A composite microbial fermentation stock solution for repairing skin immune barrier, a preparation method and a skin care composition

Abstract

The application discloses a compound microbial fermentation stock solution for repairing skin immune barrier, a preparation method and a skin care composition, relates to the field of skin care, and is prepared from a compound microbial strain, a fermentation substrate, natural nutritional additives, a pH regulator and a natural protective agent. Through an aerobic and anaerobic alternating fermentation process and in combination with a natural protective agent, the fermentation stock solution rich in active ingredients and stable is obtained, and is compounded with a skin repair agent, a moisturizing agent and the like to form the skin care composition. The application synergistically regulates skin microecological balance, enhances skin immune cell activity and repairs the stratum corneum, can effectively increase the thickness of the stratum corneum, improve the skin water content, reduce the trans-epidermal water loss, and promote the proliferation of beneficial bacteria and inhibit harmful bacteria. The product is mild and non-irritating, has a pH value consistent with human skin, is suitable for the fields of sensitive skin, barrier damaged skin and medical skin care products, and has the advantages of short repair period and long-term stability.

Description

Technical Field

[0001] This invention relates to the field of skincare technology, and in particular to a complex microbial fermentation stock solution for repairing the skin's immune barrier, its preparation method, and a skincare composition. Background Technology

[0002] The skin is the largest immune organ in the human body. Its immune barrier is mainly composed of the stratum corneum, sebum film, skin surface micro-ecosystem, and immune cells. Its function is to resist external environmental stimuli, prevent pathogen invasion, and maintain the homeostasis of the skin's internal environment. When the skin's immune barrier is damaged, symptoms such as sensitivity, redness, dryness, peeling, and itching may occur. In severe cases, it can induce skin diseases such as eczema and rosacea. With environmental degradation, increased life stress, and more improper skin care practices, the number of people with damaged barriers is increasing, and the demand for skin care products with repair functions continues to rise.

[0003] Currently, skin barrier repair products on the market are mainly divided into two categories. One category consists of chemically synthesized products, which supplement the skin's nutritional needs from the outside by adding ingredients such as ceramides and hyaluronic acid to alleviate symptoms of barrier damage. These products are mostly exogenous supplements and cannot fundamentally mobilize the body's own immune function. Furthermore, some chemical ingredients are irritating and unsuitable for sensitive skin. The other category consists of biological products, which mainly use single microbial fermentation products or plant extracts as core ingredients. While they are relatively mild, they have drawbacks such as targeting only a single point of repair, weak skin microecological regulation capabilities, and limited repair effects.

[0004] Microorganisms play a crucial role in repairing the skin's immune barrier. Beneficial microorganisms colonizing the skin surface maintain microecological balance by forming a biological barrier, secreting active metabolites such as antimicrobial peptides and short-chain fatty acids, and regulating immune cell responses, thereby promoting stratum corneum repair and enhancing immune function. In recent years, microbial fermentation skincare products have become a research hotspot. However, existing products mostly use single-strain fermentation, resulting in limited active ingredients and making it difficult to achieve the synergistic effects of microecological regulation, barrier repair, and immune enhancement. Furthermore, the active ingredients in fermentation solutions have poor stability and are easily degraded during storage and processing. Additionally, some products still exhibit some irritation, leading to long repair cycles and unsatisfactory results for severely damaged skin barriers. The combination of fermentation solutions with other auxiliary ingredients is often unreasonable, easily causing antagonism, and there is a lack of precise tailoring solutions for different degrees of damage. Moreover, existing preparation processes suffer from low fermentation efficiency, insufficient extraction of active ingredients, and a high risk of contamination by other microorganisms, resulting in high product costs and hindering large-scale production. Summary of the Invention

[0005] This invention overcomes the shortcomings of existing technologies and provides a compound microbial fermentation stock solution for repairing the skin's immune barrier, a preparation method, and a skin care composition. It uses compound strains for synergistic fermentation, combined with optimized processes and natural protectants to achieve multi-target repair, improve product stability and gentleness, and is suitable for various types of damaged skin and medical skin care products.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a compound microbial fermentation stock solution for repairing the skin's immune barrier. By weight, the compound microbial fermentation stock solution comprises: 0.5-2.0 parts of compound microbial strains, 80-95 parts of fermentation substrate, 2-8 parts of natural nutrient additives, 0.1-0.5 parts of pH adjuster, and 0.1-0.3 parts of natural protectant;

[0007] The composite microbial strain comprises Lactobacillus plantarum, Lactobacillus rhamnosus, Streptococcus thermophilus, and yeast in a mass ratio of 2-3:1-3:1-2:1.

[0008] In a preferred embodiment of the present invention, the total viable count of the composite microbial strain is ≥1×10⁻⁶. 9 CFU / mL; the *Lactobacillus plantarum* strain is the strain with accession number CGMCC 1.2469, the *Lactobacillus rhamnosus* strain is the strain with accession number CGMCC 1.2467, the *Streptococcus thermophilus* strain is the strain with accession number CGMCC 1.3920, and the yeast is the *Saccharomyces cerevisiae* strain with accession number CGMCC 2.1191.

[0009] In a preferred embodiment of the present invention, the fermentation substrate is a mixture of water, glucose, soybean peptone, and yeast extract in a mass ratio of 75-85:3-8:2-4:1-3.

[0010] In a preferred embodiment of the present invention, the natural nutritional additive is a mixture of tremella polysaccharide, oat β-glucan, and licorice extract in a mass ratio of 1-2:1-3:1; the natural protective agent is a mixture of trehalose and vitamin E in a mass ratio of 1-3:1.

[0011] In a preferred embodiment of the present invention, the pH adjuster is citric acid or lactic acid, used to adjust the pH value to 5.5-6.5.

[0012] Secondly, the present invention provides a method for preparing a compound microbial fermentation stock solution for repairing the skin's immune barrier, comprising the following steps:

[0013] S1. Activation of strains: Lactobacillus plantarum, Lactobacillus rhamnosus, Streptococcus thermophilus, and yeast were inoculated into their respective culture media and cultured at constant temperature until the logarithmic growth phase to obtain activated strain solutions.

[0014] S2. Preparation of fermentation substrate: Mix water, glucose, soybean peptone and yeast extract evenly, sterilize at 115-125℃ for 20-30 min, and cool to 30-35℃ to obtain sterile fermentation substrate;

[0015] S3. Co-fermentation: Mix the activated bacterial cultures of the four strains and inoculate them into a sterile fermentation substrate. The inoculation amount is 3-5% of the weight of the fermentation substrate. Then add natural nutrient additives. Under the conditions of 32-35 ℃ and shaking speed of 120-150 r / min, carry out aerobic fermentation, anaerobic fermentation and aerobic fermentation in sequence. The total fermentation time is 48-72 h.

[0016] S4. Filtration and purification: After fermentation, the fermentation broth is centrifuged to remove the cells and impurities. The supernatant is then filtered and sterilized through a 0.22μm filter membrane to obtain the crude fermentation broth.

[0017] S5. Adjustment and Stabilization: Add a pH adjuster to the crude fermentation broth to adjust the pH value to 5.5-6.5, then add a natural protectant, refrigerate at 2-8 ℃ and let stand for 20-28 h, then filter to obtain the compound microbial fermentation stock broth.

[0018] In a preferred embodiment of the present invention, in step S1, the *Lactobacillus plantarum* and *Lactobacillus rhamnosus* are cultured anaerobically at 35-39 °C for 24-36 h using MRS medium; the *Streptococcus thermophilus* is cultured in TSB medium with shaking at 35-39 °C for 18-24 h; and the yeast is cultured in YPD medium with shaking at 28-32 °C for 24-30 h. In step S3, the aerobic fermentation, anaerobic fermentation, and aerobic fermentation times are as follows: aerobic fermentation for the first 15-25 h, anaerobic fermentation for the middle 15-25 h, and aerobic fermentation for the last 15-25 h.

[0019] Thirdly, the present invention provides a skin care composition, which, by weight, comprises: 10-30 parts of the compound microbial fermentation stock solution as described in any one of claims 1-5, 5-15 parts of the skin repair agent, 3-10 parts of the moisturizer, 1-5 parts of the emulsifier, 0.1-0.3 parts of the preservative, and 40-70 parts of water.

[0020] In a preferred embodiment of the present invention, the skin repair agent is a mixture of ceramide NP, squalane, and panthenol in a mass ratio of 2-4:1-3:1; the moisturizer is a mixture of hyaluronic acid, glycerin, and allantoin in a mass ratio of 1-3:2-4:1; the emulsifier is a mixture of cetearyl alcohol polyether-20 and glyceryl stearate in a mass ratio of 1-2:1-2; and the preservative is phenoxyethanol.

[0021] In a preferred embodiment of the present invention, the dosage form of the skin care composition is one of toner, serum, lotion, cream or mask liquid.

[0022] This invention addresses the shortcomings of the prior art and has the following beneficial effects:

[0023] (1) This invention provides a compound microbial fermentation stock solution for repairing the skin's immune barrier, a preparation method and a skin care composition. It uses compound strains for synergistic fermentation to produce a variety of active metabolites. It also uses an aerobic-anaerobic alternating process to improve the stability of the ingredients. When combined with a skin repair agent, it can work simultaneously on three targets: regulating the microecology, enhancing immunity and repairing the stratum corneum. It effectively solves the problems of single repair and easy loss of activity in existing products. It can significantly increase the thickness of the stratum corneum and the moisture content of the skin, reduce the epidermal water loss rate, thereby achieving gentle and long-lasting repair for sensitive skin, shortening the repair cycle and improving the microecological balance.

[0024] (2) This invention uses a compound strain of Lactobacillus plantarum, Lactobacillus rhamnosus, Streptococcus thermophilus, and yeast in a specific ratio for synergistic fermentation. The metabolites of these strains can complement each other, achieving multi-target joint regulation of microecological balance, immune activity, and physical barriers. Consequently, the fermentation stock solution can simultaneously promote the proliferation of beneficial bacteria, enhance the skin's immune response, and accelerate the reconstruction of the stratum corneum. Compared with single-strain fermentation products, the compound system of this invention can avoid the defects of single repair targets, enabling the skin's immune barrier to achieve systematic repair from the inside out, thus providing a more lasting stabilizing effect on chronic barrier problems such as seasonal redness and recurring sensitivity.

[0025] (3) This invention improves the stability of the fermentation liquid by alternating aerobic and anaerobic fermentation processes and combining them with natural preservatives. The aerobic stage promotes the synthesis of antioxidant enzymes and vitamins by yeast and Streptococcus thermophilus, while the anaerobic stage induces Lactobacillus to produce large amounts of lactic acid and bacteriocins. The alternating conditions stimulate the conversion of the strain's metabolic pathways, generating more types of active small molecules. Trehalose can form a glassy protective layer on the surface of active ingredients, while vitamin E captures free radicals and prevents the oxidative degradation of unsaturated metabolites, thereby maintaining the retention rate of active ingredients in the fermentation liquid during storage. Compared with the current situation where existing microbial fermentation products require short cold chain storage due to the easy deactivation of active substances, this invention can effectively improve its stability and simplify storage and transportation requirements, thereby ensuring that the skin care composition maintains high-efficiency repair capabilities throughout its shelf life at room temperature.

[0026] (4) The skincare composition of this invention combines a complex microbial fermentation solution with a skin repair agent composed of ceramide NP, squalane, and panthenol. The immunologically active peptides and short-chain fatty acids in the fermentation solution promote the synthesis of lipids by keratinocytes from the endogenous source, while ceramide NP and squalane directly replenish the lipid components missing in the stratum corneum from the exogenous source. Panthenol accelerates epidermal healing by promoting fibroblast migration. The two pathways work simultaneously to effectively increase the thickness of the stratum corneum and the moisture content of the skin, and reduce the transepidermal water loss rate. Compared with the simple mixing or even antagonism of fermentation components and chemical repair agents in existing compositions, the formulation design of this invention can achieve synergistic effects of exogenous supplementation and endogenous repair, thereby shortening the repair cycle of severely damaged skin and reducing the probability of sensitivity recurrence.

[0027] (5) This invention uses all-natural raw materials to prepare fermentation liquid and skin care composition, and controls the pH value of the final product between 5.5 and 6.5, which is consistent with the slightly acidic environment of the surface of healthy human skin. It is suitable for highly fragile skin such as sensitive skin, infants and young children, and skin after medical aesthetic procedures. Compared with the defects of existing chemically synthesized repair products that may cause burning or stinging reactions, this invention has obvious advantages in biocompatibility, thereby broadening the application population and usage scenarios of the product. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a schematic diagram of the preparation method of a compound microbial fermentation stock solution for repairing the skin immune barrier according to the present invention. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein. Therefore, the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0032] It should be noted that the raw materials, equipment and reagents used in this invention can all be purchased from the market or obtained through existing preparation methods.

[0033] In the following embodiments and comparative examples of the present invention, the composite microbial strains and culture media are described as follows:

[0034] In this invention, *Lactobacillus plantarum* and *Lactobacillus rhamnosus* were deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 11, 2000, with accession numbers CGMCC 1.2469 and CGMCC 1.2467, respectively. *Streptococcus thermophilus* was deposited at CGMCC on April 26, 2005, with accession number CGMCC 1.3920. *Saccharomyces cerevisiae* was deposited at CGMCC on November 10, 1965, with accession number CGMCC 2.1191.

[0035] The MRS medium consisted of: 2.0% glucose, 1.0% peptone, 0.5% yeast extract, 1.0% beef extract, 0.2% diammonium citrate, 0.5% sodium acetate, 0.05% magnesium sulfate, 0.02% manganese sulfate, and 0.1% Tween-80. The solvent was water, and the pH was adjusted to 6.2-6.4.

[0036] The TSB medium consists of: 1.7% tryptone, 0.3% soybean peptone, 0.25% glucose, 0.5% sodium chloride, and 0.25% dipotassium hydrogen phosphate, with water as the solvent and the pH adjusted to 7.2-7.4.

[0037] The YPD medium consists of 2.0% glucose, 2.0% peptone, and 1.0% yeast extract, with water as the solvent and the pH adjusted to 5.8-6.0.

[0038] Example 1:

[0039] This embodiment provides a microbial fermentation stock solution that repairs the skin's immune barrier and its preparation method. The microbial fermentation stock solution of this embodiment, by weight, consists of the following components: 1.0 part of compound microbial strain, 90 parts of fermentation substrate, 5 parts of natural nutrient additives, 0.2 parts of pH adjuster, and 0.2 parts of natural protectant.

[0040] The composite microbial strain was composed of *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, *Streptococcus thermophilus*, and *Saccharomyces cerevisiae* in a mass ratio of 2:2:1:1, with a total viable count of 1.2 × 10⁻⁶. 9CFU / mL. The fermentation substrate is a mixture of water, glucose, soybean peptone, and yeast extract in a mass ratio of 80:5:3:2. Natural nutritional additives are a mixture of tremella polysaccharide, oat β-glucan, and licorice extract in a mass ratio of 2:2:1. The pH adjuster is citric acid. The natural preservative is a mixture of trehalose and vitamin E in a mass ratio of 2:1.

[0041] like Figure 1 As shown, the preparation method of this embodiment includes the following steps:

[0042] S1. Activation of strains: Lactobacillus plantarum and Lactobacillus rhamnosus were inoculated into MRS medium and cultured anaerobically at 37 ℃ for 30 h; Streptococcus thermophilus was inoculated into TSB medium and cultured with shaking at 37 ℃ and 150 r / min for 20 h; Saccharomyces cerevisiae was inoculated into YPD medium and cultured with shaking at 30 ℃ and 150 r / min for 26 h to obtain activated strain solutions.

[0043] S2. Preparation of fermentation substrate: Water, glucose, soybean peptone and yeast extract are mixed evenly, sterilized by high pressure steam at 121 ℃ for 25 min, and cooled to 32 ℃ to obtain sterile fermentation substrate.

[0044] S3. Co-fermentation: The activated bacterial cultures of the four strains were mixed in a weight ratio of 2:2:1:1 and inoculated into the sterile fermentation substrate at an inoculation rate of 4% of the fermentation substrate weight. Natural nutrient additives were added, and aerobic-anaerobic alternating fermentation was carried out at 33 ℃ and 130 r / min for a total fermentation time of 72 h. Specifically, aerobic fermentation was carried out for the first 24 h, anaerobic fermentation for the middle 24 h, and aerobic fermentation for the last 24 h.

[0045] S4. Filtration and purification: After fermentation, the fermentation broth is centrifuged at 8000 r / min for 12 min to remove bacteria and impurities. The supernatant is then filtered and sterilized through a 0.22 μm filter membrane to obtain the crude fermentation broth.

[0046] S5. Adjustment and Stabilization: Add citric acid to the crude fermentation broth to adjust the pH to 6.0, then add a natural preservative, stir well, refrigerate at 4 ℃ for 24 h, and filter to obtain the original microbial fermentation broth.

[0047] Example 2:

[0048] This embodiment is basically the same as that of embodiment 1, except that the microbial fermentation stock solution obtained in embodiment 1 is made into a skin care essence.

[0049] The skin care essence in this embodiment, by weight, consists of the following components: 22 parts of microbial fermentation stock solution, 10 parts of skin repair agent, 6 parts of moisturizer, 3 parts of emulsifier, 0.2 parts of preservative, and 58.8 parts of water, as in Example 1.

[0050] The skin repair agent is a mixture of ceramide NP, squalane, and panthenol in a weight ratio of 3:2:1. The moisturizer is a mixture of hyaluronic acid, glycerin, and allantoin in a weight ratio of 2:3:1. The emulsifier is a mixture of cetearyl alcohol polyether-20 and glyceryl stearate in a weight ratio of 1:1. The preservative is phenoxyethanol.

[0051] The preparation method of this embodiment includes the following steps:

[0052] S1. Preparation of aqueous phase: Add water and humectant to an emulsification pot, heat to 82 ℃, stir evenly, and keep warm for 25 min to obtain the aqueous phase.

[0053] S2. Preparation of oil phase: Add emulsifier and skin repair agent to another container, heat to 78 ℃, stir evenly to obtain oil phase.

[0054] S3. Emulsification and mixing: Slowly add the oil phase to the water phase, stir and emulsify at 900 r / min for 18 min, and then cool to 48 ℃.

[0055] S4. Add fermentation stock solution: Add microbial fermentation stock solution and preservative to the emulsion, stir well, and continue to cool to 32 ℃.

[0056] S5. Quality Inspection and Packaging: The pH value of the product is tested to be 6.2, and the microbial limit meets the requirements of the "Cosmetic Safety Technical Specifications". After passing the test, the product is packaged to obtain the skin care essence.

[0057] Example 3:

[0058] This embodiment is basically the same as embodiment 1, except that the microbial fermentation liquid obtained in embodiment 1 is made into a skin care cream.

[0059] The skin cream of this embodiment has the following components by weight: 12 parts of microbial fermentation stock solution, 8 parts of skin repair agent, 8 parts of moisturizer, 4 parts of emulsifier, 0.2 parts of preservative, and 67.8 parts of water.

[0060] The composition of each component is the same as that of the skin care essence in Example 2.

[0061] The preparation method in this embodiment is basically the same as the preparation method of the skin care essence in Example 2, except that: in step S1, the temperature is heated to 85 ℃ and kept warm for 30 min; in step S3, the stirring speed is 1000 r / min and the emulsification time is 20 min; in step S4, the temperature is cooled to 45 ℃, and finally a skin care cream with a uniform texture is obtained.

[0062] Performance testing: The effects of the microbial fermentation stock solution prepared in Example 1, the skin care essence prepared in Example 2, and the skin care cream prepared in Example 3 were verified by the test.

[0063] Verification of the skin immune barrier repair effect: 120 volunteers aged 18-45 with damaged skin barrier were selected. All of them showed skin sensitivity, redness, dryness and peeling. They were randomly divided into 4 groups with 30 volunteers in each group.

[0064] Example 1: Use the microbial fermentation stock solution prepared therein, apply it to the face twice a day, morning and evening after cleansing.

[0065] Example 2: Use the skin care essence prepared therein, apply it to the face twice a day, morning and evening after cleansing.

[0066] Example 3: Use the skin care cream prepared therein, apply it to the face twice a day, morning and evening after cleansing.

[0067] Control group 1: No repair skin care products were used; skin was cleansed normally.

[0068] Control group 2: Use the existing single Lactobacillus plantarum fermentation stock solution skin care essence (using Lactobacillus plantarum CGMCC1.2469 instead of the compound strain of the present invention, and the rest of the preparation method is the same as in Example 1), apply to the face twice a day, morning and evening after cleansing.

[0069] All subjects used the product continuously for 28 days. Before and after 28 days of use, the skin stratum corneum thickness, skin moisture content, and transepidermal water loss (TEWL) of each group of volunteers were measured, and the improvement of symptoms was recorded. The effective rate of symptom improvement was defined as the percentage of volunteers whose skin sensitivity, redness, dryness, and desquamation symptoms were significantly improved. The results are shown in Table 1.

[0070] Table 1:

[0071]

[0072] Verification of the skin microecological regulation effect: Ten volunteers from Example 2 and Control Group 2 in the above experiment were selected. Facial skin surface microbial samples were collected before use and 28 days after use. The number of beneficial bacteria (Lactobacillus plantarum, Lactobacillus rhamnosus) and harmful bacteria (Staphylococcus aureus, Propionibacterium acnes) on the skin surface were detected. The results are shown in Table 2.

[0073] Table 2:

[0074]

[0075] As shown in Tables 1 and 2, compared with the existing single-strain products of Control Groups 1 and 2, the products of Examples 1-3 of this invention significantly increase the thickness of the stratum corneum and the skin's moisture content, reduce transepidermal water loss, effectively improve skin sensitivity symptoms, and significantly promote the proliferation of beneficial bacteria and inhibit harmful bacteria on the skin surface. This indicates that the fermented stock solution and skincare composition provided by this invention have excellent skin immune barrier repair capabilities and microecological regulation capabilities. Among them, the skincare essence prepared in Example 2 showed the best effect.

[0076] To further simplify and make the present invention achieve its objectives and effects, the present invention will be further illustrated in conjunction with the following specific embodiments and comparative examples, but the present invention is not limited to the scope of the embodiments described herein.

[0077] Example 4:

[0078] This embodiment is basically the same as embodiment 2, except that the amount of compound microbial strain used is different. Specifically, the compound microbial strain is 0.5 parts by weight in the microbial fermentation broth.

[0079] Example 5:

[0080] This embodiment is basically the same as embodiment 2, except that the amount of compound microbial strain used is different. Specifically, the compound microbial strain is 2.0 parts by weight in the microbial fermentation broth.

[0081] Example 6:

[0082] This embodiment is basically the same as embodiment 2, except that the inoculation amount of the bacterial strain liquid is different. Specifically, the bacterial strain liquid is inoculated into the sterile fermentation substrate at an inoculation amount of 3% of the weight of the fermentation substrate.

[0083] Example 7:

[0084] This embodiment is basically the same as embodiment 2, except that the inoculation amount of the bacterial strain liquid is different. Specifically, the bacterial strain liquid is inoculated into the sterile fermentation substrate at an inoculation amount of 5% of the weight of the fermentation substrate.

[0085] Example 8:

[0086] This embodiment is basically the same as embodiment 2, except that the amount of microbial fermentation stock solution used is different. Specifically, in the skin care composition, the amount of microbial fermentation stock solution is 10 parts by weight.

[0087] Example 9:

[0088] This embodiment is basically the same as embodiment 2, except that the amount of microbial fermentation stock solution used is different. Specifically, in the skin care composition, the amount of microbial fermentation stock solution is 30 parts by weight.

[0089] Comparative Example 1:

[0090] This comparative example is basically the same as Example 2, except that the total viable count of the composite microbial strain is different. Specifically, in step S3 of the fermentation stock preparation, the total fermentation time is shortened to 24 h, and alternating fermentation (entirely aerobic fermentation) is not performed. The total viable count of the final product is 5.0 × 10⁻⁶. 8 CFU / mL.

[0091] Comparative Example 2:

[0092] This comparative example is basically the same as Example 2, except that: alternating fermentation was not used. Specifically, when preparing the fermentation stock solution, the co-fermentation did not use an aerobic-anaerobic alternating process, and the entire process was carried out with aerobic fermentation for 72 hours.

[0093] Comparative Example 3:

[0094] This comparative example is basically the same as Example 2, except that the amount of the compound microbial strain is different. Specifically, the compound microbial strain is 0.2 parts by weight in the microbial fermentation stock solution.

[0095] Comparative Example 4:

[0096] This comparative example is basically the same as Example 2, except that the amount of the compound microbial strain is different. Specifically, the compound microbial strain is 2.5 parts by weight in the microbial fermentation stock solution.

[0097] Comparative Example 5:

[0098] This comparative example is basically the same as Example 2, except that the inoculation amount of the bacterial strain liquid is different. Specifically, the bacterial strain liquid is inoculated into the sterile fermentation substrate at an inoculation amount of 1% of the weight of the fermentation substrate.

[0099] Comparative Example 6:

[0100] This comparative example is basically the same as Example 2, except that the inoculation amount of the bacterial strain liquid is different. Specifically, the bacterial strain liquid is inoculated into the sterile fermentation substrate at an inoculation amount of 7% of the weight of the fermentation substrate.

[0101] Comparative Example 7:

[0102] This comparative example is basically the same as Example 2, except that the amount of microbial fermentation stock solution used is different. Specifically, in the skin care composition, the amount of microbial fermentation stock solution is 5 parts by weight.

[0103] Comparative Example 8:

[0104] This comparative example is basically the same as Example 2, except that the amount of microbial fermentation stock solution used is different. Specifically, in the skin care composition, the amount of microbial fermentation stock solution is 35 parts by weight.

[0105] Performance testing: The skin care essences prepared in Examples 2, 4-9 and Comparative Examples 1-8 were tested for efficacy. The results are shown in Table 3.

[0106] Ex vivo skin samples with damaged skin barriers from the same batch and source (provided by the hospital and approved by the ethics committee) were selected and used to prepare samples corresponding to the examples and comparative examples.

[0107] Skin repair effect: Using the same methods and standards as the above-mentioned skin immune barrier repair effect verification, after 28 days of continuous use, the skin stratum corneum thickness, skin moisture content and TEWL were measured, and the average improvement rate of each indicator was calculated.

[0108] Microecological regulation effect: Using the same methods and standards as the above-mentioned skin immune barrier repair effect verification, the increase in the ratio of beneficial bacteria to harmful bacteria on the skin surface was detected after 28 days of use.

[0109] Table 3:

[0110]

[0111]

[0112] As shown in Table 3:

[0113] A comparison of Example 2 and Comparative Example 1 reveals that insufficient fermentation time prevented the strains from fully entering the late logarithmic growth phase and stationary phase, thus hindering the accumulation of sufficient secondary metabolites such as lactic acid, bacteriocins, and glutathione. Furthermore, the lack of an anaerobic phase severely inhibited the acid-producing and bacteriocin-producing abilities of *Lactobacillus plantarum* and *Lactobacillus rhamnosus*, significantly weakening their effects on skin surface pH regulation and harmful bacteria inhibition. It also reduced the production of signaling molecules such as short-chain fatty acids that stimulate keratinocytes to synthesize ceramides. Therefore, Comparative Example 1's performance in physical barrier repair and microecological regulation was far inferior to that of Example 2.

[0114] A comparison between Example 2 and Comparative Example 2 reveals that although Comparative Example 2 maintained sufficient fermentation time, it employed aerobic fermentation throughout, leading to a metabolic imbalance in the strains. Under these conditions, the facultative anaerobic Lactobacillus species (Lactobacillus plantarum and Lactobacillus rhamnosus) primarily engaged in aerobic respiration, inhibiting their key metabolic pathway for efficient lactic acid and bacteriocin production via glycolysis. The reduced lactic acid production weakens their ability to directly replenish lactate in the skin's natural moisturizing factor (NMF) and diminishes their role in maintaining a slightly acidic environment to promote beneficial bacterial colonization. Simultaneously, the production of bacteriocins and various immunomodulatory peptides sharply decreased, resulting in a decline in direct killing of harmful bacteria and immune regulation. Although the aerobic phase promoted the production of some antioxidant enzymes in yeast and Streptococcus thermophilus, the lack of diverse active small molecules from the anaerobic phase resulted in insufficient deep regulation of skin immune cell activity.

[0115] A comparison of Examples 2, 4-5, and Comparative Examples 3-4 shows that an appropriate amount of the compound microbial strains can ensure the formation of an optimal microbial niche and metabolic synergy network in the fermentation system. In Comparative Example 3, the dosage was too low, leading to a worse situation with extremely low concentrations of active ingredients in the fermentation broth, failing to effectively trigger skin immune responses and physical barrier reconstruction. Conversely, in Comparative Example 4, the dosage was too high, easily triggering strong interspecies competition, such as rapid nutrient consumption and accumulation of inhibitory metabolites, causing the strains to prematurely enter their decline phase. Some beneficial metabolic pathways, such as the synthesis of specific antimicrobial peptides, were even inhibited. Simultaneously, bacterial autolysis may release excessive pro-inflammatory cell wall components, posing a potential irritation risk to already damaged skin barriers, manifesting as stagnant performance improvement or even slight fluctuations in some indicators.

[0116] A comparison of Examples 2, 6-7, and Comparative Examples 5-6 reveals that an appropriate inoculum size is crucial for optimizing fermentation kinetics and metabolite profiles. Example 6 had a low inoculum size, resulting in an excessively long fermentation lag phase. Within the fixed total fermentation time, the microbial community could not reach a high-density stable phase, leading to insufficient production of active ingredients such as SOD, glutathione, and extracellular polysaccharides, which require high cell density to accumulate in large quantities. Therefore, the effects of increasing skin hydration and reducing TEWL were weaker than in Example 2. Comparative Example 5 had an extremely low inoculum size, resulting in slow microbial growth and an increased risk of contamination by other microorganisms, leading to a high risk of fermentation failure and a sharp decline in performance. In contrast, Comparative Example 6 had an excessively high inoculum size, leading to an excessive initial biomass and rapid accumulation of metabolic waste, such as excessive organic acids. The pH value dropped excessively in the early stages of fermentation, inhibiting the activity of Streptococcus thermophilus and yeast, disrupting the proper metabolic transition rhythm of aerobic-anaerobic alternating fermentation, reducing metabolite diversity, and increasing the risk of irritation from excessive cell lysis in the later stages, thus failing to achieve sustained performance improvement.

[0117] A comparison of Examples 2, 8-9, and Comparative Examples 7-8 reveals that the appropriate amount of microbial fermentation stock solution is key to balancing efficacy and safety. Example 8 used a relatively low amount in its skincare composition. While the compound fermentation stock solution provided core immune regulation and microecological control signals, its concentration was insufficient to produce a saturating effect in terms of exogenous physical barrier repair and long-term moisturizing. In particular, certain immunomodulatory peptides and β-glucan molecules, which require threshold concentrations to activate cell surface receptors (such as Toll-like receptors), were insufficiently present, failing to fully initiate the repair processes of keratinocytes and immune cells. This resulted in a lower rate of increase in stratum corneum thickness and lower microecological regulation capacity compared to Example 2, and even worse results in Comparative Example 7. Conversely, Example 9, with a slightly increased amount, achieved performance almost on par with Example 2. However, further increases to the level of Comparative Example 8 resulted in saturation of performance improvement. Excessive fermentation stock solution usage introduces excessive amounts of amino acids, sugars, and other nutrients, which may not only disrupt the stability of the formulation's preservative system and increase the risk of microbial contamination but may also provide additional nutrients for non-target harmful bacteria, ultimately hindering the long-term stability of the skin's microecology. At the same time, excessive active peptides may exceed the tolerance threshold in some individuals, turning from immune regulation to immune stimulation, which may cause a slight stinging sensation or discomfort, contrary to the product's original design intention of being gentle and non-irritating.

[0118] The above description is based on the preferred embodiments of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects. The scope of the invention is defined by the appended claims rather than the foregoing description, and all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0119] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A compound microbial fermentation stock solution for repairing the skin's immune barrier, characterized in that, By weight, the compound microbial fermentation stock solution comprises: 0.5-2.0 parts of compound microbial strains, 80-95 parts of fermentation substrate, 2-8 parts of natural nutrient additives, 0.1-0.5 parts of pH adjuster, and 0.1-0.3 parts of natural protectant; The composite microbial strain comprises Lactobacillus plantarum, Lactobacillus rhamnosus, Streptococcus thermophilus, and yeast in a mass ratio of 2-3:1-3:1-2:

1.

2. The compound microbial fermentation stock solution for repairing the skin's immune barrier according to claim 1, characterized in that, The total viable count of the composite microbial strain is ≥1×10⁻⁶. 9 CFU / mL; the *Lactobacillus plantarum* strain is the strain with accession number CGMCC1.2469, and the *Lactobacillus rhamnosus* strain is the strain with accession number CGMCC. The strain 1.2467, the thermophilic streptococcus, is preserved under accession number CGMCC. The yeast strain 1.3920 is a Saccharomyces cerevisiae strain with preservation number CGMCC 2.1191.

3. The compound microbial fermentation stock solution for repairing the skin's immune barrier according to claim 1, characterized in that, The fermentation substrate is a mixture of water, glucose, soybean peptone, and yeast extract in a mass ratio of 75-85:3-8:2-4:1-3.

4. The compound microbial fermentation stock solution for repairing the skin's immune barrier according to claim 1, characterized in that, The natural nutritional additive is a mixture of tremella polysaccharide, oat β-glucan, and licorice extract in a mass ratio of 1-2:1-3:1; the natural protective agent is a mixture of trehalose and vitamin E in a mass ratio of 1-3:

1.

5. The compound microbial fermentation stock solution for repairing the skin's immune barrier according to claim 1, characterized in that, The pH adjuster is citric acid or lactic acid, used to adjust the pH value to 5.5-6.

5.

6. A method for preparing a compound microbial fermentation stock solution for repairing the skin's immune barrier, characterized in that, Includes the following steps: S1. Activation of strains: Lactobacillus plantarum, Lactobacillus rhamnosus, Streptococcus thermophilus, and yeast were inoculated into their respective culture media and cultured at constant temperature until the logarithmic growth phase to obtain activated strain solutions. S2. Preparation of fermentation substrate: Mix water, glucose, soybean peptone and yeast extract evenly, sterilize at 115-125 ℃ for 20-30 min, and cool to 30-35 ℃ to obtain sterile fermentation substrate; S3. Co-fermentation: Mix the activated bacterial cultures of the four strains and inoculate them into a sterile fermentation substrate. The inoculation amount is 3-5% of the weight of the fermentation substrate. Then add natural nutrient additives. Under the conditions of 32-35 ℃ and shaking speed of 120-150 r / min, carry out aerobic fermentation, anaerobic fermentation and aerobic fermentation in sequence. The total fermentation time is 48-72 h. S4. Filtration and purification: After fermentation, the fermentation broth is centrifuged to remove the cells and impurities. The supernatant is then filtered and sterilized through a 0.22 μm filter membrane to obtain the crude fermentation broth. S5. Adjustment and Stabilization: Add a pH adjuster to the crude fermentation broth to adjust the pH value to 5.5-6.5, then add a natural protectant, refrigerate at 2-8 ℃ and let stand for 20-28 h, then filter to obtain the compound microbial fermentation stock broth.

7. The method for preparing a compound microbial fermentation stock solution for repairing the skin's immune barrier according to claim 6, characterized in that, In step S1, the *Lactobacillus plantarum* and *Lactobacillus rhamnosus* are cultured anaerobically at 35-39°C for 24-36 h using MRS medium; the *Streptococcus thermophilus* is cultured in TSB medium with shaking at 35-39°C for 18-24 h; and the yeast is cultured in YPD medium with shaking at 28-32°C for 24-30 h. In step S3, the aerobic fermentation, anaerobic fermentation, and aerobic fermentation times are as follows: aerobic fermentation for the first 15-25 h, anaerobic fermentation for the middle 15-25 h, and aerobic fermentation for the last 15-25 h.

8. A skincare composition, characterized in that, The skincare composition comprises, by weight, 10-30 parts of the compound microbial fermentation stock solution as described in any one of claims 1-5, 5-15 parts of the skin repair agent, 3-10 parts of the moisturizer, 1-5 parts of the emulsifier, 0.1-0.3 parts of the preservative, and 40-70 parts of water.

9. A skincare composition according to claim 8, characterized in that, The skin repair agent is a mixture of ceramide NP, squalane, and panthenol in a mass ratio of 2-4:1-3:1; the moisturizer is a mixture of hyaluronic acid, glycerin, and allantoin in a mass ratio of 1-3:2-4:1; the emulsifier is a mixture of cetearyl alcohol polyether-20 and glyceryl stearate in a mass ratio of 1-2:1-2; and the preservative is phenoxyethanol.

10. A skincare composition according to claim 8, characterized in that, The skincare composition is in the form of one of the following: toner, serum, lotion, cream, or mask.

Images