Fermented emulsion as well as preparation method and application thereof

Abstract

The invention provides a preparation method of a fermented emulsion, which specifically comprises the following steps: inoculating lactobacillus plantarum and lactobacillus bulgaricus into a fermentation substrate comprising wheat seeds, soybeans, vegetable oil and water, and carrying out fermentation culture and sterilization to obtain the fermented emulsion, wherein the mass ratio of the wheat seeds to the soybeans to the vegetable oil is 1: 1: (0.5-4); the ratio of the viable count of the lactobacillus plantarum to the viable count of the lactobacillus bulgaricus is 1 to (0.2 to 5), and the ratio of the viable count of the lactobacillus plantarum to the viable count of the lactobacillus bulgaricus is 1 to (0.2 to 5). Loss of active ingredients can be reduced through compound fermentation of lactobacillus plantarum and lactobacillus bulgaricus, a better skin care effect can be better played, good light damage repairing, anti-inflammatory and anti-aging effects can still be shown under the condition that ingredients such as an emulsifier, a thickening agent and a surfactant are not added, stability is good, and the skin care cream is suitable for being used for skin care. Sensory evaluation is good, the preparation process is simple, the production cost is saved, the use safety is high, and skin allergy is not easily caused.

Description

Technical Field

[0001] This application belongs to the field of cosmetic technology, and in particular relates to a fermented emulsion, its preparation method and application. Background Technology

[0002] With the rapid development of science and technology and the improvement of people's material living conditions, the aging population is constantly intensifying. Actively exploring and developing beauty and skincare products is of great significance in mitigating the aging of the population. As people age, their skin condition deteriorates, exacerbated by external factors such as air pollution and ultraviolet radiation. Therefore, skincare becomes crucial.

[0003] Emulsion is a liquid cream-type skincare product, named for its milky white appearance. Emulsions offer excellent moisturizing and hydrating effects, but traditional emulsion preparation is complex and requires the addition of various emulsifiers, thickeners, and neutralizers. Besides emulsifiers, many auxiliary materials are needed, such as oils (mineral oil, silicone oil) for the oil phase, carbomer and triethanolamine for thickeners, and methylisothiazolinone and diazolidinyl urea for preservatives. While creams and emulsions made from these industrially synthesized raw materials may excel in appearance, absorption, and stability, their safety and efficacy remain highly questionable, and some people may even experience allergic reactions. With societal development, only natural, highly effective, and high-tech beauty products will survive. In the 21st century, skincare products have begun to shift from traditional fine chemical beauty to bio-based beauty. The future development trend of emulsions will undoubtedly be inseparable from this bio-based skincare method.

[0004] Currently, traditional emulsions on the market often contain excessive emulsifiers and thickeners during the emulsion preparation process. These added chemicals can burden the skin and do not provide any skincare benefits. Fermentation technology, however, can avoid these drawbacks. Furthermore, the extracellular polysaccharides of lactic acid bacteria have good skincare effects.

[0005] Vegetable oils contain unsaturated fatty acids, have a low melting point, are liquid at room temperature, have a high digestibility and absorption rate, and a fat content of over 99%. In addition, they are rich in vitamin E and contain small amounts of potassium, sodium, calcium, and trace elements. Due to these properties, vegetable oils are widely used in the production of skin care products and cosmetics, especially as an important ingredient in lotions.

[0006] However, current technology involves directly applying plant oils as components in various skincare products. Since plant oils are natural high-molecular-weight compounds composed of fatty acids and glycerol, they are not composed of a single chemical structure. This means that plant oils cannot be fully absorbed by the human skin. The main reason that affects the skin feel of plant oil skincare products is that there are a small number of free macromolecular free groups in plant oils that are not easily absorbed by the skin.

[0007] Currently, there are many fermentation processes in China, but there is still a lack of a method that can simultaneously ferment vegetable oil as a fermentation substrate and microbial inoculum. As a common raw material in daily life, this process has the characteristic of being able to ferment high-quality products, and it has good potential at present. Summary of the Invention

[0008] The technical problem this application aims to solve is to overcome the shortcomings of existing technologies where traditional emulsion preparation processes are complex, requiring the addition of various industrial synthetic emulsifiers, thickeners, neutralizers, or other additives for emulsification, resulting in low safety, poor human absorption, and the potential for allergies. This application provides a fermented emulsion, its preparation method, and its applications. Compared to traditional emulsions, the fermented emulsion obtained by this application exhibits excellent moisturizing, anti-inflammatory, and anti-aging effects without the need for additives such as emulsifiers, thickeners, neutralizers, or other additives. It also demonstrates good stability, positive sensory evaluation, a simple preparation process, reduced production costs, high safety in use, and is less likely to cause skin allergies.

[0009] This application adopts the following technical solution to solve the above-mentioned technical problems:

[0010] This application provides a method for preparing a fermented emulsion, specifically including the following steps:

[0011] Lactobacillus plantarum and Lactobacillus bulgaricus were inoculated into a fermentation substrate consisting of wheat seeds, soybeans, vegetable oil and water, and the mixture was fermented, sterilized, and the fermented emulsion was obtained.

[0012] The wheat seed, soybean, and vegetable oil are in a mass ratio of 1:1:(0.5-4); the viable count ratio of Lactiplantibacillus plantarum and Lactobacillus bulgaricus is 1:(0.2-5).

[0013] In some embodiments, the wheat seeds and soybeans may be commercially available conventional wheat seeds and soybeans, such as Heilongjiang soybeans and Henan wheat.

[0014] In some embodiments, the wheat seeds may be further crushed and sieved before use, and the sieve mesh size of the wheat seed powder may be 30 to 100 mesh, preferably 50 mesh.

[0015] The soybeans may be further crushed and sieved before use, and the sieve mesh size of the soybean powder may be 30 to 100 mesh, preferably 50 mesh.

[0016] In some embodiments, the vegetable oil may include at least one of macadamia seed oil, prickly pear seed oil, peony seed oil, meadowfoam seed oil, sunflower seed oil, shea butter, crepe butter, sweet almond oil, flaxseed oil, coconut oil, and coix seed oil.

[0017] In some embodiments, the mass ratio of wheat seed, soybean and vegetable oil is 1:(0.5-4):(1-10): preferably 1:1:(1-8);

[0018] In some embodiments, the mass-to-volume ratio of the wheat seeds, soybeans, and water may be 1:1:(20-150) g / mL; more preferably, it is 1:(20-100) g / mL.

[0019] In some embodiments, the fermentation substrate may also be subjected to sterilization procedures, which are conventional in the art, before use.

[0020] The sterilization conditions and methods for the fermentation substrate can be the conventional conditions and methods for this type of operation in the art, and generally can be high-temperature sterilization.

[0021] When the fermentation substrate is sterilized using the high-temperature sterilization method, the sterilization temperature can be the conventional temperature for this type of operation in the art, preferably 110-125°C, more preferably 115-121°C, for example 121°C.

[0022] When the fermentation substrate is sterilized using the high-temperature sterilization method, the sterilization pressure can be the conventional pressure for this type of operation in the art, preferably 0.1 to 0.14 MPa, more preferably 0.1 to 0.13 MPa, for example 0.12 MPa.

[0023] When the fermentation substrate is sterilized using the high-temperature sterilization method, the sterilization time can be the conventional operation time for this type of operation in the art, preferably 20-60 min, more preferably 20-40 min, for example 30 min.

[0024] The fermentation substrate may further include a cooling process after sterilization, typically cooling to room temperature.

[0025] In some embodiments, the live count ratio of *Lactobacillus plantarum* to *Lactobacillus bulgaricus* is preferably 1:(0.5-2).

[0026] In some embodiments, the Lactiplantibacillus plantarum may include Lactiplantibacillus plantarum deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNO.30350.

[0027] In some embodiments, based on the volume of deionized water in the initial fermentation system, the *Lactobacillus plantarum* can be added in the form of a *Lactobacillus plantarum* liquid, as is customary in the art, wherein the *Lactobacillus plantarum* liquid contains 10⁻⁶ viable bacteria. 10 ~10 14 CFU / mL, preferably 10 10 ~10 12 CFU / mL.

[0028] In some embodiments, based on the volume of deionized water in the initial fermentation system, the number of *Lactobacillus plantarum* inoculated per unit volume of the fermentation substrate can be conventional in the art, preferably 10. 8 ~10 14 CFU / mL, preferably 10 8 ~10 12 CFU / mL.

[0029] In some embodiments, the Lactobacillus bulgaricus may include Lactobacillus bulgaricus deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNO.28800.

[0030] In some embodiments, based on the volume of deionized water in the fermentation substrate, the *Lactobacillus bulgaricus* can be added in the form of *Lactobacillus bulgaricus* as conventionally practiced in the art, and the viable count of the *Lactobacillus bulgaricus* culture is 10-1. 10 ~10 14 CFU / mL, preferably 10 10 ~10 12 CFU / mL.

[0031] In some embodiments, the number of *Lactobacillus bulgaricus* inoculated per unit volume of the fermentation substrate, based on the volume of deionized water in the fermentation substrate, can be conventional in the art, preferably 10. 8 ~10 14 CFU / mL, preferably 10 8 ~10 12 CFU / mL.

[0032] In some embodiments, the fermentation culture can be carried out in a shaking incubator in accordance with conventional practices in the art, wherein the rotation speed of the shaker in the shaking incubator can be 200-400 r / min, preferably 250-350 r / min, for example 300 r / min.

[0033] In some embodiments, the fermentation culture temperature may be 35–45°C, preferably 37–43°C, and more preferably 40°C.

[0034] In some embodiments, the fermentation culture time may be 12-72 h, preferably 12-60 h, and more preferably 24 h.

[0035] In some embodiments, the sterilization conditions and methods can be conventional in the art, generally high-temperature sterilization.

[0036] When the fermentation substrate is sterilized using the high-temperature sterilization method, the sterilization temperature can be the conventional temperature for this type of operation in the art, preferably 110-125°C, more preferably 115-121°C, for example 121°C.

[0037] When the fermentation substrate is sterilized using the high-temperature sterilization method, the sterilization pressure can be the conventional pressure for this type of operation in the art, preferably 0.1 to 0.14 MPa, more preferably 0.1 to 0.13 MPa, for example 0.12 MPa.

[0038] When the fermentation substrate is sterilized using the high-temperature sterilization method, the sterilization time can be the conventional operation time for this type of operation in the art, preferably 20-60 min, more preferably 20-40 min, for example 30 min.

[0039] In some embodiments, the sterilization process may further include cooling, centrifugation, and homogenization.

[0040] In accordance with conventional practice in this field, the cooling may be cooling to room temperature.

[0041] The centrifugation speed can be a speed commonly used in this type of operation in the art, preferably 3000-9000 r / min, more preferably 4000-8000 r / min, for example 5000 r / min.

[0042] The radius of the centrifugation can be a conventional radius for this type of operation in the art, preferably 8 to 15 cm.

[0043] The centrifugation time can be the conventional time for this type of operation in the art, preferably 10 to 40 minutes, more preferably 20 to 40 minutes, for example 30 minutes.

[0044] The centrifugation process may further include secondary sterilization and / or mixing with preservatives.

[0045] The conditions and methods for secondary sterilization can be the conventional conditions and methods for this type of operation in the art, and generally can be high-temperature sterilization.

[0046] When the secondary sterilization is performed using the high-temperature sterilization method, the temperature of the secondary sterilization can be the temperature conventional for this type of operation in the art, preferably 110-125°C, more preferably 115-121°C, for example 121°C.

[0047] When the secondary sterilization is performed using the high-temperature sterilization method, the pressure of the secondary sterilization can be the conventional pressure for this type of operation in the art, preferably 0.1 to 0.14 MPa, more preferably 0.1 to 0.13 MPa, for example 0.12 MPa.

[0048] When the high-temperature sterilization method is used for the secondary sterilization, the time for the secondary sterilization can be the conventional operation in this field, preferably 20 to 40 minutes, more preferably 25 to 35 minutes, for example 30 minutes.

[0049] During the mixing process with the preservative, the mixing temperature can be the temperature conventional for this type of operation in the art, preferably 50-80°C, and more preferably 70-80°C.

[0050] During the mixing process with the preservative, the preservative may include p-hydroxyacetophenone and / or 1,2-hexanediol, as is customary in the art.

[0051] When the preservative includes p-hydroxyacetophenone and 1,2-hexanediol, the mass percentage of p-hydroxyacetophenone in the supernatant obtained after centrifugation can be 0.1% to 1%, and the mass percentage of 1,2-hexanediol in the supernatant obtained after centrifugation can be 0.1% to 1%; preferably, the mass percentage of p-hydroxyacetophenone in the supernatant obtained after centrifugation is 0.5%, and the mass percentage of 1,2-hexanediol in the supernatant obtained after centrifugation is 0.5%.

[0052] The homogenization process is performed after mixing with the preservative.

[0053] The homogenization speed is preferably 8000 r / min to 14000 r / min, and more preferably 9000 rpm to 13000 rpm.

[0054] The homogenization time is preferably 3 min to 45 min, and more preferably 5 min to 25 min. After homogenization, a fermented emulsion is obtained.

[0055] This application also provides a fermented emulsion, which is prepared by the fermented emulsion preparation method described above.

[0056] This application also provides the use of the fermented emulsion as described above, either directly as a product, as an additive, or as a base in the preparation of topical skin agents.

[0057] In some embodiments, the fermented emulsion may be used as at least one of the moisturizing active ingredient, anti-inflammatory active ingredient, and anti-aging active ingredient in the topical skin agent.

[0058] The anti-inflammatory active ingredient may be an anti-inflammatory active ingredient that inhibits the production of IL-8 inflammatory factor by cells;

[0059] The anti-aging active ingredient may be an anti-aging active ingredient that promotes collagen production.

[0060] This application also provides a topical skin agent comprising the fermented emulsion described above.

[0061] In some embodiments, the topical skin agent may further include active ingredients commonly used in the art, generally including at least one of moisturizing active ingredients, anti-inflammatory active ingredients, anti-allergic active ingredients, anti-aging active ingredients, and antioxidant active ingredients.

[0062] In some embodiments, the topical skin agent may be, in accordance with the conventions of the art, including but not limited to, face masks, serums, or toners.

[0063] In some embodiments, the fermented emulsion may constitute 60% to 99% of the mass percentage of the topical skin agent, preferably 80% to 99%.

[0064] In some embodiments, the room temperature or normal temperature generally refers to 15–40°C.

[0065] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of this application.

[0066] All reagents and raw materials used in this application are commercially available.

[0067] The positive and progressive effects of this application are as follows:

[0068] Compared with traditional emulsions, the fermented emulsion prepared in this application reduces the loss of active ingredients through the combined fermentation of Lactobacillus plantarum and Lactobacillus bulgaricus, and can better exert skin care effects. Without the addition of emulsifiers, thickeners, surfactants and other ingredients, it can still show good repair of photodamage, anti-inflammatory and anti-aging effects, with good stability, good sensory evaluation, simple preparation process, saving production costs, high safety in use, and is not likely to cause skin allergies. Attached Figure Description

[0069] This application can be better understood by referring to the description given below in conjunction with the accompanying drawings. These drawings, together with the detailed description below, are included in and form part of this specification, and are used to further illustrate preferred embodiments of the application and explain its principles and advantages.

[0070] in:

[0071] Figure 1 This is a comparison chart of the hydroxyl radical scavenging abilities of the products prepared in Examples 1-4 or Comparative Examples 1-4;

[0072] Figure 2 Comparison charts showing the cell activity promotion capabilities of the products prepared in Examples 1-4 or Comparative Examples 1-4;

[0073] Figure 3 Comparison charts showing the ability of the products prepared in Examples 1-4 or Comparative Examples 1-4 to repair photodamage.

[0074] Figure 4 This is a comparison chart of the anti-inflammatory capacity (IL-8) of the products prepared in Examples 1-4 or Comparative Examples 1-4. Detailed Implementation

[0075] The present application is further illustrated below by way of embodiments, but these embodiments are not intended to limit the scope of the present application. Experimental methods not specifically described in the following embodiments are performed according to conventional methods and conditions, or as selected in accordance with the product instructions.

[0076] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0077] All raw materials used in the following examples are commercially available.

[0078] The Lactiplantibacillus plantarum used in the following examples and comparative examples was deposited on April 16, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC NO.30350.

[0079] The Lactobacillus bulgaricus used in the following examples and comparative examples was deposited on October 27, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC NO.28800.

[0080] The Saccharomyces cerevisiae in the following comparative example was deposited on March 27, 2019, at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China, accession number CGMCCNO.17452.

[0081] Example 1

[0082] (1) Wheat seeds were crushed and sieved to obtain wheat seed powder with a mesh size of 50; soybeans were crushed and sieved to obtain soybean powder with a mesh size of 50; then 4.5g wheat seed powder, 4.5g soybean powder, 24g sunflower seed oil and 300mL deionized water were mixed evenly and sterilized at a temperature of 121℃ and a pressure of 0.12MPa for 30min. After sterilization, the mixture was cooled to room temperature to obtain the fermentation substrate.

[0083] (2) Prepare a bacterial suspension of *Lactobacillus plantarum* (CGMCC NO. 30350) to a viable count of 10-1. 12 CFU / mL bacterial suspension; prepare Lactobacillus bulgaricus (CGMCC NO.28800) bacterial suspension to a viable count of 10-1. 12 CFU / mL bacterial suspension; 4.5 mL of *Lactobacillus plantarum* (CGMCC NO. 30350) bacterial suspension and 4.5 mL of *Lactobacillus bulgaricus* (CGMCC) bacterial suspension were inoculated into the fermentation substrate obtained in step (1). The culture medium (NO.28800) was fermented in a shaking incubator at 40℃ for 24 hours, with the shaking speed at 300 rpm. After fermentation, the resulting fermented emulsion was sterilized in an autoclave at 100℃ for 30 minutes to terminate fermentation. After sterilization, the emulsion was cooled to room temperature and then centrifuged at 4800 rpm for 30 minutes with a centrifugation radius of 12 cm to obtain the supernatant. The supernatant was then sterilized a second time in an autoclave at 100℃ and 0.21 MPa for 30 minutes to avoid contamination by other microorganisms during centrifugation. After sterilization, the emulsion was cooled to room temperature and then homogenized using a homogenizer at 10000 rpm for 10 minutes to obtain the fermented emulsion.

[0084] Example 2

[0085] Compared with Example 1, the only difference is that macadamia seed oil is used instead of sunflower seed oil, while other conditions and parameters are the same as in Example 1.

[0086] Example 3

[0087] Compared with Example 1, the only difference is that the mass of the wheat seed powder in the fermentation substrate is 4.5g, the mass of the soybean powder is 6g, and the mass of the vegetable oil is 18g, while other conditions and parameters are the same as in Example 1.

[0088] Example 4

[0089] Compared with Example 1, the only difference is that the amount of Lactobacillus plantarum (CGMCC NO.30350) added is 3 mL and the amount of Lactobacillus bulgaricus (CGMCC NO.28800) added is 6 mL, and the other conditions and parameters are the same as in Example 1.

[0090] Comparative Example 1

[0091] Compared with Example 1, the difference is that Lactobacillus bulgaricus (CGMCC NO.28800) was replaced with Saccharomyces cerevisiae (CGMCC NO.17452), while other conditions and parameters are the same as in Example 1. The specific operation is as follows:

[0092] (1) Wheat seeds were crushed and sieved to obtain wheat seed powder with a mesh size of 50; soybeans were crushed and sieved to obtain soybean powder with a mesh size of 50; then 4.5g wheat seed powder, 4.5g soybean powder, 24g sunflower seed oil and 300mL deionized water were mixed evenly and sterilized at a temperature of 121℃ and a pressure of 0.12MPa for 30min. After sterilization, the mixture was cooled to room temperature to obtain the fermentation substrate.

[0093] (2) Prepare a bacterial suspension of *Lactobacillus plantarum* (CGMCC NO. 30350) to a viable count of 10-1. 12 CFU / mL bacterial suspension; prepare a Saccharomyces cerevisiae (CGMCC NO.17452) bacterial suspension with a viable count of 10. 12CFU / mL bacterial culture; 4.5 mL of *Lactobacillus plantarum* (CGMCC) was inoculated into the fermentation substrate obtained in step (1). The culture of *Saccharomyces cerevisiae* (NO. 30350) and 4.5 mL of *Saccharomyces cerevisiae* (CGMCC NO. 17452) was fermented in a shaking incubator at 40℃ for 24 hours, with the shaking speed at 300 rpm. After fermentation, the resulting fermented emulsion was sterilized in an autoclave at 100℃ for 30 minutes to terminate fermentation. After sterilization, the mixture was cooled to room temperature and then centrifuged at 4800 rpm for 30 minutes with a centrifuge radius of 12 cm to obtain the supernatant. The supernatant was then sterilized a second time in an autoclave at 100℃ and 0.21 MPa for 30 minutes to avoid contamination by other microorganisms during centrifugation. After sterilization, the mixture was cooled to room temperature and then homogenized using a homogenizer at 10000 rpm for 10 minutes to obtain the fermented emulsion.

[0094] Comparative Example 2

[0095] Compared with Example 1, the difference is that the mass of the wheat seed powder is 4g, the mass of the soybean powder is 4g, and the mass of the vegetable oil is 2g, while other conditions and parameters are the same as in Example 1.

[0096] Comparative Example 3

[0097] Compared with Example 1, the only difference is that wheat seed flour is replaced with millet flour, soybean flour is replaced with corn flour, and sunflower seed oil is replaced with peanut oil. All other conditions and parameters are the same as in Example 1.

[0098] Comparative Example 4

[0099] Compared with Example 1, the only difference is the fermentation method, which is as follows:

[0100] (1) Wheat seeds were crushed and sieved to obtain wheat seed powder with a mesh size of 50; soybeans were crushed and sieved to obtain soybean powder with a mesh size of 50; then 4.5g wheat seed powder, 4.5g soybean powder, 24g sunflower seed oil and 300mL deionized water were mixed evenly and sterilized at a temperature of 121℃ and a pressure of 0.12MPa for 30min. After sterilization, the mixture was cooled to room temperature to obtain the fermentation substrate.

[0101] (2) Prepare a bacterial suspension of *Lactobacillus plantarum* (CGMCC NO. 30350) to a viable count of 10-1. 12 CFU / mL bacterial suspension; prepare Lactobacillus bulgaricus (CGMCC NO.28800) bacterial suspension to a viable count of 10-1. 12The bacterial culture was prepared at CFU / mL. 4.5 mL of *Lactobacillus plantarum* (CGMCC NO. 30350) was inoculated into the fermentation substrate obtained in step (1), and fermented in a shaking incubator at 40℃ for 24 h with a shaking speed of 300 rpm. After the fermentation of *Lactobacillus plantarum* was completed, it was sterilized in an autoclave at 100℃ for 30 min to terminate the fermentation. After sterilization, it was cooled to room temperature, and then 4.5 mL of *Lactobacillus bulgaricus* (CGMCC NO. 30350) was inoculated. The *Lactobacillus plantarum* (NO. 28800) bacterial culture was fermented in a shaking incubator at 40℃ for 24 hours, with the shaking speed at 300 rpm. After the fermentation of *Lactobacillus plantarum* was completed, it was sterilized in an autoclave at 100℃ for 30 minutes to terminate the fermentation. After sterilization, it was cooled to room temperature and then centrifuged at 4800 rpm for 30 minutes with a centrifugation radius of 12 cm to obtain the supernatant. The supernatant was then sterilized a second time in an autoclave at 100℃ and 0.21 MPa for 30 minutes to avoid contamination by other microorganisms during centrifugation. After sterilization, it was cooled to room temperature and then homogenized using a homogenizer at 10000 rpm for 10 minutes to obtain the fermented emulsion.

[0102] Example 1: Hydroxyl radical scavenging experiment

[0103] Hydroxyl radicals (·OH) are generated using the Fenton reaction. Salicylic acid is added to the reaction system, and the ·OH reacts with salicylic acid to form a colored compound, 2,3-dihydroxybenzoic acid, which exhibits characteristic absorption at 510 nm. Using a fixed reaction time method, the absorbance of the reaction solution containing the analyte is measured at 510 nm and compared with a blank solution to determine the analyte's scavenging effect on ·OH. Reagents are added to test tubes according to Table 1, including 9 mmol / L FeSO4, 9 mmol / L salicylic acid ethanol solution, the sample (products prepared in Examples 1-4 or Comparative Examples 1-6), an appropriate amount of deionized water, and 8.8 mmol / L H2O2 solution. The mixture is shaken well, heated in a 37°C water bath for 15 min, and then the absorbance A0 of the blank control (preparation method shown in Group A of Table 2) and the absorbance A0 of the added sample are measured. x (See Group B in Table 4 for preparation method) and background absorbance A of the solution without H2O2. x0 (See Group C in Table 4 for the preparation method). When measuring A0, the reference solution was a system without hydrogen peroxide; the hydroxyl radical scavenging rate of each group was calculated according to the following formula, and the results are shown in Table 2.

[0104] The products obtained in Examples 1-4 and Comparative Examples 1-4 were all diluted 100 times and used as test solutions for hydroxyl radical scavenging experiments.

[0105] The formula for calculating the hydroxyl radical scavenging rate is:

[0106] Clearance rate = (A0 - (A)x -A x0 )) / A0×100%.

[0107] Table 1

[0108] Group <![CDATA[V(FeSO4) / mL]]> V (salicylic acid) / mL V(sample) / mL V (deionized water) / mL <![CDATA[V(H2O2) / mL]]> A 0.5 0.5 0 6 0.5 B 0.5 0.5 2.5 3.5 0.5 C 0.5 0.5 2.5 4 0

[0109] Table 2

[0110] Hydroxyl radical scavenging rate (%) Example 1 84.53±1.82 Example 2 89.21±1.99 Example 3 77.2±1.85 Example 4 81.42±1.32 Comparative Example 1 56.34±2.28 Comparative Example 2 51.76±3.59 Comparative Example 3 47.12±2.6 Comparative Example 4 60.92±3.98

[0111] The results showed that the hydroxyl radical scavenging rates of the products prepared in Examples 1-4 were significantly better than those of the products prepared in Comparative Examples 1-4. Figure 1 In this context, ns indicates no statistically significant difference compared to Example 1; *p < 0.05 indicates a statistically significant difference compared to Example 1; **p < 0.01 indicates a significant statistically significant difference compared to Example 1, representing a significant reduction; ***p < 0.001 indicates an extremely significant statistically significant difference compared to Example 1, representing an extremely significant reduction.

[0112] Example 2: Human Skin Fibroblast Toxicity Experiment

[0113] This experiment used human skin fibroblasts from the Chinese Scientific Cell Bank to verify the cytotoxicity of the products prepared in Examples 1-4 and Comparative Examples 1-4.

[0114] Reagents: 0.25% (EDTA-containing) trypsin was manufactured by GIBCO, USA; DMEM medium was manufactured by GIBCO, USA; penicillin-dextrose antibody was manufactured by Corning, USA; CCK-8 was manufactured by Beijing Bairui Biotechnology Co., Ltd.; fetal bovine serum was manufactured by GIBCO, USA; phosphate buffer was manufactured by Beijing Bairui Biotechnology Co., Ltd.

[0115] Equipment: The WJ-80A-Ⅱ CO2 constant temperature incubator is manufactured by Shanghai Shengke Instrument Equipment Co., Ltd.; the Sunrise microplate reader is manufactured by Diken Trading Co., Ltd.; the TL80-2 medical centrifuge is manufactured by Jiangsu Tianli Medical Equipment Co., Ltd.; and the NUNC 96-well cell culture plate is manufactured by Thermo Fisher Scientific.

[0116] Experimental steps:

[0117] The products obtained in Examples 1-4 and Comparative Examples 1-4 were respectively prepared into test solutions with a volume percentage of 1% using serum-free DMEM medium.

[0118] Human skin fibroblasts were cultured in a solution containing 10% fetal bovine serum and 1% penicillin-dextrose antibody (1×10⁻⁶). 5Cells were cultured in DMEM medium containing 100 mg / L penicillin and 100 mg / L streptomycin. Cells were grown in an incubator at 37°C and 5% CO2 saturated humidity. When cell confluence reached 85% or higher, logarithmic growth phase cells were digested with 0.05% trypsin, and the digestion was terminated with serum-containing DMEM. Cells were counted using a cell counting chamber, and the cell suspension concentration was adjusted to 7 × 10⁻⁶ cells / mL. 4 Cells were seeded at a rate of 100 μL / well in 96-well plates and incubated at 37°C and 5% CO2 for 12 h. The old culture medium was removed, and the cells were washed twice with phosphate-buffered saline (PBS). 100 μL of the filtered and sterilized test solution was added to each well of the experimental group, with six replicates per test solution. The control group contained cells and was inoculated with serum-free DMEM medium. The blank control group contained no cells and was inoculated with 100 μL of PBS. The cells were then incubated at 37°C and 5% CO2 for 24 h. Then, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for another 3 h. The absorbance was measured at 450 nm, and the cell viability was calculated. The results are shown in Table 3. Figure 2 .

[0119] The formula for calculating cell viability is as follows:

[0120] Cell viability (%) = (A experimental group - A blank control group) / (A control group - A blank control group) × 100%.

[0121] Table 3

[0122]

[0123]

[0124] The results showed that the cell viability of the products prepared in Examples 1-4 was significantly better than that of the products prepared in Comparative Examples 1-4. Figure 2 In this context, ns indicates no statistically significant difference compared to Example 1; *p < 0.05 indicates a statistically significant difference compared to Example 1; **p < 0.01 indicates a significant statistically significant difference compared to Example 1, representing a significant reduction; ***p < 0.001 indicates an extremely significant statistically significant difference compared to Example 1, representing an extremely significant reduction.

[0125] Example 3: Cell viability (repair of photodamage)

[0126] The products obtained in Examples 1-4 and Comparative Examples 1-4 were respectively prepared into 1% (v / v) experimental test solutions using serum-free DMEM medium. The experimental test solutions were filtered through a 0.22 μm sterile filter membrane.

[0127] HaCaT cells were cultured in a solution containing 10% fetal bovine serum and 1% penicillin-dextrin (1×10⁻⁶).5 Cells were cultured in DMEM medium containing 100 mg / L penicillin and 100 mg / L streptomycin. Cells were grown in an incubator at 37°C and 5% CO2 saturated humidity. When cell confluence reached 85% or higher, logarithmic growth phase cells were digested with 0.05% trypsin, and the digestion was terminated with serum-containing DMEM. Cells were counted using a cell counting chamber, and the cell suspension concentration was adjusted to 7 × 10⁻⁶ cells / mL. 4 Cells were seeded at a rate of 100 μL per well in 96-well plates and incubated at 37°C with 5% CO2 for 12 h. After incubation, the old culture medium was removed, and the cells were washed twice with phosphate-buffered saline (PBS). Then, 100 μL of PBS was added. Both the model and experimental groups were irradiated with UVB at a dose of 40 mJ / cm². 2 The irradiation time was 80 seconds, and the negative control group was not irradiated. PBS was discarded, and serum-free DMEM medium was added to the model group and negative control group. 100 μL of the filtered and sterilized experimental test solution was added to each well of the experimental group, with 6 replicates per test solution. The blank control group was cell-free and contained 100 μL of PBS. The cells were then incubated at 37°C and 5% CO2 for 24 hours. Then, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for another 3 hours. The absorbance was measured at 450 nm, and the cell viability of each group was calculated. The results are shown in Table 4. Figure 3 .

[0128] The formula for calculating cell viability is as follows:

[0129] Cell viability (%) = (A experimental group or model group - A blank control group) / (A negative control group - A blank control group) × 100%.

[0130] Table 4

[0131] Cell viability (%) Blank group 100±4.9 Model group 54.78±3.31 Example 1 71.95±2.27 Example 2 75.25±3.56 Example 3 70.28±3.15 Example 4 72.34±3.64 Comparative Example 1 62.63±2.19 Comparative Example 2 61.41±3.12 Comparative Example 3 61.71±3.61 Comparative Example 4 64.56±3.75

[0132] The results showed that the cell viability of the products prepared in Examples 1-4 was significantly better than that of the products prepared in Comparative Examples 1-4. Figure 3 A p < 0.001 indicates a highly statistically significant difference compared to the model group, representing a highly significant decrease. ### p < 0.001 indicates a highly statistically significant difference compared to the control group, meaning a highly significant decrease.

[0133] Example 4: Anti-inflammatory effect (determination of inflammatory factor IL-8 content)

[0134] Preparation of test solution for experimental group: The products prepared in the above examples and comparative examples were used to prepare the test solution of experimental group with a volume fraction of 1% in serum-free DMEM medium and filtered through a sterile filter membrane.

[0135] HaCaT cells in the logarithmic growth phase were seeded at a density of 250,000 cells / mL into 6-well cell culture plates, with 2 mL of cell suspension added to each well. The plates were incubated for 12 hours. Experimental, control, and model groups were set up. The experimental and model groups were treated with 40 mJ / cm²... 2 Cells were irradiated with UVB for 80 seconds. After irradiation, the supernatant from the experimental group, blank group, and model group was discarded. 2 mL of DMEM solution was added to the blank group and model group, and 2 mL of test solution was added to the experimental group (the product prepared in the above examples or comparative examples was prepared to a volume percentage of 2% using DMEM; the test solution needs to be filtered through a 0.22 μm sterile membrane before use). Cells were added and treated for 24 hours. The supernatant was discarded, and the cells were washed 2-3 times with PBS. Cells were then treated with cell lysis buffer and transferred to centrifuge tubes. The cells were centrifuged at 10000 r / min and 4℃ for 10 min, and the supernatant was collected to obtain the cell lysis buffer. 20 μL of the cell lysis buffer was used to detect the total protein content in the sample using a BCA kit. The determination of inflammatory factors was performed according to the ELISA kit instructions, and the OD values ​​were measured at 450 nm.

[0136] The expression level (A) of inflammatory factors was calculated based on the standard curve, and corrected for by protein content (B) to obtain C = A / B. The relative expression level was calculated as C / C (for the blank group). The results are shown in Table 5. Figure 4 .

[0137] Table 5

[0138] IL-8 relative expression level Blank group 1±0.15 Model group 1.83±0.07 Example 1 0.69±0.05 Example 2 0.56±0.12 Example 3 0.87±0.21 Example 4 0.77±0.23 Comparative Example 1 1.37±0.16 Comparative Example 2 1.25±0.15 Comparative Example 3 1.45±0.14 Comparative Example 4 1.33±0.18

[0139] The results showed that the anti-inflammatory activity of the products prepared in Examples 1-4 was significantly better than that of the products prepared in Comparative Examples 1-4, and the relative expression level of IL-8 in the products prepared in Examples 1-4 was significantly lower than that in Comparative Examples 1-4. Figure 4 In the table, *p < 0.05 indicates a statistically significant difference compared to the control group; **p < 0.01 indicates a statistically significant difference compared to the model group, indicating a significant decrease; ***p < 0.001 indicates an extremely statistically significant difference compared to the model group, indicating a highly significant decrease. ## p < 0.001 indicates a highly statistically significant difference compared to the control group, meaning a highly significant increase.

[0140] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof in this application are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0141] Although this application has been disclosed above through the description of specific embodiments, it should be understood that those skilled in the art can devise various modifications, improvements, or equivalents to this application within the spirit and scope of the appended solutions. Such modifications, improvements, or equivalents should also be considered to be included within the scope of protection claimed in this application.

Claims

1. A method for preparing a fermented emulsion, characterized in that, Specifically, it includes the following steps: Lactobacillus plantarum and Lactobacillus bulgaricus were inoculated into a fermentation substrate consisting of wheat seeds, soybeans, vegetable oil and water, and the mixture was fermented, sterilized, and the fermented emulsion was obtained. The wheat seed, soybean, and vegetable oil are in a mass ratio of 1:1:(1-10); the viable count ratio of Lactiplantibacillus plantarum to Lactobacillus bulgaricus is 1:(0.2-5).

2. The method for preparing fermented emulsion as described in claim 1, characterized in that, The method for preparing the fermented emulsion satisfies at least one of the following conditions: The wheat seeds are crushed and sieved before use. The sieved wheat seed powder has a mesh size of 30 to 100 mesh, preferably 50 mesh. The soybeans are crushed and sieved before use. The soybean powder is sieved through a mesh size of 30 to 100, preferably 50. The plant oils include at least one of the following: macadamia seed oil, sea buckthorn seed oil, peony seed oil, meadowfoam seed oil, sunflower seed oil, shea butter, crepe oil, sweet almond oil, flaxseed oil, coconut oil, and coix seed oil. The water is deionized water; The mass ratio of the wheat seeds, the soybeans, and the vegetable oil is 1:1:(1-8); The mass-to-volume ratio of the wheat seeds, the soybeans, and the water is 1:1:(20-150)g / mL, preferably 1:(20-100)g / mL.

3. The method for preparing fermented emulsion as described in claim 1, characterized in that, The fermentation substrate further includes a sterilization process before use; Preferably, the sterilization conditions and method for the fermentation substrate are high-temperature sterilization; when the fermentation substrate is sterilized using the high-temperature sterilization method, the sterilization temperature is 110–125°C, preferably 115–121°C, for example 121°C; when the fermentation substrate is sterilized using the high-temperature sterilization method, the sterilization pressure is 0.1–0.14 MPa, preferably 0.1–0.13 MPa, for example 0.12 MPa; when the fermentation substrate is sterilized using the high-temperature sterilization method, the sterilization time is 20–60 min, preferably 20–40 min, for example 30 min. Preferably, the fermentation substrate, after the sterilization process, further includes a cooling process to room temperature.

4. The method for preparing fermented emulsion as described in claim 1, characterized in that, The method for preparing the fermented emulsion satisfies at least one of the following conditions: The viable count ratio of *Lactiplantibacillus plantarum* to *Lactobacillus bulgaricus* is 1:(0.5-2). The Lactiplantibacillus plantarum mentioned includes the Lactiplantibacillus plantarum deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.30350; The *Lactobacillus plantarum* was added in the form of a *Lactobacillus plantarum* liquid, and the viable count of the *Lactobacillus plantarum* liquid was 10-1. 10 ~10 14 CFU / mL, preferably 10 10 ~10 12 CFU / mL; The number of *Lactobacillus plantarum* inoculated per unit volume of the fermentation substrate is preferably 10. 8 ~10 14 CFU / mL, preferably 10 8 ~10 12 CFU / mL; The Lactobacillus bulgaricus mentioned includes the Lactobacillus bulgaricus deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.28800; The Lactobacillus bulgaricus was added in the form of a Lactobacillus bulgaricus bacterial solution, wherein the viable count of the Lactobacillus bulgaricus bacterial solution was 10. 10 ~10 14 CFU / mL, preferably 10 10 ~10 12 CFU / mL; The number of *Lactobacillus bulgaricus* inoculated per unit volume of the fermentation substrate is 10. 8 ~10 14 CFU / mL, preferably 10 8 ~10 12 CFU / mL.

5. The method for preparing fermented emulsion according to any one of claims 1 to 4, characterized in that, The method for preparing the fermented emulsion satisfies at least one of the following conditions: The fermentation culture is an aerobic fermentation culture, preferably carried out in a shaking incubator; Preferably, when the fermentation culture is an aerobic fermentation culture, the rotation speed of the shaker in the shaking incubator is 200-400 r / min, more preferably 250-350 r / min, for example 300 r / min; The fermentation culture temperature is 35–45°C, preferably 37–43°C, and more preferably 40°C; The fermentation time is 12-72 hours, preferably 12-60 hours, and more preferably 24 hours.

6. The method for preparing fermented emulsion as described in claim 1, characterized in that, The sterilization process is further followed by cooling and / or centrifugation, and collection of the supernatant. Preferably, the cooling is cooling to room temperature; Preferably, the centrifugation speed is 3000-9000 r / min, more preferably 4000-8000 r / min, for example 5000 r / min; Preferably, the radius of the centrifuge is 8–15 cm; Preferably, the centrifugation time is 10 to 40 minutes, more preferably 20 to 40 minutes, for example 30 minutes.

7. The method for preparing fermented emulsion as described in claim 6, characterized in that, The centrifugation operation further includes at least one of the following operations: homogenization, secondary sterilization, and mixing with a preservative; Preferably, the conditions and method for the secondary sterilization are high-temperature sterilization. Preferably, when the secondary sterilization is performed using the high-temperature sterilization method, the temperature of the secondary sterilization is 110-125°C, more preferably 115-121°C, for example 121°C; Preferably, when the secondary sterilization is performed using the high-temperature sterilization method, the pressure of the secondary sterilization is 0.1 to 0.14 MPa, more preferably 0.1 to 0.13 MPa, for example 0.12 MPa; Preferably, when the secondary sterilization is performed using the high-temperature sterilization method, the secondary sterilization time is 20-40 minutes, more preferably 25-35 minutes, for example 30 minutes; Preferably, during the mixing process with the preservative, the mixing temperature is more preferably 50-80°C, for example 70-80°C; Preferably, during the mixing process with the preservative, the preservative may include p-hydroxyacetophenone and / or 1,2-hexanediol; Preferably, when the preservative comprises p-hydroxyacetophenone and 1,2-hexanediol, the p-hydroxyacetophenone accounts for 0.1% to 1% of the mass of the supernatant obtained after centrifugation, and the 1,2-hexanediol accounts for 0.1% to 1% of the mass of the supernatant obtained after centrifugation; more preferably, the p-hydroxyacetophenone accounts for 0.5% of the mass of the supernatant obtained after centrifugation, and the 1,2-hexanediol accounts for 0.5% of the mass of the supernatant obtained after centrifugation. Preferably, the homogenization process is performed after mixing with the preservative. Preferably, the homogenization speed is 8000 r / min to 14000 r / min, and more preferably 9000 rpm to 13000 rpm; Preferably, the homogenization time is 3 min to 45 min, more preferably 5 min to 25 min, and the homogenization is completed to obtain the fermented emulsion.

8. A fermented emulsion, characterized in that, It is prepared by the method for preparing fermented emulsion according to any one of claims 1 to 7.

9. The use of the fermented emulsion as described in claim 8, directly as a product, as an additive, or as a base in the preparation of topical skin agents; Preferably, the fermented emulsion is at least one of the moisturizing active ingredient, anti-inflammatory active ingredient, and anti-aging active ingredient in the topical skin agent; More preferably, the anti-inflammatory active ingredient is an anti-inflammatory active ingredient that inhibits the production of IL-8 inflammatory factor by cells; More preferably, the anti-aging active ingredient is an anti-aging active ingredient that promotes collagen production.

10. A topical skin agent, characterized in that, It includes the fermented emulsion as described in claim 8; Preferably, the topical skin agent further includes at least one of the following: moisturizing active ingredient, anti-inflammatory active ingredient, anti-allergic active ingredient, anti-aging active ingredient, and antioxidant active ingredient; Preferably, the topical skin agent includes, but is not limited to, a face mask, serum, or toner; Preferably, the fermented emulsion accounts for 60% to 99% of the mass percentage of the topical skin agent, more preferably 80% to 99%.

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