Sequoia sempervirens stem fermentation liquor as well as preparation method and application thereof

Abstract

The invention provides sequoia sempervirens stem fermentation liquor and a preparation method and application thereof, and the preparation method specifically comprises the following steps: (1) crushing dried bark of sequoia sempervirens, adding inulin, a carbon source, a nitrogen source, inorganic salt and water, and sterilizing to obtain a dried bark culture medium of sequoia sempervirens; and (2) inoculating saccharomyces cerevisiae and bacillus subtilis into the sequoia sempervirens dry bark culture medium for fermentation, and then sterilizing and centrifuging to obtain sequoia sempervirens stem fermentation liquor. Experiments show that the sequoia sempervirens stem fermentation liquor prepared by the method provided by the invention is high in active ingredient content, and has various effects of resisting inflammation, relieving and resisting allergy, promoting wound healing, resisting aging and the like. The fermentation liquor prepared by the method provided by the invention can be widely applied to preparation of cosmetics with anti-aging, anti-allergy and repairing effects.

Description

Technical Field

[0001] The invention relates to a fermented liquor of North American redwood stems and a preparation method and application thereof, belonging to the technical field of biological fermentation. Background Art

[0002] With the development of economy, the disposable income of Chinese residents is increasing, the awareness of skin care is increasing, and the concept of healthy skin is gradually forming. 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 limited. The emergence of natural plant anti-allergic active ingredients fills this gap and is favored by the market and consumers.

[0003] Sequoia sempervirens, scientific name, is a large evergreen tree of the Taxodiaceae family and Sequoia genus. It has a long rapid growth period and a fast growth rate, and has excellent characteristics for cultivating large-diameter timber. Sequoia stems can promote wound healing, improve skin elasticity and luster, inhibit inflammatory response, and have 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. It is widely used in personal care such as cosmetics.

[0004] Traditional extraction methods of natural plant raw materials have some defects. For example, in the hot water extraction method of North American redwood stems, the total sugar content extracted is 1.2-4.6 mg / g, the polyphenol content is 0.20-0.69 mg / g, and the protein content is 70-100 mg / g. The extraction rate of effective ingredients is low, and the extraction time is long and the extraction is difficult. In the prior art, Chinese patent document CN217139296U discloses a highly efficient extraction structure of North American redwood stem extract, which solves the problems of slow extraction efficiency and difficult residue cleaning of North American redwood stems, but fails to fundamentally solve the problems of extraction rate and purity of North American redwood stems, which seriously restricts its application in the beauty industry.

[0005] Plant fermentation helps to solve the above difficulties. For example, Chinese patent document CN117431275A provides a lactobacillus fermentation liquid to obtain the effective ingredients of flower plants, and extracts the active substances bound in the cell wall very well, which increases the anthocyanin and total phenol content by 21% and 19% more than the ordinary method; Chinese patent document CN117987285A provides a brewer's yeast BYBC1.21100 and a South China Sea Bacillus BYBC1.20001, and provides the preparation of yellow wine lees fermentation extract by these two strains and their application in cosmetics, realizing the high-value utilization of yellow wine lees. Combined with the difficulties in the current extraction process of North American redwood, the present invention greatly improves the extraction rate of the effective ingredients of North American redwood through the fermentation process, making its soothing, anti-allergic and repairing effects more significant, and can give North American redwood a wider application in the cosmetics market. Summary of the invention

[0006] The invention aims to provide a fermented liquor of North American redwood stems and a preparation method and application thereof. The fermented liquor of North American redwood stems has anti-aging, soothing, anti-allergic and repairing effects.

[0007] All “parts” mentioned in this application are parts by weight.

[0008] The technical scheme of the fermented liquid of North American redwood stem provided by the present invention is as follows:

[0009] Saccharomyces cerevisiae and Bacillus subtilis are inoculated into a culture medium of North American redwood stem bark for fermentation to obtain a North American redwood stem fermentation liquid.

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

[0011] Preferably according to the present invention, the components of the North American redwood dry bark culture medium are as follows: 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.

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

[0013] Preferably according to the present invention, the crushed North American redwood dry bark is passed through a 40-80 mesh sieve.

[0014] Further preferably, the crushed North American redwood dry bark is passed through a 60-mesh sieve.

[0015] The preparation method of the above-mentioned North American redwood stem fermentation liquid comprises the following specific steps:

[0016] (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;

[0017] (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.

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

[0019] Preferably, according to the present invention, the fermentation in step (2) is to inoculate 2-5% by mass of the total bacterial cell inoculation amount of Saccharomyces cerevisiae and Bacillus subtilis into a North American redwood dry bark culture medium, and ferment at 30-32°C, pH 5.0-6.5, and 140-200 r / min in a shake flask for 18-48 hours.

[0020] Further preferably, the fermentation in step (2) is to inoculate Saccharomyces cerevisiae and Bacillus subtilis at a total cell inoculation rate of 3% by mass into a North American redwood dry bark culture medium, and ferment at 31°C, pH 6.0, and 160 r / min in a shake flask for 30 hours.

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

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

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

[0024] Further preferably, the centrifugation in step (2) is performed at 10,000 r / min for 2 min.

[0025] According to a preferred embodiment of the present invention, in step (2), 2-3 portions of the saccharomyces cerevisiae and 1-2 portions of the Bacillus subtilis are taken, and the bacterial concentration is 10 5 -10 7 CFU / mL.

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

[0027] Another object of the present invention is to provide a fermented liquid of North American redwood stems prepared by the above-mentioned preparation method.

[0028] Preferably, the effective components of the fermented liquid of North American redwood stems include monosaccharides, polysaccharides, proteins and polyphenols. Preferably, the content of polyphenols is 0.40-0.89 mg / g, the content of total sugars is 3.2-6.6 mg / g, and the content of protein is 100-150 mg / g.

[0029] Another object of the present invention is to provide the use of the above-mentioned North American redwood stem fermentation liquid in the preparation of cosmetics.

[0030] Preferably, the above-mentioned fermented liquor of North American redwood stems is used in the preparation of cosmetics with soothing, anti-allergic and repairing effects.

[0031] Beneficial effects:

[0032] 1. The fermented liquid of redwood stems prepared by fermentation with Saccharomyces cerevisiae and Bacillus subtilis contains rich monosaccharides, polysaccharides, proteins, polyphenols and other effective ingredients. Compared with the traditional method, the content of polyphenols, polysaccharides and proteins has increased by 28%, 43% and 50% respectively. Redwood stem polyphenols have the functions of immunomodulation, scavenging free radicals and improving oxidative stress, which helps to maintain cell health and enhance the body's immunity; polysaccharides have the characteristics of water locking and water absorption, which can better moisturize the skin and improve the skin's barrier function; proteins have certain antioxidant, anti-free radical and anti-aging effects, which can relieve allergic symptoms and enhance the skin's barrier function. The efficient extraction of these effective ingredients gives the fermented liquid of redwood stems better anti-aging, soothing, anti-allergic and repair effects, and can be widely used in the preparation of cosmetics.

[0033] 2. Compared with the existing technology, plant fermentation utilizes the metabolism and transformation of microorganisms, uses plant raw materials as substrates for fermentation, and optimizes the fermentation process to achieve large-scale preparation of fermentation actives. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The hyaluronidase activity inhibition rate curve of the fermented liquid of the stem of North American redwood provided by the present invention;

[0035] Figure 2 The DPPH free radical scavenging rate curve of the fermented liquid of the stem of North American redwood provided by the present invention;

[0036] Figure 3A superoxide anion free radical scavenging rate curve of the fermented liquid of the North American redwood stem provided by the present invention;

[0037] Figure 4 A hydroxyl radical scavenging rate curve of the fermented liquid of the stem of North American redwood provided by the present invention;

[0038] Figure 5 A bar graph showing the effect of the fermented liquid of North American redwood stems provided by the present invention on the survival rate of cells;

[0039] Figure 6 This is a picture of the effect of the fermented liquid of the stem of North American redwood on cell migration provided by the present invention;

[0040] Figure 7 A bar graph showing the effect of the fermented liquid of North American redwood stems provided by the present invention on the content of inflammatory factors in cells;

[0041] Figure 8 A bar graph showing the effect of the fermented liquid of North American redwood stems provided by the present invention on the tail area of ​​zebrafish. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described below in conjunction with specific embodiments:

[0043] In the following examples, comparative examples or test examples, the dry bark of North American redwood was purchased from Sushan Seedlings (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 purchased from the China General Microbiological Culture Collection Center with a collection number of CGMCC No. 28276 or CGMCC No. 23880, and the cell concentration was 10 5 -10 7 CFU / mL; Bacillus subtilis was Bacillus subtilis YK006, purchased from Yokomeigu (Shandong) Biotechnology Co., Ltd. or Jinan Ruibeike Biotechnology Co., Ltd., and the bacterial concentration was 10 5 -10 7 CFU / mL; the Lactobacillus plantarum (L. plantarum) is deposited as CGMCC No. 29289, and the bacterial concentration is 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 North American redwood stems, comprising the following steps:

[0046] (1) Crush the dry bark of North American redwood and pass it through a 60-mesh sieve, take 1 kg of the crushed dry bark of North American redwood, 0.05 kg of inulin, 0.25 kg of a carbon source, 0.15 kg of a nitrogen source, and 0.01 kg of an inorganic salt, add water to a total weight of 10.0 kg, and sterilize at 121° C. for 20 minutes to prepare a dry bark culture medium of North American redwood;

[0047] (2) 0.30 kg of Saccharomyces cerevisiae (strain number CGMCC No. 28276) and 0.20 kg of Bacillus subtilis (Bacillus subtilis YK006) were inoculated into the North American redwood stem bark culture medium prepared in step (1) at a total bacterial inoculum amount of 5% by mass, and the culture medium was sealed with gauze. The culture medium was shaken and fermented at 31° C., pH 6.0, and 160 r / min for 30 h, and then the temperature was raised to 80° C. for sterilization for 20 min. The culture medium was centrifuged at 10,000 r / min for 2 min, and the upper clear liquid was taken, which was the North American redwood stem fermentation liquid.

[0048] Example 2

[0049] A method for preparing a fermented liquid of North American redwood stems, comprising the following steps:

[0050] (1) Crush the dry bark of North American redwood and pass it through an 80-mesh sieve, take 1 kg of the crushed dry bark of North American redwood, 0.05 kg of inulin, 0.2 kg of a carbon source, 0.1 kg of a nitrogen source, and 0.01 kg of an inorganic salt, add water to a total weight of 10.0 kg, and sterilize at 121° C. for 20 minutes to prepare a dry bark culture medium of North American redwood;

[0051] (2) 0.20 kg of Saccharomyces cerevisiae (strain number CGMCC No. 28276) and 0.10 kg of Bacillus subtilis (Bacillus subtilis YK006) were inoculated into the North American redwood stem bark culture medium prepared in step (1) at a total bacterial inoculum amount of 3% by mass, and the culture medium was sealed with gauze. The culture medium was shaken and fermented at 31° C., pH 6.0, and 160 r / min for 30 h, and then the temperature was raised to 80° C. for sterilization for 20 min. The culture medium was centrifuged at 10,000 r / min for 2 min, and the upper clear liquid was taken, which was the North American redwood stem fermentation liquid.

[0052] Example 3

[0053] A method for preparing a fermented liquor of North American redwood stems, which differs from that of Example 1 in that the CGMCC No. 23880 strain of brewer's yeast is selected.

[0054] Example 4

[0055] A method for preparing a fermented liquor of North American redwood stems, which differs 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 North American redwood stem bark culture medium prepared in step (1) at a total bacterial inoculum amount of 4% by mass.

[0056] Comparative Example 1

[0057] A method for preparing a fermented liquid of North American redwood stems, which differs from Example 1 in that Bacillus subtilis is replaced by Saccharomyces cerevisiae (strain number CGMCC No. 28276).

[0058] Comparative Example 2

[0059] A method for preparing a fermented liquid of North American redwood stems, which differs from that of Example 1 in that Saccharomyces cerevisiae is replaced by Lactobacillus plantarum (strain number CGMCC No. 29289).

[0060] Test Example 1

[0061] The effective components of the fermented liquid of the stem of Sequoia gmelinii obtained in the above embodiments 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; and the total phenol content was determined by the sodium nitrite-aluminum nitrate method. The contents of the effective components were shown in Table 1.

[0062] Table 1. Content of active ingredients in fermented broth of redwood stems

[0063]

[0064] As shown in Table 1, the content of effective ingredients in the fermentation liquid of North American redwood stems is significantly improved compared with the traditional extraction method, among which the total sugar and total phenol contents in Example 4 are the highest, the protein content in Example 1 is the highest, and the effective ingredient contents in the comparative examples are all lower than those 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 effective ingredients in the fermentation liquid is higher than when Bacillus subtilis or Saccharomyces cerevisiae is not added; therefore, the combination of Saccharomyces cerevisiae and Bacillus subtilis is better than other strain combinations or their use alone.

[0065] Test Example 2

[0066] Hyaluronidase inhibition assay

[0067] In this method, a hyaluronidase in vitro inhibition experiment is used to determine the inhibitory effect of the fermented liquid of the stems of North American redwood on the activity of hyaluronidase, and the experimental concentrations are 5, 10, 15, 20, and 50 mg / mL (the mass concentration of the fermented liquid of the stems of North American redwood in the system).

[0068] Preparation of solutions required for the experiment:

[0069] Acetate buffer solution: Measure 1.155 mL of glacial acetic acid, dilute to 100 mL and mix well, of which 4.8 mL is solution A; weigh 2.72 g of sodium acetate crystals, dissolve in water and dilute to 100 mL and mix well, of which 45.2 mL is solution B; mix solutions A and B, dilute to 100 mL with water and mix well.

[0070] Hyaluronidase solution: weigh 10 mg of hyaluronidase into a beaker and add 4 mL of acetate buffer solution.

[0071] Sodium hyaluronate solution: weigh 5 mg of sodium hyaluronate into a beaker and add 10 mL of acetate 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 anhydrous 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 should be prepared before use.

[0074] Experimental steps:

[0075] Take 0.25mmol / L CaCl 2 0.1mL of solution and 0.5mL of hyaluronidase solution were incubated at 37℃ for 20min; 0.5mL of fermented broth of North American redwood stems at different concentrations was added and the culture was continued at 37℃ for 20min; 0.5mL of sodium hyaluronate solution was added and the mixture was incubated at 37℃ for 30min and placed at room temperature for 5min; 0.1mL of 0.4mol / L NaOH solution and 0.5mL of acetylacetone solution were added and the mixture was heated in a boiling water bath for 15min and immediately cooled with ice water for 5min; 1.0mL of Ehrlich reagent was added and diluted with 3.0mL of anhydrous ethanol and the mixture was placed for color development for 20min. The absorbance value was determined by a spectrophotometer, and the inhibition rate of hyaluronidase activity by fermented broth of different concentrations was calculated as shown in Table 2.

[0076] The calculation formula of the sample inhibition rate on hyaluronidase activity is:

[0077] Hyaluronidase activity inhibition rate (%) = [(A1-A2)-(B1-B2)] / (A1-A2)×100%

[0078] Among them, A1 is the absorbance value of the reference solution, in which deionized water is used instead of the fermentation broth; A2 is the absorbance value of the reference blank solution, in which deionized water is used instead of the fermentation broth, and acetate buffer solution is used instead of the hyaluronidase solution and the sodium hyaluronate solution; B1 is the absorbance value of the sample solution; B2 is the absorbance value of the sample blank solution, in which acetate buffer solution is used instead of the hyaluronidase solution and the sodium hyaluronate solution.

[0079] During the experiment, the sample is first scanned in the wavelength range of 450-700nm to determine the maximum absorption wavelength, and then the measurement is performed at the maximum absorption wavelength.

[0080] Table 2. Hyaluronidase activity inhibition rate of fermentation broth at different concentrations, %

[0081]

[0082] As shown in Table 2, Example 4 has the strongest hyaluronidase inhibitory activity, while the comparative example has the weakest inhibitory effect. The hyaluronidase activity inhibition rate of the fermented liquid of the stem of the redwood obtained in Example 4 is shown in Table 2. Figure 1 .like Figure 1 As shown, with the increase of the concentration of the fermented broth of North American redwood stems (SSP), its inhibitory effect on the activity of hyaluronidase gradually increased, indicating that the fermented broth of North American redwood stems obtained in Example 4 can effectively inhibit the activity of hyaluronidase, thereby playing a good soothing and anti-allergic effect.

[0083] Test Example 3

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

[0085] The sample solution was prepared by dissolving and diluting the fermented broth of the stem of North American redwood with deionized water, and setting 5 concentration gradients, namely 5, 10, 15, 20, and 50 mg / mL. The sample group, blank group, and control group were set in a 96-well plate.

[0086] Table 3. Reagent preparation

[0087]

[0088] According to the above table, the 96-well plate with the solution added was shaken on a shaker under light-shielding conditions for 30 minutes, and its absorbance at a wavelength of 517 nm was immediately measured using an ELISA reader. The DPPH radical scavenging rates of the fermented liquids of North American redwood stems at different concentrations were calculated as shown in Table 4.

[0089] Table 4. DPPH radical scavenging rate of fermented broth of North American redwood stems at different concentrations, %

[0090]

[0091] As shown in Table 4, the DPPH radical scavenging activity of Example 4 is the strongest, and the DPPH radical scavenging rate of the comparative example is the lowest. The DPPH radical scavenging rate of the fermented liquid of the stem of the redwood obtained in Example 4 is shown in Table 4. Figure 2 .like Figure 2 As shown, with the increase of the concentration of the fermented liquid from the stems of North American redwood, its scavenging effect on DPPH free radicals gradually increases, indicating that the fermented liquid from the stems of North American redwood obtained in Example 4 can effectively scavenges DPPH free radicals, thereby playing a good anti-aging role.

[0092] Test Example 4

[0093] Superoxide anion free radical scavenging assay

[0094] Take an appropriate amount of sample, dissolve and dilute with deionized water, set 5 concentration gradients, 5, 10, 15, 20, 50 mg / mL respectively. Prepare reagents according to Table 5, A solution: first add 0.6 mL 0.1 mol / L Tris-HCl buffer and 0.58 mL distilled water, mix well and place in a water bath at 25 ° C for 20 minutes, add 0.58 mL sample; B solution: preheated 0.02 mL 5 mmol / L pyrogallol solution. After the preparation of A and B solutions, quickly mix and shake well, measure the absorbance of the solution at 325 nm as the first absorbance value, measure once every 1 minute, and continuously measure for 4 minutes to calculate the increase in absorbance per minute within the linear range. Use 10 mmol / L HCl solution to replace the pyrogallol solution for zero absorbance. The superoxide anion radical scavenging rate of different concentrations of North American redwood stem fermentation broth is shown in Table 6.

[0095] Table 5. Reagent preparation

[0096]

[0097] Table 6. Superoxide anion radical scavenging rate of fermented broth of different concentrations of North American redwood stems, %

[0098]

[0099] As shown in Table 6, the superoxide anion radical scavenging activity of Example 4 is the strongest, and the superoxide anion radical scavenging rate of the comparative example is the lowest. The superoxide anion radical scavenging rate of the fermented liquid of the stem of the redwood obtained in Example 4 is shown in Table 6. Figure 3 .like Figure 3 As shown, with the increase of the concentration of the fermented liquid of North American redwood stems, its superoxide anion radical scavenging effect gradually increases, indicating that the fermented liquid of North American redwood stems obtained in Example 4 can effectively scavenge superoxide anion 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 1.5mL colorimetric tube system. Take an appropriate amount of the fermentation broth sample of North American redwood stem, 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 7, add 0.1mL 9mmol / L ferrous sulfate solution, 0.1mL 9mmol / L salicylic acid-ethanol solution, add an appropriate amount of deionized water, and finally add 0.1mL 8.8mmol / L hydrogen peroxide solution to the colorimetric tube, shake well, and bathe at 37℃ for 15min. Measure its absorbance, and calculate the hydroxyl radical scavenging rate of North American redwood stem fermentation broth of different concentrations as shown in Table 8.

[0103] Table 7. Reagent preparation

[0104]

[0105] Table 8. Hydroxyl radical scavenging rate of fermented broth of North American redwood stem at different concentrations, %

[0106]

[0107] As shown in Table 8, Example 4 has the strongest hydroxyl radical scavenging activity, and the comparative example has the lowest hydroxyl radical scavenging rate. The hydroxyl radical scavenging rate of the fermented liquid of the stem of the redwood obtained in Example 4 is shown in Table 8. Figure 4 .like Figure 4 As shown, with the increase of the concentration of the fermented liquid from the stems of North American redwood, its scavenging effect on hydroxyl radicals gradually increases, indicating that the fermented liquid from the stems of North American redwood obtained in Example 4 can effectively scavenging hydroxyl radicals, thereby playing a good anti-aging role.

[0108] Test Example 6

[0109] Cytotoxicity assay

[0110] Press 6-8×10 4 Keratinocytes (HaCaT) were seeded into 96-well plates at a seeding density of 10 cells / well and incubated in an incubator (37°C, 5% CO 2 ) and incubate overnight.

[0111] When the cell plating rate in the 96-well plate reaches 40-60%, the drug is administered. Wash the 96-well plate before administration. Add 100 μL of culture solution to each well of the blank control group; add 100 μL of cell solution to each well of the solvent control group and culture for 24 hours before administration. After administration, place the 96-well plate in an incubator (37°C, 5% CO 2) for 24 hours. After the cells were incubated for 24 hours, 10 μL CCK-8 was added and incubated at 37°C in the dark for 2-4 hours. After the incubation, the OD value was read at 490 nm, and the effects of different concentrations of North American redwood stem fermentation liquid on cell survival rate were calculated as shown in Table 9.

[0112] Table 9. Effects of different concentrations of fermented broth from North American redwood stems on cell survival rate, %

[0113]

[0114] As shown in Table 9, the fermented liquid of North American redwood stems has weak cytotoxicity at low concentrations and good safety. The cell survival rate of the fermented liquid of North American redwood stems obtained in Example 4 is shown in Table 9. Figure 5 The fermented liquid of the stem of Sequoia obtained in Example 4 has substantially no toxic effect on cells.

[0115] Test Example 7

[0116] Cell scratch assay

[0117] Before the experiment, use a marker pen to mark three horizontal lines on the back of the 6-well plate. Take logarithmic phase HaCaT cells at 6-8×10 4 The cells were seeded into 6-well plates at a density of 1000 cells / well and incubated in an incubator (37°C, 5% CO 2 ) overnight, and the cells were cultured to monolayer adherent cells before the experiment began. The experiment was divided into two groups, blank control group (Ctrl) and sample group (Sample). The compounds to be tested were selected and 5 test concentrations were set to 5, 10, 15, 20, and 50 mg / mL. Before administration, a 200 μL sterile pipette tip was used to evenly draw a line perpendicular to the marker line in the center of the cell plate, washed 3 times with PBS, added fresh serum-free culture medium, and photographed. After the drug treatment, the cells were cultured for 24 hours and photographed after the experiment.

[0118] The results of the effect of the fermented liquid of the stems of North American redwood obtained in Example 4 on cell migration are shown in Figure 6 .like Figure 6 As shown, the fermented liquid of North American redwood stems has the ability to promote the migration of keratinocytes at low concentrations, and the effect is best at a concentration of 10 mg / mL. This indicates that the fermented liquid of North American redwood stems can promote cell migration and accelerate wound healing.

[0119] Test Example 8

[0120] Inflammatory factor inhibition experiment

[0121] Mouse tumor necrosis factor-α (TNF-α) Elisa kit and interleukin-6 (IL-6) Elisa kit were used to determine the anti-inflammatory effect of the fermented liquid of North American redwood stems in vitro. The fermented liquid of North American redwood stems was mixed with DMEM high-glucose medium containing 15% fetal serum at 37°C and 5% CO 2 The cells were incubated in an incubator for 24 h to obtain the fermentation broth extract of the stem of North American redwood. Mouse mononuclear macrophages (RAW264.7 cells) were cultured at 2×10 5 The cells were inoculated into a 24-well cell culture plate at a density of 500 μL per well and cultured for 24 hours. After incubation in the incubator for 24 hours, the cell supernatant was taken and 100 ng / mL of lipopolysaccharide (LPS) was added to each well. The group without any treatment was the negative control (Ctrl), and the group with only LPS was the positive control. Three replicates were set for each group. After 24 hours, the cell supernatant was collected and the mouse IL-6 and TNF-α Elisa kits were used to detect the IL-6 and TNF-α concentrations in each group.

[0122] Table 10. Effects of different concentrations of fermented broth from North American redwood stems on changes in TNF-α concentration, pg / mL

[0123]

[0124] Table 11. Effects of different concentrations of fermented broth from North American redwood stems on IL-6 concentration changes, pg / mL

[0125]

[0126]

[0127] As shown in Tables 10 and 11, Example 4 has the best anti-inflammatory effect. The results of the inflammatory factor release of the fermented liquid of the stems of the redwood obtained in Example 4 are shown in Tables 10 and 11. Figure 7 .like Figure 7 As shown in the figure, the effect of the fermented liquid of North American redwood stem on the release of TNF-α in macrophages stimulated by LPS 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 minimum point at a concentration of 10 mg / mL, with the best anti-inflammatory effect, and has a good significance (**P<0.05). This shows that the fermented liquid of North American redwood stem can inhibit the release of TNF-α and IL-6 in cells, effectively slow down the inflammatory response, alleviate the occurrence of inflammatory aging, and play an important role in skin aging.

[0128] Test Example 9

[0129] Zebrafish anti-tail fin wrinkling experiment

[0130] Normally developed 2dpf zebrafish eggs were selected and randomly distributed into six-well plates, with 15 eggs per well. The standard dilution water in the six-well cell culture plate was removed without harming the young fish, and then 3mL of the test solution was quickly added to each well; among them, the control group (Ctrl) was the initial tail fin area, the model group (Model) was the tail fin area after the test without adding the test substance, the positive control group (Positive) was the tail fin area after adding 10mg / mL tea polyphenols, and the sample group was the tail fin area after adding the fermentation liquid of North American redwood stems. The sample concentrations were 5, 10, and 15mg / mL, respectively. First, cover the culture plate panel after fully mixing, incubate in dark for 2h, and then irradiate under UVB for 3 times, each irradiation time was 15min, with an interval of 30min. The irradiation dose each time was (2.07±0.18)J / cm 2 The total radiation dose is (6.21±0.54) J / cm 2 Finally, continue to incubate in a (28.5±1.0)℃ biochemical incubator in the dark for 22h.

[0131] Observe the phenotype and behavior of the zebrafish after hatching to ensure that they are normal zebrafish. Then randomly select at least 12 zebrafish using zebrafish sampling forceps. Place the selected zebrafish in a container containing 3% methylcellulose solution and immerse it completely. Let it stand for a while to allow the methylcellulose solution to fully contact the zebrafish and fix the zebrafish. Observe and take pictures under a microscope. All zebrafish photos are completed within 2 hours under the same instrument and environmental conditions.

[0132] After taking the photos, the image analysis software was used to analyze the obtained zebrafish images. The quantitative area was selected as the zebrafish tail fin, and the analysis parameter was set to area. The images were analyzed to obtain data. 10 valid data of tail fin area were taken for each group. The effects of different concentrations of North American redwood stem fermentation liquid on the zebrafish tail fin area were calculated as shown in Table 12.

[0133] Table 12. Effects of different concentrations of fermented broth from redwood stems on the tail fin area of ​​zebrafish, μm 2

[0134]

[0135] The zebrafish tail fin area of ​​the fermented liquid of the stem of North American redwood obtained in Example 4 is shown in Figure 8 .like Figure 8 As shown, with the increase of the concentration of the fermented liquid from the stems of North American redwood, its inhibitory effect on the wrinkling of the zebrafish tail fin became stronger, indicating that the fermented liquid from the stems of North American redwood can effectively resist the effects of photoaging.

[0136] Test Example 10

[0137] Human sensitivity test

[0138] Twenty volunteers were selected, half of them were male and half were female. Before the experiment, the volunteers cleaned their left and right forearms with clean water respectively, and sat quietly in an environment with a temperature of 20-22℃ and a relative humidity of 50-60%. The skin sensitivity experiment was conducted without the subjects applying any cosmetics.

[0139] The subjects were smeared with the fermented liquid of redwood stems on their left forearms, once every half an hour for a total of 6 times, with an area of ​​3×3 cm 2 The right forearm was used as a blank control without any product (Ctrl). Then, 5% sodium dodecyl sulfate solution (SDS) was applied to the left and right forearms of the subjects respectively. The reaction of the subjects' arms was observed after 0.5 hours. The statistical results of the sensitization rate are shown in Table 13.

[0140] Table 13. Effect of fermented broth of North American redwood stems under different conditions on the sensitization rate of irritants

[0141]

[0142] As shown in Table 13, after applying the fermented liquid of the stem of North American redwood, the irritant was then applied, and the tingling and redness of the left forearm were not obvious, and the sensitization rate was greatly reduced. However, the right forearm, which served as the blank control, had a strong reaction to the irritant, and 0.5 hours after applying the irritant, the skin had obvious redness and swelling, accompanied by tingling, and the sensitization rate of the blank control was high. This shows that the fermented liquid of the stem of North American redwood has a good anti-allergic effect.

[0143] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a fermented liquid of North American redwood stems, characterized in that: Saccharomyces cerevisiae and Bacillus subtilis are inoculated into a culture medium of North American redwood stem bark for fermentation to obtain a North American redwood stem fermentation liquid.

2. The method for preparing the fermented liquid of North American redwood stems according to claim 1, characterized in that: The Saccharomyces cerevisiae has a collection number of CGMCC No. 28276 or CGMCC No. 23880, which is purchased from the China General Microbiological Culture Collection Center; the Bacillus subtilis is Bacillus subtilis YK006, which is purchased from Yokomeigu (Shandong) Biotechnology Co., Ltd. or Jinan Ruibeike Biotechnology Co., Ltd.

3. The method for preparing the fermented liquid of North American redwood stems according to claim 1, characterized in that: 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.

4. The method for preparing the fermented liquid of North American redwood 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.

5. The method for preparing the fermented liquid of North American redwood stems as claimed in claim 4, characterized in that: The fermentation in step (2) is to inoculate 2-5% by mass of total bacterial cell inoculum of Saccharomyces cerevisiae and Bacillus subtilis into a culture medium of dry bark of North American redwood, and ferment at 30-32°C, pH 5.0-6.5, and 140-200 r / min in a shaking flask for 18-48 hours.

6. The method for preparing the fermented liquid of North American redwood stems according to claim 4, characterized in that: The sterilization in step (2) is performed at 80-85°C for 20 minutes.

7. The method for preparing the fermented liquid of North American redwood stems according to claim 4, characterized in that: In step (2), 2-3 weight parts of the brewer's yeast and 1-2 weight parts of the Bacillus subtilis were used, and the bacterial concentration was 10 5 -10 7 CFU / mL.

8. The fermented liquid of North American redwood stems prepared by the method for preparing the fermented liquid of North American redwood stems according to any one of claims 1 to 7 is characterized in that: The effective ingredients contain monosaccharides, polysaccharides, proteins and polyphenols, wherein the content of the polyphenols is 0.40-0.89 mg / g, the content of the total sugars is 3.2-6.6 mg / g, and the content of the proteins is 100-150 mg / g.

9. Use of the fermented liquor of the stem of Sequoia gmelinii according to claim 8 in the preparation of cosmetics.

10. The use according to claim 9, characterized in that The cosmetics have anti-aging, anti-allergy and repairing effects.

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