Galangal leavening, skin external preparation containing same, and preparation method and application of galangal leavening

Abstract

The invention discloses a preparation method of a galangal fermentation product, which comprises the following steps: inoculating Saccharomyces cerevisiae into a fermentation substrate, and carrying out fermentation culture and sterilization, thereby obtaining the galangal fermentation product. Wherein the fermentation substrate comprises galangal and water; the saccharomyces cerevisiae comprises yellow wine saccharomycetes and / or fruit wine saccharomycetes. The prepared galangal leavening has ideal effects of resisting oxidation and allergy, repairing damaged cells and the like, the preparation process is simple, the fermentation condition is mild, the energy consumption is low, resources are saved, no organic reagent is adopted, the use safety of the product is high, the requirements of modern society for the functionality and safety of skin external preparations are met, and the galangal leavening is suitable for popularization and application. The composition can be widely applied to the field of skin external preparations.

Description

Technical Field

[0001] The present application belongs to the technical field of cosmetics, and in particular relates to a galangal fermented product, a skin external preparation containing the same, and a preparation method and application thereof. Background Art

[0002] With the rapid development of science and technology, the improvement of human material living conditions, people's requirements for quality of life are constantly increasing. Actively exploring and developing antioxidant and anti-aging products are of great significance to improving the quality of life. Alpinia offcinarum Hance is the dried rhizome of the ginger plant Alpinia offcinarum Hance. It was first listed as a medium-grade product and recorded in the "Famous Doctors' Records". It is hot in nature and spicy in taste, and has the effects of warming the stomach and stopping vomiting, dispelling cold and relieving pain. At present, a large number of studies have shown that galangin has multiple pharmacological activities such as anti-inflammatory, anti-tumor, skin protection, anti-diabetes and its complications, anti-fibrosis, anti-pathogenic microorganisms, and neuroprotection. The many advantages of galangin can be applied to antioxidant and anti-aging products to improve people's quality of life.

[0003] At present, the research on galangal products is focused on separation and purification. Excessive separation and purification operations will lead to the continuous loss of active ingredients in galangal. In addition, existing technologies mostly use organic solvents to extract active ingredients in galangal, for example, alcohol precipitation method to extract galangin, combined with adsorbents for purification. However, organic solvents can only dissolve active ingredients in galangal that are soluble in organic solvents, resulting in the loss of other insoluble components of galangal, and reducing the resource utilization rate of galangal; and the organic solvent needs to be removed after extraction, which makes the process complicated and will also have adverse effects on the environment. There will be solvent residues in the final product, reducing the safety of product use.

[0004] Therefore, there is an urgent need in the art to develop a method for preparing a galangal extract that can efficiently utilize the active ingredients in galangal, is simple to operate, can be used in the cosmetics field, has good cosmetic effects, and is highly safe to use. Summary of the invention

[0005] The technical problem to be solved by the present application is to overcome the defects that the preparation method of galangal products in the prior art cannot achieve the extraction of all ingredients, resulting in reduced resource utilization of galangal ingredients, and the use of organic solvents in the extraction process, which pollutes the environment, is complicated to operate, and reduces the safety of use due to solvent residues, and to provide a galangal fermentation product, a skin topical preparation containing it, and a preparation method and application thereof. The galangal fermentation product of the present application has ideal antioxidant properties, and the preparation process is simple, the energy consumption is low, no organic solvent is required, and the final product is highly safe to use, which meets the requirements of modern society for the efficacy and safety of cosmetics.

[0006] The present invention adopts the following technical solutions to solve the above technical problems:

[0007] The invention provides a method for preparing galangal fermented product, which comprises the following steps: a fermentation substrate comprises galangal and water; inoculating Saccharomyces cerevisiae into the fermentation substrate, fermenting and culturing, and sterilizing;

[0008] Wherein, the brewing yeast includes rice wine yeast and / or fruit wine yeast.

[0009] In some embodiments, the galangal is galangal powder.

[0010] The galangal powder is crushed and sieved to a mesh size of 80 to 200 meshes, preferably 100 meshes.

[0011] In some embodiments, the number of the Saccharomyces cerevisiae inoculated per unit volume of the fermentation substrate is 10 6 ~10 10 CFU / mL, preferably 10 7 ~10 9 CFU / mL.

[0012] In some embodiments, the rice wine yeast may include rice wine yeast AS2.1392 purchased from Beijing Food Brewing Research Institute.

[0013] The yellow wine yeast AS2.1392 can be added in the form of yellow wine yeast AS2.1392 bacterial solution according to the conventional art, and the viable bacterial count concentration of the yellow wine yeast AS2.1392 in the yellow wine yeast AS2.1392 bacterial solution can be 10 9 ~10 13 CFU / mL, preferably 10 10 ~10 12 CFU / mL.

[0014] In some embodiments, the fruit wine yeast may include yeast SY purchased from Angel Yeast Co., Ltd. and / or yeast RW purchased from Angel Yeast Co., Ltd.

[0015] Wherein, the yeast SY is inoculated in the form of bacterial powder; preferably, the number of live bacteria per unit volume of the yeast SY is 10 6 ~10 10 CFU / mL, preferably 10 7 ~10 9 CFU / mL.

[0016] Wherein, the yeast RW is inoculated in the form of bacterial powder; the number of viable bacteria per unit volume of the yeast RW is 10 6 ~10 10 CFU / mL, preferably 10 7 ~109 CFU / mL.

[0017] In some embodiments, the mass ratio of the galangal to the water in the fermentation substrate may be 1:(100-500), preferably 1:(150-300).

[0018] In some embodiments, the fermentation substrate may be sterilized according to conventional procedures in the art before use. The method for sterilizing the fermentation medium may be a high temperature sterilization method conventionally used in the art.

[0019] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature may be a conventional temperature for such operations in the art, preferably 90-130°C, more preferably 115-125°C, for example 121°C.

[0020] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time can be the conventional time for such operation in the art, preferably 10 to 40 minutes, such as 30 minutes.

[0021] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization pressure is 0.1-0.14 MPa, preferably 0.1-0.13 MPa, and more preferably 0.12 MPa.

[0022] According to the common practice in the art, the sterilization operation of the fermentation substrate may further include a cooling operation, which may generally be cooling to room temperature.

[0023] In some embodiments, the fermentation culture temperature may be 25-37°C, preferably 25-35°C.

[0024] In some embodiments, the fermentation culture time may be 24 to 120 hours, preferably 36 to 96 hours.

[0025] In some embodiments, the fermentation culture is aerobic fermentation, which can generally be carried out under oscillation conditions according to the conventional art, and the oscillation speed can be 150-250 rpm, preferably 200 rpm.

[0026] In some embodiments, the sterilization method may be a method conventionally used for such operations in the art, generally a high-temperature sterilization method.

[0027] When the high temperature sterilization method is used for the sterilization, the sterilization temperature may be 110-125°C, preferably 115-121°C.

[0028] When the high temperature sterilization method is used for the sterilization, the sterilization pressure may be 0.1-0.14 MPa, preferably 0.1-0.13 MPa, and more preferably 0.12 MPa.

[0029] When the high temperature sterilization method is used for the sterilization, the sterilization time may be 15 to 35 minutes, preferably 25 to 35 minutes, and more preferably 30 minutes.

[0030] In some embodiments, the sterilization operation may further include cooling and / or centrifugation, and collecting the supernatant.

[0031] Wherein, according to the common practice in the art, the cooling may be cooling to room temperature.

[0032] The centrifugal rotation speed may be a conventional rotation speed for such operation in the art, preferably 3000-9000 rpm, more preferably 4000-6000 rpm, for example 4800 rpm.

[0033] 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.

[0034] Wherein, the centrifugation operation may further include secondary sterilization and / or mixing with a preservative.

[0035] The secondary sterilization method may be a high temperature sterilization method conventionally used in this field for such operations.

[0036] 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.

[0037] When the high temperature sterilization method is used for the secondary sterilization, the time of the secondary sterilization can be the conventional time for such operation in the art, preferably 20 to 40 minutes, more preferably 25 to 35 minutes.

[0038] When the high temperature sterilization method is used for the secondary sterilization, the pressure of the secondary sterilization may be a conventional pressure for such operations in the art, preferably 0.1 to 0.14 MPa, more preferably 0.1 to 0.13 MPa.

[0039] During the mixing process with the preservative, the mixing temperature can be a conventional temperature for such operations in the art, preferably 50-80°C.

[0040] In the process of mixing with the preservative, the preservative may include p-hydroxyacetophenone and / or 1,2-hexanediol as is conventional in the art. In the process of mixing with the preservative, the preservative may include p-hydroxyacetophenone and / or 1,2-hexanediol as is conventional in the art. When the preservative includes p-hydroxyacetophenone and 1,2-hexanediol, the mass percentage of p-hydroxyacetophenone in the supernatant obtained after centrifugation may be 0.25% to 1%, and the mass percentage of 1,2-hexanediol in the supernatant obtained after centrifugation may be 0.25% to 1%.

[0041] The present application also provides a galangal fermented product, which is prepared by the preparation method of the galangal fermented product as described above.

[0042] The present application also provides a use of the above-mentioned galangal fermented product directly as a product, as an additive or as a base in the preparation of a skin external preparation.

[0043] In some embodiments, the fermented product of Alpinia officinalis can be used as at least one of an antioxidant active ingredient, an anti-allergic active ingredient, and a skin repairing active ingredient in the skin external preparation.

[0044] The antioxidant active ingredient is an antioxidant active ingredient with DPPH free radical scavenging effect.

[0045] The anti-allergic active ingredient is an anti-allergic active ingredient with hyaluronidase inhibitory effect.

[0046] The anti-aging active ingredient is an anti-aging active ingredient that has the function of promoting cell repair.

[0047] The anti-inflammatory active ingredient is an anti-inflammatory active ingredient that can reduce the relative expression amount of the intracellular inflammatory factor IL-8.

[0048] The present invention also provides a skin external preparation comprising the above-mentioned galangal fermented product.

[0049] In some embodiments, the skin topical preparation may further include active ingredients commonly used in the art, generally including at least one of anti-inflammatory active ingredients, anti-allergic active ingredients and antioxidant active ingredients.

[0050] In some embodiments, the skin topical preparation may include but is not limited to a facial mask, essence or toner according to conventional methods in the art.

[0051] In some embodiments, the room temperature generally refers to 15-40°C.

[0052] In some embodiments, the fermented product of Alpinia officinalis accounts for 1% to 50% of the external skin preparation.

[0053] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present application.

[0054] The reagents and raw materials used in this application are commercially available.

[0055] The positive progressive effect of the present application is that the fermented product of Alpinia officinalis prepared in the present application has ideal antioxidant, anti-allergic and damaged cell repairing effects, the preparation process is simple, the fermentation conditions are mild, the energy consumption is low, resources are saved, no organic reagents are used, the product is highly safe to use, and meets the needs of modern society for the functionality and safety of skin topicals, and can be widely used in the field of skin topicals. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] 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.

[0057] in:

[0058] Figure 1 This is a comparison chart of the DPPH free radical scavenging ability of the products obtained in Examples 1 to 4 and Comparative Examples 1 to 4;

[0059] Figure 2 This is a comparison chart of the hyaluronidase inhibition ability of the products obtained in Examples 1 to 4 and Comparative Examples 1 to 4;

[0060] Figure 3 This is a comparison chart of the repairing ability of the products obtained in Examples 1 to 4 and Comparative Examples 1 to 4 on light-damaged cells;

[0061] Figure 4 This is a comparison chart of the effects of the products prepared in Examples 1 to 4 and Comparative Examples 1 to 4 on reducing the relative expression of the inflammatory factor IL-8 in damaged cells. DETAILED DESCRIPTION

[0062] 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 in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product instructions.

[0063] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0064] The galangal in the following examples and comparative examples is commercially available.

[0065] The yellow wine yeast AS2.1392 in the following examples was purchased from Beijing Food Brewing Research Institute.

[0066] The yeast SY in the following examples was purchased from Angel Yeast Co., Ltd.

[0067] The yeast RW in the following examples was purchased from Angel Yeast Co., Ltd.

[0068] The Angel high-sugar yeast powder in the following comparative examples was purchased from Angel Yeast Co., Ltd.

[0069] The Lactobacillus helveticus in the following comparative example was purchased from China Industrial Microbiological Culture Collection Center with a collection number of CICC 20243.

[0070] Example 1

[0071] Clean and intact galangal was screened, dried, crushed and sieved to 100 mesh, 1.5 g of galangal powder was mixed evenly with 300 mL of deionized water, and then sterilized in an autoclave at a temperature of 121° C. and a pressure of 0.12 MPa for 30 min, and cooled to room temperature after sterilization to obtain a fermentation substrate.

[0072] The preparation method of rice wine yeast AS2.1392 bacterial solution is as follows: the rice wine yeast AS2.1392 is inoculated into YPD solid medium for streaking activation, cultured in a 28°C incubator for 48 hours to obtain a single colony, and the single colony obtained is picked up by an inoculation loop and placed in YPD liquid medium, and cultured in a shaking incubator at a temperature of 28°C and a rotation speed of 180 rpm to obtain a live bacteria concentration of 10 10 CFU / mL of rice wine yeast AS2.1392 bacterial liquid is set aside.

[0073] 15 mL of yellow wine yeast AS2.1392 bacterial liquid was inoculated into the fermentation substrate prepared above. After the fermentation substrate was inoculated with the brewer's yeast bacterial liquid, it was shaken and fermented in a constant temperature incubator with a shaker speed of 200 rpm and a temperature of 30°C for 48 hours. After the fermentation and cultivation, it was placed in a high-pressure sterilizer at a temperature of 121°C and a pressure of 0.12 MPa for sterilization for 30 minutes; after the sterilization was completed, the fermentation liquid was cooled to room temperature and then centrifuged to collect the supernatant. The centrifugal speed was 4800 rpm and the centrifugal time was 30 minutes. The supernatant obtained after centrifugation was secondary sterilized in an autoclave at a temperature of 121°C and a pressure of 0.12 MPa for 30 minutes, and then p-hydroxyacetophenone and 1,2-hexanediol were added at 75°C, the mass percentage of p-hydroxyacetophenone in the supernatant was 0.25%, and the mass percentage of 1,2-hexanediol in the supernatant was 0.25%, and the mixture was mixed evenly to obtain the galangal fermentation product.

[0074] Example 2

[0075] Compared with Example 1, the only difference is that the fermentation strain is different. The yellow wine yeast AS2.1392 is replaced by 0.3g of yeast SY powder, and the number of live bacteria of yeast SY inoculated in a unit volume of fermentation substrate is 10 7 CFU / mL, other parameters were the same as in Example 1.

[0076] Example 3

[0077] Compared with Example 1, the only difference is that the fermentation strain is different. The yellow wine yeast AS2.1392 is replaced by yeast RW. Specifically, 0.3g of yeast RW powder is added, and the number of live bacteria of yeast SY inoculated in a unit volume of the fermentation substrate is 10 7 CFU / mL, other parameters were the same as in Example 1.

[0078] Example 4

[0079] Compared with Example 1, the only difference is that the fermentation strains are different. A composite strain fermentation culture is adopted, 0.15 g of yeast SY and yeast RW are inoculated respectively, and the total inoculation amount is 0.3 g. Other condition parameters are the same as in Example 1.

[0080] Comparative Example 1

[0081] Compared with Example 1, the only difference is that fermentation is not performed, and the specific operations are as follows:

[0082] Screen clean and intact galangal, take 1.5g of galangal and mix it evenly with 300mL of deionized water, then sterilize it in an autoclave at a temperature of 121°C and a pressure of 0.12MPa for 30min, cool it to room temperature after sterilization, and then centrifuge it at a centrifugal speed of 4800rpm for 30min. The centrifugal end temperature is 121°C and a pressure of 0.12MPa in an autoclave for 30min, and then add 0.5% p-hydroxyacetophenone and 0.5% 1,2-hexanediol at 75°C.

[0083] Comparative Example 2

[0084] Compared with Example 1, the only difference is that the fermentation strain is different, the strain is Angel high-sugar yeast powder, the addition amount is 0.3g, and the other condition parameters are the same as Example 1.

[0085] Comparative Example 3

[0086] Compared with Example 1, the only difference is that the fermentation strains are different, namely, yeast RW and Lactobacillus helveticus CICC20243, both of which are inoculated in the form of bacterial powder, 0.15 g of each, and other condition parameters are the same as in Example 1.

[0087] Comparative Example 4

[0088] Compared with Example 1, the only difference is that the fermentation culture time is different, specifically 144 hours, and the other condition parameters are the same as Example 1.

[0089] Effect Example 1

[0090] DPPH free radical scavenging experiment

[0091] 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.

[0092] The specific experimental steps of the DPPH free radical scavenging experiment are as follows:

[0093] (1) Take an equal volume (1 mL) of the test solution (the example and the prepared product) and Mix the DPPH solution (tube A1);

[0094] (2) Take an equal volume (1 mL) of anhydrous ethanol (solvent for the test substance) and Mix the DPPH solution (tube A2);

[0095] (3) Take an equal volume (1 mL) of anhydrous ethanol and mix it with the test solution (tube A3);

[0096] (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:

[0097]

[0098] In this experiment, the products obtained in the examples and comparative examples were subjected to DPPH free radical scavenging experimental tests. The results are shown in Table 1 and Figure 1.

[0099] Table 1

[0100] DPPH free radical scavenging rate (%) Example 1 78.44±0.44 Example 2 82.34±0.79 Example 3 81.34±0.49 Example 4 76.78±1.17 Comparative Example 1 57.69±1.68 Comparative Example 2 62.58±2.68 Comparative Example 3 59.31±1.52 Comparative Example 4 51.28±1.86

[0101] From Table 1 and Figure 1 The results show that the DPPH radical scavenging rates of the products prepared in Examples 1 to 4 are much higher than those of the products prepared in Comparative Examples 1 to 4. It can be seen that the products prepared in the examples of the present application have ideal antioxidant effects, and the fermentation time and strains have a great influence on the antioxidant properties of the final products. Figure 1In the table, ns means no statistical difference compared with Example 1; ***p<0.001 means there is an extremely significant statistical difference compared with Example 1; ## p<0.01, indicating that there is a very significant statistical difference compared with the blank control group.)

[0102] Effect Example 2

[0103] Hyaluronidase clearance assay

[0104] Hyaluronidase is one of the lysosomes that can decompose polysaccharides. It can hydrolyze potassium hyaluronate to generate β-N-acetylglucosamine. This substance condenses with acetylacetone under alkaline conditions to generate the chromogen 2-methyl-3-diacetylpyrrole derivative. The chromogen and Ehrlich reagent develop color in concentrated hydrochloric acid and ethanol. Hyaluronidase is strongly correlated with inflammation and allergy and is a participant in type I allergic reactions. Therefore, the hyaluronidase in vitro inhibition test can be used as a quick anti-allergic determination method.

[0105] Reagents: hyaluronidase, sodium hyaluronate, anhydrous ethanol, sodium hydroxide, anhydrous sodium carbonate, concentrated hydrochloric acid, p-dimethylaminobenzaldehyde, acetylacetone, glacial acetic acid, anhydrous calcium chloride.

[0106] Equipment: Sunrise microplate reader manufactured by Tecan Trading Co., Ltd.; digital constant temperature water bath from Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory;

[0107] Take 0.1mL CaCl2 solution (0.25mmol / L) and 0.5mL hyaluronidase solution (100U / mL) and incubate in a water bath at 37°C for 20min; add 0.5mL of the test sample solution (the product prepared in the embodiment and the comparative example was diluted 5 times), and continue to keep warm for 20min; then add 0.5mL sodium hyaluronate solution (0.5mg / mL), incubate in a water bath at 37°C for 30min, then take out and place at room temperature for 5min; add 0.1mL NaOH solution (0.4 mol / L) and 0.5 mL of acetylacetone solution (3.5 mL of acetylacetone dissolved in 50 mL of 1.0 mol / L sodium carbonate solution) were heated in a boiling water bath for 15 min and then immediately transferred to an ice water bath for cooling for 5 min; 1.0 mL of Ehrlich reagent (0.8 g of p-dimethylaminobenzaldehyde dissolved in 15 mL of concentrated hydrochloric acid and 15 mL of anhydrous ethanol) was added dropwise and diluted with 3.0 mL of anhydrous ethanol, and placed at room temperature for 20 min for color development, and the absorbance value at a wavelength of 540 nm was measured by a spectrophotometer. Three parallels were made for each of the above groups, and the results were averaged, and the hyaluronidase inhibition rate was calculated according to the following formula. The results are shown in Tables 2 and Figure 2 .

[0108] Hyaluronidase inhibition rate = [(AB) - (CD)] / (AB) × 100%, test results are shown in Table 2 and Figure 1 ( Figure 1 ***p<0.001, indicating that there is an extremely significant statistical difference compared with Example 1, and it is extremely significantly reduced; **p<0.01, indicating that there is a significant statistical difference compared with Example 1, and it is significantly reduced;);

[0109] In the formula: A—absorbance value of control solution (acetic acid buffer solution is used instead of test sample solution); B—absorbance value of control blank solution (acetic acid buffer solution is used instead of test sample solution and enzyme solution); C—absorbance value of test sample solution; D—absorbance value of test sample blank solution (acetic acid buffer solution is used instead of enzyme solution).

[0110] Table 2

[0111]

[0112]

[0113] From Table 2 and Figure 2 The results show that the hyaluronidase inhibition rate of the galangal fermented product obtained in Examples 1 to 4 of the present application is significantly higher than that in Comparative Examples 1 to 4, which shows that the product prepared in the present application has an ideal anti-allergic effect. Figure 2 In the table, ns means no statistical difference compared with Example 1; *p<0.05 means there is a statistical difference compared with Example 1; # p<0.05, indicating that there is an extremely significant statistical difference compared with the blank group, extremely significant increase).

[0114] Effect Example 3 Anti-cell damage (repair)

[0115] 1. Experimental steps:

[0116] The products obtained in the above examples and comparative examples were respectively prepared into 2% 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.

[0117] 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 , irradiation time was 80s, negative control group was not irradiated; PBS was discarded, serum-free DMEM medium was added to the model group and negative control group, 100μL of the above-configured filtered and sterilized experimental group test solution was added to each well of the experimental group, and 6 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 3 and Figure 3 ,

[0118] The cell viability was calculated as follows:

[0119] 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%.

[0120] Table 3

[0121] Cell survival rate (%) Negative control group 100.00±5.53 Model Group 56.50±2.09 Example 1 70.00±3.29 Example 2 71.67±3.18 Example 3 72.33±2.89 Example 4 68.38±3.43 Comparative Example 1 60.58±3.25 Comparative Example 2 61.64±1.99 Comparative Example 3 60.44±1.99 Comparative Example 4 59.51±2.28

[0122] From Table 3 and Figure 3 It can be seen that after treating sun-exposed cells with the fermented products of Alpinia officinalis prepared in Examples 1 to 4 of the present application, the cell survival rate is significantly improved, and Comparative Examples 1 to 4 also improve the survival rate of damaged cells, but the improvement effect is not as good as that of the embodiments. It can be seen that the product prepared in the present application has an ideal after-sun repair effect.

[0123] Effect Example 4

[0124] Determination of inflammatory factor IL-8 content

[0125] The fermented oils prepared in the above examples and comparative examples were diluted with DMSO to a volume fraction of 10%, and then prepared with serum-free DMEM medium to prepare a test solution of the experimental group with a volume percentage of 0.1%. The test solution needs to be filtered through a 0.22 μm sterile filter membrane.

[0126] HaCaT cells in logarithmic phase were taken and the cell suspension was inoculated into a 6-well cell culture plate at a density of 250,000 cells / mL. 2 mL of cell suspension was added to each well and cultured for 12 h. The experimental group and the model group were treated with 40 mJ / cm 2After UVB irradiation of cells, the supernatant was discarded. 2 mL of DMEM solution was added to the blank group and the model group, and 2 mL of DMEM solution containing the sample (sample volume percentage was 0.1%) was added to the experimental group to treat the cells for 24 h. After the treatment, 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, centrifuged at 10000 rpm and 4 ° C for 10 min, and the supernatant was obtained to obtain the cell lysis supernatant. 20 μL of cell lysate was taken to detect the total protein content in the sample using the BCA kit; the determination of inflammatory factors was carried out according to the instructions of the ELISA kit, and the OD values ​​were measured at 450 nm.

[0127] 2. Detection steps

[0128] 1) 30 minutes before the experiment, take out the test kit and return to room temperature. Before adding the standard / sample, wash the plate three times and spin dry.

[0129] 2) Add 100 μL of standard / sample to the reaction wells, seal the plate, incubate at 37°C for 90 min, tap and wash the plate 4 times.

[0130] 3) Add 100 μL of biotinylated antibody working solution to the reaction wells, seal the plate, incubate at 37°C for 60 min, tap and wash the plate 4 times.

[0131] 4) Add 100 μL of enzyme conjugate working solution to the reaction wells, seal the plate, incubate at 37°C for 30 min, tap and wash the plate 5 times.

[0132] 5) Add 50 μL of color development substrate to the reaction wells, seal the plate and incubate in a 37°C incubator in the dark for 15 min.

[0133] 6) Add 50 μL of stop solution and immediately measure the OD value at 450 nm using an ELISA reader (within 5 min).

[0134] According to the OD value obtained from the above test, the release level of IL-8 was calculated. The results are shown in Table 4 below. The release amount of each inflammatory factor needs to be corrected by BCA content. The expression amount A of the inflammatory factor was calculated according to the standard curve, and corrected with the protein content B to obtain C = A / B, and the relative expression amount = C / C 空白组 .

[0135] Table 4

[0136] Absorbance value Relative expression of IL-8 Blank Group 0.152 1±0.055 Model Group 0.181 1.639±0.081 Example 1 0.142 0.948±0.064 Example 2 0.136 0.889±0.133 Example 3 0.132 0.826±0.129 Example 4 0.148 0.951±0.07 Comparative Example 1 0.157 1.342±0.13 Comparative Example 2 0.16 1.269±0.127 Comparative Example 3 0.164 1.285±0.132 Comparative Example 4 0.173 1.38±0.068

[0137] From Table 4 and Figure 4 It can be seen that the fermented products of Alpinia officinalis obtained in Examples 1 to 4 of the present application can significantly reduce the relative expression of IL-8 in cells. Although the products obtained in Comparative Examples 1 to 4 can also reduce the relative expression of IL-8 in damaged cells, the reduction effect is much lower than that of the Examples. Figure 4 In the table, *p<0.05 indicates that there is a statistical difference compared with the model group; **p<0.01 indicates that there is a significant statistical difference compared with the model group; ***p<0.001 indicates that there is an extremely significant statistical difference compared with the model group; ### p<0.001, indicating that there is an extremely significant statistical difference compared with the blank group).

[0138] 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.

[0139] 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 method for preparing a fermented product of galangal, characterized in that: The method comprises the following steps: inoculating Saccharomyces cerevisiae into a fermentation substrate, culturing the fermentation substrate, and sterilizing the fermentation substrate; Wherein, the fermentation substrate includes galangal and water; the brewing yeast includes rice wine yeast and / or fruit wine yeast.

2. The method for preparing the fermented product of Alpinia officinalis according to claim 1, characterized in that: The preparation method of the galangal fermented product meets at least one of the following conditions: The number of the brewer's yeast inoculated in the fermentation substrate per unit volume is 10 6 ~10 10 CFU / mL, preferably 10 7 ~10 9 CFU / mL; The rice wine yeast includes rice wine yeast AS2.1392 purchased from Beijing Food Brewing Research Institute; The fruit wine yeast includes yeast SY purchased from Angel Yeast Co., Ltd. and / or yeast RW purchased from Angel Yeast Co., Ltd.; The mass ratio of the galangal to the water in the fermentation substrate is 1:(100-500), preferably 1:(150-300); The galangal is galangal powder; preferably, the galangal powder is crushed and sieved with a mesh size of 80 to 200 meshes, such as 100 meshes.

3. The method for preparing the fermented product of Alpinia officinalis according to claim 2, characterized in that: The preparation method of the galangal fermented product meets at least one of the following conditions: When the yeast includes the yellow wine yeast, the yellow wine yeast is added in the form of a yellow wine yeast liquid; preferably, the number of live bacteria in the yellow wine yeast liquid is 10 9 ~10 13 CFU / mL, preferably 10 10 ~10 12 CFU / mL; When the yeast includes the yeast SY, the yeast SY is added in the form of yeast SY powder; preferably, the number of live bacteria of the yeast SY inoculated in a unit volume of the fermentation substrate is 10 6 ~10 10 CFU / mL, preferably 10 7 ~10 9 CFU / mL; When the yeast includes the yeast RW, the yeast RW is added in the form of yeast RW powder; preferably, the number of live bacteria of the yeast RW inoculated in a unit volume of the fermentation substrate is 10 6 ~10 10 CFU / mL, preferably 10 7 ~10 9 CFU / mL.

4. The method for preparing the fermented product of Alpinia officinalis according to claim 1, characterized in that: The fermentation substrate also includes a further 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 90-130°C, preferably 115-125°C, and more preferably 121°C; when the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time is 10-40min, preferably 30min; when the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization pressure is 0.1-0.14MPa, preferably 0.1-0.13MPa, and more preferably 0.12Mpa; Preferably, the sterilization operation further includes cooling to room temperature.

5. The method for preparing the fermented product of Alpinia officinalis according to claim 1, characterized in that: The preparation method of the galangal fermented product meets at least one of the following conditions: The fermentation culture temperature is 25-37°C, preferably 25-35°C; The fermentation culture time is 24 to 120 hours, preferably 36 to 96 hours; The fermentation culture is aerobic fermentation; preferably, it is carried out under oscillation conditions, and more preferably, the rotation speed of the oscillation is 150-250rpm, preferably 200rpm.

6. The method for preparing the fermented product of Alpinia officinalis according to claim 1, characterized in that: The sterilization operation further includes cooling and / or centrifuging, and collecting the supernatant; Preferably, the cooling is cooling to room temperature; Preferably, the centrifugal speed is 3000-9000 rpm, more preferably 4000-6000 rpm, for example 4800 rpm; Preferably, the centrifugation time is 10 to 40 min, more preferably 20 to 40 min, for example 30 min.

7. The method for preparing the fermented product of Alpinia officinalis according to claim 6, characterized in that: The centrifugal operation further includes secondary sterilization and / or mixing with a preservative; Preferably, the secondary sterilization method is a high temperature sterilization method; when the high temperature sterilization method is used for the secondary sterilization, the temperature of the secondary sterilization is 110-125°C, more preferably 115-121°C; when the high temperature sterilization method is used for the secondary sterilization, the time of the secondary sterilization is 20-40 minutes, more preferably 25-35 minutes; when the high temperature sterilization method is used for the secondary sterilization, the pressure of the secondary sterilization is 0.1-0.14MPa, more preferably 0.1-0.13MPa; Preferably, during the mixing process with the preservative, the mixing temperature is 50 to 80°C; Preferably, in the process of mixing with the preservative, the preservative includes p-hydroxyacetophenone and / or 1,2-hexanediol; when the preservative includes p-hydroxyacetophenone and / or 1,2-hexanediol, the mass percentage of p-hydroxyacetophenone in the supernatant obtained after centrifugation is 0.25% to 1%; when the preservative includes 1,2-hexanediol, the mass percentage of 1,2-hexanediol in the supernatant obtained after centrifugation is 0.25% to 1%.

8. A fermented product of galangal, characterized in that: The product is prepared by the method for preparing the galangal fermented product according to any one of claims 1 to 7.

9. A use of the fermented product of Alpinia officinalis according to claim 8 directly as a product, as an additive or as a base in the preparation of a skin external preparation; Preferably, the galangal fermented product is used as at least one of the antioxidant active ingredient, anti-allergic active ingredient, anti-aging active ingredient and anti-inflammatory active ingredient of the skin external preparation; More preferably, the antioxidant active ingredient is an antioxidant active ingredient with DPPH free radical scavenging effect; the anti-allergic active ingredient is an anti-allergic active ingredient with hyaluronidase inhibitory effect; the anti-aging active ingredient is an anti-aging active ingredient with cell repair promoting effect; the anti-inflammatory active ingredient is an anti-inflammatory active ingredient with the ability to reduce the relative expression of intracellular inflammatory factor IL-8.

10. A skin external preparation, characterized in that: The galangal fermented product according to claim 8; Preferably, the skin external preparation further comprises at least one of an antioxidant active ingredient, an anti-allergic active ingredient, an anti-aging active ingredient and an anti-inflammatory active ingredient; Preferably, the skin external preparation comprises a facial mask, an essence or a toner; Preferably, the mass percentage of the galangal fermented product in the skin external preparation is 1% to 50%.

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