Multi-function bio-fermented essence cream containing skin external agent and preparation method and application thereof

Abstract

The application discloses a multi-effect bio-fermentation essence cream containing a skin external use agent and a preparation method and application thereof, and specifically comprises the following steps: inoculating yeast into a fermentation substrate composed of coix seed, sorghum seed, vegetable oil and water, carrying out fermentation culture, sterilizing, and preparing the fermentation essence cream; wherein the yeast is composed of yellow rice wine yeast and saccharomyces cerevisiae, and the ratio of the viable cell count of the yellow rice wine yeast to the saccharomyces cerevisiae is 1:(0.5-5). The application utilizes the yellow rice wine yeast and the saccharomyces cerevisiae to effectively utilize the active ingredients of coix seed and vegetable oil through fermentation, and obtains a fermentation essence cream containing natural plant active ingredients, so that the loss of the active ingredients is reduced, the fermentation essence cream has good repair, tightening and anti-aging effects, and has no side effects, and in the case that no additional auxiliary agent is needed, the fermentation essence cream has ideal cold resistance and heat resistance stability, and can be widely used in the field of cosmetics.

Description

Technical Field

[0001] This application belongs to the field of cosmetic technology, and in particular relates to a multifunctional bio-fermented essence emulsion containing a topical skin agent, its preparation method and application. Background Technology

[0002] As we age, our skin naturally deteriorates, and its condition worsens. External factors such as air pollution and UV radiation exacerbate this damage. Furthermore, the cosmetics industry increasingly uses chemically synthesized ingredients. While these ingredients have advantages such as well-defined structures, efficacy, and mature quality control methods, consumers have gradually discovered that they can cause adverse effects on the human body, such as sensitization, redness, and irritation. Therefore, reducing the adverse reactions caused by chemically synthesized substances has become a key direction for cosmetic development, making effective skin care crucial.

[0003] Job's tears, also known as coix seed, is commonly called "the king of medicinal rice" and is mainly produced in Fujian, Liaoning, and Shaanxi provinces in my country. Starch accounts for over 60% of its dry weight, making it a high-starch agricultural product. Its carbohydrate content is around 65%, equivalent to that of ordinary hybrid corn; its protein content is around 17%, 1.3 times higher than rice; its lipid content is around 5%, equivalent to 18 times that of rice, and is mostly unsaturated fatty acids; it also contains minerals such as calcium, phosphorus, and iron, as well as various vitamins, dietary fiber, and amino acids, making it highly nutritious and beneficial for health. In recent years, scholars both domestically and internationally have conducted in-depth research on the structure and physicochemical properties of Job's tears, resulting in numerous research reports on its pharmacological activities, including anti-tumor, analgesic and anti-inflammatory effects, immune enhancement, regulation of glucose and lipid metabolism, blood pressure reduction, antioxidant effects, and anti-aging properties.

[0004] Sorghum is rich in nutrients such as starch and protein, and also contains various active ingredients including polyphenols, phytosterols, higher alkanols, and lipids. Currently, sorghum is mainly used as a grain in food processing and brewing, with relatively limited applications; its use in topical skincare products is rarely reported. Existing methods for obtaining the active ingredients from sorghum mostly involve water extraction or organic solvent extraction, which is not only cumbersome and inefficient, but also costly and wasteful of resources, leading to environmental pollution. Plant oils are natural high-molecular-weight compounds composed of fatty acids and glycerol. They are not composed of a single chemical structure, therefore, not all plant oils can be absorbed by the human skin. The main reason affecting the skin feel of plant oil-based skincare products is the presence of small amounts of free macromolecular groups in plant oils that are not easily absorbed by the skin.

[0005] Currently, there are separate fermentation processes for Job's tears and oils in China, but a method is still lacking that can simultaneously ferment Job's tears and vegetable oil as fermentation substrates and inoculum. Job's tears and vegetable oil are common ingredients in daily life and possess the characteristic of being able to ferment excellent products, making this process highly promising at present. Summary of the Invention

[0006] The technical problem this application aims to solve is to overcome the shortcomings of existing technologies, such as the complex composition and rich nutrition of Job's tears, the inability to effectively control the fermentation process, the difficulty in absorbing and utilizing the large molecular free groups contained in plant oils, and the lack of fermentation processes for Job's tears and oils. This application provides a multi-functional bio-fermented essence emulsion containing topical skin agents, its preparation method, and its application. This application exhibits good stability without the addition of any functional additives and possesses excellent repairing, firming, anti-aging, and antioxidant effects.

[0007] This application adopts the following technical solution to solve the above-mentioned technical problems:

[0008] This application provides a method for preparing a multifunctional bio-fermented essence emulsion containing a topical skin agent, specifically including the following steps: inoculating yeast into a fermentation substrate composed of Job's tears, sorghum seeds, vegetable oil and water, fermenting and culturing, sterilizing, and obtaining the fermented essence emulsion;

[0009] The yeast strain is composed of rice wine yeast and brewing yeast.

[0010] In some embodiments, the Job's tears are commercially available regular Job's tears.

[0011] In some embodiments, the sorghum seeds are commercially available sorghum seeds.

[0012] The mass ratio of the Job's tears to the sorghum seeds is 1:(0.8-1.2), preferably 1:1;

[0013] In some embodiments, the Job's tears are ground and sieved to obtain Job's tears powder, wherein the sieve mesh size of the Job's tears powder is 50-150 mesh, preferably 50-100 mesh.

[0014] In some embodiments, the sorghum seeds are ground and sieved to obtain Job's tears powder, wherein the sieve mesh size is 50-150 mesh, preferably 50-100 mesh;

[0015] In some embodiments, the mass ratio of the Job's tears to the sorghum seeds is 1:(0.8-1.2), preferably 1:1.

[0016] In some embodiments, the vegetable oil is composed of at least one of the following: macadamia seed oil, prickly pear fruit oil, peony seed oil, meadowfoam seed oil, sunflower seed oil, shea butter, crepe oil, sweet almond oil, jojoba oil, peony seed oil, baobab seed oil, small-fruit coffee seed oil, wheat germ oil, Chinese kiwi seed oil, flaxseed oil, perilla seed oil, Ganoderma lucidum spore oil, borage seed oil, camellia seed oil, deep-sea shepherd's purse seed oil, milk thistle seed oil, and olive fruit oil.

[0017] In some embodiments, the total mass ratio of the Job's tears and sorghum seeds to the mass ratio of the vegetable oil is 1:(0.5-4), preferably 1:(0.5-3).

[0018] In some embodiments, the mass-to-volume ratio of the Job's tears to the water may be 1:(80-120)g / mL; more preferably 1:(90-110)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 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.

[0023] The fermentation substrate may further include a cooling process after sterilization, typically cooling to room temperature.

[0024] In some embodiments, the yeast is inoculated in the form of a bacterial solution.

[0025] The preparation of the yeast culture includes: picking a single colony and culturing it in YPD liquid medium for 48 hours at a temperature of 28°C and a shaking speed of 200 r / min.

[0026] In some embodiments, the live cell ratio of the rice wine yeast and the brewing yeast is 1:(0.5-2).

[0027] In some embodiments, the fermentation substrate may also be subjected to sterilization procedures, which are conventional in the art, before use.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] The fermentation substrate may further include a cooling process after sterilization, typically cooling to room temperature.

[0032] In some embodiments, the rice wine yeast may include rice wine yeast AS2.1392 purchased from the Beijing Institute of Food and Beverage.

[0033] In some embodiments, based on the volume of deionized water in the initial fermentation system, the rice wine yeast can be added in the form of a rice wine yeast culture solution, as is conventional in the art, wherein the live cell count in the rice wine yeast culture solution is 10-1. 10 ~10 14 CFU / mL, preferably 10 10 ~10 12 CFU / mL.

[0034] In some embodiments, based on the volume of deionized water in the initial fermentation system, the number of rice wine yeasts inoculated per unit volume of the fermentation substrate can be conventional in the art, preferably 10. 8 ~10 12 CFU / mL, preferably 10 9 ~10 11 CFU / mL.

[0035] In some embodiments, the brewer's yeast may include brewer's yeast with accession number CGMCC No. 17452.

[0036] In some embodiments, based on the volume of deionized water in the initial fermentation system, the brewer's yeast can be added in the form of a brewer's yeast culture, as is conventional in the art, wherein the number of viable cells in the brewer's yeast culture is 10-1. 10 ~10 14 CFU / mL, preferably 10 10 ~10 12 CFU / mL.

[0037] In some embodiments, based on the volume of deionized water in the initial fermentation system, the number of *Saccharomyces cerevisiae* inoculated per unit volume of the fermentation substrate can be conventional in the art, preferably 10. 10 ~10 14 CFU / mL, preferably 10 10 ~10 12 CFU / mL.

[0038] In some embodiments, the fermentation culture is aerobic fermentation, which can be carried out in a shaking incubator in accordance with conventional practices in the art. 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.

[0039] In some embodiments, the fermentation culture temperature may be 25–35°C, preferably 25–30°C, and more preferably 28°C.

[0040] In some embodiments, the fermentation culture time may be 12-72 h, preferably 12-60 h, and more preferably 48 h.

[0041] In some embodiments, the sterilization conditions and methods can be conventional in the art, generally high-temperature sterilization.

[0042] 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.

[0043] 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.

[0044] In some embodiments, the sterilization process may further include cooling and / or centrifugation, and collection of the supernatant.

[0045] In this context, according to conventional practice, the cooling may be cooling to room temperature.

[0046] 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.

[0047] The radius of the centrifugation can be a conventional radius for this type of operation in the art, preferably 8 to 15 cm.

[0048] 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.

[0049] The centrifugation operation may further include at least one of the following operations: secondary sterilization, preservative mixing, and homogenization.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] During the mixing process with the preservative, the preservative may include p-hydroxyacetophenone and / or 1,2-hexanediol, as is customary in the art.

[0055] 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%.

[0056] The homogenization speed is 8000 r / min to 14000 r / min, preferably 9000 r / min to 13000 r / min. The homogenization time is 3 to 45 min, preferably 5 to 25 min.

[0057] This application also provides a multifunctional bio-fermented essence emulsion containing a topical skin agent, which is prepared by the fermented essence emulsion preparation method described above.

[0058] This application also provides the use of the multifunctional bio-fermented essence emulsion containing skin topical agents as described above, directly as a product, as an additive, or as a base in the preparation of skin topical agents.

[0059] In some embodiments, the fermented essence emulsion may be used as at least one of the antioxidant and anti-aging active ingredients in the topical skin agent.

[0060] The antioxidant active ingredient may be an antioxidant active ingredient that can scavenge DPPH free radicals.

[0061] The anti-aging active ingredient is an anti-aging active ingredient that has the ability to promote collagen production and / or promote cell repair.

[0062] This application also provides a topical skin agent comprising the multifunctional bio-fermented essence emulsion containing the topical skin agent as described above.

[0063] In some embodiments, the topical skin agent may further include active ingredients commonly used in the art, generally including at least one of repairing active ingredients, firming active ingredients, anti-inflammatory active ingredients, and anti-aging active ingredients.

[0064] 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.

[0065] In some embodiments, the fermented essence emulsion may account for 5% to 99% of the mass percentage of the topical skin agent, preferably 60% to 99%.

[0066] In some embodiments, the room temperature or normal temperature generally refers to 15–40°C.

[0067] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of this application.

[0068] All reagents and raw materials used in this application are commercially available.

[0069] This application utilizes fermentation to effectively utilize the active ingredients of Job's tears and plant oils, employing rice wine yeast and brewing yeast, to obtain a multi-functional bio-fermented essence milk containing natural plant active ingredients. This not only reduces the loss of active ingredients but also has excellent repair, firming, and anti-aging effects, without any side effects. Furthermore, it exhibits ideal cold and heat resistance stability without the need for external additives, making it widely applicable in the cosmetics field. Attached Figure Description

[0070] 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.

[0071] in:

[0072] Figure 1 This is a comparison chart showing the DPPH free radical scavenging capabilities of the products prepared in Examples 1-5 and Comparative Examples 1-5;

[0073] Figure 2 The diagram shows a comparison of the repair capabilities of the products prepared in Examples 1-5 and Comparative Examples 1-5 on damaged skin.

[0074] Figure 3 A comparative diagram showing the effect of the products prepared in Examples 1-5 and Comparative Examples 1-5 on the growth of human skin fibroblasts;

[0075] Figure 4 This is a comparison chart showing the ability of the products prepared in Examples 1-5 and Comparative Examples 1-5 to repair damaged cells.

[0076] Figure 5 This is a comparison chart of the stability (cold resistance) of the products obtained in Examples 1-5 and Comparative Examples 1-5;

[0077] Figure 6 The graph shows a comparison of the stability (heat resistance) of the products obtained in Examples 1-5 and Comparative Examples 1-5. Detailed Implementation

[0078] 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.

[0079] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0080] In the following examples and comparative examples, the preparation method of the rice wine yeast culture (hereinafter referred to as culture 1) includes the following steps: rice wine yeast AS2.1392 purchased from Beijing Food Brewing Research Institute was inoculated into YPD solid medium for streaking activation and cultured in an incubator at 28°C for 48 hours to obtain single colonies; the single colonies were inoculated into YPD liquid medium and cultured in a shaker at 28°C for 48 hours at a shaking speed of 200 rpm.

[0081] In the following examples and comparative examples, Saccharomyces cerevisiae YWY1 was deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, with a deposit date of March 27, 2019, and accession number CGMCCNo.17452. The preparation method of the Saccharomyces cerevisiae liquid (hereinafter referred to as liquid 2) of CGMCC No.17452 was the same as that of the rice wine yeast, except that the strain was different.

[0082] The Lactobacillus helveticus in the following comparative examples was purchased from the China Industrial Microbial Culture Collection Center, with accession number CICC 20243.

[0083] The *Saccharomycopsis fibuligera* strain in the following comparative example was deposited on June 7, 2021, 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. 22671.

[0084] Example 1

[0085] (1) Coix seed is ground into powder and then passed through a 100-mesh sieve to obtain coix seed powder; sorghum seeds are ground into powder and then passed through a 100-mesh sieve to obtain sorghum seed powder. Weigh 12g of coix seed powder, 12g of sorghum seed powder, 12g of meadowfoam seed oil and 12g of peony seed oil, and then mix them with 300mL of deionized water. Sterilize at a temperature of 121℃ and a pressure of 0.12MPa for 30min. After sterilization, cool to room temperature to obtain fermentation substrate.

[0086] (2) The fermentation substrate was inoculated with rice wine yeast culture AS2.21392 and brewer's yeast culture CGMCCNo.17452, with a viable cell count of 10 in both cultures. 12 CFU / mL, 7.5 mL of each of the two bacterial cultures were added, and fermentation was carried out in an incubator at 28℃ for 48 h, with the shaking speed at 300 r / min. After fermentation, the resulting fermented essence was sterilized in an autoclave at 100℃ for 30 min to terminate fermentation. After sterilization, the mixture was cooled to room temperature and then centrifuged at 4800 r / min for 30 min to obtain the supernatant. The supernatant was then sterilized in an autoclave at 100℃ for 30 min to avoid contamination by other microorganisms during centrifugation. After sterilization, the mixture was cooled to room temperature and then homogenized at 12000 r / min for 10 min to prepare the multifunctional bio-fermented essence.

[0087] Example 2

[0088] Compared with Example 1, the only difference is that the fermentation temperature is 35°C and the fermentation time is 28 hours, while all other conditions are the same as in Example 1.

[0089] Example 3

[0090] Compared with Example 1, the only difference is that the amount of Job's tears powder is 12g, the amount of sorghum seed powder is 12g, the amount of meadowfoam seed oil is 10g, and the amount of peony seed oil is 10g. All other conditions are the same as in Example 1.

[0091] Example 4

[0092] Compared with Example 1, the only difference is that the ratio of live cells of rice wine yeast AS2.1392 to brewer's yeast CGMCC No.17452 is 1:2, 5 mL of rice wine yeast AS2.1392 culture is added, and 10 mL of brewer's yeast CGMCC No.17452 culture is added. All other conditions are the same as in Example 1.

[0093] Example 5

[0094] Compared with Example 2, the only difference is that the ratio of viable cells of rice wine yeast AS2.21392 to brewer's yeast CGMCCNo.17452 is 2:1; 10 mL of rice wine yeast AS2.21392 culture solution is added and 5 mL of brewer's yeast CGMCCNo.17452 culture solution is added. All other conditions are the same as in Example 1.

[0095] Comparative Example 1

[0096] Compared with Example 1, the only difference is that the amount of Job's tears powder is 12g, the amount of sorghum seed powder and meadowfoam seed oil is 3g, and the amount of peony seed oil is 3g. All other conditions are the same as in Example 1.

[0097] Comparative Example 2

[0098] Compared with Example 1, the only difference is that the fermentation strains are Saccharomyces cerevisiae CGMCC No.17452 and Lactobacillus helveticus CICC 20243. All other conditions are the same as in Example 1.

[0099] Comparative Example 3

[0100] Compared with Example 1, the only difference is that the fermentation strains are Saccharomyces cerevisiae CGMCC No.17452 and Saccharomyces cerevisiae CGMCC No.22671, and all other conditions are the same as in Example 1.

[0101] Comparative Example 4

[0102] Compared with Example 1, the only difference is that the fermentation substrate is replaced with rice instead of Job's tears and sorghum seeds are replaced with corn, while all other conditions are the same as in Example 1.

[0103] Comparative Example 5

[0104] Compared with Example 1, the only difference is that after the initial fermentation with the yellow rice wine yeast AS2.1392, the fermentation liquid is then inoculated with the brewing yeast AS2.1392 for a secondary fermentation. All other conditions are the same as in Example 1. The specific operation is as follows:

[0105] (1) Coix seed is ground into powder and then passed through a 100-mesh sieve to obtain coix seed powder; sorghum seeds are ground into powder and then passed through a 100-mesh sieve to obtain sorghum seed powder. 12g of coix seed powder, 12g of sorghum seed powder, 12g of meadowfoam seed oil, 12g of peony seed oil and 300mL of deionized water are weighed and mixed. The mixture is sterilized at a temperature of 121℃ and a pressure of 0.12MPa for 30min. After sterilization, it is cooled to room temperature to obtain the fermentation substrate.

[0106] (2) 7.5 mL of rice wine yeast culture AS2.21392 was inoculated into the fermentation substrate. The viable count of the rice wine yeast culture AS2.21392 was 10. 12 CFU / mL, fermented once in an incubator at 28℃ for 24 hours, with the shaker speed at 300 rpm. After fermentation, the resulting fermented essence was sterilized in an autoclave at 100℃ for 30 minutes to terminate fermentation. Then, 7.5 mL of rice wine yeast culture AS2.21392 with a viable count of 10⁻⁶ was added again. 12 The emulsion was fermented at CFU / mL in a 28℃ incubator for 24 hours, with the shaking speed at 300 rpm. After fermentation, the resulting emulsion was sterilized at 100℃ for 30 minutes to terminate the secondary fermentation. After sterilization, the emulsion was cooled to room temperature and then centrifuged at 4800 rpm for 30 minutes to obtain the supernatant. The supernatant was then sterilized at 100℃ for 30 minutes to avoid contamination by other microorganisms during centrifugation. After sterilization, the emulsion was cooled to room temperature and then homogenized at 12000 rpm for 10 minutes to prepare a multi-functional bio-fermented essence emulsion.

[0107] Example 1

[0108] DPPH free radical scavenging experiment

[0109] DPPH is an early synthesized organic free radical often used to evaluate the hydrogen-donating capacity of antioxidants. It is very stable in organic solvents, exhibits a purple color, and has a characteristic absorption peak at 517 nm. When it encounters a free radical scavenger, the lone pair electrons of DPPH are paired, causing it to fade, meaning the absorbance at the maximum absorption wavelength decreases. Therefore, the scavenging effect of a sample on DPPH free radicals can be evaluated by measuring the change in absorbance.

[0110] The specific experimental steps of the DPPH free radical scavenging experiment are as follows:

[0111] (1) Take an equal volume (1 mL) of the test solution (diluted 10 times with deionized water) and 2 × 10 -4 Mix the mol / L DPPH solution thoroughly (tube A1);

[0112] (2) Take an equal volume (1 mL) of anhydrous ethanol (the solvent for the analyte) and 2 × 10⁻⁶ mol / L. -4 Mix the mol / L DPPH solution thoroughly (tube A2);

[0113] (3) Take an equal volume (1 mL) of anhydrous ethanol and mix it with the test solution (A3 tube);

[0114] (4) After reacting in the dark for 30 minutes, measure the absorbance values ​​of tubes A1, A2 and A3 at 517 nm; the clearance rate is calculated as follows: clearance rate = [(A2+A3)-A1] / A2×100%.

[0115] This experiment tested the DPPH free radical scavenging assay on the examples and comparative examples. The results are shown in Table 1 and... Figure 1 .

[0116] Table 1

[0117]

[0118]

[0119] From Table 1 and Figure 1 The results showed that the DPPH radical scavenging ability of the products prepared in Examples 1-5 was significantly higher than that of the products prepared in Comparative Examples 1-5.

[0120] Example 2

[0121] Determination of human type I collagen (COL-I) content

[0122] This experiment verifies the repair ability of the products prepared in the above examples and comparative examples on damaged skin by measuring the COL-I content in human skin fibroblasts.

[0123] Logarithmic-phase human skin fibroblasts 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 18 mJ / cm²... 2 Cells were irradiated with UVA for 40 min. 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 h. 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 collagen content was determined according to the ELISA kit instructions, with OD values ​​measured at 450 nm. The standard curve equation was (Y = 0.1989X - 0.0283, R...). 2 =0.9993), the collagen expression level x was calculated based on the standard curve, and corrected using the total protein content b to obtain A = x / b. Then, the relative expression level of COL-I was calculated: COL-I relative expression level = A experimental group / model group / A blank group. The results are shown in […]. Figure 2 ( Figure 2 In the table, p < 0.001 indicates a highly significant decrease compared to the control group; p > 0.05 indicates no significant increase compared to the model group; *p < 0.05 indicates an increase compared to the model group; **p < 0.01 indicates a significant increase compared to the model group; ***p < 0.001 indicates a highly significant increase compared to the model group (see Table 2).

[0124] Table 2

[0125]

[0126]

[0127] Depend on Figure 2 The results in Table 2 show that the COL-I content produced by the products prepared in Examples 1-5 after cell treatment was higher than that produced by the products prepared in Comparative Examples 1-5 after cell treatment.

[0128] Example 3

[0129] Human skin fibroblast toxicity test

[0130] This experiment used human skin fibroblasts from the Chinese Scientific Cell Bank to verify the cytotoxicity of the products prepared in the above examples and comparative examples.

[0131] 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.

[0132] 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.

[0133] 1. Experimental steps:

[0134] The products obtained in the above examples and comparative examples were respectively prepared into experimental test solutions with a volume percentage of 2% using serum-free DMEM medium.

[0135] Human skin fibroblasts were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin (1×10⁵ U / L) and streptomycin (100 mg / L). Cells were grown in an incubator at 37°C and 5% CO₂ saturated humidity. When cell confluence reached 85% or higher, the 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 7×10⁵ 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). In each experimental group, 100 μL of the pre-filtered and sterilized test solution of different concentrations was added to each well, with three 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 below. Figure 3 And Table 3.

[0136] The formula for calculating cell viability is as follows:

[0137] Cell viability (%) = (A experimental group - A blank control group) / (A control group - A blank control group) × 100%.

[0138] Table 3

[0139] Cell viability (%) Example 1 116.63±3.71 Example 2 105.08±5.2 Example 3 112.29±0.91 Example 4 110.3±7.17 Example 5 109.72±4.28 Comparative Example 1 84.92±2.45 Comparative Example 2 85.83±3.19 Comparative Example 3 92.99±4.07 Comparative Example 4 95.75±6.68 Comparative Example 5 92.93±4.59

[0140] Depend on Figure 3 The results in Table 3 show that the cell survival rate after treatment with the products prepared in Examples 1-5 is higher than that after treatment with the products prepared in Comparative Examples 1-5; and Examples 1-5 of this application have a certain cell growth promoting effect.

[0141] Example 4

[0142] Repair capacity (cell survival rate)

[0143] 1. Experimental steps:

[0144] The products obtained in Examples 1-5 and Comparative Examples 1-5 were respectively prepared into 1% (v / v) test solutions using serum-free DMEM medium. The test solutions were filtered through a 0.22 μm sterile filter membrane.

[0145] 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, the 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 the negative control group. 100 μL of the filtered and sterilized experimental test solution was added to each well of the experimental group, with each test solution prepared in triplicate. 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 4 , ( Figure 4In the table, ###p < 0.001 indicates a highly significant decrease compared to the control group; p > 0.05 indicates no significant increase compared to the model group; *p < 0.05 indicates an increase compared to the model group; **p < 0.01 indicates a significant increase compared to the model group; and ***p < 0.001 indicates a highly significant increase compared to the model group.

[0146] The formula for calculating cell viability is as follows:

[0147] Cell survival rate of experimental group or model group = (A experimental group or model group - A blank control group) / (A negative control group - A blank control group) × 100%.

[0148] Table 4

[0149]

[0150]

[0151] Depend on Figure 4 The results in Table 4 show that the cell survival rate after treatment with the products prepared in Examples 1-5 is higher than that after treatment with the products prepared in Comparative Examples 1-5. This indicates that the cell repair ability of Examples 1-5 in this application is higher than that of Comparative Examples 1-5.

[0152] Example 5

[0153] Stability observation.

[0154] Cold resistance test was conducted according to national standard GBT29665-2013—maintaining a temperature of (-8±2)℃ for 24 hours:

[0155] Examples 1-5 and Comparative Examples 1-5 were placed at -8°C for 24 hours.

[0156] Depend on Figure 5 It is evident that Examples 1-5 showed no delamination or discoloration after being placed at -8℃ for 24 hours and then returning to room temperature; while Comparative Examples 1-5 showed yellowing and slight delamination. This demonstrates that the cold resistance of Examples 1-5 in this application is higher than that of Comparative Examples 1-5.

[0157] Heat resistance test was conducted according to national standard GB / T29665-2013—maintaining a temperature of (40±1)℃ for 24 hours:

[0158] Examples 1-5 and Comparative Examples 1-5 were placed at 40°C for 24 hours:

[0159] Depend on Figure 6It is evident that Examples 1-5 showed no delamination or discoloration after being placed at 40°C for 24 hours and then returning to room temperature, while Comparative Examples 1-5 showed yellowing and slight delamination. This demonstrates that the heat resistance of Examples 1-5 in this application is higher than that of Comparative Examples 1-5.

[0160] 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.

[0161] 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 multi-functional bio-fermented essence cream containing a skin external agent, characterized in that, Specifically comprising the following steps: inoculating yeast into a fermentation substrate composed of Job's tears, sorghum seeds, vegetable oil and water, carrying out fermentation culture, sterilization, and preparing the fermentation essence milk. The yeast is composed of yellow wine yeast and Saccharomyces cerevisiae, and the ratio of the viable cell count of the yellow wine yeast to the Saccharomyces cerevisiae is 1:(0.5-5). The yellow wine yeast includes yellow wine yeast AS2.1392 purchased from the Beijing Food Brewing Institute Strain Preservation Center. The Saccharomyces cerevisiae includes Saccharomyces cerevisiae with the preservation number CGMCC No.17452. The mass ratio of the total mass of the Job's tears and the sorghum seeds to the mass of the vegetable oil is 1:(0.5-4).

2. The method of claim 1, wherein the skin-external agent-containing multi-functional biofermented essence cream is prepared by adding 0.1 to 10 parts by weight of the skin-external agent to 100 parts by weight of the biofermented essence cream. The preparation method of the fermentation essence milk satisfies at least one of the following conditions: The Job's tears are ground and sieved to obtain Job's tears powder, and the sieving mesh number is 50-150 meshes. The sorghum seeds are ground and sieved to obtain Job's tears powder, and the sieving mesh number is 50-150 meshes. The mass ratio of the Job's tears to the sorghum seeds is 1:(0.8-1.2). The vegetable oil is composed of at least one of the following: macadamia seed oil, Chinese olive oil, peony seed oil, white pool flower seed oil, sunflower seed oil, shea butter, thousand-layer fruit oil, sweet almond oil, jojoba oil, peony seed oil, monkey bread tree seed oil, small coffee seed oil, wheat germ oil, Chinese kiwi seed oil, rapeseed oil, perilla seed oil, ganoderma spore oil, glass flaxseed oil, camellia seed oil, deep-sea two-joint rapeseed oil, milk thistle seed oil, and olive fruit oil. The mass ratio of the total mass of the Job's tears and the sorghum seeds to the mass of the vegetable oil is 1:(0.5-3). The mass-volume ratio of the Job's tears to the water is 1:(80-120) g / mL. The fermentation substrate further includes sterilization before use. The ratio of the viable cell count of the yellow wine yeast to the Saccharomyces cerevisiae is 1:(0.5-2).

3. The method of claim 2, wherein the skin-external agent-containing multi-functional biofermented essence cream is prepared by adding 0.1 to 10 parts by weight of the skin-external agent to 100 parts by weight of the biofermented essence cream. The preparation method of the fermentation essence milk satisfies at least one of the following conditions: The Job's tears are ground and sieved to obtain Job's tears powder, and the sieving mesh number is 50-100 meshes. The sorghum seeds are ground and sieved to obtain Job's tears powder, and the sieving mesh number is 50-100 meshes. The mass ratio of the Job's tears to the sorghum seeds is 1:

1. The mass-volume ratio of the Job's tears to the water is 1:(90-110) g / mL. The sterilization method is high-temperature sterilization, and when the high-temperature sterilization is used to sterilize the fermentation substrate, the sterilization temperature is 110-125°C, and the sterilization time is 20-60 min. The ratio of the viable cell count of the yellow wine yeast to the Saccharomyces cerevisiae is 1:(0.5-2).

4. The method of claim 3, wherein the skin-external agent-containing multi-functional biofermented essence cream is prepared by adding 0.1 to 5% of the skin-external agent to the biofermented essence cream of claim 1. The preparation method of the fermentation essence milk satisfies at least one of the following conditions: When the high-temperature sterilization is used to sterilize the fermentation substrate, the sterilization temperature is 115-121°C, and the sterilization time is 20-40 min.

5. The method of claim 4, wherein the skin external agent-containing multi-functional biofermented essence cream is prepared by adding 0.1 to 5 parts by weight of the skin external agent to 100 parts by weight of the biofermented essence cream. The preparation method of the fermentation essence milk satisfies at least one of the following conditions: When the high-temperature sterilization is used to sterilize the fermentation substrate, the sterilization temperature is 121°C, and the sterilization time is 30 min.

6. The preparation method of the multi-functional bio-fermented essence emulsion containing a topical skin agent as described in claim 1, characterized in that, The preparation method of the fermented essence milk satisfies at least one of the following conditions: The yellow rice wine yeast is added in the form of a yellow rice wine yeast liquid, the number of viable bacteria in the yellow rice wine yeast liquid being 10 10 ~10 14 CFU / mL, based on the volume of deionized water in the initial fermentation system. The number of the inoculated yellow rice wine yeast in unit volume of the fermentation substrate is 10 8 ~10 12 CFU / mL; Said Saccharomyces cerevisiae is added in the form of a Saccharomyces cerevisiae liquid, the viable cell count of which is 10 10 ~10 14 CFU / mL, based on the volume of deionized water in the initial fermentation system. The number of the inoculated Saccharomyces cerevisiae bacteria in unit volume of the fermentation substrate is 10 10 ~10 14 CFU / mL, based on the volume of deionized water in the initial fermentation system.

7. The preparation method of the multi-functional bio-fermented essence emulsion containing a topical skin agent as described in claim 6, characterized in that, The preparation method of the fermented essence milk satisfies at least one of the following conditions: The number of viable bacteria in the yellow rice wine yeast bacterial solution is 10 10 ~10 12 CFU / mL; The number of the inoculated yellow rice wine yeast in unit volume of the fermentation substrate is 10 9 ~10 11 CFU / mL, based on the volume of deionized water in the initial fermentation system. The viable cell count of the Saccharomyces cerevisiae bacterial solution is 10 10 ~10 12 CFU / mL; The number of the inoculated S. cerevisiae cells in unit volume of the fermentation substrate is 10 10 ~10 12 CFU / mL.

8. The preparation method of the multi-functional bio-fermented essence emulsion containing a topical skin agent as described in claim 1, characterized in that, The preparation method of the fermented essence milk satisfies at least one of the following conditions: The fermentation culture is aerobic fermentation; The temperature of the fermentation culture is 25-35℃; The time of the fermentation culture is 12-72h.

9. The method of claim 8, wherein the skin-external agent-containing multi-functional bio-fermented essence cream is prepared by adding 0.1 to 5% of the skin-external agent to the bio-fermented essence cream of claim 1. The preparation method of the fermented essence milk satisfies at least one of the following conditions: The aerobic fermentation is performed in a shaking incubator, and the rotation speed of the shaking bed in the shaking incubator is 200-400r / min; The temperature of the fermentation culture is 25-30℃; The time of the fermentation culture is 12-60h.

10. The method for preparing a multi-functional bio-fermented essence emulsion containing a topical skin agent as described in claim 9, characterized in that, The preparation method of the fermented essence milk satisfies at least one of the following conditions: The rotation speed of the shaking bed in the shaking incubator is 250-350r / min; The temperature of the fermentation culture is 28℃; The time of the fermentation culture is 48h.

11. The method for preparing a multi-functional bio-fermented essence emulsion containing a topical skin agent as described in claim 10, characterized in that, The rotation speed of the shaking bed in the shaking incubator is 300r / min.

12. The preparation method of the multi-functional bio-fermented essence emulsion containing a topical skin agent as described in claim 1, characterized in that, The operation of sterilization further comprises the operations of cooling and / or centrifugation and collection of the supernatant.

13. The method for preparing a multi-functional bio-fermented essence emulsion containing a topical skin agent as described in claim 12, characterized in that, The cooling is cooling to room temperature; The rotation speed of the centrifugation is 3000-9000r / min; The radius of the centrifugation is 8-15cm; The time of the centrifugation is 10-40min.

14. The method for preparing a multi-functional bio-fermented essence emulsion containing a topical skin agent as described in claim 13, characterized in that, The rotation speed of the centrifugation is 4000-8000r / min; and the time of the centrifugation is 20-40min.

15. The method for preparing a multi-functional bio-fermented essence emulsion containing a topical skin agent as described in claim 14, characterized in that, The rotation speed of the centrifugation is 5000r / min; and the time of the centrifugation is 30min.

16. The method for preparing a multi-functional bio-fermented essence emulsion containing a topical skin agent as described in any one of claims 12-15, characterized in that, The operation of centrifugation further comprises at least one of the operations of secondary sterilization, preservative mixing and homogenization.

17. The method for preparing a multi-functional bio-fermented essence emulsion containing a topical skin agent as described in claim 16, characterized in that, The secondary sterilization is high-temperature sterilization; In the process of mixing with the preservative, the temperature of the mixing is 50-80℃; in the process of mixing with the preservative, the preservative comprises p-hydroxyacetophenone and / or 1,2-hexanediol; when the preservative comprises the p-hydroxyacetophenone and the 1,2-hexanediol, the mass percentage of the p-hydroxyacetophenone in the supernatant prepared after the centrifugation is 0.1%-1%, and the mass percentage of the 1,2-hexanediol in the supernatant prepared after the centrifugation is 0.1%-1%; The rotation speed of the homogenization is 8000r / min-14000r / min; The time of the homogenization is 3-45min.

18. The method for preparing a multi-functional bio-fermented essence emulsion containing a topical skin agent as described in claim 17, characterized in that, In the process of mixing with the preservative, the temperature of the mixing is 70-80℃; the mass percentage of the p-hydroxyacetophenone in the supernatant prepared after the centrifugation is 0.5%, and the mass percentage of the 1,2-hexanediol in the supernatant prepared after the centrifugation is 0.5%; The rotation speed of the homogenization is 9000r / min-13000r / min; The time of the homogenization is 5-25min. 19.A multi-effect biological fermented essence milk containing a skin external preparation, which is prepared by the preparation method of the multi-effect biological fermented essence milk containing a skin external preparation according to any one of claims 1-18.

20. Use of the multi-functional bio-fermented essence cream containing a skin external agent according to claim 19 for preparing a skin external agent directly as a product, as an additive or as a base.

21. Use of the skin external agent-containing multi-functional biofermented essence cream according to claim 20, directly as a product, as an additive or as a base in the preparation of a skin external agent, characterized in that, The fermented essence cream is at least one of an antioxidant component and an anti-aging active component in the skin external agent.

22. Use of the skin external agent-containing multi-functional biofermented essence cream of claim 21 in the preparation of a skin external agent directly as a product, as an additive, or as a base, characterized in that, The antioxidant component is an antioxidant active component having a DPPH radical scavenging ability; and the anti-aging active component is an anti-aging active component having a collagen production promoting ability and / or a cell repair promoting ability.

23. A multi-functional bio-fermented essence cream containing a skin external agent, characterized in that, The fermented essence cream according to claim 19.

24. The multi-functional bio-fermented essence emulsion containing a topical skin agent as described in claim 23, characterized in that, The multi-functional bio-fermented essence cream satisfies at least one of the following conditions: The skin external agent further comprises at least one of a repair active component, a firming active component, an anti-inflammatory active component and an anti-aging active component; The skin external agent further comprises a mask, an essence or a toner; The fermented essence cream accounts for 70% to 99% of the mass percentage of the skin external agent.

25. The multi-functional bio-fermented essence emulsion containing a topical skin agent as described in claim 24, characterized in that, The fermented essence cream accounts for 80% to 99% of the mass percentage of the skin external agent.

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