is composed of Lactobacillus plantarum, Lactobacillus rhamnosus, and Bifidobacterium adolescentis at a volume ratio of 2-4:1-3:1-2; the soothing agent is composed of bisabolol, allantoin, and Centella asiatica extract at a mass ratio of 2-3:1-2:3-4.

[0006] When this repair agent is working, the microbial enzymes produced by Lactobacillus plantarum, Lactobacillus rhamnosus, and Bifidobacterium adolescentis dissociate the large-molecule triglycerides in camellia oil into small-molecule free fatty acids and diglycerides, reducing the molecular weight of the oil and making it easier for it to penetrate the stratum corneum and enter the skin. The metabolites of the compound probiotics and the original active ingredients of camellia oil work synergistically on the skin surface and inside to promote the synthesis of skin ceramides and barrier lipids. The soothing aid composed of bisabolol, allantoin, and Centella asiatica extract comes into contact with the nerve endings of the epidermis, inhibits the release of inflammatory factors, and accelerates the fading of redness.

[0007] Furthermore, in the above technical solution, the preparation process of compound probiotic fermented camellia oil is as follows: preheat the camellia oil to 35-40℃, inoculate with compound probiotic liquid, and ferment in a sealed environment at a constant temperature of 35-38℃ and a rotation speed of 120-150 rpm for 48-72 hours. After fermentation, adjust the pH to 5.5-6.0, let it stand and age for 12-24 hours, and take the upper oil phase to obtain compound probiotic fermented camellia oil.

[0008] In practice, the temperature and rotation speed conditions maintain the kinetic balance of the enzymatic hydrolysis reaction of the compound probiotic community, which allows the triglycerides to dissociate continuously and stably. After fermentation, the pH is adjusted and the mixture is allowed to stand and age, which causes the aqueous phase and oil phase in the fermentation system to demulsify and separate due to the density difference. The upper oil phase is enriched with small molecule free fatty acids and active metabolites.

[0009] Furthermore, in the above technical solution, the compound probiotic solution needs to be activated and expanded before being inoculated with camellia oil. Lactobacillus plantarum, Lactobacillus rhamnosus, and Bifidobacterium adolescentis are anaerobically activated in lactic acid bacteria liquid culture medium at 37°C for 24 hours, and then transferred to a new lactic acid bacteria liquid culture medium at an inoculation rate of 3%-5%, and expanded at 37°C until the bacterial concentration reaches 1×10⁹ colony-forming units per milliliter. Subsequently, they are mixed according to the volume ratio to obtain the compound probiotic solution.

[0010] In practice, the anaerobic activation and expanded culture process restored the activity of endogenous metabolic enzymes in the strain, enabling the cell membrane permeability and enzyme expression to reach a stable logarithmic growth phase, ensuring that lipase and esterase can immediately exert their catalytic hydrolysis effect after inoculation with camellia oil.

[0011] Furthermore, in the above technical solution, the Centella asiatica extract in the soothing agent is prepared by a compound solvent extraction method. Centella asiatica is pulverized and passed through a 60-mesh sieve. An ethanol aqueous solution with a volume concentration of 50%-60% is added at a material-to-liquid ratio of 1:10-15. The extract is ultrasonically extracted 2-3 times at 45-50℃, each time for 30-40 minutes. The extracts are combined and concentrated under reduced pressure to one-fifth of the original volume. Then, it is mixed evenly with bisabolol and allantoin at the mass ratio mentioned above to obtain the soothing agent.

[0012] In practice, a specific concentration of ethanol aqueous solution disrupts the lipid bilayer of Centella asiatica plant cells, and the microscopic shear force generated by ultrasonic cavitation accelerates cell wall rupture, causing triterpenoid saponins such as asiaticoside to dissolve. Reduced pressure concentration removes the ethanol solvent, allowing the extract to form a homogeneous soothing system with bisabolol and allantoin.

[0013] Furthermore, in the above technical solution, the emulsifier is composed of polyglycerol-10 oleate, cetearyl oleate and sorbitan oleate in a mass ratio of 1-2:2-3:1-1.5, the moisturizer is composed of sodium hyaluronate, glycerin and butylene glycol in a mass ratio of 0.1-0.5:3-5:5-8, and the antioxidant is composed of tocopherol and rosemary extract in a mass ratio of 3-5:1-2.

[0014] In practice, three emulsifiers in specific proportions are oriented at the oil-water interface to form a liquid crystal network structure, which is similar to the lipid bilayer structure of the stratum corneum of the skin, thus reducing transepidermal water evaporation. Tocopherol and sarsaparilla acid in rosemary extract work synergistically to capture free radicals generated by the oxidation of camellia oil by providing hydrogen atoms, thereby blocking the auto-oxidation chain reaction of unsaturated fatty acids.

[0015] Furthermore, in the above technical solution, the thickener is composed of carbomer and xanthan gum in a mass ratio of 1:2-3. Before use, carbomer and xanthan gum need to be soaked in deionized water to swell for 8-12 hours. The final pH of the repair agent system is adjusted to 5.8-6.2, and the pH of the system is adjusted by triethanolamine or citric acid.

[0016] In practice, during the swelling process, the carboxyl groups on the polyacrylic acid molecular chains of carbomer are fully hydrated. When the pH of the system is adjusted to 5.8-6.2, the carboxyl groups partially dissociate and generate electrostatic repulsion, causing the molecular chains to extend and physically entangle with the helical structure of xanthan gum, thus constructing a three-dimensional network structure and achieving suspension stability of small molecule droplets of fermented camellia oil.

[0017] Furthermore, in the above technical solution, a method for preparing a compound probiotic fermented camellia oil sensitive skin repair agent includes the following steps: Step 1: Camellia oil pretreatment: heating the camellia oil to 80-85℃ and sterilizing for 20-30 minutes, then cooling to 35-40℃; Step 2: Compound probiotic fermentation: adding compound probiotic bacterial solution to the pretreated camellia oil and fermenting at a constant temperature of 35-38℃ for 48-72 hours; Step 3: Centrifugal purification: centrifuging the fermentation broth at 6000-8000 rpm for 15-20 minutes, taking the upper clear oil phase, and filtering it through a microporous membrane for sterilization to obtain... Compound probiotic fermented camellia oil; Step 4: Aqueous phase preparation: Mix humectant, thickener and deionized water, heat to 75-80℃ and stir to dissolve; Step 5: Oil phase preparation: Mix compound probiotic fermented camellia oil, emulsifier and antioxidant, heat to 75-80℃ and stir to dissolve; Step 6: Homogenization: Slowly add oil phase to aqueous phase, homogenize at a shear rate of 3000-4000 rpm for 5-8 minutes, cool to 40-45℃ and add soothing agent, continue stirring for 15-20 minutes; Step 7: Sterilization and filling: Cool to below 35℃, adjust pH, sterilize and fill to obtain the final product.

[0018] In practice, this dynamic process eliminates the interference of miscellaneous bacteria in camellia oil on the fermentation system through high-temperature pretreatment, completes the enzymatic hydrolysis of macromolecular oils during the constant-temperature fermentation stage, intercepts dead bacteria and macromolecular proteins through centrifugation and microporous filtration membranes, reduces the interfacial tension between the water and oil phases by heating at 75-80℃ to facilitate emulsification, disperses fermented camellia oil in the aqueous phase as micron-sized droplets through high-speed shearing, and adds soothing agents at low temperature to avoid the degradation of heat-sensitive active ingredients.

[0019] Furthermore, in the above technical solution, in the second step of compound probiotic fermentation, the amount of compound probiotic liquid added is 80%-120% of the mass of camellia oil. The viable bacterial concentrations of Lactobacillus plantarum, Lactobacillus rhamnosus, and Bifidobacterium adolescentis in the compound probiotic liquid must all reach more than 1×10⁹ colony-forming units per milliliter. During the fermentation process, sterile nitrogen gas needs to be introduced into the fermentation system to maintain an anaerobic environment, and the fermentation time is controlled at 60-72 hours.

[0020] In practice, the high concentration of bacterial solution and the high substrate ratio ensured the substrate saturation of the enzymatic hydrolysis reaction, the introduction of sterile nitrogen eliminated the inhibition of anaerobic lipase activity by oxygen, and the extended fermentation time made the dissociation of triglycerides more thorough and the conversion rate of free fatty acids higher.

[0021] Furthermore, in the above technical solution, in the centrifugal purification step three, the pore size of the microporous filter membrane is 0.22-0.45 micrometers, the centrifugation temperature is controlled at 4-8℃, and after the upper clear oil phase is filtered, tocopherol equivalent to 0.01%-0.02% of its mass needs to be added under sterile conditions, and then it is transferred to the oil phase preparation step five and mixed with the emulsifier.

[0022] In practice, low-temperature centrifugation inhibits the continuous catalytic activity of residual enzymes from fermentation, preventing changes in the composition of the oil phase during separation. Micron-sized pores physically remove microbial cells, and trace amounts of tocopherol are added immediately after filtration. Their antioxidant activity preferentially consumes the trace amounts of dissolved oxygen remaining in the filtered oil phase, thus blocking the initiation of unsaturated fatty acid oxidation.

[0023] Furthermore, in the above technical solution, in the sixth homogenization mixing step, the drip rate when the oil phase is added to the water phase is 2-3 ml per minute, and the cooling rate after homogenization is controlled at 1-2℃ per minute. In the seventh sterilization and filling step, triethanolamine or citric acid is used to adjust the pH of the system to 5.8-6.2, sterilization is carried out by ultraviolet irradiation in the dark for 15-20 minutes, and the relative humidity is controlled below 40% during filling.

[0024] In practice, the combination of slow drip acceleration and high-speed shearing allows the oil phase droplets to disperse layer by layer in the aqueous phase, avoiding demulsification caused by local oil phase overload; the cooling rate of 1-2℃ per minute gives the emulsifier molecules sufficient time to orient themselves at the oil-water interface to form a robust interfacial film; the light-shielding ultraviolet light inactivates the residual microbial vegetative cells in the system, and the low-humidity filling environment prevents moisture from invading and disrupting the established emulsion balance.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention uses a compound probiotic composed of *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, and *Bifidobacterium adolescentis* to ferment and modify camellia oil. Microbial enzymes dissociate large-molecule triglycerides into small-molecule free fatty acids and diglycerides, reducing the molecular weight of the oil and improving transdermal absorption. Fermentation metabolites synergistically promote the synthesis of skin ceramides and barrier lipids with the original active ingredients of camellia oil. The fermentation process consumes easily oxidized free substrates in camellia oil, and combined with antioxidants composed of tocopherol and rosemary extract, it inhibits the formation of peroxides, reducing the risk of irritation to sensitive skin.

[0026] 2. This invention uses a soothing agent composed of bisabolol, allantoin, and Centella asiatica extract in a specific ratio. Based on the improved transdermal absorption of fermented camellia oil, the small molecule active ingredients and the soothing agent contact the nerve endings of the epidermis, inhibiting the release of inflammatory factors and accelerating the reduction of redness. Polyglycerol-10 oleate, cetearyl alcohol oleate, and sorbitan oleate are used as emulsifiers to construct a liquid crystal emulsion system similar to the structure of the skin's sebum film, reducing the damage of the emulsifier to the damaged stratum corneum and maintaining the safety of the repair process. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to embodiments. Those skilled in the art can reproduce the technical solution of the present invention and achieve its claimed technical effects based on the content disclosed in this specification. It should be noted that the following embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention. Any non-substantial improvements and adjustments made based on the core concept of the present invention should fall within the scope of protection of the present invention.

[0028] Example 1: This example provides a compound probiotic fermented camellia oil sensitive skin repair agent. The formula, by weight percentage, is: 50% compound probiotic fermented camellia oil, 10% soothing agent, 5% emulsifier, 1% thickener, 15% moisturizer, 1% antioxidant, and 18% deionized water.

[0029] The compound probiotic fermented camellia oil is prepared by fermenting camellia oil and compound probiotic liquid in a mass ratio of 1:1. The compound probiotic liquid is composed of Lactobacillus plantarum, Lactobacillus rhamnosus, and Bifidobacterium adolescentis in a volume ratio of 3:2:1. The soothing agent is composed of bisabolol, allantoin, and Centella asiatica extract in a mass ratio of 2:1:3. The emulsifier is composed of polyglycerol-10 oleate, cetearyl alcohol olive oil ester, and sorbitan olive oil ester in a mass ratio of 1:2:1. The moisturizer is composed of sodium hyaluronate, glycerin, and butylene glycol in a mass ratio of 0.2:4:10.8. The antioxidant is composed of tocopherol and rosemary extract in a mass ratio of 3:1. The thickener is composed of carbomer and xanthan gum in a mass ratio of 1:2.

[0030] The preparation method is as follows: Step 1: Camellia oil pretreatment: Heat the camellia oil to 85℃ and keep it at that temperature for 25 minutes to sterilize it, then cool it down to 38℃; Step 2, Compound Probiotic Fermentation: Lactobacillus plantarum, Lactobacillus rhamnosus, and Bifidobacterium adolescentis were anaerobically activated in lactic acid bacteria liquid culture medium at 37°C for 24 hours. Then, they were transferred to new lactic acid bacteria liquid culture medium at an inoculation rate of 4% and cultured at 37°C until the bacterial concentration reached 1×10⁹ colony-forming units per milliliter. The bacterial solutions were then mixed in proportion to obtain a compound probiotic solution. 100% of the compound probiotic solution was added to the pretreated camellia oil and fermented at a constant temperature of 38°C for 72 hours. During the fermentation process, sterile nitrogen gas was introduced into the fermentation system to maintain an anaerobic environment. Step 3, centrifugal purification: Centrifuge the fermentation broth at 4℃ and 8000 rpm for 15 minutes, take the upper clear oil phase, filter it through a 0.45-micron microporous membrane for sterilization, and add tocopherol equivalent to 0.015% of the mass of the upper clear oil phase under sterile conditions to obtain compound probiotic fermented camellia oil. Step 4, Aqueous phase preparation: Mix the humectant, the thickener soaked and swollen in deionized water for 10 hours, and deionized water, and heat to 75°C while stirring to dissolve; Step 5, Oil phase preparation: Mix the compound probiotic fermented camellia oil, emulsifier and antioxidant, heat to 75℃ and stir to dissolve; Step 6, Homogenization: Slowly add the oil phase to the aqueous phase at a rate of 3 ml per minute, homogenize for 5 minutes at a shear rate of 4000 rpm, then cool to 45°C at a cooling rate of 1°C per minute, add the soothing agent, and continue stirring for 20 minutes. Step 7, Sterilization and Filling: Cool down to below 35℃, adjust the pH of the system to 6.0 using triethanolamine, sterilize by ultraviolet irradiation in the dark for 20 minutes, and fill in an environment with a relative humidity of 35%.

[0031] Example 2: The only difference from Example 1 is the formulation ratio. The formula consists of 45% fermented camellia oil with compound probiotics, 8% soothing agent, 4% emulsifier, 0.8% thickener, 18% moisturizer, 0.8% antioxidant, and 23.4% deionized water. All other conditions are the same as in Example 1.

[0032] Example 3: The only difference from Example 1 is the formulation ratio. It contains 55% compound probiotic fermented camellia oil, 12% soothing agent, 6% emulsifier, 1.5% thickener, 12% moisturizer, 1.5% antioxidant, and 12% deionized water. All other conditions are the same as in Example 1.

[0033] Example 4: The only difference from Example 1 is that the compound probiotic solution contains Lactobacillus plantarum, Lactobacillus rhamnosus, and Bifidobacterium adolescentis in a volume ratio of 4:1:2. All other conditions are the same as in Example 1.

[0034] Example 5: The only difference from Example 1 is that the compound probiotic solution contains Lactobacillus plantarum, Lactobacillus rhamnosus, and Bifidobacterium adolescentis in a volume ratio of 2:3:1. All other conditions are the same as in Example 1.

[0035] Example 6: The only difference from Example 1 is that the soothing agent consists of bisabolol, allantoin, and Centella asiatica extract in a mass ratio of 3:2:4. All other conditions are the same as in Example 1.

[0036] Example 7: The only difference from Example 1 is that when preparing the Centella asiatica extract using the compound solvent extraction method, the Centella asiatica is pulverized and passed through a 60-mesh sieve. A 50% (v / v) ethanol aqueous solution is added at a material-to-liquid ratio of 1:10. The mixture is then ultrasonically extracted twice at 45°C for 30 minutes each time. The extracts are combined and concentrated under reduced pressure to one-fifth of their original volume. All other conditions are the same as in Example 1.

[0037] Example 8: The only difference from Example 1 is that the emulsifier is composed of polyglycerol-10 oleate, cetearyl alcohol oleate, and sorbitan oleate in a mass ratio of 2:3:1.5. All other conditions are the same as in Example 1.

[0038] Example 9: The only difference from Example 1 is that the antioxidant is composed of tocopherol and rosemary extract in a mass ratio of 5:1. All other conditions are the same as in Example 1.

[0039] Example 10: The only difference from Example 1 is the preparation method parameters: in step two, the fermentation temperature is 35℃ and the fermentation time is 60 hours; in step three, the centrifugation speed is 6000 rpm and the centrifugation time is 20 minutes. All other conditions are the same as in Example 1.

[0040] Example 11: The only difference from Example 1 is the preparation method parameters: in step six, the oil phase was added to the aqueous phase at a dropping rate of 2 ml / min, homogenized for 8 minutes at a shear rate of 3000 rpm, and cooled at a rate of 2°C / min. All other conditions were the same as in Example 1.

[0041] Example 12: The only difference from Example 1 is the preparation method parameters: in step seven, the pH of the system is adjusted to 5.8, and sterilized by ultraviolet irradiation for 15 minutes. The other conditions are the same as in Example 1.

[0042] Comparative Example 1: The only difference from Example 1 is that the fermented camellia oil with compound probiotics was omitted, and it was replaced with an equal mass of unfermented camellia oil. That is, the camellia oil was directly added to the oil phase preparation in step five without undergoing the compound probiotic fermentation and subsequent purification steps. All other conditions were the same as in Example 1.

[0043] Comparative Example 2: Traditional physical mixed repair agent: Unfermented camellia oil, glycerin, sodium hyaluronate, and polyoxyethylene sorbitan monooleate were mixed, heated to 80°C for emulsification, without the addition of soothing agent complex and antioxidant complex, without fermentation treatment or liquid crystal emulsification system construction, and then filled after cooling.

[0044] Comparative Example 3: The only difference from Example 1 is that the fermentation temperature in step two was set to 42°C, which is outside the specified range. All other conditions are the same as in Example 1.

[0045] Comparative Example 4: The only difference from Example 1 is that the step of "adding tocopherol equivalent to 0.015% of the mass of the upper clear oil phase under aseptic conditions" in step three is omitted, and the filtered upper clear oil phase is directly transferred to step five. All other conditions are the same as in Example 1.

[0046] Test method: Free fatty acid conversion rate test: The free fatty acid content in fermented camellia oil was determined by acid-base titration, and the dissociation conversion rate of macromolecular triglycerides was calculated.

[0047] (1) Accurately weigh 2.0 g of the oil phase sample to be tested into a 250 mL Erlenmeyer flask, add 50 mL of neutral ethanol-ether mixed solution (volume ratio 1:1), and shake until the sample is completely dissolved; (2) Add 2-3 drops of phenolphthalein indicator and titrate with 0.05 mol / L potassium hydroxide standard solution until the solution turns slightly red and does not fade for 30 seconds. Record the volume of potassium hydroxide standard solution consumed, V1. (3) At the same time, perform a blank test and record the volume of potassium hydroxide standard solution consumed in the blank test, V0; (4) Calculate the free fatty acid content (calculated as oleic acid), and calculate the free fatty acid conversion rate (%) based on the theoretical free fatty acid content of complete hydrolysis of triglycerides in camellia oil. Three parallel samples are tested in each group, and the arithmetic mean is taken.

[0048] Ceramide synthesis promotion rate test: The test was performed using an in vitro cell culture method combined with enzyme-linked immunosorbent assay (ELISA).

[0049] (1) Take human immortalized epidermal cells (HaCaT cells) in the logarithmic growth phase, and use 1×10 5 The cells were seeded at a density of cells / well in 6-well plates and incubated at 37°C in a 5% CO2 incubator for 24 hours. (2) Discard the original culture medium and add serum-free culture medium containing 10% (volume fraction) of the test repair agent, and continue to culture for 48h; at the same time, set up a blank control group (only serum-free culture medium was added). (3) Collect cells, add cell lysis buffer to extract total protein, and use ceramide ELISA kit to detect the ceramide content in cells of each group; (4) Calculate the ceramide synthesis promotion rate (%) according to the formula: Promotion rate = (Ceramide content in experimental group - Ceramide content in blank group) / Ceramide content in blank group × 100. Three parallel samples are tested in each group, and the arithmetic mean is taken.

[0050] Peroxide value test: Perform the titration method according to GB5009.227-2016 "National Food Safety Standard for Determination of Peroxide Value in Food".

[0051] (1) Accurately weigh 5.0g of the sample to be tested into a 250mL iodine flask, add 30mL of chloroform-glacial acetic acid mixed solution (volume ratio 2:3), and shake to completely dissolve the sample; (2) Add 1.00 mL of saturated potassium iodide solution, seal tightly, shake well, and place in the dark to react for 3 min; (3) Immediately add 100 mL of distilled water, shake well, and titrate with 0.002 mol / L sodium thiosulfate standard solution until pale yellow. Add 1 mL of starch indicator and continue titrating until the blue color disappears. Record the volume of sodium thiosulfate standard solution consumed, V2. (4) At the same time, perform a blank test and record the volume of sodium thiosulfate standard solution consumed in the blank test, V3; (5) Calculate the peroxide value of the sample in milliequivalents per kilogram. Three parallel samples are tested in each group, and the arithmetic mean is taken.

[0052] Interleukin-1 alpha (IL-1α) inhibition rate assay: The assay was performed using a lipopolysaccharide (LPS)-induced cell inflammation model combined with ELISA.

[0053] (1) Take HaCaT cells in the logarithmic growth phase and use 1×10 5 The cells were seeded at a density of 1 cell / well in 6-well plates and cultured for 24 hours until the cell confluence reached 80%. (2) Discard the original culture medium and divide it into three groups: blank control group (serum-free culture medium), model group (serum-free culture medium containing 1 μg / mL LPS), and experimental group (serum-free culture medium containing 1 μg / mL LPS + 10% test repair agent). Each group has 3 replicates. (3) After culturing for another 24 hours, the cell supernatant was collected, and the IL-1α content in the supernatant of each group was detected by human IL-1α ELISA kit; (4) Calculate the IL-1α inhibition rate (%) according to the formula: Inhibition rate = (IL-1α content in the model group - IL-1α content in the experimental group) / (IL-1α content in the model group - IL-1α content in the blank group) × 100, and take the arithmetic mean as the final result.

[0054] Transdermal water loss rate test: The human skin test method was used to measure the transdermal water loss rate using a transdermal water loss tester.

[0055] (1) Select 30 healthy volunteers (aged 18-35 years, with no history of skin diseases). Do not use any skin care products on the test site for 24 hours before the test. The test environment temperature is controlled at 22℃±2℃ and the relative humidity is controlled at 50%±5%. (2) After the volunteers sat still for 30 minutes, three 3cm×3cm test areas were marked on the inner side of the forearm of the subjects. The initial transdermal water loss T0 in the blank area (without sample) and the sample area were tested respectively. (3) Apply 0.05g of the repair agent to be tested evenly to the test area, and test the transdermal water loss T at 2h, 4h and 8h after application. t ; (4) Calculate the rate of decrease in transdermal water loss (%) using the formula: Rate of decrease = (T0 - T t ) / T0×100, take the decrease rate at 8h as the final test result, and calculate the average value of all volunteers.

[0056] The test results are shown in Table 1.

[0057] Table 1 Performance test results of each embodiment and comparative example.

[0058] Results analysis: Example 1 showed the best performance, verifying that under this specific ratio and process conditions, the microbial enzymatic hydrolysis reaction was sufficient, converting large-molecule oils into small-molecule free fatty acids, and significantly improving transdermal absorption and ceramide synthesis. The liquid crystal system constructed with a specific emulsifier synergistically with the soothing agent achieved an optimal balance between the reduction in water loss and the inhibition of inflammation. Examples 2-9, by adjusting the formulation ratio and the types of excipients, maintained a high level of free fatty acid conversion rate and various indicators, proving the robustness of this technical solution within the limited ratio range. Examples 10-12 adjusted the preparation process parameters. The data showed that within the limited process parameter range, the preparation method could achieve oil modification and emulsification stability. However, deviations from the optimal parameters (such as the lower fermentation temperature and shorter time in Example 10) led to insufficient dissociation and a decrease in conversion rate.

[0059] Comparative Example 1 used unfermented camellia oil, resulting in extremely low free fatty acid conversion and a significant decrease in ceramide synthesis promotion. Furthermore, the lack of fermentation to consume easily oxidizable substrates led to a peroxide value as high as 6.8, demonstrating that probiotic fermentation modification is a necessary means to achieve oil molecule reduction and reduce oxidative stimulation. Comparative Example 2 used traditional physical mixing without fermentation or specific excipient formulation, resulting in the worst performance across all indicators, especially peroxide value and inflammation inhibition rate, proving that conventional methods cannot solve the problem of sensitive skin irritation. Comparative Example 3 increased the fermentation temperature to 42℃, exceeding the suitable range for anaerobic bacteria. Bacterial inactivation caused a sharp drop in free fatty acid conversion to 21.5%, and the high temperature accelerated oil oxidation, raising the peroxide value to 9.2, demonstrating that limiting the fermentation temperature is crucial for maintaining enzymatic hydrolysis kinetic balance. Comparative Example 4 omitted the step of adding tocopherol after filtration. Although the degree of fermentation dissociation was not affected, the lack of pre-intervention with antioxidants led to a peroxide value increase to 5.2, proving that this step is indispensable for blocking the chain reaction of unsaturated fatty acid auto-oxidation.

Claims

1. A compound probiotic fermented camellia oil sensitive skin repair agent, characterized in that, It consists of the following components by mass percentage: Compound probiotic fermented camellia oil 45%-55%, soothing agent 8%-12%, emulsifier 4%-6%, thickener 0.8%-1.5%, moisturizer 12%-18%, antioxidant 0.8%-1.5%, deionized water balance; The compound probiotic fermented camellia oil is prepared by fermenting camellia oil and compound probiotic liquid at a mass ratio of 1:0.8-1.

2. The compound probiotic liquid is composed of Lactobacillus plantarum, Lactobacillus rhamnosus and Bifidobacterium adolescentis at a volume ratio of 2-4:1-3:1-2. The soothing agent is composed of bisabolol, allantoin and centella asiatica extract in a mass ratio of 2-3:1-2:3-4.

2. The compound probiotic fermented camellia oil sensitive skin repair agent according to claim 1, characterized in that, The preparation process of the compound probiotic fermented camellia oil is as follows: The camellia oil is preheated to 35-40℃, and the compound probiotic liquid is inoculated. It is then fermented in a sealed environment at a constant temperature of 35-38℃ and a rotation speed of 120-150 rpm for 48-72 hours. After fermentation, the pH is adjusted to 5.5-6.0, and the mixture is allowed to stand and age for 12-24 hours. The upper oil phase is then taken to obtain the compound probiotic fermented camellia oil.

3. The compound probiotic fermented camellia oil sensitive skin repair agent according to claim 2, characterized in that, Before inoculating the camellia oil, the compound probiotic solution needs to be activated and expanded. The Lactobacillus plantarum, Lactobacillus rhamnosus, and Bifidobacterium adolescentis are anaerobically activated in MRS liquid medium at 37°C for 24 hours, and then transferred to a new MRS liquid medium at an inoculation rate of 3%-5% and expanded at 37°C until the bacterial concentration reaches 1×10^9 CFU / mL. Subsequently, they are mixed according to the volume ratio to obtain the compound probiotic solution.

4. The compound probiotic fermented camellia oil sensitive skin repair agent according to claim 1, characterized in that, The Centella asiatica extract in the soothing agent is prepared by a compound solvent extraction method. Centella asiatica is pulverized and passed through a 60-mesh sieve. An ethanol aqueous solution with a volume concentration of 50%-60% is added at a material-to-liquid ratio of 1:10-15. The extract is ultrasonically extracted 2-3 times at 45-50℃, each time for 30-40 minutes. The extracts are combined and concentrated under reduced pressure to 1 / 5 of the original volume. Then, it is mixed evenly with the bisabolol and allantoin at the mass ratio to obtain the soothing agent.

5. The compound probiotic fermented camellia oil sensitive skin repair agent according to claim 1, characterized in that, The emulsifier is composed of polyglycerol-10 oleate, cetearyl oleate and sorbitan oleate in a mass ratio of 1-2:2-3:1-1.5; the moisturizer is composed of sodium hyaluronate, glycerin and butylene glycol in a mass ratio of 0.1-0.5:3-5:5-8; and the antioxidant is composed of tocopherol and rosemary extract in a mass ratio of 3-5:1-2.

6. The compound probiotic fermented camellia oil sensitive skin repair agent according to claim 1, characterized in that, The thickener is composed of carbomer and xanthan gum in a mass ratio of 1:2-3. Before use, the carbomer and xanthan gum need to be soaked and swollen in the deionized water for 8-12 hours. The final pH value of the repair agent system is adjusted to 5.8-6.

2. The pH value of the system is adjusted by triethanolamine or citric acid.

7. A method for preparing a compound probiotic fermented camellia oil sensitive skin repair agent, characterized in that, Includes the following steps: S1. Camellia oil pretreatment: Heat the camellia oil to 80-85℃ and keep it at that temperature for 20-30 minutes to sterilize it, then cool it down to 35-40℃; S2. Compound probiotic fermentation: Compound probiotic liquid is added to the pretreated camellia oil and fermented at a constant temperature of 35-38℃ for 48-72 hours; S3. Centrifugal purification: Centrifuge the fermentation broth at 6000-8000 rpm for 15-20 min, take the upper clear oil phase, filter it through a microporous membrane to remove bacteria, and obtain compound probiotic fermented camellia oil; S4. Aqueous phase preparation: Mix the humectant, thickener and deionized water, heat to 75-80℃ and stir to dissolve; S5. Oil phase preparation: Mix the compound probiotic fermented camellia oil, emulsifier and antioxidant, and heat to 75-80℃ while stirring to dissolve; S6. Homogenization: Slowly add the oil phase to the aqueous phase, homogenize for 5-8 minutes at a shear rate of 3000-4000 rpm, add the soothing agent when the temperature drops to 40-45℃, and continue stirring for 15-20 minutes. S7. Sterilization and filling: Cool to below 35℃, adjust pH, sterilize, and then fill.

8. The preparation method of a compound probiotic fermented camellia oil sensitive skin repair agent according to claim 7, characterized in that, In the S2 compound probiotic fermentation step, the amount of compound probiotic solution inoculated is 80%-120% of the mass of camellia oil. The viable bacterial concentrations of Lactobacillus plantarum, Lactobacillus rhamnosus, and Bifidobacterium adolescentis in the compound probiotic solution must all reach 1×10^9 CFU / mL or higher. During the fermentation process, sterile nitrogen gas needs to be introduced into the fermentation system to maintain an anaerobic environment, and the fermentation time is controlled at 60-72 hours.

9. The preparation method of a compound probiotic fermented camellia oil sensitive skin repair agent according to claim 7, characterized in that, In the S3 centrifugal purification step, the pore size of the microporous filter membrane is 0.22-0.45 μm, the centrifugation temperature is controlled at 4-8℃, and after the upper clear oil phase is filtered, tocopherol equivalent to 0.01%-0.02% of its mass is added under aseptic conditions, and then it is transferred to the S5 oil phase preparation step to be mixed with the emulsifier.

10. The preparation method of a compound probiotic fermented camellia oil sensitive skin repair agent according to claim 7, characterized in that, In the S6 homogenization mixing step, the drop rate of the oil phase when added to the aqueous phase is 2-3 mL / min, and the cooling rate after homogenization is controlled at 1-2℃ / min. In the S7 sterilization and filling step, the pH of the system is adjusted to 5.8-6.2 using triethanolamine or citric acid. The sterilization is carried out by irradiating with ultraviolet light in the dark for 15-20 min, and the relative humidity of the filling environment is controlled below 40%.

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