A Sequoia sempervirens stem fermentation broth, its preparation method and application

Abstract

The present invention provides a fermented liquid of Sequoia sempervirens stems, a preparation method thereof and an application thereof, which specifically comprise the following steps: (1) pulverize dry Sequoia sempervirens bark, add inulin, carbon source, nitrogen source, inorganic salts and water, and perform sterilization to obtain a dry Sequoia sempervirens bark culture medium; (2) inoculate Saccharomyces cerevisiae and Bacillus subtilis into the dry Sequoia sempervirens bark culture medium for fermentation, and then perform sterilization and centrifugation to obtain a fermented liquid of Sequoia sempervirens stems. Experimental tests show that the fermented liquid of Sequoia sempervirens stems prepared according to the method provided by the present invention has a high content of active ingredients and has various effects such as anti-inflammatory, soothing and anti-allergic, promoting wound healing, and anti-aging. The fermented liquid prepared by using the method provided by the present invention can be widely applied in the preparation of cosmetics with anti-aging, anti-allergic and repair effects.

Description

Technical Field

[0001] The present invention relates to a fermented liquid of Sequoia sempervirens stem, a preparation method thereof and an application thereof, belonging to the technical field of biological fermentation. Background Art

[0002] With the development of the economy, the disposable income of Chinese residents has been continuously increasing, the skin care awareness has been continuously enhanced, and the concept of healthy skin has gradually taken shape. The Chinese cosmetics market is booming at an unprecedented speed. With the gradual improvement of living conditions, people's requirements for the performance of cosmetics are gradually getting higher. The application of general soothing, anti-allergic and repairing ingredients in cosmetics is restricted. The emergence of natural plant anti-allergic active ingredients has filled this gap and is favored by the market and consumers.

[0003] Sequoia sempervirens, scientific name Sequoia sempervirens, is an evergreen large tree of the family Taxodiaceae and the genus Sequoia. It has a long fast-growing period and a fast growth rate, and has excellent characteristics for cultivating large-diameter timber. The stem of Sequoia sempervirens has the effects of promoting wound healing, improving skin elasticity and luster, and inhibiting inflammatory reactions, and has a good therapeutic effect on skin injuries such as burns and ulcers. At the same time, it has the advantages of scavenging free radicals, protecting cells from oxidative damage, and delaying cell aging, and is widely used in personal care such as cosmetics.

[0004] There are some defects in the traditional extraction methods of natural plant raw materials. For example, in the hot water extraction method of Sequoia sempervirens stem, the content of total sugar extracted is 1.2 - 4.6 mg / g, the content of polyphenols is 0.20 - 0.69 mg / g, and the protein content is 70 - 100 mg / g. The extraction rate of active ingredients is low, the extraction time is long, and the extraction difficulty is large. In the prior art, Chinese patent document CN217139296U discloses an extraction structure for Sequoia sempervirens stem extract with high efficiency, which solves the problems of slow extraction efficiency and difficult residue cleaning of Sequoia sempervirens stem, but fails to fundamentally solve the extraction rate and purity problems of Sequoia sempervirens stem, which severely restricts its application in the beauty industry.

[0005] Plant fermentation helps to solve the above difficulties. For example, Chinese patent document CN117431275A provides a method for obtaining the effective components of flower plants by fermenting Lactobacillus, which can well extract the bound active substances in the cell wall. The contents of anthocyanins and total phenols extracted by this method are increased by more than 21% and 19% respectively compared with the ordinary method. Chinese patent document CN117987285A provides a Saccharomyces cerevisiae BYBC1.21100 and a Bacillus marinus BYBC1.20001, and provides the preparation of yellow rice wine lees fermentation extract by these two strains and its application in cosmetics, realizing the high-value utilization of yellow rice wine lees. Considering the current difficulties in the extraction process of Sequoia sempervirens, the present invention greatly improves the extraction rate of the effective components of Sequoia sempervirens through the fermentation process, making its soothing, anti-allergic and repair effects more significant, and endowing Sequoia sempervirens with a broader application in the cosmetics market. Summary of the Invention

[0006] The purpose of the present invention is to provide a fermentation broth of Sequoia sempervirens stem, a preparation method and an application thereof, and the fermentation broth of Sequoia sempervirens stem has anti-aging, soothing, anti-allergic and repair effects.

[0007] All "parts" involved in this application are parts by weight.

[0008] The technical solution of the fermentation broth of Sequoia sempervirens stem provided by the present invention is as follows:

[0009] Inoculate Saccharomyces cerevisiae and Bacillus subtilis into the Sequoia sempervirens dry bark medium for fermentation to obtain the fermentation broth of Sequoia sempervirens stem.

[0010] The preservation number of the Saccharomyces cerevisiae is CGMCC No.28276 or CGMCC No.23880, and it is purchased from the China General Microbiological Culture Collection Center; the Bacillus subtilis is Bacillus subtilis YK006, which is purchased from Youkemegu (Shandong) Biotechnology Co., Ltd. or Jinan Ruibeike Biotechnology Co., Ltd.

[0011] Preferably according to the present invention, the components of the Sequoia sempervirens dry bark medium are as follows: 5-10 parts of crushed Sequoia sempervirens dry bark, 0.5-1.0 part of inulin, 0.5-2.5 parts of carbon source, 0.5-1.5 parts of nitrogen source, 0.05-0.2 part of inorganic salt, and water is added to a total weight of 100 parts.

[0012] More preferably, the components of the Sequoia sempervirens dry bark medium are as follows: 10 parts of crushed Sequoia sempervirens dry bark, 0.5 part of inulin, 2 parts of glucose, 1 part of peptone, 0.1 part of sodium chloride, and water is added to a total weight of 100 parts.

[0013] Preferably according to the present invention, the crushed dry bark of Sequoia sempervirens passes through a 40-80 mesh sieve.

[0014] More preferably, the crushed dry bark of Sequoia sempervirens passes through a 60 mesh sieve.

[0015] The preparation method of the above-mentioned fermentation broth of Sequoia sempervirens stem is as follows:

[0016] (1) Add inulin, carbon source, nitrogen source, inorganic salt, and water to the crushed dry bark of Sequoia sempervirens, and perform sterilization to obtain a culture medium of dry bark of Sequoia sempervirens.

[0017] (2) Inoculate Saccharomyces cerevisiae and Bacillus subtilis into the culture medium of dry bark of Sequoia sempervirens prepared in step (1) for fermentation, and then perform sterilization and centrifugation to obtain the fermentation broth of Sequoia sempervirens stem.

[0018] Preferably according to the present invention, the sterilization in step (1) is carried out at 121 °C for 20 min.

[0019] Preferably according to the present invention, in step (2), the fermentation is carried out by inoculating Saccharomyces cerevisiae and Bacillus subtilis into the culture medium of dry bark of Sequoia sempervirens with a total cell inoculation amount of 2-5% by mass percentage, and performing shaking flask fermentation at 30-32 °C and pH 5.0-6.5 for 18-48 h at 140-200 r / min.

[0020] More preferably, in step (2), the fermentation is carried out by inoculating Saccharomyces cerevisiae and Bacillus subtilis into the culture medium of dry bark of Sequoia sempervirens with a total cell inoculation amount of 3% by mass percentage, and performing shaking flask fermentation at 31 °C and pH 6.0 for 30 h at 160 r / min.

[0021] Preferably according to the present invention, the sterilization in step (2) is carried out at 80-85 °C for 20 min.

[0022] More preferably, the sterilization in step (2) is carried out at 80 °C for 20 min.

[0023] Preferably according to the present invention, the centrifugation in step (2) is carried out at 8000-11000 r / min for 2 min.

[0024] More preferably, the centrifugation in step (2) is carried out at 10000 r / min for 2 min.

[0025] Preferably according to the present invention, in step (2), 2-3 parts of Saccharomyces cerevisiae are taken, and 1-2 parts of Bacillus subtilis are taken, and the cell concentration is 10 5 -10 7 CFU / mL.

[0026] Further preferably, in step (2), 3 parts of the Saccharomyces cerevisiae are taken, and 2 parts of the Bacillus subtilis are taken, and the cell concentrations are both 10 5 -10 7 CFU / mL.

[0027] Another object of the present invention is to provide a fermented liquid of Sequoia sempervirens stems prepared by using the above preparation method.

[0028] Preferably, the active ingredients of the fermented liquid of Sequoia sempervirens stems include monosaccharides, polysaccharides, proteins and polyphenols. Preferably, the content of the polyphenols is 0.40 - 0.89 mg / g, the content of the total sugar is 3.2 - 6.6 mg / g, and the protein content is 100 - 150 mg / g.

[0029] Another object of the present invention is to provide the application of the above fermented liquid of Sequoia sempervirens stems in the preparation of cosmetics.

[0030] Preferably, the above fermented liquid of Sequoia sempervirens stems is applied in the preparation of cosmetics with the efficacy of soothing, anti-allergic and repair.

[0031] Beneficial effects:

[0032] 1. The fermented liquid of Sequoia sempervirens stems prepared by fermenting with Saccharomyces cerevisiae and Bacillus subtilis contains rich active ingredients such as monosaccharides, polysaccharides, proteins and polyphenols. Compared with the traditional method, the contents of polyphenols, polysaccharides and proteins are increased by more than 28%, 43% and 50% respectively. The polyphenols in Sequoia sempervirens stems have the functions of immune regulation, free radical scavenging and improvement of oxidative stress, which helps to maintain cell health and enhance the body's immunity; the polysaccharides have the characteristics of water locking and water absorption, can better moisturize the skin and improve the skin barrier function; the proteins have certain antioxidant, anti-free radical and anti-aging effects, can relieve allergic symptoms and enhance the skin barrier function. The efficient extraction of these active ingredients endows the fermented liquid of Sequoia sempervirens stems with better anti-aging, soothing, anti-allergic and repair effects, and can be widely used in the preparation of cosmetics.

[0033] 2. Compared with the prior art, the plant fermentation utilizes the microbial metabolism and transformation, ferments with plant raw materials as substrates, and through the optimization of the fermentation process, can realize the preparation of large-scale fermented active substances. Description of the drawings

[0034] Figure 1 It is the inhibition rate curve of hyaluronidase activity of the fermented liquid of Sequoia sempervirens stems provided by the present invention;

[0035] Figure 2 It is the DPPH free radical scavenging rate curve of the fermented liquid of Sequoia sempervirens stems provided by the present invention;

[0036] Figure 3Superoxide anion radical scavenging rate curve of the fermented liquid of the stems of Sequoia sempervirens provided by the present invention;

[0037] Figure 4 Hydroxyl radical scavenging rate curve of the fermented liquid of the stems of Sequoia sempervirens provided by the present invention;

[0038] Figure 5 Bar graph of the effect of the fermented liquid of the stems of Sequoia sempervirens provided by the present invention on cell viability;

[0039] Figure 6 Picture of the effect of the fermented liquid of the stems of Sequoia sempervirens provided by the present invention on cell migration;

[0040] Figure 7 Bar graph of the effect of the fermented liquid of the stems of Sequoia sempervirens provided by the present invention on the content of inflammatory factors in cells;

[0041] Figure 8 Bar graph of the effect of the fermented liquid of the stems of Sequoia sempervirens provided by the present invention on the tail fin area of zebrafish. Detailed implementation manners

[0042] The following combines specific embodiments to further describe the technical solutions of the present invention:

[0043] In the following embodiments, comparative examples or test examples, the dry bark of Sequoia sempervirens was purchased from Sushan Nursery (Yancheng) Co., Ltd.; the carbon source used was glucose, the nitrogen source was peptone, and the inorganic salt was sodium chloride; the Saccharomyces cerevisiae was preserved under the accession number CGMCC No. 28276 or CGMCC No. 23880, purchased from the China General Microbiological Culture Collection Center, and the cell concentration was 10 5 -10 7 CFU / mL; the Bacillus subtilis was Bacillus subtilis YK006, purchased from Youkemegu (Shandong) Biotechnology Co., Ltd. or Jinan Ruibeike Biotechnology Co., Ltd., and the cell concentration was 10 5 -10 7 CFU / mL; the Lactobacillus plantarum was preserved under the accession number CGMCC No. 29289, and the cell concentration was 10 5 -10 7 CFU / mL, purchased from Jinan Ruibeike Biotechnology Co., Ltd.

[0044] Example 1

[0045] A method for preparing a fermented liquid of the stems of Sequoia sempervirens, the steps are as follows:

[0046] (1) Crush the dry bark of Sequoia sempervirens through a 60-mesh sieve. Take 1 kg of the crushed dry bark of Sequoia sempervirens, 0.05 kg of inulin, 0.25 kg of carbon source, 0.15 kg of nitrogen source, 0.01 kg of inorganic salts, and add water to a total weight of 10.0 kg. After sterilizing at 121 °C for 20 minutes, prepare the dry bark medium of Sequoia sempervirens;

[0047] (2) Inoculate 0.30 kg of Saccharomyces cerevisiae (strain number CGMCC No. 28276) and 0.20 kg of Bacillus subtilis (Bacillus subtilis YK006) into the dry bark medium of Sequoia sempervirens prepared in step (1) with a total cell inoculation amount of 5% by mass. Seal it with gauze and perform shake flask fermentation at 31 °C, pH 6.0, and 160 r / min for 30 h, then raise the temperature to 80 °C and sterilize for 20 min; centrifuge at 10000 r / min for 2 min, and take the upper clear liquid, which is the fermentation broth of Sequoia sempervirens stem.

[0048] Example 2

[0049] A method for preparing the fermentation broth of Sequoia sempervirens stem is as follows:

[0050] (1) Crush the dry bark of Sequoia sempervirens through an 80-mesh sieve. Take 1 kg of the crushed dry bark of Sequoia sempervirens, 0.05 kg of inulin, 0.2 kg of carbon source, 0.1 kg of nitrogen source, 0.01 kg of inorganic salts, and add water to a total weight of 10.0 kg. After sterilizing at 121 °C for 20 minutes, prepare the dry bark medium of Sequoia sempervirens;

[0051] (2) Inoculate 0.20 kg of Saccharomyces cerevisiae (strain number CGMCC No. 28276) and 0.10 kg of Bacillus subtilis (Bacillus subtilis YK006) into the dry bark medium of Sequoia sempervirens prepared in step (1) with a total cell inoculation amount of 3% by mass. Seal it with gauze and perform shake flask fermentation at 31 °C, pH 6.0, and 160 r / min for 30 h, then raise the temperature to 80 °C and sterilize for 20 min; centrifuge at 10000 r / min for 2 min, and take the upper clear liquid, which is the fermentation broth of Sequoia sempervirens stem.

[0052] Example 3

[0053] A method for preparing the fermentation broth of Sequoia sempervirens stem, which is different from Example 1 in that the Saccharomyces cerevisiae strain CGMCC No. 23880 is selected.

[0054] Example 4

[0055] A preparation method of Sequoia sempervirens stem fermentation broth, which is different from Example 1 in that 0.30 kg of Saccharomyces cerevisiae (strain number CGMCC No. 28276) and 0.10 kg of Bacillus subtilis (Bacillus subtilis YK006) are inoculated into the Sequoia sempervirens dry bark medium prepared in step (1) with a total cell inoculation amount of 4% by mass.

[0056] Comparative Example 1

[0057] A preparation method of Sequoia sempervirens stem fermentation broth, which is different from Example 1 in that Bacillus subtilis is replaced by Saccharomyces cerevisiae (strain number CGMCC No. 28276).

[0058] Comparative Example 2

[0059] A preparation method of Sequoia sempervirens stem fermentation broth, which is different from Example 1 in that Saccharomyces cerevisiae is replaced by Lactobacillus plantarum (strain number CGMCC No. 29289).

[0060] Test Example 1

[0061] The active ingredients of the Sequoia sempervirens stem fermentation broth obtained in the above examples and comparative examples were determined. The total sugar content was determined by the phenol-sulfuric acid method; the monosaccharide content was determined by the 3,5-dinitrosalicylic acid (DNS) method; the protein content was determined by the Bradford method; the total phenol content was determined by the sodium nitrite-aluminum nitrate method. The contents of each active ingredient are shown in Table 1.

[0062] Table 1. Contents of active ingredients in Sequoia sempervirens stem fermentation broth

[0063]

[0064] According to Table 1, the content of active ingredients in the Sequoia sempervirens stem fermentation broth is significantly higher than that of the traditional extraction method. Among them, the total sugar and total phenol contents in Example 4 are the highest, the protein content in Example 1 is the highest, and the content of active ingredients in the comparative examples is lower than that in the examples. That is, when Saccharomyces cerevisiae and Bacillus subtilis are added under different conditions, even if different strains are selected, the content of active ingredients in the fermentation broth is higher than that without adding Bacillus subtilis or Saccharomyces cerevisiae; therefore, the combined use of Saccharomyces cerevisiae and Bacillus subtilis is superior to other strain combinations or single use.

[0065] Test Example 2

[0066] Hyaluronidase activity inhibition experiment

[0067] This method uses an in vitro inhibition experiment of hyaluronidase to determine the inhibitory effect of the fermentation broth of Sequoia sempervirens stems on hyaluronidase activity. The experimental concentrations are 5, 10, 15, 20, and 50 mg / mL (the mass concentration of the fermentation broth of Sequoia sempervirens stems in the system), respectively.

[0068] Preparation of solutions required for the experiment:

[0069] Acetic acid buffer solution: Measure 1.155 mL of glacial acetic acid, dilute it to 100 mL and mix well. Take 4.8 mL of it as solution A; weigh 2.72 g of sodium acetate crystals, dissolve them in water and make up the volume to 100 mL and mix well. Take 45.2 mL of it as solution B; mix solutions A and B, and make up the volume to 100 mL with water and mix well.

[0070] Hyaluronidase solution: Weigh 10 mg of hyaluronidase in a beaker and add 4 mL of acetic acid buffer solution.

[0071] Sodium hyaluronate solution: Weigh 5 mg of sodium hyaluronate in a beaker and add 10 mL of acetic acid buffer solution.

[0072] Ehrlich reagent: Weigh 0.8 g of p-dimethylaminobenzaldehyde and dissolve it in 15 mL of concentrated hydrochloric acid and 15 mL of absolute ethanol.

[0073] Acetylacetone solution: Take 3.5 mL of acetylacetone and dissolve it in 50 mL of 1.0 mol / L sodium carbonate solution. This solution is prepared before use.

[0074] Experimental steps:

[0075] Take 0.1 mL of 0.25 mmol / L CaCl2 solution and 0.5 mL of hyaluronidase solution, incubate at 37 °C for 20 min; add 0.5 mL of fermentation broth of Sequoia sempervirens stems at different concentrations, and continue to incubate at 37 °C for 20 min; add 0.5 mL of sodium hyaluronate solution and incubate at 37 °C for 30 min, then let it stand at room temperature for 5 min; add 0.1 mL of 0.4 mol / L NaOH solution and 0.5 mL of acetylacetone solution, heat in a boiling water bath for 15 min, immediately cool with ice water for 5 min; add 1.0 mL of Ehrlich reagent and dilute it with 3.0 mL of absolute ethanol, let it stand for color development for 20 min, measure its absorbance value with a spectrophotometer, and calculate the inhibitory rate of the fermentation broth at different concentrations on hyaluronidase activity as shown in Table 2.

[0076] Calculation formula for the inhibitory rate of the sample on hyaluronidase activity:

[0077] Inhibitory rate of hyaluronidase activity (%) = [(A1 - A2) - (B1 - B2)] / (A1 - A2) × 100%

[0078] Among them, A1 is the absorbance value of the reference solution, using deionized water to replace the fermentation broth; A2 is the absorbance value of the reference blank solution, using deionized water to replace the fermentation broth and acetic acid buffer solution to replace the hyaluronidase solution and sodium hyaluronate solution; B1 is the absorbance value of the sample solution; B2 is the absorbance value of the sample blank solution, using acetic acid buffer solution to replace the hyaluronidase solution and sodium hyaluronate solution.

[0079] During the experiment, first perform a wavelength scan on the sample in the range of 450 - 700 nm to determine the maximum absorption wavelength, and measure at the maximum absorption wavelength.

[0080] Table 2. Inhibition rate of hyaluronidase activity of fermentation broth with different concentrations, %

[0081]

[0082] According to Table 2, the hyaluronidase inhibitory activity of Example 4 is the strongest, and the inhibitory effect of the comparative example is the weakest. For the fermentation broth of Sequoia sempervirens stem obtained in Example 4, the results of the inhibition rate of hyaluronidase activity are shown in Figure 1 . As Figure 1 shown, with the increase of the concentration of the fermentation broth of Sequoia sempervirens stem (SSP), its inhibitory effect on hyaluronidase activity gradually increases. It shows that the fermentation broth of Sequoia sempervirens stem obtained in Example 4 can effectively inhibit the activity of hyaluronidase, thus playing a good role in soothing and anti-allergy.

[0083] Test Example 3

[0084] DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) free radical scavenging experiment

[0085] Prepare the sample solution: Dissolve and dilute the fermentation broth of Sequoia sempervirens stem with deionized water, and set 5 concentration gradients, which are 5, 10, 15, 20, 50 mg / mL respectively. Set the sample group, blank group, and control group in a 96-well plate.

[0086] Table 3. Reagent preparation

[0087]

[0088] Shake the 96-well plate with the added solution on a shaker for 30 min under light-shielded conditions according to the above table, immediately measure its absorbance value at a wavelength of 517 nm with an enzyme-labeled instrument, and calculate the DPPH free radical scavenging rate of the fermentation broth of Sequoia sempervirens stem with different concentrations as shown in Table 4.

[0089] Table 4. DPPH free radical scavenging rate of the fermentation broth of Sequoia sempervirens stem with different concentrations, %

[0090]

[0091] As described in Table 4, the DPPH free radical scavenging activity of Example 4 is the strongest, and the DPPH free radical scavenging rate of the comparative example is the lowest. For the fermented liquid of Sequoia sempervirens stems obtained in Example 4, the results of its DPPH free radical scavenging rate are shown in Figure 2 . As Figure 2 shown, as the concentration of the fermented liquid of Sequoia sempervirens stems increases, its effect on scavenging DPPH free radicals gradually enhances. This indicates that the fermented liquid of Sequoia sempervirens stems obtained in Example 4 can effectively scavenge DPPH free radicals, thereby playing a good anti-aging role.

[0092] Test Example 4

[0093] Superoxide anion free radical scavenging test

[0094] Take an appropriate amount of the sample, dissolve and dilute it with deionized water, and set 5 concentration gradients, namely 5, 10, 15, 20, and 50 mg / mL. Prepare the reagents according to Table 5. Solution A: First add 0.6 mL of 0.1 mol / L Tris-HCl buffer solution and 0.58 mL of distilled water, mix well, and place in a water bath at 25 °C for 20 min, then add 0.58 mL of the sample; Solution B: 0.02 mL of preheated 5 mmol / L pyrogallol solution. After preparing Solution A and Solution B, quickly mix them, shake well, measure the absorbance of the solution at 325 nm as the first absorbance value, measure it once every 1 min, and continuously measure for 4 min to calculate the increase in absorbance per minute within the linear range. Use 10 mmol / L HCl solution to replace the pyrogallol solution for absorbance zero adjustment. The superoxide anion free radical scavenging rates of the fermented liquid of Sequoia sempervirens stems at different concentrations are shown in Table 6.

[0095] Table 5. Reagent preparation

[0096]

[0097] Table 6. Superoxide anion free radical scavenging rates of the fermented liquid of Sequoia sempervirens stems at different concentrations, %

[0098]

[0099] As described in Table 6, the superoxide anion free radical scavenging activity of Example 4 is the strongest, and the superoxide anion free radical scavenging rate of the comparative example is the lowest. For the fermented liquid of Sequoia sempervirens stems obtained in Example 4, the results of its superoxide anion free radical scavenging rate are shown in Figure 3 . As Figure 3 shown, as the concentration of the fermented liquid of Sequoia sempervirens stems increases, its effect on scavenging superoxide anion free radicals gradually enhances. This indicates that the fermented liquid of Sequoia sempervirens stems obtained in Example 4 can effectively scavenge superoxide anion free radicals, thereby playing a good anti-aging role.

[0100] Test Example 5

[0101] Hydroxyl radical scavenging assay

[0102] The experiment was carried out in a 1.5 mL colorimetric tube system. An appropriate amount of the fermented liquid sample of Sequoia sempervirens stems was taken and dissolved and diluted with deionized water, and 5 concentration gradients were set, namely 5, 10, 15, 20, and 50 mg / mL. According to Table 7, the reagents were prepared. In the colorimetric tube, 0.1 mL of 9 mmol / L ferrous sulfate solution, 0.1 mL of 9 mmol / L salicylic acid-ethanol solution were added in sequence, an appropriate amount of deionized water was added, and finally 0.1 mL of 8.8 mmol / L hydrogen peroxide solution was added. After shaking well, it was incubated in a water bath at 37 °C for 15 min, and its absorbance was measured. The hydroxyl radical scavenging rates of the fermented liquid of Sequoia sempervirens stems at different concentrations were calculated as shown in Table 8.

[0103] Table 7. Reagent preparation

[0104]

[0105] Table 8. Hydroxyl radical scavenging rates of the fermented liquid of Sequoia sempervirens stems at different concentrations, %

[0106]

[0107] According to Table 8, the hydroxyl radical scavenging activity of Example 4 was the strongest, and the hydroxyl radical scavenging rate of the comparative example was the lowest. For the fermented liquid of Sequoia sempervirens stems obtained in Example 4, the results of its hydroxyl radical scavenging rate are shown in Figure 4 . As Figure 4 shown, with the increase in the concentration of the fermented liquid of Sequoia sempervirens stems, its effect on scavenging hydroxyl radicals gradually increases. It shows that the fermented liquid of Sequoia sempervirens stems obtained in Example 4 can effectively scavenge hydroxyl radicals, thus playing a good anti-aging role.

[0108] Test Example 6

[0109] Cytotoxicity test

[0110] Keratinocytes (HaCaT) were inoculated into a 96-well plate at an inoculation density of 6 - 8×10 4 cells / well and incubated overnight in an incubator (37 °C, 5% CO2).

[0111] When the cell plating rate in the 96-well plate reached 40 - 60%, drug administration was carried out. Before drug administration, the 96-well plate was washed first. In the blank control group, 100 μL of culture medium was added to each well; in the solvent control group, 100 μL of cell solution was added to each well, and after culturing for 24 h, drug administration was carried out. After the drug administration was completed, the 96-well plate was placed in an incubator (37 °C, 5% CO2) and cultured for 24 h. After the cells were incubated and cultured for 24 h, 10 μL of CCK-8 was added, and it was incubated in the dark at 37 °C for 2 - 4 h. After the incubation was completed, the OD value was read at 490 nm, and the effects of the fermented liquid of Sequoia sempervirens stems at different concentrations on cell viability were calculated as shown in Table 9.

[0112] Table 9. Effects of Sequoia sempervirens stem fermentation broth at different concentrations on cell viability, %

[0113]

[0114] As described in Table 9, the Sequoia sempervirens stem fermentation broth has weak cytotoxicity to cells and good safety at low concentrations. For the Sequoia sempervirens stem fermentation broth obtained in Example 4, the results of cell viability are shown in Figure 5 , and the Sequoia sempervirens stem fermentation broth obtained in Example 4 has basically no toxic effect on cells.

[0115] Test Example 7

[0116] Cell scratch assay

[0117] Before the experiment, use a marker pen to mark 3 horizontal lines on the back of a 6-well plate. Take HaCaT cells in the logarithmic phase and inoculate them into the 6-well plate at an inoculation density of 6 - 8×10 4 cells / well, incubate overnight in an incubator (37°C, 5% CO2), and start the experiment when the cells are cultured to a monolayer of adherent cells. The experiment is divided into two groups, a blank control group (Ctrl) and a sample group (Sample). Select the compound to be tested and set 5 test concentrations at 5, 10, 15, 20, and 50 mg / mL respectively. Before administration, use a 200 μL sterile pipette tip to uniformly draw a line perpendicular to the marker line in the center of the cell plate, wash 3 times with PBS, add fresh serum-free medium, take a photo, continue to culture for 24 h after drug administration treatment, and take a photo at the end of the experiment.

[0118] For the Sequoia sempervirens stem fermentation broth obtained in Example 4, the results of its effect on cell migration are shown in Figure 6 . As Figure 6 shown, the Sequoia sempervirens stem fermentation broth has the ability to promote the migration of keratinocytes at low concentrations, and the effect is best at a concentration of 10 mg / mL. It shows that the Sequoia sempervirens stem fermentation broth can promote cell migration and accelerate wound healing.

[0119] Test Example 8

[0120] Inflammatory factor inhibition experiment

[0121] Use a mouse tumor necrosis factor-α (TNF-α) ELISA kit and an interleukin-6 (IL-6) ELISA kit to measure the anti-inflammatory effect of the Sequoia sempervirens stem fermentation broth in vitro. Incubate the Sequoia sempervirens stem fermentation broth with DMEM high-glucose medium containing 15% fetal serum in an incubator at 37°C with 5% CO2 for 24 h to obtain an extract of the Sequoia sempervirens stem fermentation broth. Seed mouse mononuclear macrophages (RAW264.7 cells) at 2×10 5Inoculate into a 24-well cell culture plate at a density of [[ID=]], 500 μL per well, and culture for 24 h. Incubate in an incubator for 24 h, take the cell supernatant, and at the same time add 100 ng / mL lipopolysaccharide (LPS) into each well. The group without any treatment is the negative control (Ctrl), and the group with only LPS added is the positive control. Each group is set with 3 replicates. After 24 h, collect the cell supernatant, and use a mouse IL-6 and TNF-α ELISA kit to detect the concentrations of IL-6 and TNF-α in each group.

[0122] Table 10. Effects of different concentrations of Sequoia sempervirens stem fermentation broth on the change in TNF-α concentration, pg / mL

[0123]

[0124] Table 11. Effects of different concentrations of Sequoia sempervirens stem fermentation broth on the change in IL-6 concentration, pg / mL

[0125]

[0126]

[0127] As shown in Table 10 and Table 11, the anti-inflammatory effect of Example 4 is the best. For the Sequoia sempervirens stem fermentation broth obtained in Example 4, the results of the release amount of inflammatory factors are shown in Figure 7 . As Figure 7 shown, the effect of Sequoia sempervirens stem fermentation broth on the release of TNF-α from LPS-stimulated macrophages is concentration-dependent. As the concentration increases, the content of TNF-α gradually decreases. It shows a large V-shaped dose response to IL-6, reaching a lowest point at a concentration of 10 mg / mL, with the best anti-inflammatory effect and good significance (**P < 0.05). This indicates that Sequoia sempervirens stem fermentation broth can inhibit the release of TNF-α and IL-6 in cells, effectively slow down the progress of the inflammatory reaction, relieve the occurrence of inflammatory aging, and play an important role in skin aging.

[0128] Test Example 9

[0129] Zebrafish anti-caudal fin shrinkage experiment

[0130] Select zebrafish eggs at 2 dpf with normal development, randomly distribute them into six-well plates, with 15 tails in each well. Remove the standard dilution water in the six-well cell culture plates without harming the larvae, and then quickly add 3 mL of the test substance solution to each well. Among them, the control group (Ctrl) is the initial caudal fin area, the model group (Model) is the caudal fin area after the test without adding the test substance, the positive control group (Positive) is the caudal fin area after adding 10 mg / mL tea polyphenols, and the sample group is the caudal fin area after adding the fermentation broth of Sequoia sempervirens stems. The sample concentrations are 5, 10, and 15 mg / mL respectively. First, mix well and then cover the culture plate panel, incubate in the dark for 2 h, and then irradiate 3 times under UVB, with each irradiation time of 15 min and an interval of 30 min. The irradiation dose per time is (2.07 ± 0.18) J / cm 2 , and the total irradiation dose is (6.21 ± 0.54) J / cm 2 . Finally, continue to incubate in the dark in a biochemical incubator at (28.5 ± 1.0) °C for 22 h.

[0131] Among the zebrafish after hatching, observe their phenotypes and behaviors to ensure they are normal zebrafish. Then randomly select at least 12 zebrafish with zebrafish sampling forceps. Put the selected zebrafish into a container containing 3% methylcellulose solution, completely immerse them, let it stand for a period of time, allow the methylcellulose solution to fully contact with the zebrafish, and fix the zebrafish. Observe and take pictures under a microscope, and complete the photographing of all zebrafish within 2 h under the same instrument and environmental conditions.

[0132] After photographing, use image analysis software to analyze the obtained zebrafish pictures. Select the quantitative area as the caudal fin of the zebrafish, set the analysis parameter as the area, analyze the pictures to obtain data, take 10 valid data of the caudal fin area in each group, and calculate the influence of the fermentation broth of Sequoia sempervirens stems at different concentrations on the caudal fin area of zebrafish as shown in Table 12.

[0133] Table 12. Influence of the fermentation broth of Sequoia sempervirens stems at different concentrations on the caudal fin area of zebrafish, μm 2

[0134]

[0135] For the fermentation broth of Sequoia sempervirens stems obtained in Example 4, the results of the caudal fin area of zebrafish are shown in Figure 8 . As Figure 8 shown, with the increase in the concentration of the fermentation broth of Sequoia sempervirens stems, its inhibitory effect on the shrinkage of the caudal fin of zebrafish is stronger, indicating that the fermentation broth of Sequoia sempervirens stems can effectively resist photoaging.

[0136] Test Example 10

[0137] Human sensitivity test

[0138] Twenty volunteers were selected, with an equal number of men and women. Before the experiment, the left and right forearms of the volunteers were cleaned with clean water, and they sat quietly in an environment with a temperature of 20-22 °C and a relative humidity of 50-60%. The skin sensitivity test was carried out without the use of any cosmetics by the subjects.

[0139] The left forearm of the subject was smeared with the fermented liquid of Sequoia sempervirens stem, once every half hour, for a total of 6 times, and the smearing area was 3×3 cm 2 , and the right forearm was used as a blank control without smearing any product (Ctrl). Then, a sodium dodecyl sulfate solution (SDS) with a mass percentage content of 5% was smeared on the left and right forearms of the subjects respectively. After 0.5 h, the reactions of the subjects' arms were observed, and the statistical results of the sensitization rate are shown in Table 13.

[0140] Table 13. Influence of the fermented liquid of Sequoia sempervirens stem under different conditions on the sensitization rate of the irritant

[0141]

[0142] As described in Table 13, after smearing the fermented liquid of Sequoia sempervirens stem and then smearing the irritant, the stinging pain and swelling on the left forearm were not obvious, and the sensitization rate was greatly reduced. However, the right forearm used as the blank control had a strong reaction to the irritation of the irritant. After 0.5 h of smearing the irritant, the swelling in the skin was obvious and accompanied by stinging pain, and the sensitization rate of the blank control was high. This shows that the fermented liquid of Sequoia sempervirens stem has good anti-allergic efficacy.

[0143] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Any person skilled in the art may use the disclosed technical content to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution content of the present invention still belong to the protection scope of the technical solution of the present invention.

Claims

1. A preparation method of the fermentation broth of Sequoia sempervirens stems, characterized in that, Inoculating saccharomyces cerevisiae and Bacillus subtilis into a culture medium of North American redwood stem bark for fermentation to obtain a North American redwood stem fermentation liquid; The Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) has a deposit number of CGMCC No. 28276 or CGMCC No. 23880 and is purchased from the China General Microbiological Culture Collection Center; the Bacillus subtilis is Bacillus subtilis ( Bacillus subtilis ) YK006, which is purchased from Youke Meigu (Shandong) Biotechnology Co., Ltd. or Jinan Ruibeike Biotechnology Co., Ltd.; The components of the North American redwood dry bark culture medium are as follows, all in parts by weight: 5-10 parts of crushed North American redwood dry bark, 0.5-1.0 parts of inulin, 0.5-2.5 parts of carbon source, 0.5-1.5 parts of nitrogen source, 0.05-0.2 parts of inorganic salt, and water is added to a total weight of 100 parts; wherein the crushed North American redwood dry bark is sieved through a 40-80 mesh sieve; Take 2-3 parts by weight of the Saccharomyces cerevisiae and 1-2 parts by weight of the Bacillus subtilis, and the cell concentration of both is 10 5 -10 7 CFU / mL.

2. The preparation method of the fermented liquid of Sequoia sempervirens stems according to claim 1, characterized in that, Here are the steps: (1) adding inulin, a carbon source, a nitrogen source, an inorganic salt, and water to crushed North American redwood dry bark, sterilizing the mixture, and preparing a North American redwood dry bark culture medium; (2) Inoculating Saccharomyces cerevisiae and Bacillus subtilis into the North American redwood stem bark culture medium prepared in step (1) for fermentation, and then sterilizing and centrifuging to obtain North American redwood stem fermentation liquid.

3. The preparation method of the fermented liquid of the stem of Sequoia sempervirens as described in claim 2, characterized in that, The fermentation in step (2) is to inoculate 2-5% by mass of the total bacterial cell inoculum of Saccharomyces cerevisiae and Bacillus subtilis into a North American redwood dry bark culture medium, and ferment at 140-200 r / min at 30-32° C. and pH 5.0-6.5 for 18-48 h.

4. The preparation method of the fermented liquid of Sequoia sempervirens stems according to claim 2, characterized in that, The sterilization in step (2) is performed at 80-85°C for 20 min.

5. Use of the fermentation liquid prepared by the method for preparing the fermentation liquid of North American redwood stems according to claim 1 in preparing cosmetics.

6. The application according to claim 5, wherein, The cosmetics have anti-aging, anti-allergy and repairing effects.

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