A high galangal ferment, a skin external preparation containing the same, and a preparation method and application thereof

Abstract

The application discloses a preparation method of high galangal fermentation product, comprising the following steps: inoculating Saccharomyces cerevisiae into a fermentation substrate, carrying out fermentation culture, and sterilizing, and the process ends; wherein the fermentation substrate comprises high galangal and water; and the Saccharomyces cerevisiae comprises yellow wine yeast and / or fruit wine yeast. The high galangal fermentation product prepared by the application has ideal effects of antioxidation, anti-allergy and damaged cell repair, the preparation process is simple, the fermentation condition is mild, energy consumption is low, resources are saved, no organic reagent is used, the product has high use safety, meets the demand of modern society on the functionality and safety of skin external agents, and can be widely applied in the field of skin external agents.

Description

Technical Field

[0001] The present application belongs to the field of cosmetic technology, and in particular relates to a fermented product of galangal, 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, people's material living conditions have improved, and people's demands for quality of life are constantly increasing. Actively exploring and developing antioxidant and anti-aging products is of great significance to improving the quality of life. Alpinia offcinarum Hance, the dried rhizome of the ginger family, was first listed as a medium-grade product and recorded in the "Ming Yi Bie Lu". It is hot in nature and pungent in taste, and has the effects of warming the stomach, stopping vomiting, and dispelling cold and relieving pain. Currently, a large number of studies have shown that galangin has multiple pharmacological activities, including 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] Currently, research on galangal products focuses on separation and purification. Excessive separation and purification operations can lead to the continuous loss of galangal's active ingredients. Existing technologies often use organic solvents to extract galangin, for example, using alcohol precipitation to extract galangin, which is then purified using adsorbents. However, organic solvents can only dissolve those active ingredients in galangal that are soluble in the organic solvent, resulting in the loss of other insoluble components and reducing the resource utilization of galangal. Furthermore, the organic solvent must be removed after extraction, which complicates the process and has adverse environmental impacts. Solvent residues may remain in the final product, reducing its safety.

[0004] Therefore, there is an urgent need in the art to develop a method for preparing an Alpinia officinalis extract that can efficiently utilize the active ingredients in Alpinia officinalis, 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 this application is to overcome the defects of the existing methods for preparing galangal products, such as the inability to extract all ingredients, resulting in reduced resource utilization of galangal ingredients, the use of organic solvents in the extraction process, environmental pollution, complex operation, and reduced safety due to solvent residues. The present application provides a galangal fermented product, a skin topical preparation containing the same, and a preparation method and application thereof. The galangal fermented product of this application has ideal antioxidant properties, a simple preparation process, low energy consumption, and does not require any organic solvents. The final product is highly safe to use and meets the efficacy and safety requirements of modern society for cosmetics.

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

[0007] The application provides a preparation method of high galangal fermentation product, which comprises the following steps: fermenting a fermentation substrate comprising high galangal and water; inoculating Saccharomyces cerevisiae into the fermentation substrate, carrying out fermentation culture, sterilizing, and then obtaining the high galangal fermentation product.

[0008] The Saccharomyces cerevisiae comprises yellow wine yeast and / or fruit wine yeast.

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

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

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

[0012] In some embodiments, the yellow wine yeast can comprise yellow 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 liquid according to the conventional method in the art, and the viable cell concentration of the yellow wine yeast AS2.1392 in the yellow wine yeast AS2.1392 liquid can be 10 9 -10 13 CFU / mL, preferably 10 10 -10 12 CFU / mL.

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

[0015] The yeast SY is inoculated in the form of yeast powder; preferably, the viable cell number of the yeast SY per unit volume is 10 6 -10 10 CFU / mL, more preferably 10 7 -10 9 CFU / mL.

[0016] The yeast RW is inoculated in the form of yeast powder; the viable cell number of the yeast RW per unit volume is 10 6 -10 10 CFU / mL, more preferably 10 7 -109 CFU / mL.

[0017] In some embodiments, the mass ratio of the Alpinia oxyphylla and the water in the fermentation substrate can be 1:(100-500), preferably 1:(150-300).

[0018] In some embodiments, the fermentation substrate can further comprise a sterilization operation before use according to the conventional operation in the art. The method for sterilizing the fermentation medium can be the autoclaving method commonly used in the art.

[0019] When the autoclaving method is used to sterilize the fermentation substrate, the temperature of the sterilization can be the temperature commonly used in the operation in the art, preferably 90-130°C, more preferably 115-125°C, for example 121°C.

[0020] When the autoclaving method is used to sterilize the fermentation substrate, the time of the sterilization can be the time commonly used in the operation in the art, preferably 10-40 min, for example 30 min.

[0021] When the autoclaving method is used to sterilize the fermentation substrate, the pressure of the sterilization can be 0.1-0.14 MPa, preferably 0.1-0.13 MPa, more preferably 0.12 MPa.

[0022] According to the conventional operation in the art, the operation of sterilizing the fermentation substrate can further comprise a cooling operation, which can generally be cooling to room temperature.

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

[0024] In some embodiments, the time of the fermentation culture can be 24-120 h, preferably 36-96 h.

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

[0026] In some embodiments, the method for sterilization can be the method commonly used in the operation in the art, which can generally be the autoclaving method.

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

[0028] When the sterilization is performed by the high-temperature sterilization method, the sterilization pressure can be 0.1-0.14 MPa, preferably 0.1-0.13 MPa, and more preferably 0.12 MPa.

[0029] When the sterilization is performed by the high-temperature sterilization method, the sterilization time can be 15-35 min, preferably 25-35 min, and more preferably 30 min.

[0030] In some embodiments, the sterilization operation can further include cooling and / or centrifugation, and collection of supernatant.

[0031] In the cooling, the cooling can be to room temperature according to the convention in the art.

[0032] In the centrifugation, the centrifugation speed can be the speed conventionally used in the art for such operation, preferably 3000-9000 rpm, and more preferably 4000-6000 rpm, for example 4800 rpm.

[0033] In the centrifugation, the centrifugation time can be the time conventionally used in the art for such operation, preferably 10-40 min, and more preferably 20-40 min, for example 30 min.

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

[0035] The secondary sterilization method can be the high-temperature sterilization method conventionally used in the art for such operation.

[0036] In the secondary sterilization by the high-temperature sterilization method, the secondary sterilization temperature can be the temperature conventionally used in the art for such operation, preferably 110-125°C, and more preferably 115-121°C.

[0037] In the secondary sterilization by the high-temperature sterilization method, the secondary sterilization time can be the time conventionally used in the art for such operation, preferably 20-40 min, and more preferably 25-35 min.

[0038] In the secondary sterilization by the high-temperature sterilization method, the secondary sterilization pressure can be the pressure conventionally used in the art for such operation, preferably 0.1-0.14 MPa, and more preferably 0.1-0.13 MPa.

[0039] In the mixing with the preservative, the mixing temperature can be the temperature conventionally used in the art for such operation, preferably 50-80°C.

[0040] The preservative can include p-hydroxyacetophenone and / or 1,2-hexanediol during mixing with the preservative. The preservative can include p-hydroxyacetophenone and / or 1,2-hexanediol during mixing with the preservative. When the preservative includes the p-hydroxyacetophenone and the 1,2-hexanediol, the p-hydroxyacetophenone can be included in an amount of 0.25% to 1% by mass of the supernatant prepared after centrifugation, and the 1,2-hexanediol can be included in an amount of 0.25% to 1% by mass of the supernatant prepared after centrifugation.

[0041] The present application also provides a galangal ferment prepared by the method for preparing a galangal ferment as described above.

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

[0043] In some embodiments, the galangal ferment can be used as at least one of an antioxidant active ingredient, a skin repair active ingredient, and an anti-allergic active ingredient in the skin external agent.

[0044] The antioxidant active ingredient is an antioxidant active ingredient having a DPPH radical scavenging effect.

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

[0046] The anti-aging active ingredient is an anti-aging active ingredient having a cell repair promoting effect.

[0047] The anti-inflammatory active ingredient is an anti-inflammatory active ingredient having a reducing effect on the relative expression amount of an inflammatory factor IL-8 in cells.

[0048] The present application also provides a skin external agent including the galangal ferment as described above.

[0049] In some embodiments, the skin external agent can further include an active ingredient generally used in the art, and can include at least one of an anti-inflammatory active ingredient, an anti-allergic active ingredient, and an antioxidant active ingredient.

[0050] In some embodiments, the skin external agent can include, but is not limited to, a pack, an essence, or a toner, as in the art.

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

[0052] In some embodiments, the galangal ferment can be included in an amount of 1% to 50% of the skin external agent.

[0053] The above-mentioned preferred conditions can be combined arbitrarily to obtain preferred examples of the present application.

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

[0055] The positive progress effect of the present application is that the high galangal ferment prepared by 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 reagent is used, the product has high safety in use, meets the needs of modern society for the functionality and safety of skin external agents, and can be widely applied in the field of skin external agents. BRIEF DESCRIPTION OF DRAWINGS

[0056] The present application can be better understood by referring to the following description in conjunction with the accompanying drawings. The drawings are incorporated in and constitute a part of the specification and are meant to further exemplify preferred embodiments of the present application and to explain the principles and advantages of the present application.

[0057] wherein:

[0058] Figure 1 The product prepared in Examples 1-4 and Comparative Examples 1-4 is compared in terms of DPPH free radical scavenging ability.

[0059] Figure 2 The product prepared in Examples 1-4 and Comparative Examples 1-4 is compared in terms of hyaluronidase inhibitory ability.

[0060] Figure 3 The product prepared in Examples 1-4 and Comparative Examples 1-4 is compared in terms of repair ability on light-damaged cells.

[0061] Figure 4 The product prepared in Examples 1-4 and Comparative Examples 1-4 is compared in terms of reducing the relative expression amount of inflammatory factor IL-8 in damaged cells. DETAILED DESCRIPTION

[0062] The present application will be further described by way of examples below, but the present application is not limited in the scope of the examples. The experimental methods in the following examples are not specified, and are selected according to conventional methods and conditions, or according to the instructions of the goods.

[0063] The experimental methods used in the following examples are conventional methods unless otherwise specified.

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

[0065] The yellow rice wine yeast AS2.1392 in the following examples is 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 type yeast powder in the following comparative examples was purchased from Angel Yeast Co., Ltd.

[0069] The Lactobacillus helveticus in the following comparative examples was purchased from China Industrial Microbial Culture Collection Center, and the preservation number was CICC 20243.

[0070] Example 1

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

[0072] The preparation method of the Saccharomyces cerevisiae AS2.1392 bacterial liquid was as follows: the Saccharomyces cerevisiae AS2.1392 was inoculated into YPD solid culture medium for streaking activation, and cultured in a 28 ℃ incubator for 48 h to obtain single colonies. The single colonies were picked up with an inoculation loop and cultured in YPD liquid culture medium in a shaking incubator at a temperature of 28 ℃ and a speed of 180 rpm to prepare a Saccharomyces cerevisiae AS2.1392 bacterial liquid with a viable bacterial concentration of 1010CFU / mL, which was ready for use.

[0073] 15 mL of the Saccharomyces cerevisiae AS2.1392 bacterial liquid was inoculated into the fermentation substrate prepared above, and the inoculated fermentation substrate was shaken and then cultured in a shaking incubator at a speed of 200 rpm and a temperature of 30 ℃ for 48 h. After the culture, the fermentation substrate was sterilized in an autoclave at a temperature of 121 ℃ and a pressure of 0.12 MPa for 30 min. After sterilization, the fermentation liquid was cooled to room temperature, and then centrifuged to collect the supernatant. The centrifugation was performed at a speed of 4800 rpm for 30 min. The supernatant prepared after centrifugation was sterilized in an autoclave at a temperature of 121 ℃ and a pressure of 0.12 MPa for 30 min, and then p-hydroxyacetophenone and 1,2-hexanediol were added at 75 ℃. 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%. The mixture was uniformly mixed to prepare a galangal fermentation product.

[0074] Example 2

[0075] The difference compared with example 1 is only that the fermentation strain is different, and the Saccharomyces cerevisiae AS2.1392 is replaced by 0.3 g of Saccharomyces cerevisiae SY powder, and the number of live Saccharomyces cerevisiae SY in the unit volume of fermentation substrate is 10 7 CFU / mL, and other condition parameters are the same as those in example 1.

[0076] Example 3

[0077] The difference compared with example 1 is only that the fermentation strain is different, and the Saccharomyces cerevisiae AS2.1392 is replaced by Saccharomyces cerevisiae RW, specifically 0.3 g of Saccharomyces cerevisiae RW powder is added, and the number of live Saccharomyces cerevisiae SY in the unit volume of fermentation substrate is 10 7 CFU / mL, and other condition parameters are the same as those in example 1.

[0078] Example 4

[0079] The difference compared with example 1 is only that the fermentation strain is different, and the Saccharomyces cerevisiae SY and Saccharomyces cerevisiae RW are each inoculated with 0.15 g, and the total amount of inoculation is 0.3 g, and other condition parameters are the same as those in example 1.

[0080] Comparative example 1

[0081] The difference compared with example 1 is only that no fermentation is performed, and the specific operation is as follows:

[0082] Clean and intact Alpinia officinarum Hance is selected, 1.5 g of Alpinia officinarum Hance is mixed with 300 mL of deionized water to obtain a mixture, and then the mixture is sterilized in a high-pressure sterilization pot at a temperature of 121 ℃ and a pressure of 0.12 MPa for 30 min. After sterilization, the mixture is cooled to room temperature, and then centrifuged at a speed of 4800 rpm for 30 min. After centrifugation, the temperature is sterilized in a high-pressure sterilization pot at a temperature of 121 ℃ and a pressure of 0.12 MPa for 30 min, and then 0.5% of p-hydroxyacetophenone and 0.5% of 1,2-hexanediol are added at 75 ℃.

[0083] Comparative example 2

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

[0085] Comparative example 3

[0086] The difference compared with example 1 is only that the fermentation strain is different, and the strains are Saccharomyces cerevisiae RW and Lactobacillus helveticus CICC20243, which are inoculated in the form of powder, and each is inoculated with 0.15 g, and other condition parameters are the same as those in example 1.

[0087] Comparative example 4

[0088] The only difference between Example 1 and the present example is that the fermentation culture time is different, specifically 144 h, and other conditions are the same as in Example 1.

[0089] 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 ability of antioxidants. It is very stable in organic solvents, has a purple color, and has a characteristic absorption peak at 517 nm. When a free radical scavenger is encountered, the lone pair of electrons of DPPH is paired, causing it to lose color, that is, the absorbance value at the maximum absorption wavelength decreases. Therefore, the effect of the sample on the scavenging of 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) Mix equal volumes (1 mL) of the test solution (Example and product prepared) and 2 x 10 ﹣4 mol / L DPPH solution (A1 tube);

[0094] (2) Mix equal volumes (1 mL) of anhydrous ethanol (solvent for the test substance) and 2 x 10 ﹣4 mol / L DPPH solution (A2 tube);

[0095] (3) Mix equal volumes (1 mL) of anhydrous ethanol and the test solution (A3 tube);

[0096] (4) After 30 min of light-free reaction, measure the absorbance values of the A1 tube, A2 tube, and A3 tube at 517 nm; the calculation formula for the scavenging rate is:

[0097] Scavenging rate = [(A2 + A3) - A1] / A2 x 100%.

[0098] The products prepared in the examples and comparative examples were tested by the DPPH free radical scavenging experiment, and the results are shown in Table 1 and Figure 1 .

[0099] Table 1

[0100] DPPH 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 the results in Table 1 and Figure 1 , it can be seen that the DPPH free radical scavenging rates of the products prepared in Examples 1-4 are much higher than those of the products prepared in Comparative Examples 1-4. It can be seen that the products prepared in the present application have ideal antioxidant efficacy, and the fermentation culture time and strain have a great influence on the antioxidant performance of the final product. Figure 1ns indicates no statistical difference compared with Example 1; ***p<0.001 indicates extremely significant statistical difference compared with Example 1; ## ***p<0.001 indicates extremely significant statistical difference compared with the blank control group.

[0102] Example 2

[0103] Hyaluronidase elimination experiment

[0104] Hyaluronidase is one of the lysosomes capable of decomposing polysaccharides, and can hydrolyze potassium hyaluronate to generate β-N-acetylglucosamine. The substance is condensed with acetylacetone under alkaline conditions to generate a chromogenic 2-methyl-3-diacetyl pyrrole derivative. The chromogenic agent is colored with Ehrlich's reagent in concentrated hydrochloric acid ethanol. Hyaluronidase has a strong correlation with inflammation and allergy, and is a participant of type I allergic reaction. Therefore, the in vitro inhibition experiment of hyaluronidase can be used as a quick anti-allergy determination method.

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

[0106] Equipment: Sunrise microplate reader, manufacturer: Dikin Trade Co., Ltd.; digital constant temperature water bath, Shanghai Boxin Industrial Co., Ltd. Medical Equipment Factory;

[0107] Take 0.1 mL of CaCl2 solution (0.25 mmol / L) and 0.5 mL of hyaluronidase solution (100 U / mL) in a 37°C water bath for 20 min; add 0.5 mL of test sample solution (the product prepared in the examples and comparative examples is diluted 5 times), continue to incubate for 20 min; add 0.5 mL of sodium hyaluronate solution (0.5 mg / mL), incubate in a 37°C water bath for 30 min, then take out and place at room temperature for 5 min; add 0.1 mL of NaOH solution (0.4 mol / L) and 0.5 mL of acetylacetone solution (3.5 mL of acetylacetone is dissolved in 50 mL of 1.0 mol / L sodium carbonate solution), heat in a boiling water bath for 15 min, then immediately transfer to an ice water bath for cooling for 5 min; add 1.0 mL of Ehrlich's reagent (0.8 g of p-dimethylamino benzaldehyde is dissolved in 15 mL of concentrated hydrochloric acid and 15 mL of anhydrous ethanol), and dilute with 3.0 mL of anhydrous ethanol, place at room temperature for 20 min for color development, and measure the absorbance value at a wavelength of 540 nm with a spectrophotometer. Each group is performed in triplicate, the results are averaged, and the hyaluronidase inhibition rate is calculated according to the following formula, and the results are shown in Table 2 and Figure 2 .

[0108] Hyaluronidase inhibition rate = [(A-B)-(C-D)] / (A-B) x 100%, the test results are shown in Table 2 and Figure 1 Figure 1 Among them, ***p<0.001 indicates that there is a very significant statistical difference compared with Example 1, and the decrease is very significant; **p<0.01 indicates that there is a significant statistical difference compared with Example 1, and the decrease is significant;).

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

[0110] Table 2

[0111]

[0112]

[0113] From the results of Table 2 and Figure 2 It can be seen from the results that the hyaluronidase inhibition rate of the high galangal ferment prepared in Examples 1-4 of the present application is significantly higher than that of Comparative Examples 1-4, and it can be seen that the product prepared in the present application has ideal anti-allergic effect. Figure 2 Among them, ns indicates no statistical difference compared with Example 1; *p<0.05 indicates that there is a statistical difference compared with Example 1. # p<0.05 indicates that there is a very significant statistical difference compared with the blank group, and the increase is very significant.

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

[0115] 1. Experimental steps:

[0116] The products prepared in the above examples and comparative examples were respectively configured into a 2% volume percentage test solution in a serum-free DMEM medium. The test solution needs to pass through a 0.22 μm sterile filter membrane.

[0117] HaCaT cells were cultured in a DMEM medium containing 10% fetal bovine serum and 1% double-antibiotic (1x10 5 U / L of penicillin, 100 mg / L of streptomycin). The cells were grown in a 37°C, 5% CO2 saturated humidity incubator, and when the cell fusion reached more than 85%, the logarithmic growth period cells were digested with 0.05% trypsin, and the digestion reaction was terminated with serum-containing DMEM. The cell suspension concentration was adjusted to 7x10 4 ​mL, the cell suspension was inoculated into a 96-well plate at a ratio of 100 μL per well, and incubated at 37°C in a 5% CO2 incubator for 12 h. After incubation, the old culture solution was removed, the cells were washed twice with phosphate buffer solution, and 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 , for 80 s, and the negative control group was not irradiated. The PBS was aspirated, and the model group and the negative control group were added with serum-free DMEM medium, and the experimental group was added with 100 μL of the filtered and sterilized experimental group test solution prepared above, with 6 replicate wells for each test solution. The blank control group had no cells and was added with 100 μL of PBS. Then, it was incubated at 37°C in a 5% CO2 incubator for 24 h. Then, 10 μL of CCK-8 solution was added per well, and it was incubated for another 3 h. The absorbance value was measured at a wavelength of 450 nm, and the cell survival rate of each group was calculated. The results are shown in Table 3 and Figure 3 ,

[0118] The calculation formula of the cell survival rate is as follows:

[0119] The cell survival rate of the experimental group or the model group = (A experimental group or model group - A blank control group) / (A negative control group - A blank control group) x 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] As can be seen from Table 3 and Figure 3 , the cell survival rate of the post-sun cells treated with the high galangal ferment prepared in Examples 1 to 4 is significantly improved, and the 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 examples. It can be seen that the product prepared in the present application has ideal post-sun repair effect.

[0123] Effect Example 4

[0124] Inflammation factor IL-8 content determination

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

[0126] Logarithmic phase HaCaT cells were taken, and the cell suspension was inoculated on a 6-well cell culture plate at a density of 25 million / mL, 2 mL of cell suspension was added per well, and it was cultured for 12 h. The experimental group and the model group were irradiated with UVB at a dose of 40 mJ / cm 2After irradiating the cells with UVB, the supernatant was discarded. The blank and model groups were treated with 2 mL of DMEM solution, while the experimental group was treated with 2 mL of DMEM solution containing the sample (0.1% by volume) for 24 hours. The supernatant was discarded and the cells were washed two to three times with PBS. The cells were then treated with cell lysis buffer and centrifuged at 10,000 rpm for 10 minutes at 4°C. The supernatant was collected to obtain the cell lysis supernatant. 20 μL of the cell lysate was used to assay the total protein content in the sample using a BCA kit. Inflammatory factors were assayed according to the ELISA kit instructions, and the OD values ​​were measured at 450 nm.

[0127] 2. Detection steps

[0128] 1) 30 minutes before the experiment, remove the test kit and return to room temperature. Before adding standards / samples, 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 the plate, and wash it four 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 the plate, and wash it four 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 the plate, and wash it five times.

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

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

[0134] Based on the OD values ​​obtained from the above test, the IL-8 release level 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 IL-8 relative expression amount 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 **p<0.01, indicates significant statistical difference compared with model group; ***p<0.001, indicates extremely significant statistical difference compared with model group; ### ***p<0.001, indicates extremely significant statistical difference compared with blank group).

[0138] Finally, it should be noted that the terms "comprising", "comprising" or any other variant thereof in the present application are intended to cover non-exclusive inclusion, so that processes, methods, articles or devices including a series of elements not only include those elements, but also include other elements not explicitly listed or inherent to such processes, methods, articles or devices.

[0139] Although the present application has been disclosed by the description of the specific embodiments of the present application above, it should be understood that those skilled in the art can design various modifications, improvements or equivalents of the present application within the spirit and scope of the appended claims. These modifications, improvements or equivalents should also be considered to be included in the scope of the present application.

Claims

1. A method for preparing a high-quality ginger ferment, characterized by, The process comprises the following steps: Saccharomyces cerevisiae ) is inoculated into the fermentation substrate, fermented and cultured, and sterilized; The fermentation substrate comprises high galangal and water; the Saccharomyces cerevisiae comprises Saccharomyces cerevisiae and / or Saccharomyces cerevisiae; The temperature of the fermentation culture is 25-37℃; The time of the fermentation culture is 24-120h; The fermentation culture is aerobic fermentation; and the fermentation culture is carried out under oscillation.

2. The preparation method of the ginger ferment as claimed in claim 1, characterized by, The preparation method of the high galangal fermentation product satisfies at least one of the following conditions: the number of the inoculated Saccharomyces cerevisiae bacteria in the fermentation substrate per unit volume is 10 6 CFU / mL 10 CFU / mL The Saccharomyces cerevisiae comprises Saccharomyces cerevisiae AS2.1392 purchased from Beijing Food Brewing Research Institute; The Saccharomyces cerevisiae comprises Saccharomyces cerevisiae SY purchased from Angel Yeast Co., Ltd. and / or Saccharomyces cerevisiae RW purchased from Angel Yeast Co., Ltd.; The mass ratio of the high galangal to the water in the fermentation substrate is 1:(100-500); The high galangal is high galangal powder.

3. The preparation method of the ginger ferment as claimed in claim 2, characterized by, The preparation method of the high galangal fermentation product satisfies at least one of the following conditions: the number of the inoculated S. cerevisiae cells in the fermentation substrate per unit volume is 10 7 ~10 9 CFU / mL; The mass ratio of the high galangal to the water in the fermentation substrate is 1:(150-300); The high galangal powder is crushed and sieved to a mesh number of 80-200 mesh.

4. The preparation method of the ginger ferment as claimed in claim 3, characterized by, The preparation method of the high galangal fermentation product satisfies that the high galangal powder is crushed and sieved to a mesh number of 100 mesh.

5. The method of preparing a zingiber officinale ferment according to any one of claims 2 to 4, wherein the zingiber officinale ferment is prepared by the method comprising the steps of: The preparation method of the high galangal fermentation product satisfies at least one of the following conditions: ​ When the Saccharomyces cerevisiae comprises the Saccharomyces cerevisiae, the Saccharomyces cerevisiae is added in the form of Saccharomyces cerevisiae liquid; When the Saccharomyces cerevisiae comprises the Saccharomyces cerevisiae SY, the Saccharomyces cerevisiae SY is added in the form of Saccharomyces cerevisiae SY powder; When the Saccharomyces cerevisiae comprises the Saccharomyces cerevisiae RW, the Saccharomyces cerevisiae RW is added in the form of Saccharomyces cerevisiae RW powder.

6. The preparation method of the ginger ferment as claimed in claim 5, characterized by, The preparation method of the high galangal fermentation product satisfies at least one of the following conditions: The viable cell count of the yellow rice wine yeast bacterial liquid is 10 9 ~10 13 CFU / mL; The number of viable yeast SY inoculated in the fermentation substrate per unit volume is 10 6 ~10 10 CFU / mL; The number of viable yeast RW cells inoculated per volume of the fermentation substrate is 10 6 ~10 10 CFU / mL.

7. The preparation method of the ginger ferment as claimed in claim 6, characterized by, The preparation method of the high galangal fermentation product satisfies at least one of the following conditions: The viable cell count of the yellow rice wine yeast bacterial liquid is 10 10 ~10 12 CFU / mL; The number of viable yeast SY inoculated in the fermentation substrate per unit volume is 10 7 ~10 9 CFU / mL The number of viable yeast RW cells inoculated per volume of the fermentation substrate is 10 7 ~10 9 CFU / mL.

8. The preparation method of the ginger ferment as claimed in claim 1, characterized by, The fermentation substrate further comprises sterilization before use.

9. The preparation method of the ginger ferment as claimed in claim 8, characterized by, The sterilization method is high-temperature sterilization; when the high-temperature sterilization is used for the sterilization of the fermentation substrate, the sterilization temperature is 90-130℃; when the high-temperature sterilization is used for the sterilization of the fermentation substrate, the sterilization time is 10-40min; when the high-temperature sterilization is used for the sterilization of the fermentation substrate, the sterilization pressure is 0.1-0.14MPa.

10. The preparation method of the ginger ferment as claimed in claim 9, characterized by, When the high-temperature sterilization is used for the sterilization of the fermentation substrate, the sterilization temperature is 115-125℃; when the high-temperature sterilization is used for the sterilization of the fermentation substrate, the sterilization time is 30min; when the high-temperature sterilization is used for the sterilization of the fermentation substrate, the sterilization pressure is 0.1-0.13MPa; The sterilization is followed by cooling to room temperature.

11. The preparation method of the ginger ferment as claimed in claim 10, wherein, When the high-temperature sterilization is used for the sterilization of the fermentation substrate, the sterilization temperature is 121℃; when the high-temperature sterilization is used for the sterilization of the fermentation substrate, the sterilization pressure is 0.12MPa. ​ 12. The preparation method of the ginger ferment as claimed in claim 1, characterized by, The preparation method of the high galangal fermentation product satisfies at least one of the following conditions: The temperature of the fermentation culture is 25-35℃. The time of the fermentation culture is 36-96h. The rotation speed of the oscillation is 150-250rpm.

13. The preparation method of the ginger ferment as claimed in claim 12, characterized by, The rotation speed of the oscillation is 200rpm.

14. The preparation method of the ginger ferment as claimed in claim 1, wherein, The sterilization is followed by cooling and / or centrifugation, and collection of the supernatant. ​ 15. The method of claim 14, wherein, The cooling is to room temperature. The rotation speed of the centrifugation is 3000-9000rpm, and the time of the centrifugation is 10-40min.

16. The method of claim 15, wherein the preparation of the zingiber officinale ferment is characterized by, The rotation speed of the centrifugation is 4000-6000rpm, and the time of the centrifugation is 20-40min.

17. The method of claim 16, wherein the preparation of the zingiber officinale ferment is characterized by, The rotation speed of the centrifugation is 4800rpm, and the time of the centrifugation is 30min.

18. The method of producing a Zingiber officinale ferment according to any one of claims 14 to 17, wherein The centrifugation is followed by secondary sterilization and / or mixing with a preservative.

19. The method of claim 18, wherein, The secondary sterilization is by autoclaving, and when the secondary sterilization is by autoclaving, the temperature of the secondary sterilization is 110-125℃, the time of the secondary sterilization is 20-40min, and the pressure of the secondary sterilization is 0.1-0.14MPa. The temperature of the mixing with the preservative is 50-80℃. The preservative comprises p-hydroxyacetophenone and / or 1,2-hexanediol, and when the preservative comprises p-hydroxyacetophenone, the mass percentage of the p-hydroxyacetophenone in the supernatant obtained after the centrifugation is 0.25%-1%, and when the preservative comprises 1,2-hexanediol, the mass percentage of the 1,2-hexanediol in the supernatant obtained after the centrifugation is 0.25%-1%.

20. The method for preparing the fermented product of Alpinia officinalis according to claim 19, wherein: When the secondary sterilization is by autoclaving, the temperature of the secondary sterilization is 115-121℃, the time of the secondary sterilization is 25-35min, and the pressure of the secondary sterilization is 0.1-0.13MPa.

21. A fermented product of Alpinia galanga, characterized by, The high ginger ferment is prepared by the method of any one of claims 1-20.

22. Use of the high ginger ferment of claim 21 as a product, an additive, or a substrate in the preparation of a skin external agent.

23. Use of the Zingiber officinale fermentate according to claim 22 for the manufacture of a skin preparation, directly as a product, as an additive or as a substrate, characterized in that, The high ginger ferment is at least one of an antioxidant active ingredient, an anti-allergic active ingredient, an anti-aging active ingredient, and an anti-inflammatory active ingredient of the skin external agent.

24. Use of the Zingiber officinale fermentate according to claim 23 for the manufacture of a skin preparation, directly as a product, as an additive or as a substrate, characterized in that, The antioxidant active ingredient is an antioxidant active ingredient having a DPPH free radical scavenging effect; the anti-allergic active ingredient is an anti-allergic active ingredient having a hyaluronidase inhibitory effect; the anti-aging active ingredient is an anti-aging active ingredient having a cell repair promoting effect; and the anti-inflammatory active ingredient is an anti-inflammatory active ingredient having a relative expression amount reducing effect on inflammatory factor IL-8 in cells.

25. A skin external preparation, characterized by comprising: The galangal ferment includes the galangal ferment of claim 21.

26. The external agent for the skin as claimed in claim 25, wherein The skin external agent includes at least one of an antioxidant active ingredient, an anti-allergic active ingredient, an anti-aging active ingredient, and an anti-inflammatory active ingredient.

27. The external agent for the skin as claimed in claim 25, wherein The skin external agent includes a mask, an essence, or a toner.

28. The external agent for the skin as claimed in claim 25, wherein The galangal ferment accounts for 1% to 50% of the mass percentage of the skin external agent.

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