Multi-effect biological fermentation essence milk containing skin external preparation as well as preparation method and application of multi-effect biological fermentation essence milk

Abstract

The invention discloses a multi-effect biological fermentation essence milk containing a skin external preparation and a preparation method and application thereof, and the preparation method specifically comprises the following steps: inoculating saccharomycetes into a fermentation substrate composed of coix seeds, sorghum seeds, vegetable oil and water, and carrying out fermentation culture and sterilization to obtain the fermentation essence milk, wherein the saccharomycetes are composed of yellow wine saccharomycetes and saccharomyces cerevisiae, and the viable count ratio of the yellow wine saccharomycetes to the saccharomyces cerevisiae is 1: (0.5-5). According to the present invention, the active components of coix seed and vegetable oil are effectively utilized by using the yellow wine yeast and the saccharomyces cerevisiae in the fermentation manner so as to obtain the fermented essence milk containing the active components of natural plants, such that the loss of the active components is reduced while the fermented essence milk has good repairing, tightening and anti-aging effects, and does not have any side effect; under the condition that no additive is added, the cold-resistant and heat-resistant oil has ideal cold-resistant and heat-resistant stability, and can be widely applied to the field of cosmetics.

Description

Technical Field

[0001] The present application belongs to the technical field of cosmetics, and in particular relates to a multifunctional bio-fermented essence milk containing a skin external preparation, and a preparation method and application thereof. Background Art

[0002] As we age, our skin ages naturally and our skin condition deteriorates. External factors such as air pollution and ultraviolet radiation can exacerbate skin damage. Moreover, the cosmetics industry uses more chemical synthetic raw materials. Although they have advantages such as clear ingredient structure, clear efficacy, and mature quality control methods, people gradually find that chemical synthetic products can have adverse effects on the human body when using these cosmetics, such as sensitization, redness, and irritation. Therefore, reducing the adverse reactions caused by chemical synthetics has become the direction of cosmetics development, and how to effectively care for the skin has become very critical.

[0003] Job's tears, also known as coix seed, coix seed, coix rice, etc., is commonly known as "medicine king rice". In my country, the production is relatively high in Fujian, Liaoning, Shaanxi and other provinces. Starch accounts for more than 60% of the dry content in coix, and it is a high-starch agricultural product. The carbohydrate content is about 65%, which is equivalent to that of ordinary hybrid corn; the protein content is about 17%, 1.3 times higher than that of rice; the lipid content is about 5%, which is equivalent to 18 times that of rice, and most of it is unsaturated fatty acids; it contains calcium, phosphorus, iron, and other mineral elements and a variety of vitamins, dietary fiber and amino acids, and has high health effects and nutritional value. In recent years, scholars at home and abroad have gradually conducted in-depth research on the structure and physical and chemical properties of coix, and relevant research reports on the pharmacological activities of coix in anti-tumor, analgesia and anti-inflammatory, immune enhancement, regulation of glucose and lipid metabolism, lowering blood pressure, anti-oxidation, and anti-aging have been published one after another.

[0004] Sorghum is rich in nutrients such as starch and protein, and also contains a variety of active ingredients such as polyphenol compounds, phytosterols, higher alkanols and lipids. At present, sorghum is mainly used as a cereal in the fields of food processing and brewing. Its application field is relatively limited, and its application in the field of skin topical preparations is rarely reported. In the prior art, the methods for obtaining the active ingredients of sorghum mostly use water extraction or organic solvent extraction, which not only has cumbersome operating steps, low extraction efficiency, and high extraction cost, but also causes waste of resources and environmental pollution. Plant oils are natural high-molecular compounds composed of fatty acids and glycerol. They themselves are not composed of a single chemical structure, so plant oils cannot be fully absorbed by human skin. The main reason that affects the skin feel of plant oil skin care products is that there are a small amount of free macromolecular free groups in plant oils that are not easily absorbed by the skin.

[0005] At present, there are preparation processes for fermenting coix seeds and oil separately in China, but there is still a lack of a process that can ferment coix seeds and vegetable oil as fermentation substrates with bacteria at the same time. Coix seeds and vegetable oil are common raw materials in daily life, and they have the characteristics of being able to ferment excellent products. At this stage, this process has good potential. Summary of the invention

[0006] The technical problem to be solved by this application is to overcome the defects of the prior art, such as the complex composition and rich nutrition of coix seeds, the inability to effectively grasp the fermentation process, the difficulty in absorbing and utilizing the macromolecular free groups contained in the vegetable oil, and the lack of the process for fermenting coix seeds and oils, and to provide a multi-functional bio-fermented essence milk containing a skin topical agent and its preparation method and application. This application can show good stability without adding any functional additives, and has excellent repair, firming, anti-aging and antioxidant effects.

[0007] This application adopts the following technical solutions to solve the above technical problems:

[0008] The present application provides a method for preparing a multifunctional bio-fermented essence milk containing a skin external preparation, which specifically comprises the following steps: inoculating yeast into a fermentation substrate composed of coix seeds, sorghum seeds, vegetable oil and water, fermenting and culturing, and sterilizing to obtain the fermented essence milk;

[0009] Wherein, the yeast is composed of yellow wine yeast and brewer's yeast.

[0010] In some embodiments, the coix seeds are commercially available conventional coix seeds.

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

[0012] The mass ratio of the coix seed to the sorghum seed is 1:(0.8-1.2), preferably 1:1;

[0013] In some embodiments, the coix seeds are ground and sieved to obtain coix seed powder, and the coix seed powder has a sieve mesh number of 50 to 150 meshes, preferably 50 to 100 meshes.

[0014] In some embodiments, the sorghum seeds are ground and sieved to obtain coix seed powder, and the sieve mesh number is 50-150 mesh, preferably 50-100 mesh;

[0015] In some embodiments, the mass ratio of the coix seeds 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 macadamia seed oil, pruinosa fruit oil, peony seed oil, white meadowfoam seed oil, sunflower seed oil, shea butter, melaleuca oil, sweet almond oil, jojoba oil, peony seed oil, baobab seed oil, small-fruit coffee seed oil, wheat germ oil, actinidia chinensis seed oil, camelina seed oil, perilla seed oil, ganoderma lucidum spore oil, borage seed oil, camellia seed oil, shea butter seed oil, milk thistle seed oil, and olive oil.

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

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

[0019] In some embodiments, the fermentation substrate may be sterilized according to conventional procedures in the art before use.

[0020] The conditions and methods for sterilizing the fermentation substrate may be conventional conditions and methods for such operations in the art, generally high temperature sterilization.

[0021] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature can be conventional for such operations in the art, preferably 110-125°C, more preferably 115-121°C, for example 121°C.

[0022] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time can be conventional for such operations in the art, preferably 20 to 60 minutes, more preferably 20 to 40 minutes, for example 30 minutes.

[0023] The fermentation substrate may be further cooled after the sterilization operation, generally to room temperature.

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

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

[0026] In some embodiments, the ratio of the number of live cells of the rice wine yeast to the number of live cells of the brewer's yeast is 1:(0.5-2).

[0027] In some embodiments, the fermentation substrate may be sterilized according to conventional procedures in the art before use.

[0028] The conditions and methods for sterilizing the fermentation substrate may be conventional conditions and methods for such operations in the art, generally high temperature sterilization.

[0029] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature can be conventional for such operations in the art, preferably 110-125°C, more preferably 115-121°C, for example 121°C.

[0030] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time can be conventional for such operations in the art, preferably 20 to 60 minutes, more preferably 20 to 40 minutes, for example 30 minutes.

[0031] The fermentation substrate may be further cooled after the sterilization operation, generally to room temperature.

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

[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 liquid according to the conventional art, and the number of live bacteria in the rice wine yeast liquid is 10 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 the rice wine yeast inoculated in the fermentation substrate per unit volume 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 Saccharomyces cerevisiae may include Saccharomyces cerevisiae with a deposit number of CGMCC No.17452.

[0036] In some embodiments, based on the volume of deionized water in the initial fermentation system, the saccharomyces cerevisiae can be added in the form of a saccharomyces cerevisiae bacterial solution according to the conventional art, and the number of live bacteria in the saccharomyces cerevisiae bacterial solution is 10 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 the Saccharomyces cerevisiae inoculated in the fermentation substrate per unit volume 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 according to the conventional 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 to 72 hours, preferably 12 to 60 hours, and more preferably 48 hours.

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

[0042] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature can be conventional for such operations in the art, preferably 110-125°C, more preferably 115-121°C, for example 121°C.

[0043] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time can be conventional for such operations in the art, preferably 20 to 60 minutes, more preferably 20 to 40 minutes, for example 30 minutes.

[0044] In some embodiments, the sterilization operation may further include cooling and / or centrifugation to collect the supernatant.

[0045] Wherein, according to the common practice in the art, the cooling may be cooling to room temperature.

[0046] The centrifugal speed may be a conventional speed for such operation in the art, preferably 3000-9000 r / min, more preferably 4000-8000 r / min, for example 5000 r / min.

[0047] The centrifugal radius may be a conventional radius for such operations in the art, preferably 8 to 15 cm.

[0048] The centrifugation time may be the conventional time for such operation in the art, preferably 10 to 40 min, more preferably 20 to 40 min, for example 30 min.

[0049] Wherein, the centrifugal operation may further include at least one of secondary sterilization, preservative mixing and homogenization.

[0050] The conditions and methods for the secondary sterilization may be conventional conditions and methods for such operations in the art, and may generally be high-temperature sterilization.

[0051] When the high temperature sterilization method is used for the secondary sterilization, the temperature of the secondary sterilization can be a conventional temperature for such operations 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 of the secondary sterilization can be conventional for such operations in the art, 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 a conventional temperature for such operations in the art, preferably 50 to 80°C, more preferably 70 to 80°C.

[0054] During the mixing with the preservative, the preservative may include p-hydroxyacetophenone and / or 1,2-hexanediol according to conventional practices in the art.

[0055] When the preservative includes the p-hydroxyacetophenone and the 1,2-hexanediol, the mass percentage of the p-hydroxyacetophenone in the upper layer liquid obtained after centrifugation may be 0.1% to 1%, and the mass percentage of the 1,2-hexanediol in the upper layer liquid obtained after centrifugation may be 0.1% to 1%; preferably, the mass percentage of the p-hydroxyacetophenone in the upper layer liquid obtained after centrifugation is 0.5%, and the mass percentage of the 1,2-hexanediol in the upper layer liquid obtained after centrifugation is 0.5%.

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

[0057] The present application also provides a multifunctional bio-fermented essence milk containing a skin external preparation, which is prepared by the preparation method of the fermented essence milk as described above.

[0058] The present application also provides a use of the multifunctional bio-fermented essence milk containing the skin external preparation as described above directly as a product, as an additive or as a base in the preparation of the skin external preparation.

[0059] In some embodiments, the fermented milk essence can be used as at least one of an antioxidant active ingredient and an anti-aging active ingredient in the skin external preparation.

[0060] Wherein, the antioxidant active ingredient may be an antioxidant active ingredient having the ability to scavenge DPPH free radicals.

[0061] Wherein, the anti-aging active ingredient is an anti-aging active ingredient having the ability to promote collagen production and / or promote cell repair.

[0062] The present application also provides a skin external preparation, which includes the multi-functional bio-fermented essence milk containing the skin external preparation as described above.

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

[0064] In some embodiments, the topical skin preparation may include but is not limited to a facial mask, an essence or a toner according to conventional methods in the art.

[0065] In some embodiments, the fermented essence milk may account for 5% to 99% by mass of the skin external preparation, preferably 60% to 99% by mass.

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

[0067] On the basis of conforming to the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present application.

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

[0069] The present application uses rice wine yeast and brewer's yeast to effectively utilize the active ingredients of coix seed and vegetable oil through fermentation to obtain a multi-functional bio-fermented essence milk containing natural plant active ingredients. It not only reduces the loss of active ingredients, but also has good repairing, firming and anti-aging effects without any side effects. It also has ideal cold and heat stability without the need for external additives and can be widely used in the field of cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] The present application can be better understood by referring to the description given below in conjunction with the accompanying drawings. The accompanying drawings, together with the following detailed description, are included in this specification and form a part of this specification, and are used to further illustrate the preferred embodiments of the present application and explain the principles and advantages of the present application.

[0071] in:

[0072] Figure 1 A comparison chart of the DPPH free radical scavenging abilities of the products obtained in Examples 1 to 5 and Comparative Examples 1 to 5;

[0073] Figure 2 This is a comparison chart of the repairing ability of the products obtained in Examples 1 to 5 and Comparative Examples 1 to 5 on damaged skin;

[0074] Figure 3 This is a comparison chart of the effects of the products obtained in Examples 1 to 5 and Comparative Examples 1 to 5 on the growth of human skin fibroblasts;

[0075] Figure 4 This is a comparison chart of the damaged cell repair capabilities of the products obtained in Examples 1 to 5 and Comparative Examples 1 to 5;

[0076] Figure 5 The stability (cold resistance) comparison chart of the products obtained in Examples 1 to 5 and Comparative Examples 1 to 5;

[0077] Figure 6 The following is a comparison chart of the stability (heat resistance) of the products obtained in Examples 1 to 5 and Comparative Examples 1 to 5. DETAILED DESCRIPTION

[0078] The present application is further described below by way of examples, but the present application is not limited to the scope of the examples. The experimental methods without specific conditions in the following examples are selected according to conventional methods and conditions, or according to 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 liquid (referred to as culture liquid 1) comprises the following steps: the rice wine yeast AS2.1392 purchased from Beijing Food Brewing Research Institute is inoculated into the YPD solid culture medium for streak activation, and cultured in an incubator at a temperature of 28°C for 48 hours to obtain a single colony; the single colony is inoculated into the YPD liquid culture medium, and cultured in a shaker at a temperature of 28°C for 48 hours, and the shaker speed is 200 rpm.

[0081] In the following embodiments and comparative examples, brewer's yeast (Saccharomyces cerevisiae) YWY1, the depository unit is the General Microbiological Center of China Microorganism Culture Collection Administration (referred to as CGMCC), the address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, zip code 100101, the deposit date is March 27, 2019, and the deposit number is CGMCC No. 17452; the preparation method of the brewer's yeast solution (referred to as solution 2) of CGMCC No. 17452 is the same as that of the rice wine yeast, except that the strains are different.

[0082] The Lactobacillus helveticus in the following comparative example was purchased from China Industrial Microbiological Culture Collection Center with a collection number of CICC 20243.

[0083] The Saccharomycopsis fibuligera strain in the following comparative example was deposited at the General Microbiology Center of China Culture Collection Administration on June 7, 2021. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, and the deposit number is CGMCC No. 22671.

[0084] Example 1

[0085] (1) Grind coix seeds and then pass through a 100-mesh sieve to obtain coix seed powder; grind sorghum seeds and then pass through a 100-mesh sieve to obtain sorghum seed powder; weigh 12 g of coix seed powder, 12 g of sorghum seed powder, 12 g of meadowfoam seed oil, and 12 g of peony seed oil; then mix them with 300 mL of deionized water, sterilize them at a temperature of 121° C. and a pressure of 0.12 MPa for 30 min, and cool them to room temperature after sterilization to obtain a fermentation substrate.

[0086] (2) The fermentation substrate was inoculated with the culture liquid of yellow wine yeast AS2.21392 and the culture liquid of brewer's yeast CGMCC No.17452. The number of viable bacteria in the two culture liquids was 10 12 CFU / mL, 7.5mL of each of the two bacterial liquids was added, and fermented in a 28℃ incubator for 48h, with the shaker speed at 300r / min. After the fermentation culture was completed, the fermented essence milk was sterilized in an autoclave at a temperature of 100℃ for 30min to terminate the fermentation. After sterilization, it was cooled to room temperature and then centrifuged at 4800r / min for 30min to obtain the upper layer liquid, which was sterilized in an autoclave at a temperature of 100℃ for 30min to avoid contamination by other bacteria during the centrifugation process. After sterilization, it was cooled to room temperature and homogenized in a homogenizer at 12000r / min for 10min to prepare a multi-functional biological fermented essence milk.

[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 h. Other conditions are the same as those in Example 1.

[0089] Example 3

[0090] Compared with Example 1, the only difference is that 12g of coix seed powder, 12g of sorghum seed powder, 10g of white meadowfoam seed oil, and 10g of peony seed oil are used, and other conditions are the same as those in Example 1.

[0091] Example 4

[0092] Compared with Example 1, the only difference is that the ratio of the number of live bacteria of yellow wine yeast AS2.1392 to that of brewer's yeast CGMCC No.17452 is 1:2, 5 mL of yellow wine yeast AS2.1392 bacterial solution is inoculated, and 10 mL of brewer's yeast CGMCC No.17452 bacterial solution is inoculated, and other conditions are the same as those in Example 1.

[0093] Example 5

[0094] Compared with Example 2, the only difference is that the ratio of the number of live bacteria of rice wine yeast AS2.21392 to that of brewer's yeast CGMCC No.17452 is 2:1; 10 mL of rice wine yeast AS2.21392 culture solution is inoculated, and 5 mL of brewer's yeast CGMCC No.17452 culture solution is inoculated, and other conditions are the same as those in Example 1.

[0095] Comparative Example 1

[0096] Compared with Example 1, the only difference is that 12g of coix seed powder, 3g of sorghum seed powder, 3g of meadowfoam seed oil, and 3g of peony seed oil are used, and other conditions are the same as those 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, and the other conditions are the same as those 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 the other conditions are the same as those in Example 1.

[0101] Comparative Example 4

[0102] Compared with Example 1, the only difference is that the fermentation substrate is replaced by rice instead of coix seed, and the sorghum seed is replaced by corn, and the other conditions are the same as those in Example 1.

[0103] Comparative Example 5

[0104] Compared with Example 1, the only difference is that after the yellow wine yeast AS2.1392 bacterial liquid is inoculated for primary fermentation, the fermentation liquid is then inoculated with the brewer's yeast AS2.1392 bacterial liquid for secondary fermentation. The other conditions are the same as those in Example 1. The specific operations are as follows:

[0105] (1) Grind coix seeds and then pass through a 100-mesh sieve to obtain coix seed powder; grind sorghum seeds and then pass through a 100-mesh sieve to obtain sorghum seed powder, weigh 12 g of coix seed powder, 12 g of sorghum seed powder, 12 g of meadowfoam seed oil, 12 g of peony seed oil and 300 mL of deionized water, mix them, sterilize them at a temperature of 121° C. and a pressure of 0.12 MPa for 30 min, and cool them to room temperature after sterilization to obtain a fermentation substrate.

[0106] (2) Inoculate 7.5 mL of yellow wine yeast AS2.21392 into the fermentation substrate. The number of live bacteria in the yellow wine yeast AS2.21392 is 10 12 CFU / mL, and fermented in a 28℃ incubator for 24h, with the shaker speed at 300r / min. After the fermentation, the fermented essence milk was sterilized in an autoclave at 100℃ for 30min to terminate the fermentation. Then 7.5mL of rice wine yeast AS2.21392 was added again, and the number of live bacteria was 10 12 CFU / mL, secondary fermentation in a 28℃ incubator for 24h, where the shaker speed is 300r / min. After the fermentation culture is completed, the obtained secondary fermentation emulsion is sterilized in a sterilizer at a temperature of 100℃ for 30min to terminate the secondary fermentation. After sterilization, it is cooled to room temperature and then centrifuged at 4800r / min for 30min to obtain the upper layer liquid, which is sterilized in an autoclave at a temperature of 100℃ for 30min to avoid contamination by other bacteria during the centrifugation process. After sterilization, it is cooled to room temperature and homogenized in a homogenizer at 12000r / min for 10min to prepare a multi-functional biological fermentation essence milk.

[0107] Effect Example 1

[0108] DPPH free radical scavenging experiment

[0109] DPPH is an early synthesized organic free radical, which is often used to evaluate the hydrogen-donating capacity of antioxidants. It is very stable in organic solvents, purple, and has a characteristic absorption peak at 517nm. When encountering free radical scavengers, the lone pair of electrons of DPPH is paired and it fades, that is, the absorbance at the maximum absorption wavelength becomes smaller. Therefore, the scavenging effect of the 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 mol / L DPPH solution and mix well (tube A1);

[0112] (2) Take an equal volume (1 mL) of anhydrous ethanol (solvent for the test substance) and 2×10 -4 mol / L DPPH solution and mix well (tube A2);

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

[0114] (4) After 30 minutes of reaction in the dark, measure the absorbance 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 experiment on the embodiments and comparative examples. The results are shown in Tables 1 and Figure 1 .

[0116] Table 1

[0117]

[0118]

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

[0120] Effect Example 2

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

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

[0123] Human skin fibroblasts in logarithmic phase were taken and the cell suspension was inoculated on a 6-well cell culture plate at a density of 250,000 / mL. 2 mL of cell suspension was added to each well and cultured for 12 hours. Experimental group, blank group and model group were set up. Among them, the experimental group and model group were treated with 18mJ / cm 2 The cells were irradiated with UVA for 40 minutes. After irradiation, the supernatant in 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 the test solution was added to the experimental group (the product prepared in the above embodiment or comparative example was configured into a test solution with a volume percentage of 2% using DMEM, and the test solution needed to be filtered through a 0.22 μm sterile filter membrane before use), and the cells were treated for 24 hours, the supernatant was discarded, and the cells were washed 2 to 3 times with PBS, and then the cells were treated with cell lysis solution and transferred to a centrifuge tube, centrifuged at 10000 r / min and 4°C for 10 minutes, and the supernatant was taken to obtain the cell lysate. 20 μL of cell lysate was taken to detect the total protein content in the sample using the BCA kit; the determination of collagen content was carried out according to the instructions of the ELISA kit, and each OD value was measured at 450nm. The standard curve equation was (Y=0.1989X-0.0283, R 2 =0.9993), the collagen expression x was calculated according to the standard curve, and corrected with the total protein content b to obtain A=x / b, and then the relative expression of COL-I was calculated, COL-I relative expression = A experimental group / model group / A blank group, the results are shown in Figure 2 ( Figure 2 In the table, ###p<0.001, indicating an extremely significant decrease compared with the blank group; p>0.05, ns indicates no significant increase compared with the model group; *p<0.05, indicating an increase compared with the model group, **p<0.01, indicating a significant increase compared with the model group, ***p<0.001, indicating an extremely significant increase compared with the model group) and Table 2.

[0124] Table 2

[0125]

[0126]

[0127] Depend on Figure 2 The results of Table 2 show that the COL-I content produced by treating cells with the products prepared in Examples 1 to 5 is higher than the COL-I content produced by treating cells with the products prepared in Comparative Examples 1 to 5.

[0128] Effect Example 3

[0129] Human skin fibroblast toxicity test

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

[0131] Reagents: 0.25% (containing EDTA) trypsin was produced by GIBCO, USA; DMEM culture medium was produced by GIBCO, USA; double antibody was produced by Corning, USA; CCK-8 was produced by Beijing Biode Biotech Co., Ltd.; fetal bovine serum was produced by GIBCO, USA; phosphate buffer was produced by Beijing Bioregi Biotech Co., Ltd.

[0132] Equipment: The manufacturer of WJ-80A-Ⅱ CO2 constant temperature incubator is Shanghai Shengke Instrument Equipment Co., Ltd.; the manufacturer of Sunrise microplate reader is Tecan Trading Co., Ltd.; the manufacturer of TL80-2 medical centrifuge is Jiangsu Tianli Medical Equipment Co., Ltd.; the manufacturer of NUNC 96-well cell culture plate is Thermo Fisher Scientific.

[0133] 1. Experimental steps:

[0134] The products obtained in the above examples and comparative examples were respectively prepared with serum-free DMEM culture medium to form a 2% volume percentage of the experimental group test solution.

[0135] Human skin fibroblasts were cultured in DMEM medium containing 10% fetal bovine serum and 1% double-antibody (1×105U / L penicillin, 100mg / L streptomycin). The cells were grown in an incubator at 37°C and 5% CO2 saturated humidity. When the cell fusion reached more than 85%, the logarithmic growth phase cells were digested with 0.05% trypsin and the digestion reaction was terminated with serum-containing DMEM. The cell count was performed on a cell counting plate and the cell suspension concentration was adjusted to 7×10 4 / mL, inoculate the cell suspension into a 96-well plate at a ratio of 100μL per well, and incubate at 37℃, 5% CO2 for 12h. Remove the old culture medium and wash the cells twice with phosphate buffer. Add 100μL of the above-configured, filtered and sterilized test solution of different concentrations to each well in the experimental group, and make 3 replicate wells for each test solution; the control group contains cells, and serum-free DMEM culture medium is added; the blank control group has no cells, and 100μL of PBS is added. Incubate for another 24h at 37℃, 5% CO2. Then add 10μL of CCK-8 solution to each well, incubate for another 3h, measure the absorbance at a wavelength of 450nm, and calculate the cell survival rate of each group. The results are shown in Figure 3 and Table 3.

[0136] The cell viability was calculated as follows:

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

[0138] Table 3

[0139] Cell survival rate (%) 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 of Table 3 show that the survival rate of cells treated with the products prepared in Examples 1 to 5 is higher than that of cells treated with the products prepared in Comparative Examples 1 to 5; and Examples 1 to 5 of the present application have a certain effect of promoting cell growth.

[0141] Effect Example 4

[0142] Repair capacity (cell survival rate)

[0143] 1. Experimental steps:

[0144] The products obtained in the above Examples 1 to 5 and Comparative Examples 1 to 5 were respectively prepared into 1% test solution in the experimental group using serum-free DMEM culture medium. The test solution was required to be filtered through a 0.22 μm sterile filter membrane.

[0145] HaCaT cells were cultured in a medium containing 10% fetal bovine serum and 1% double antibody (1×10 5 U / L penicillin, 100 mg / L streptomycin) in DMEM medium. The cells were grown in an incubator at 37°C and 5% CO2 saturated humidity. When the cell fusion reached more than 85%, the cells in the logarithmic growth phase were digested with 0.05% trypsin and the digestion reaction was terminated with serum-containing DMEM. The cell count was performed on a cell counting plate and the cell suspension concentration was adjusted to 7×10 4 / mL, the cell suspension was inoculated into a 96-well plate at a ratio of 100 μL per well and incubated in a 37°C, 5% CO2 incubator for 12 hours. After incubation, the old culture medium was removed, the cells were washed twice with phosphate buffer, and then 100 μL of PBS solution was added. The model group and the experimental group were irradiated with UVB at a dose of 40 mJ / cm 2 , the irradiation time was 80s, and the negative control group was not irradiated; PBS was aspirated and discarded, serum-free DMEM medium was added to the model group and the negative control group, 100μL of the above-configured filtered and sterilized test solution of the experimental group was added to each well of the experimental group, and 3 replicates were made for each test solution; the blank control group had no cells and 100μL of PBS was added. Then incubate in an incubator at 37°C and 5% CO2 for 24h. Then 10μL of CCK-8 solution was added to each well, and incubated for another 3h. The absorbance value was measured at a wavelength of 450nm, and the cell survival rate of each group was calculated. The results are shown in Table 4 and Figure 4 , ( Figure 4In the table, ###p<0.001, indicating an extremely significant decrease compared with the blank group; p>0.05, ns, indicating no significant increase compared with the model group; *p<0.05, indicating an increase compared with the model group, **p<0.01, indicating a significant increase compared with the model group, ***p<0.001, indicating an extremely significant increase compared with the model group).

[0146] The cell viability was calculated as follows:

[0147] The cell survival rate of the 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 survival rate of cells treated with the products prepared in Examples 1 to 5 is higher than that of cells treated with the products prepared in Comparative Examples 1 to 5. This shows that the cell repair capabilities of Examples 1 to 5 of the present application are higher than those of Comparative Examples 1 to 5.

[0152] Effect Example 5

[0153] Stability observation.

[0154] Cold resistance test according to national standard GBT29665-2013 - (-8±2)℃ for 24h:

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

[0156] Depend on Figure 5 It is obvious that Examples 1 to 5 were placed at -8°C for 24 hours and then returned to room temperature without stratification or discoloration; Comparative Examples 1 to 5 were found to have yellowing and slight stratification. It can be seen that the cold-resistant stability of Examples 1 to 5 of the present application is higher than that of Comparative Examples 1 to 5.

[0157] According to the national standard GBT29665-2013, heat resistance test is carried out at (40±1)℃ for 24h:

[0158] Examples 1 to 5 and Comparative Examples 1 to 5 were placed at 40° C. for 24 h:

[0159] Depend on Figure 6It is obvious that Examples 1 to 5 were placed at 40°C for 24 hours and then returned to room temperature without stratification or discoloration, while Comparative Examples 1 to 5 were found to have yellowing and slight stratification. It can be seen that the heat stability of Examples 1 to 5 of the present application is higher than that of Comparative Examples 1 to 5.

[0160] Finally, it should be noted that in this application, the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements that are not explicitly listed, or also includes elements that are inherent to such process, method, article or apparatus.

[0161] Although the present application has been disclosed above through the description of the specific embodiments of the present application, it should be understood that those skilled in the art can design various modifications, improvements or equivalents to the present application within the spirit and scope of the attached scheme. These modifications, improvements or equivalents should also be considered to be included in the scope of protection claimed in the present application.

Claims

1. A multifunctional bio-fermented essence milk containing a skin external preparation, characterized in that: The method specifically comprises the following steps: inoculating yeast into a fermentation substrate composed of coix seeds, sorghum seeds, vegetable oil and water, fermenting and culturing, and sterilizing to obtain the fermented essence milk; The yeast is composed of yellow wine yeast and brewer's yeast, and the ratio of the number of live bacteria of the yellow wine yeast to that of the brewer's yeast is 1:(0.5-5).

2. The method for preparing the multifunctional bio-fermented essence milk containing a skin external preparation according to claim 1, characterized in that: The method for preparing the fermented essence milk satisfies at least one of the following conditions: The coix seeds are ground and sieved to obtain coix seed powder, wherein the sieve mesh number is 50 to 150 meshes, preferably 50 to 100 meshes; The sorghum seeds are ground and sieved to obtain coix seed powder, wherein the sieve mesh number is 50-150 meshes, preferably 50-100 meshes; The mass ratio of the coix seed to the sorghum seed is 1:(0.8-1.2), preferably 1:1; The vegetable oil is composed of at least one of macadamia seed oil, prune fruit oil, peony seed oil, white meadowfoam seed oil, sunflower seed oil, shea butter, melaleuca oil, sweet almond oil, jojoba oil, peony seed oil, baobab seed oil, coffee seed oil, wheat germ oil, kiwi seed oil, camelina seed oil, perilla seed oil, ganoderma lucidum spore oil, borage seed oil, camellia seed oil, shea butter seed oil, milk thistle seed oil, and olive oil; The mass ratio of the total mass of the coix seeds and sorghum seeds to the mass ratio of the vegetable oil is 1:(0.5-4), preferably 1:(0.5-3); The mass volume ratio of the coix seed to the water is 1: (80-120) g / mL; preferably 1: (90-110) g / mL; The fermentation substrate also includes a sterilization operation before use; preferably, the sterilization method is a high temperature sterilization method; when the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature is preferably 110-125°C, more preferably 115-121°C, for example, 121°C; when the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time may be preferably 20-60 minutes, more preferably 20-40 minutes, for example, 30 minutes; In some embodiments, the ratio of the number of live cells of the rice wine yeast to the number of live cells of the brewer's yeast is 1:(0.5-2).

3. The method for preparing the multifunctional bio-fermented essence milk containing a skin external preparation according to claim 1, characterized in that: The method for preparing the fermented essence milk satisfies at least one of the following conditions: The rice wine yeast includes rice wine yeast AS2.1392 purchased from the strain collection center of Beijing Food and Brewing Research Institute; Based on the volume of deionized water in the initial fermentation system, the yellow wine yeast was added in the form of a yellow wine yeast liquid, and the number of live bacteria in the yellow wine yeast liquid was 10 10 ~10 14 CFU / mL, preferably 10 10 ~10 12 CFU / mL; Based on the volume of deionized water in the initial fermentation system, the number of the rice wine yeast inoculated in the fermentation substrate per unit volume is 10 8 ~10 12 CFU / mL, preferably 10 9 ~10 11 CFU / mL; The saccharomyces cerevisiae includes the saccharomyces cerevisiae with a deposit number of CGMCC No. 17452; Based on the volume of deionized water in the initial fermentation system, the brewer's yeast is added in the form of the brewer's yeast liquid, and the number of live bacteria in the brewer's yeast liquid is 10 10 ~10 14 CFU / mL, preferably 10 10 ~10 12 CFU / mL; In some embodiments, based on the volume of deionized water in the initial fermentation system, the number of the Saccharomyces cerevisiae inoculated in the fermentation substrate per unit volume is 10 10 ~10 14 CFU / mL, preferably 10 10 ~10 12 CFU / mL.

4. The method for preparing the multifunctional bio-fermented essence milk containing a skin external preparation according to claim 1, characterized in that: The method for preparing the fermented essence milk satisfies at least one of the following conditions: The fermentation culture is aerobic fermentation. Preferably, the aerobic fermentation is carried out in a shaking incubator. 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 temperature is 25-35°C, preferably 25-30°C, more preferably 28°C; The fermentation culture time is 12 to 72 hours, preferably 12 to 60 hours, and more preferably 48 hours; The sterilization is a high-temperature sterilization method; preferably, the sterilization method is a high-temperature sterilization method; when the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature is 110-125°C, preferably 115-121°C, and more preferably 121°C; when the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time is 20-60min, preferably 20-40min, and more preferably 30min.

5. The method for preparing the multifunctional bio-fermented essence milk containing a skin external preparation according to claim 1, characterized in that: The sterilization operation further includes cooling and / or centrifuging to collect the supernatant; Preferably, the cooling is cooling to room temperature; Preferably, the centrifugal speed is 3000-9000 r / min, preferably 4000-8000 r / min, for example 5000 r / min; Preferably, the radius of the centrifuge is 8 to 15 cm; Preferably, the centrifugation time is 10 to 40 min, more preferably 20 to 40 min, for example 30 min.

6. The method for preparing the multifunctional bio-fermented essence milk containing a skin external preparation according to claim 5, characterized in that: The centrifugal operation further includes at least one of secondary sterilization, preservative mixing and homogenization; Preferably, the secondary sterilization is a high temperature sterilization method; when the high temperature sterilization method is used for the secondary sterilization, the sterilization method is a high temperature sterilization method; when the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature is 110-125°C, preferably 115-121°C, and more preferably 121°C; when the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time is 20-60 minutes, preferably 20-40 minutes, and more preferably 30 minutes; Preferably, during the mixing process with the preservative, the mixing temperature is 50-80°C, more preferably 70-80°C; during the mixing process with the preservative, the preservative includes p-hydroxyacetophenone and / or 1,2-hexanediol; when the preservative includes p-hydroxyacetophenone and 1,2-hexanediol, the mass percentage of p-hydroxyacetophenone in the upper liquid obtained after centrifugation is 0.1%-1%, and the mass percentage of 1,2-hexanediol in the upper liquid obtained after centrifugation is 0.1%-1%; preferably, the mass percentage of p-hydroxyacetophenone in the upper liquid obtained after centrifugation is 0.5%, and the mass percentage of 1,2-hexanediol in the upper liquid obtained after centrifugation is 0.5%; Preferably, the homogenizing speed is 8000r / min to 14000r / min, preferably 9000r / min to 13000r / min; Preferably, the homogenization time is 3 to 45 minutes, more preferably 5 to 25 minutes.

7. A multifunctional bio-fermented essence milk containing a skin external preparation, which is prepared by the preparation method of the multifunctional bio-fermented essence milk containing a skin external preparation according to any one of claims 1 to 6.

8. Use of the multifunctional bio-fermented essence milk containing a skin external preparation as claimed in claim 7 directly as a product, as an additive or as a base in the preparation of a skin external preparation.

9. The multifunctional bio-fermented essence milk containing a skin external preparation according to claim 7, characterized in that: The fermented essence milk is used as at least one of the antioxidant component and the anti-aging active component in the skin external preparation; Preferably, the antioxidant component is an antioxidant active component having the ability to scavenge DPPH free radicals; Preferably, the anti-aging active ingredient is an anti-aging active ingredient that has the ability to promote collagen production and / or promote cell repair.

10. A multifunctional bio-fermented essence milk containing a skin external preparation, characterized in that: The fermented essence milk according to claim 7; Preferably, the skin topical preparation comprises at least one of a repairing active ingredient, a firming active ingredient, an anti-inflammatory active ingredient and an anti-aging active ingredient; Preferably, the skin external preparation further comprises a facial mask, an essence or a toner; Preferably, the fermented essence milk accounts for 70% to 99% by mass of the skin external preparation, and more preferably 80% to 99% by mass.

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