Multi-enzyme synergistic skin care composition suitable for sensitive skin and application thereof

Abstract

The invention relates to a multi-enzyme synergistic skin care composition suitable for sensitive skin and application thereof, the skin care composition comprises the following components: crithmum maritimum extract, pomegranate peel extract, lysozyme and coenzyme Q10, the crithmum maritimum extract is obtained by anaerobic fermentation of crithmum maritimum through leuconostoc mesenteroides and lactobacillus plantarum and is rich in SOD enzyme; the pomegranate peel extract is obtained by performing anaerobic fermentation on pomegranate fruits through lactobacillus helveticus and is rich in protease and lipase. The crithmum maritimum extract, the pomegranate peel extract, the lysozyme and the coenzyme Q10 are compounded for use, a multi-dimensional synergistic skin care system is constructed, and repairing, anti-inflammatory and anti-aging integrated efficient improvement is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cosmetics, in particular to a multi-enzyme synergistic skin care composition suitable for sensitive skin and application thereof. BACKGROUND

[0002] Oily sensitive skin is a complex facial skin syndrome, and its core problem lies in the interweaving of impaired skin barrier function, neurovascular hyperreactivity and immune inflammation activation. Traditional single components often have difficulty in achieving multi-target intervention and fundamental repair. Moreover, the "inflammation-sensitivity-aging" problems of sensitive skin are interwoven, forming a vicious cycle. For example, chronic inflammation can activate melanocytes and accelerate collagen degradation, while barrier damage can exacerbate inflammatory response. Single components often have difficulty in dealing with such complexity. Enzymes have natural advantages in sensitive skin care: On the one hand, there are many types of enzyme components, each with different functions, providing the possibility for systematic intervention. Superoxide dismutase (SOD) can eliminate free radicals from the source and block the inflammation chain triggered by oxidative stress. Protease can gently promote the metabolism of old and waste keratin and improve barrier function; and lysozyme can regulate skin microecology and inhibit immune inflammation caused by harmful bacteria colonization, etc. By rational compounding of different enzymes, a multi-target regulation network can be constructed to simultaneously cope with multiple challenges such as inflammation, barrier damage and aging.

[0003] On the other hand, the physiological activities of the skin, such as collagen synthesis, production of inflammatory factors, and cell energy metabolism, are the result of a series of enzymatic reactions. Exogenous supplementation of specific enzymes or activation of endogenous enzyme activity is expected to fundamentally affect the health status of the skin like a "regulation switch". The use of enzyme skin care ingredients not only has exogenous efficacy, but also focuses on its potential for regulating endogenous enzymatic reactions in the skin.

[0004] In addition, the enzyme component also has unique advantages in maintaining the skin barrier. The integrity of the skin barrier depends on the orderly arrangement of keratinocytes in the stratum corneum and the stable structure of intercellular lipids. Enzymes such as proteases are involved in the normal differentiation of keratinocytes and the natural shedding of aged keratin. If old and worn-out keratin accumulates, the skin becomes rough and dull, and the barrier function is also weakened. Traditional "harsh" components for assisting desquamation, such as acids and high-concentration retinol, although they have a quick effect, their strong exfoliating or irritating properties can easily cause discomfort such as redness and stinging in sensitive skin with damaged barrier, and the risk is high. The mechanism of action of the enzyme component is more gentle. Its catalytic reaction usually occurs at room temperature and near neutral pH, and has high compatibility with the skin microecology. In particular, enzymes have high specificity and can accurately recognize and act on specific "substrates" like "keys", such as proteases that only break down peptide bonds in aged keratin, without indiscriminately attacking healthy skin cells and tissues, thereby effectively exfoliating the skin while significantly reducing the risk of damage to the skin barrier. These gentle and specific properties make enzymes an ideal choice for sensitive skin to achieve effective care. SUMMARY

[0005] The present application aims to overcome the shortcomings of the prior art and provide a multi-enzyme synergistic skin care composition suitable for sensitive skin and its application.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: In a first aspect, the present application provides a multi-enzyme synergistic skin care composition suitable for sensitive skin, comprising the following components: sea fennel extract, pomegranate peel extract, lysozyme and coenzyme Q10, the weight ratio of the sea fennel extract, pomegranate peel extract, lysozyme and coenzyme Q10 being (0.5-3):(0.6-2):(0.5-2):(0.5-3); wherein the sea fennel extract is obtained by anaerobic fermentation of sea fennel with Leuconostoc mesenteroides and Lactobacillus plantarum, the strain number of the Lactobacillus plantarum being CICC 23185, purchased from China Industrial Microbial Culture Collection Center; the strain number of the Leuconostoc mesenteroides being CICC 6055, purchased from China Industrial Microbial Culture Collection Center; the pomegranate peel extract is obtained by anaerobic fermentation of pomegranate fruit with Lactobacillus helveticus; the strain number of the Lactobacillus helveticus being CICC 6024, purchased from China Industrial Microbial Culture Collection Center.

[0007] The skin care composition of the present application has the following effects: The sea fennel extract is subjected to anaerobic fermentation by Leuconostoc mesenteroides and Lactobacillus plantarum. Lactobacillus plantarum rapidly produces acid to reduce the pH to 3.6-4.0, creating an acidic stress environment to induce the expression of SOD; Leuconostoc mesenteroides metabolizes sucrose to produce dextran, forming a natural microcapsule network in the fermentation system. These dextran microcapsules not only protect SOD enzyme from degradation in the acidic environment, but also stabilize the spatial conformation of the enzyme through intermolecular forces. Therefore, the sea fennel extract can provide stable SOD enzyme. The core activity of SOD enzyme is to specifically scavenge superoxide anion free radicals by catalyzing dismutation reaction to generate hydrogen peroxide and oxygen, thereby effectively blocking the damage of oxidative stress to skin cells, reducing inflammatory response, and enhancing the resistance of skin barrier to environmental stress such as ultraviolet light, thereby delaying the aging process from the root. In addition, the fermentation process biotransforms the complex polyphenols (such as chlorogenic acid, caffeoylquinic acid, etc.) in sea fennel into phenolic acids with smaller molecular weight, higher biological activity, and better skin absorption, such as protocatechuic acid and vanillic acid. These ingredients have excellent anti-inflammatory and antioxidant capacity, can effectively inhibit the release of inflammatory factors, neutralize free radicals, and alleviate sensitive skin and resist photoaging from the source. At the same time, the trace amount of lactic acid produced during fermentation can maintain the slightly acidic environment of the skin, and dextran also has the functions of moisturizing and barrier repair, together forming a multi-dimensional anti-aging repair system.

[0008] Pomegranate peel extract is a source of protease and lipase, because the abundant fructose and organic acids in pomegranate provide carbon source for Lactobacillus helveticus, promoting it to secrete extracellular protease and lipase. The lactic acid produced during fermentation reduces the environmental pH, activates the conversion of protease zymogen in the bacterial body to active form, and at the same time promotes the catalytic activity of lipase at the oil-water interface. Protease can specifically hydrolyze protein peptide bonds, gently degrade keratin in the aging stratum corneum, promote the renewal of collagen and elastin, and thus improve skin roughness and luster; at the same time, it can inhibit the colonization of harmful bacteria by decomposing the protein structure of pathogenic bacteria, and reduce the release of inflammatory mediators, thereby synergistically maintaining the balance of skin microecology. Lipase can efficiently hydrolyze triglycerides into fatty acids and glycerol, accurately decompose excess sebum on the skin surface, unblock pores and reduce acne occurrence; its unique oil-water interface catalytic activity can also promote the reconstruction of sebum film, indirectly inhibit the reproduction of harmful bacteria by regulating lipid metabolism balance, and enhance the water-locking and defense functions of skin barrier. At the same time, the pomegranate peel extract after fermentation also generates gamma-aminobutyric acid to relieve nerve sensitivity, and the bacteriocin secreted by it can accurately regulate the microecology together with lysozyme. Fermentation also converts the original ellagic acid and punicalagins into high-activity small-molecule polyphenols, enhancing the antioxidant and anti-inflammatory efficacy. In combination with the produced short-chain fatty acids and amino acids, they together promote keratin renewal and lipid barrier repair, forming a multi-dimensional repair network.

[0009] Lysozyme naturally exists in saliva, tears and egg white, is a basic globulin, can directly destroy the structure of pathogenic bacteria such as Staphylococcus aureus by hydrolyzing the β-1, 4-glycosidic bond of bacterial cell wall peptidoglycan, leading to its death; this mechanism not only can selectively inhibit harmful bacteria without affecting beneficial bacteria, but also can reduce the stimulation of bacterial toxins on keratinocytes, thereby repairing the imbalance of microecology and relieving the inflammation and itching reaction caused thereby. Coenzyme Q10 is mainly extracted from animal viscera or fish, and can also be synthesized by the human body itself. As a key coenzyme of cell energy metabolism, it can strongly scavenge free radicals and synergistically inhibit lipid peroxidation with vitamin E to protect mitochondrial function from oxidative damage. At the same time, by regulating the nuclear factor-κB signaling pathway, it can reduce the expression of inflammatory factors, enhance the vitality of skin cells, improve barrier integrity, and inhibit the transmission of itching-related nerve signals from the source.

[0010] Preferably, the weight ratio of the sea fennel extract, pomegranate peel extract, lysozyme and coenzyme Q10 is (1-2):(1-1.5):(0.8-1.5):(1-1.5).

[0011] Specifically, the preparation method of the sea fennel extract comprises the following steps: S1, after the sea fennel is washed, 5-10 times the mass of water is added for crushing and pulping to obtain sea fennel slurry, which is sterilized; S2, a mixed bacterial solution of Lactobacillus plantarum and Leuconostoc mesenteroides is inoculated in the sterilized sea fennel slurry, and anaerobic fermentation is carried out at 36-38℃ for 60-72h to obtain a fermentation product; S3, the fermentation product is filtered to obtain a fermentation liquor, which is vacuum freeze-dried to obtain the sea fennel extract.

[0012] Preferably, the inoculation amount of the mixed bacterial solution is 10-30% v / v of the sea fennel slurry, the viable bacterial count of the mixed bacterial solution is 1×10 9 9 CFU / mL, and the viable bacterial count ratio of Lactobacillus plantarum to Leuconostoc mesenteroides in the mixed bacterial solution is 1:(1-3).

[0013] Specifically, the preparation method of the sea fennel extract comprises the following steps: T1, after the sea fennel is washed, 5-10 times the mass of water is added for crushing and pulping to obtain sea fennel slurry, which is sterilized; T2, a mixed bacterial solution of Lactobacillus plantarum and Leuconostoc mesenteroides is inoculated in the sterilized sea fennel slurry, and anaerobic fermentation is carried out at 36-38℃ for 60-72h to obtain a fermentation product; T3, the fermentation product is filtered to obtain a fermentation liquor, which is vacuum freeze-dried to obtain the sea fennel extract. ​

[0014] Preferably, the inoculation amount of Lactobacillus helveticus liquid is 2-10% v / v of pomegranate peel pulp; the viable bacterial count of the Lactobacillus helveticus liquid is 2.5 x 10 8 x 10 8 CFU / mL.

[0015] In the second aspect, the present application provides the use of the multi-enzyme synergistic skin care composition suitable for sensitive skin in the preparation of cosmetics, wherein the cosmetics are cosmetic water, emulsion, cream, mask, serum or spray, and the addition amount of the composition is 0.5%-5% of the total weight of the cosmetics.

[0016] In the third aspect, the present application provides a serum, comprising the following raw materials by weight percentage: 0.5%-5% of the multi-enzyme synergistic skin care composition suitable for sensitive skin in the first aspect, 0.5%-10% of a humectant, 0.5%-1% of a thickening agent, 0.05%-0.3% of a preservative and 0.01%-0.3% of a pH regulator, and the balance is deionized water.

[0017] Preferably, the thickening agent comprises at least one of xanthan gum, carbomer, hydroxyethyl acrylate / acryloyl dimethyl taurine sodium copolymer, ammonium acryloyl dimethyl taurine / VP copolymer and microbacterial gum.

[0018] Preferably, the humectant comprises at least one of allantoin, betaine, beta-glucan, trehalose, glycol, dipropylene glycol, sodium hyaluronate, 1,3-butanediol, glycerol, D-panthenol and ceramide.

[0019] Preferably, the pH regulator comprises at least one of arginine, tromethamine and EDTA-disodium.

[0020] Preferably, the preservative comprises at least one of 1,3-propanediol, 1,2-hexanediol and p-hydroxyacetophenone.

[0021] In the fourth aspect, the present application provides a preparation method of the serum in the third aspect, comprising the following steps: (1) mixing the humectant, the thickening agent and part of the deionized water, homogenizing at 75-85℃, then adding the preservative and stirring uniformly to obtain a mixture; (2) after the temperature of the mixture in (1) is reduced to 35-45℃, adding each component in the multi-enzyme synergistic skin care composition suitable for sensitive skin in the first aspect and the remaining deionized water, stirring uniformly, and finally adding the pH regulator to adjust the pH to obtain the serum.

[0022] Compared with the prior art, the present application has the following beneficial effects: The components of the skin care composition of the present application include sea fennel extract, pomegranate peel extract, lysozyme and coenzyme Q10, wherein the sea fennel extract is obtained by anaerobic fermentation of sea fennel by Enterococcus casseliflavus and Lactobacillus plantarum, can provide SOD enzyme, and also generates small molecule phenolic acid (such as protocatechuic acid), dextran and lactic acid, thereby synergistically exerting the multiple effects of anti-inflammatory, moisturizing, barrier repair and stabilization of SOD activity, the pomegranate peel extract is obtained by anaerobic fermentation of pomegranate peel by Lactobacillus helveticus, is the main source of protease and lipase, and also generates gamma-aminobutyric acid, bacteriocin, small molecule polyphenol and short-chain fatty acid in the fermentation process, thereby endowing it with the comprehensive ability of soothing nerves, precise regulation of microecology, enhanced antioxidant and promotion of barrier repair. Therefore, the skin care composition of the present application constructs a multi-dimensional synergistic skin care system: the SOD enzyme in the sea fennel extract plays a strong antioxidant effect, the small molecule phenolic acid and lactic acid therein play a key role in source anti-inflammatory and maintenance of the healthy slightly acidic environment of the skin, the protease in the pomegranate peel extract can gently renew the keratin, the lipase can decompose excess sebum, and the gamma-aminobutyric acid and bacteriocin therein can also play the role of soothing nerve sensitivity and precisely inhibiting harmful bacteria and maintaining the balance of microecology; the lysozyme regulates the microecology, the coenzyme Q10 enhances the cell energy, and more importantly, the four components in the composition can deeply regulate the endogenous enzymatic reaction network in the skin: by inhibiting the activity of pro-inflammatory factors such as cyclooxygenase (COX-2), the inflammatory response is reduced from the source; at the same time, the cell energy metabolism is enhanced, the collagen synthesis enzyme activity is activated, and the dermal layer structure reconstruction is promoted; finally, the vicious cycle of “barrier damage-inflammation-aging” of sensitive skin is broken, and integrated efficient improvement of repair, anti-inflammatory and anti-aging is achieved. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The contrast chart before and after the use of the serum of application example 1. DETAILED DESCRIPTION

[0024] In order to better illustrate the purpose, technical scheme and advantages of the present application, the present application will be further described below in combination with specific examples.

[0025] The raw materials used in the following examples and comparative examples are as follows: Sea fennel: provided by Sabinsa Corporation.

[0026] Pomegranate peel: purchased on the market and used after peeling the peel.

[0027] Lactobacillus helveticus 1 (Lactobacillus helveticus): purchased from China Industrial Microbial Culture Collection Center (CICC), strain number CICC 6024; Lactobacillus helveticus 2: purchased from China General Microbiological Culture Collection Center (CICC), with strain number CICC 20289; Lactobacillus plantarum 1: purchased from China General Microbiological Culture Collection Center (CICC), with strain number CICC 23185; Lactobacillus plantarum 2: purchased from China General Microbiological Culture Collection Center (CICC), with strain number CICC 25125; Leuconostoc mesenteroides 1: purchased from China General Microbiological Culture Collection Center (CICC), with strain number CICC 6055; Leuconostoc mesenteroides 2: purchased from China General Microbiological Culture Collection Center (CICC), with strain number CICC 25245; Lysozyme: purchased from Zhejiang Aijies Biotechnology Co., Ltd., with trade name Lysozyme; Coenzyme Q10: purchased from Suzhou Nakang Biotechnology Co., Ltd., with trade name Coenzyme Q10 Liposome; SOD enzyme: purchased from Active Concepts, USA, with trade name ACB Ox-Red SOD; Sea fennel extract 2: purchased from Boson Industry Co., Ltd., with trade name Sea Fennel Water Sea Fennel Extract.

[0028] Pomegranate peel extract 2: purchased from SLIAB Co., Ltd., with trade name ACNESIUM ® .

[0029] Other materials, reagents, etc. used in the examples can be obtained from commercial channels if not otherwise specified.

[0030] Examples 1-5 and Comparative Examples 1-10 Specifically, the preparation method of the sea fennel extract 1 comprises the following steps: S1, after washing the sea fennel, 10 times the mass of water is added for crushing and pulping to obtain sea fennel pulp, and the sea fennel pulp is sterilized at 135℃ for 15s; S2, inoculating the mixed bacterial solution of Lactobacillus plantarum 1 and Leuconostoc mesenteroides 1 into the sterilized sea fennel slurry, and anaerobically fermenting at 37°C for 65h to obtain a fermentation product; wherein the inoculation amount of the mixed bacterial solution is 15% v / v of the sea fennel slurry, the viable bacterial count of the mixed bacterial solution is 1.5x10 9 CFU / mL, and the viable bacterial count ratio of Lactobacillus plantarum to Leuconostoc mesenteroides in the mixed bacterial solution is 1:2; S3, filtering the fermentation product using a filter membrane of 0.22μm to obtain a fermentation liquor, and vacuum freeze-drying to obtain the sea fennel extract; wherein the vacuum freeze-drying process is continuously completed through programmed control, first deep pre-freezing, rapidly cooling the sample to -40℃±2℃ and maintaining for 2-4h to ensure complete solidification of the material. Subsequently, after the vacuum degree is stabilized below 30 Pa, entering the stepwise temperature rising sublimation stage, gradually rising from -40℃ to -20℃ at a rate not exceeding 10℃ / h (maintaining for 2h), continuing to rise to -5℃ (maintaining for 10h), and finally rising to 5℃ until the ice nucleus completely disappears through the pressure rise test, and this stage takes about 15-25h in total. Finally, entering the resolution drying stage, raising the temperature to 25℃±5℃ and maintaining for 4-8h to stabilize the residual moisture content of the final product to below 3%.

[0031] The preparation method of the pomegranate peel extract 1 comprises the following steps: T1, after washing the pomegranate peel, adding 3 times the mass of water to break and pulp to obtain a pomegranate peel slurry, and sterilizing the pomegranate peel slurry at 135℃ for 15s; T2, inoculating Lactobacillus helveticus 1 bacterial solution into the sterilized pomegranate peel slurry, and anaerobically fermenting at 40℃ for 72h to obtain a fermentation product; wherein the addition amount of the Lactobacillus helveticus bacterial solution is 6% v / v of the pomegranate peel slurry; and the viable bacterial count of the Lactobacillus helveticus bacterial solution is 3.5x10 8 CFU / mL; T3, filtering the fermentation product using a filter membrane of 0.22μm to obtain a fermentation liquor, and vacuum freeze-drying to obtain the pomegranate peel extract; wherein the vacuum freeze-drying process is continuously completed through programmed control, first deep pre-freezing, rapidly cooling the sample to -40℃±2℃ and maintaining for 2-4h to ensure complete solidification of the material. Subsequently, after the vacuum degree is stabilized below 30 Pa, entering the stepwise temperature rising sublimation stage, gradually rising from -40℃ to -20℃ at a rate not exceeding 10℃ / h (maintaining for 2h), continuing to rise to -5℃ (maintaining for 10h), and finally rising to 5℃ until the ice nucleus completely disappears through the pressure rise test, and this stage takes about 15-25h in total. Finally, entering the resolution drying stage, raising the temperature to 25℃±5℃ and maintaining for 4-8h to stabilize the residual moisture content of the final product to below 3%.

[0032] The weight ratio between the components of the multi-enzyme synergistic skin care compositions for sensitive skin of Examples 1-5 and Comparative Examples 1-4 is shown in Table 1, and the total mass of each skin care composition is equal.

[0033] The preparation method of the multi-enzyme synergistic skin care compositions for sensitive skin of Examples 1-5 and Comparative Examples 1-10 comprises the following steps: Mix each component to obtain each group of skin care compositions.

[0034] Table 1 Weight ratio of each component in each group of multi-enzyme synergistic skin care compositions for sensitive skin

[0035] Comparative Example 5 Comparative Example 5 differs from Example 1 in that Lactobacillus plantarum 2 bacterial solution is used instead of Lactobacillus plantarum 1 bacterial solution in the preparation process of Sea Fennel Extract 1.

[0036] Comparative Example 6 Comparative Example 6 differs from Example 1 in that Leuconostoc mesenteroides 2 bacterial solution is used instead of Leuconostoc mesenteroides 1 bacterial solution in the preparation process of Sea Fennel Extract 1.

[0037] Comparative Example 7 Comparative Example 7 differs from Example 1 in that Lactobacillus helveticus 2 bacterial solution is used instead of Lactobacillus helveticus 1 bacterial solution in the preparation process of Pomegranate Peel Extract 1.

[0038] Comparative Example 8 Comparative Example 8 differs from Example 1 in that an equal amount of Sea Fennel Extract 2 is used instead of Sea Fennel Extract 1.

[0039] Comparative Example 9 Comparative Example 9 differs from Example 1 in that an equal amount of Pomegranate Peel Extract 2 is used instead of Pomegranate Peel Extract 1.

[0040] Comparative Example 10 Comparative Example 10 differs from Example 1 in that an equal amount of SOD enzyme is used instead of Sea Fennel Extract 1.

[0041] Test Example 1: Cyclooxygenase COX-2 Inhibition Test Cyclooxygenase is mainly involved in inflammatory response and prostaglandin synthesis, and its production often means that the skin is stimulated by inflammation or cell damage. By detecting the size of the inhibition of COX-2 by the composition, the strength of the anti-inflammatory effect of the composition can be obtained.

[0042] Experimental method: The effect example explores the inhibition of the enzyme that causes inflammation in the body by the multi-enzyme synergistic skin care composition prepared in Examples 1-5 and Comparative Examples 1-10.

[0043] The cell line used is human keratinocytes HaCaT (BeNa Biotech), and the test conditions are: incubator temperature 37±1℃, humidity 90±5%, carbon dioxide 5±1%; the cells are cultured and treated according to the grouping, and then tested, and the specific test is COX-2 content detection, and the test method is as follows: (1) The cell suspension is inoculated in a 96-well cell culture plate at a density of 2000 cells per well, 100 μL of DMEM culture medium is added to each well, and the cells are cultured for 24 h; the multi-enzyme synergistic compositions of Examples 1-5 and Comparative Examples 1-10 are diluted with DMEM medium to an experimental final concentration of 1% (v / v) of the composition in the medium.

[0044] (2) The supernatant is discarded, the control group is added with 100 μL of DMEM medium containing 10 μg / mL of LPS (2105341, Gibco), and the sample group is added with 100 μL of DMEM medium containing 10 μg / mL of LPS and containing 1% of the multi-enzyme synergistic skin care composition prepared in Examples 1-5 and Comparative Examples 1-10; (3) After 24 h of incubation, the culture medium and cells are collected respectively; (4) COX-2 detection: The COX-2 content in the keratinocytes is detected according to the experimental steps of the human cyclooxygenase 2 (COX-2) ELISA kit (Jianglai Biological, JL19470). First, 50 μL of standard or sample to be tested is accurately added to the standard sample well and the sample well of the pre-coated plate, followed by the addition of 100 μL of HRP-labeled detection antibody, and then sealed and incubated at 37℃ for 60 minutes to allow the antigen and antibody to fully bind. Subsequently, the key washing procedure is performed, which involves five repeated steps of liquid injection, standing and shaking to completely remove unbound impurities, thereby laying a clean background for the color development reaction. In the color development stage, 50 μL of substrate A and B are added to each well in turn, and the solution is reacted at 37℃ for 15 minutes in the dark, at which time the enzyme catalyzes the substrate to produce a blue signal; finally, 50 μL of stop solution is added, and the solution instantly changes from blue to yellow, indicating the termination of the reaction.

[0045] The inhibition rate of COX-2 = | (control group content - sample group content) | / control group content x 100%; The higher the inhibition rate of COX-2, the stronger the anti-inflammatory ability of the composition, and the better the improvement effect on sensitive skin. The results calculated are shown in Table 2.

[0046] Test Example 2: Collagen promotion test Collagen provides support and firmness to the skin, keeping it plump; elastin gives the skin the ability to bounce back, preventing sagging. Both proteins work together to maintain the elasticity and youthful state of the skin. These two proteins are synthesized by collagen synthetase and elastin synthetase, so by detecting the amount of elastin and collagen in the dermal cells, we can know the strength of the composition's promotion of protein synthesis.

[0047] Experimental method: This effect example explores the promotion of collagen and elastin in the dermis of skin cells by the multi-enzyme synergistic skin care compositions prepared in Examples 1-5 and Comparative Examples 1-10.

[0048] The cell line used is human dermal fibroblasts HSF (Guangzhou Geniobio Biotechnology Co., Ltd.), and the test conditions are: incubator temperature 37±1℃, humidity 90±5%, carbon dioxide 5±1%; the cells are cultured and treated according to the grouping, and then tested, the test method is as follows: (1) The cell suspension was inoculated in a 96-well cell culture plate, with a density of 2000 cells per well, and 100 μL of DMEM + 10% fetal bovine serum (FBS) + 1% penicillin / streptomycin culture solution was added to each well, and the cells were cultured for 24 h; the multi-enzyme synergistic compositions of Examples 1-5 and Comparative Examples 1-10 were diluted with DMEM medium to an experimental final concentration of 0.1% (v / v) of skin care composition in the medium.

[0049] (2) The cells were discarded and the supernatant was discarded, and divided into 1 blank group and 10 experimental groups, and the blank group was added with 100 μL of DMEM medium. The experimental groups were added with 100 μL of medium containing 0.1% of the corresponding compositions of Examples 1-5 and Comparative Examples 1-10. Each was cultured for 24 h, and the cells were collected, the medium was discarded, and PBS was washed twice. Add 1 mL of TRIzol lysis buffer, scrape the cells, transfer to an EP tube, and store at -80℃ for later use.

[0050] (3) ELISA detection of protein content: use human collagen type I (Col I) ELISA kit (Shanghai Enzyme Link, item number ml057630) to detect the content of collagen type I in HSF cells. Use human elastin ELISA kit (Enzyme Link Biological, ml038411) to detect the content of elastin in HSF.

[0051] The protein content promotion rate = | (protein content sample group - protein content blank group) | / protein content blank group x 100%; Wherein, the higher the protein content promotion rate, the stronger the ability of the composition to endogenously promote collagen and elastin. The results calculated are shown in Table 2.

[0052] Table 2 COX-2 inhibition rate and elastin and collagen promotion rate of samples in each group Group / property COX-2 inhibition rate / % Collagen promotion rate / % Elastin promotion rate / % Example 1 62.3 73.2 53.2 Example 2 61.7 71.3 51.5 Example 3 62.0 70.1 50.5 Example 4 55.2 64.5 47.2 Example 5 57.9 65.4 47.7 Comparative Example 1 34.9 45.1 36.8 Comparative Example 2 32.5 39.7 31.4 Comparative Example 3 37.8 42.0 33.7 Comparative Example 4 36.4 38.2 30.1 Comparative Example 5 50.8 62.7 45.7 Comparative Example 6 53.4 61.5 44.7 Comparative Example 7 46.1 57.9 42.4 Comparative Example 8 39.4 44.0 35.1 Comparative Example 9 36.2 43.8 34.0 Comparative Example 10 54.8 60.5 44.3 As can be seen from Table 2, in combination with the data of Examples 1-5, when the weight ratio of sea fennel extract, pomegranate peel extract, lysozyme and coenzyme Q10 is in the range of (1-2):(1-1.5):(0.5-1.5):(1-1.5), the anti-inflammatory and collagen synthesis promoting performance of the composition is at a relatively optimal level.

[0053] In combination with the data of Example 1 and Comparative Examples 1-4, it can be seen that the anti-inflammatory and collagen synthesis promoting performance of the compositions of Comparative Examples 1-4, each of which lacks one of sea fennel extract, pomegranate peel extract, lysozyme and coenzyme Q10, is significantly lower than that of Example 1, which may be because sea fennel extract, pomegranate peel extract, lysozyme and coenzyme Q10 can inhibit the activity of pro-inflammatory factors such as cyclooxygenase (COX-2) by synergistic inhibition, thereby reducing the inflammatory response from the source; and at the same time, enhance cell energy metabolism and activate collagen synthesis enzyme activity.

[0054] In combination with the data of Example 1 and Comparative Examples 5-6, it can be seen that in Comparative Example 5, Lactobacillus plantarum 1 is replaced by Lactobacillus plantarum 2 in the fermentation step of sea fennel extract, and in Comparative Example 6, Leuconostoc mesenteroides 1 is replaced by Leuconostoc mesenteroides 2 in the fermentation step of sea fennel extract, and the anti-inflammatory and collagen synthesis promoting performance of the compositions of Comparative Examples 5-6 is lower than that of Example 1, which may be because the selection of the species of Lactobacillus plantarum and Leuconostoc mesenteroides has a significant influence on the generation of SOD enzyme and small molecular phenolic substances in sea fennel extract, indicating that the selection of Lactobacillus plantarum 1 and Leuconostoc mesenteroides 1 for fermentation of sea fennel in the present application can not only improve the yield and stability of SOD enzyme, but also promote the generation of phenolic substances, thereby improving the anti-inflammatory effect and collagen synthesis promoting effect.

[0055] Similarly, in combination with the data of Example 1 and Comparative Example 7, it can be seen that in Comparative Example 7, Lactobacillus helveticus 1 is replaced by Lactobacillus helveticus 2 in the fermentation step of pomegranate peel extract, and its performance is also lower than that of Example 1, indicating that the selection of the species of Lactobacillus helveticus is also a major factor affecting the generation of lipase, protease and small molecular phenolic substances in pomegranate peel extract, so the selection of Lactobacillus helveticus 1 as the fermentation bacteria of pomegranate peel extract in the present application can further improve the anti-inflammatory and collagen synthesis activating effect of the composition.

[0056] From the data of Example 1 and Comparative Examples 8-9, it can be seen that the anti-inflammatory and collagen synthesis-activating effects of the compositions of Comparative Examples 8-9 are significantly lower than those of Example 1, which may be because Comparative Example 8 replaces Sea Fennel Extract 1 with Sea Fennel Extract 2, lacks a fermentation step, and thus the content of SOD enzyme and small-molecule phenolic substances is significantly reduced, and Comparative Example 9 replaces Pomegranate Fruit Extract 1 with Pomegranate Fruit Extract 2, resulting in a reduction in the content of protease, lipase, and gamma-aminobutyric acid, bacteriocin, thereby affecting the performance of the composition.

[0057] From the data of Example 1 and Comparative Example 10, it can be seen that Comparative Example 10 directly adds SOD enzyme to replace Sea Fennel Extract, and the anti-inflammatory and collagen synthesis-activating performance of the composition of Comparative Example 10 is lower than that of Example 1, which may be because Sea Fennel Extract can provide SOD enzyme in addition to phenolic substances, which can play an anti-inflammatory, antioxidant, and anti-early aging role.

[0058] Application Examples 1-5 and Comparative Application Examples 1-10 The multi-enzyme synergistic skin care compositions of Examples 1-5 and Comparative Examples 1-10 are added to the serum at a concentration of 3 wt%, to obtain the serum of Application Examples 1-5 and Comparative Application Examples 1-10, and the formula is shown in Table 3.

[0059] The preparation method of the serum of Application Examples 1-5 and Comparative Application Examples 1-10 comprises the following steps: S1, mix the humectant, thickening agent, and 1 / 2 amount of deionized water, and homogenize at 80°C; S2, after the temperature of the aqueous solution in S1 is reduced to 60°C, add the preservative, and when the system temperature is reduced to 40°C, add each component of the multi-enzyme synergistic skin care composition suitable for sensitive skin and the remaining deionized water, stir uniformly, and finally add the pH adjuster to obtain the serum.

[0060] Table 3 Formula of the serum of Application Examples 1-5 and Comparative Application Examples 1-10

[0061] Comparative Application Example 11 The serum of Comparative Application Example 11 does not add the multi-enzyme synergistic composition, and an equal amount of deionized water is used instead of the composition, and the preparation method is the same as that of Application Example 1.

[0062] Test Example 3: Sensitive Skin Redness Relief, Barrier Improvement, Anti-aging, and Sensitivity Reduction Test Experimental method: According to the General Principles of Safety Testing of Cosmetics, volunteers were enrolled. 64 Asian adult volunteers aged 18-60 years who felt sensitive to their skin were selected, and the lactic acid stinging score was ≥ 3 points. They were randomly divided into 16 groups, 4 people in each group. On the day of the visit, the test area was washed with water, and no product was applied. The volunteers sat in an air-conditioned room with a temperature of 21±1℃ and a humidity of 50±10% for 20 minutes. The Tewameter TM Hex probe and Colorimeter CL440 skin color probe were used to test the zygomatic transdermal water loss (TEWL) and the degree of facial redness a* value. Then the VISIA instrument was used to take pictures of the face of the subjects, and the number of under-eye fine lines was evaluated. After using a 10% lactic acid solution patch (diameter 0.8 cm double-layer filter paper) on the nasolabial fold of each subject for 5 minutes, the facial sensitivity to lactic acid stimulation was evaluated, and the sensitivity score was evaluated using a 4-point questionnaire. The questionnaire content is: How do you think the facial stinging score is now? 0-no feeling, 1-mild stinging, 2-obvious stinging, 3-severe stinging.

[0063] Test time point: The subjects used the serum twice a day for 28 days, and then returned for a visit. The zygomatic TEWL and moisture content were tested.

[0064] The improvement rate of each skin parameter before and after use = | mean after use - mean before use | / mean before use x 100%; The improvement effect on the barrier is represented by the TEWL improvement rate, the improvement effect on the redness is represented by the a* value improvement rate, the improvement effect on facial aging is represented by the number of under-eye fine lines improvement rate, and the improvement effect on skin sensitivity is represented by the lactic acid stinging improvement rate. The calculation results are shown in Table 4.

[0065] Table 4 Human test results of each group of serum Group / property TEWL improvement rate / % Erythema improvement rate / % Fine wrinkle improvement rate / % Lactic acid stinging improvement rate / % Application Example 1 21.6 24.8 21.8 47.8 Application Example 2 19.3 22.5 17.3 44.0 Application Example 3 21.8 23.1 20.4 48.2 Application Example 4 17.2 19.0 14.7 33.9 Application Example 5 18.7 20.3 15.2 35.6 Comparative Application Example 1 6.1 6.2 9.5 13.2 Comparative Application Example 2 5.6 6.8 5.2 12.1 Comparative Application Example 3 9.3 10.4 7.6 16.7 Comparative Application Example 4 8.1 9.2 6.4 14.6 Comparative Application Example 5 14.0 16.2 13.7 30.9 Comparative Application Example 6 15.7 17.6 12.8 32.1 Comparative Application Example 7 12.3 14.9 11.8 24.6 Comparative Application Example 8 10.9 12.2 8.5 20.2 Comparative Application Example 9 6.5 8.8 8.1 13.9 Comparative Application Example 10 16.3 18.4 12.4 32.7 Comparative Application Example 11 3.2 2.1 1.3 5.2 As can be seen from Table 4, compared with the serum of Comparative Application Example 11 (blank control group without adding the composition), the repair, improvement of erythema and fine lines, and soothing effect of each serum are improved.

[0066] According to the data of Application Examples 1-5, when the weight ratio of the sea fennel extract, pomegranate peel extract, lysozyme and coenzyme Q10 is (1-2):(1-1.5):(0.5-1.5):(1-1.5), the serum has better soothing, repair, anti-inflammatory and anti-aging effects.

[0067] It can be seen from the data of application example 1 and comparative application examples 1-4 that the anti-inflammatory, repair and anti-aging properties of the composition in the serum are reduced if one of the components is missing, which may be because the present application builds a multi-dimensional synergistic skin care system through the synergy of sea fennel extract, pomegranate peel extract, lysozyme and coenzyme Q10, and realizes the soothing, repair, anti-inflammatory and anti-aging multiple functions of sensitive skin through multi-enzyme synergy and endogenous enzyme regulation.

[0068] It can be seen from the data of application example 1 and comparative application examples 5-6 that the soothing, repair, anti-inflammatory and anti-aging properties of the serum composition in comparative application examples 5-6 are lower than those in example 1, which indicates that Lactobacillus plantarum 1 and Leuconostoc mesenteroides 1 can further improve the yield and stability of SOD enzyme and generate phenolic substances, thereby improving the performance of the serum.

[0069] It can be seen from the data of application example 1 and comparative application example 7 that the soothing, repair, anti-inflammatory and anti-aging properties of the serum composition in comparative application example 7 are lower than those in example 1, which indicates that the species of Lactobacillus helveticus is also a main parameter affecting the performance of the serum.

[0070] It can be seen from the data of application example 1 and comparative application examples 8-9 that the soothing, repair, anti-inflammatory and anti-aging properties of the serum in comparative examples 8-9 are lower than those in example 1, which may indicate that if sea fennel is not fermented, the yield of SOD enzyme and phenolic substances is low, and if pomegranate is not fermented, it cannot successfully secrete extracellular protease and lipase, and the content of gamma-aminobutyric acid and bacteriocin is also reduced, thereby affecting the soothing, repair, anti-inflammatory and anti-aging effects of the serum.

[0071] It can be seen from the data of application example 1 and comparative application example 10 that the anti-inflammatory and collagen synthesis promoting properties of the composition in comparative example 10 are lower than those in example 1, which may be because sea fennel extract contains not only SOD enzyme but also phenolic substances, which can play an anti-inflammatory and antioxidant role, so it is verified that sea fennel extract, pomegranate peel extract, lysozyme and coenzyme Q10 can synergistically improve the soothing, repair, anti-inflammatory and anti-aging effects of the serum.

[0072] It can be seen from the data of application example 1 and comparative application example 10 that the anti-inflammatory and collagen synthesis promoting properties of the composition in comparative example 10 are lower than those in example 1, which may be because sea fennel extract contains not only SOD enzyme but also phenolic substances, which can play an anti-inflammatory and antioxidant role, so it is verified that sea fennel extract, pomegranate peel extract, lysozyme and coenzyme Q10 can synergistically improve the soothing, repair, anti-inflammatory and anti-aging effects of the serum. Figure 1 It can be seen that the serum of example 1 can significantly improve the facial redness and under-eye fine lines of the subjects within 28 days of use, and has soothing, repair and anti-aging effects.

[0073] In summary, the present application realizes the multiple functions of soothing, repairing, anti-inflammatory and anti-aging of sensitive skin by the composition through the regulation of endogenous enzyme by multi-enzyme synergy and enzymatic reaction.

[0074] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, but not to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A multi-enzyme synergistic skincare composition suitable for sensitive skin and its application, characterized in that, The product comprises the following components: sea fennel extract, pomegranate peel extract, lysozyme, and coenzyme Q10, wherein the weight ratio of sea fennel extract, pomegranate peel extract, lysozyme, and coenzyme Q10 is (0.5-3):(0.6-2):(0.5-2):(0.5-3); wherein the sea fennel extract is obtained by anaerobic fermentation of sea fennel with Leuconostoc mesenteroides and Lactobacillus plantarum, wherein the strain number of Lactobacillus plantarum is CICC 23185 and it was purchased from the China Industrial Microbial Culture Collection Center; the strain number of Leuconostoc mesenteroides is CICC 6055 and it was purchased from the China Industrial Microbial Culture Collection Center; the pomegranate peel extract is obtained by anaerobic fermentation of pomegranate fruit with Lactobacillus helveticus; the strain number of Lactobacillus helveticus is CICC 6024 and it was purchased from the China Industrial Microbial Culture Collection Center.

2. The multi-enzyme synergistic skincare composition for sensitive skin as described in claim 1, characterized in that, The weight ratio of the sea fennel extract, pomegranate peel extract, lysozyme and coenzyme Q10 is (1-2):(1-1.5):(0.8-1.5):(1-1.5).

3. The multi-enzyme synergistic skincare composition for sensitive skin as described in claim 1, characterized in that, The preparation method of the sea fennel extract includes the following steps: S1. After washing the sea fennel, add 5-10 times the weight of water to crush and pulp it to obtain sea fennel pulp, and then sterilize it. S2. Inoculate a mixed bacterial culture of Lactobacillus plantarum and Leuconostoc mesenteroides into the sterilized sea fennel pulp, and anaerobic ferment at 36-38℃ for 60-72 hours to obtain the fermentation product. S3. Filter the fermentation product to obtain the fermentation broth, and freeze-dry it under vacuum to obtain the sea fennel extract.

4. The multi-enzyme synergistic skincare composition for sensitive skin as described in claim 3, characterized in that, The inoculation amount of the mixed bacterial solution is 10-30% v / v of sea fennel paste, and the viable count of the mixed bacterial solution is 1×10⁻⁶. 9 ×10 9 CFU / mL, the ratio of viable Lactobacillus plantarum to Leuconostoc mesenteroides in the mixed bacterial solution is 1:(1-3).

5. The multi-enzyme synergistic skincare composition for sensitive skin as described in claim 1, characterized in that, The preparation method of the pomegranate peel extract includes the following steps: T1. After washing the pomegranate peel, add 2.5-3.5 times the weight of water to crush and pulp it to obtain pomegranate peel pulp, and then sterilize it. T2. Inoculate the sterilized pomegranate peel pulp with Lactobacillus helveticus culture and anaerobic ferment at 36-42℃ for 48-96 hours to obtain the fermentation product. T3. Filter the fermentation product to obtain the fermentation broth, and freeze-dry it under vacuum to obtain the pomegranate peel extract.

6. The multi-enzyme synergistic skincare composition for sensitive skin as described in claim 5, characterized in that, The inoculation amount of the *Lactobacillus helveticus* culture was 2-10% v / v of pomegranate peel pulp, and the viable count of the *Lactobacillus helveticus* culture was 2.5 × 10⁻⁶. 8 ×10 8 CFU / mL.

7. The use of the multi-enzyme synergistic skincare composition for sensitive skin according to any one of claims 1-6 in the preparation of cosmetics, characterized in that, The cosmetic is a toner, lotion, cream, mask, serum, or spray, and the amount of the composition added is 0.5%-5% of the total weight of the cosmetic.

8. An essence, characterized in that, The raw materials include the following weight percentages: 0.5%-5% of the multi-enzyme synergistic skin care composition suitable for sensitive skin according to any one of claims 1-6, 0.5%-10% of moisturizer, 0.5%-1% of thickener, 0.05%-0.3% of preservative and 0.01%-0.3% of pH adjuster, with the balance being deionized water.

9. The essence as described in claim 8, characterized in that, The raw material is selected from at least one of (a)-(d): (a) The thickener comprises at least one of xanthan gum, carbomer, hydroxyethyl acrylate / sodium acryloyldimethyl taurate copolymer, ammonium acryloyldimethyl taurate / VP copolymer and sclerotium gum; (b) The moisturizer comprises at least one of allantoin, betaine, beta-glucan, trehalose, caprylyl glycol, dipropylene glycol, sodium hyaluronate, 1,3-butanediol, glycerin, D-panthenol and ceramide; (c) The pH adjuster includes at least one of arginine, tromethamine, and disodium EDTA; (d) The preservative includes at least one of 1,3-propanediol, 1,2-hexanediol and p-hydroxyacetophenone.

10. The method for preparing the essence according to claim 8, characterized in that, Includes the following steps: (1) Mix the humectant, thickener and part of deionized water, homogenize at 75-85℃, add the preservative, stir evenly to obtain a mixture; (2) When the temperature of the mixture in (1) drops to 35-45℃, add each component of the multi-enzyme synergistic skin care composition suitable for sensitive skin and the remaining deionized water, stir evenly, and finally add pH adjuster to adjust pH to obtain the essence.

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