Mother-of-pearl vitriol fermentation product, and preparation method and application of mother-of-pearl vitriol fermentation product

Abstract

The invention discloses a mother-of-pearl glassy yeast as well as a preparation method and application thereof, and the preparation method of the mother-of-pearl glassy yeast specifically comprises the following steps: inoculating lactobacillus into a fermentation substrate, and carrying out fermentation culture and sterilization, so as to obtain the mother-of-pearl glassy yeast, wherein the fermentation substrate comprises nacre mother of pearl, botryn, a carbon source, a nitrogen source and water, and the lactobacillus is composed of mucus fermentum and lactobacillus helveticus. Through a compound fermentation method, active ingredients in the mother-of-pearl are extracted and are effectively combined with the vitriol in the fermentation process, the effects of the mother-of-pearl and the vitriol are fully played, the skin care effect is played, and meanwhile, the prepared product can be directly used as a cosmetic and also can be used as a cosmetic. And the composition can also be added into a cosmetic basic formula for use, and has a wide application prospect.

Description

Technical Field

[0001] The present application belongs to the field of fermentation technology, and in particular relates to a pearl mother-of-pearl fermentation product, a preparation method thereof, and an application thereof. Background Art

[0002] Mother of pearl, the shell of the clams of the family Hyriopsis cumingii, the crown of the clams, or the pearl oyster of the family Pearl Oyster, is a traditional Chinese medicine in my country. Its main component is calcium carbonate, and it also contains a small amount of trace elements and amino acids, as well as oligopeptide chemical components. Mother of pearl is mostly used in processed products, mainly used in Chinese medicine clinics, and can treat symptoms such as insomnia, neurasthenia, dizziness, tinnitus, and heart palpitations. At present, there are relatively few studies on the use of mother of pearl in the field of cosmetics. In China, water extraction is mostly used to extract active ingredients from mother of pearl, but the extraction efficiency is low and resources are seriously wasted.

[0003] Pro-Xylane is a xylose derivative with anti-aging active ingredients. It can promote the synthesis of collagen, make the skin stronger and more elastic, significantly improve fine lines on the neck, and achieve the effect of preventing aging. Since Pro-Xylane is generally artificially synthesized, it is easy to cause allergic reactions such as redness and itching during long-term use.

[0004] Therefore, there is an urgent need for a method that can combine the advantages of mother-of-pearl and bosacene and alleviate the adverse reactions of artificial synthetics during use. Summary of the invention

[0005] The technical problem to be solved by the present application is to overcome the defects of the prior art such as low efficiency of extracting mother-of-pearl components leading to waste of resources, artificially synthesized phosphatase that is prone to allergic reactions when used for a long time, and the lack of effective means to combine mother-of-pearl with phosphatase at present, and to provide a mother-of-pearl phosphatase fermentation product, a preparation method thereof, and application. The present application utilizes lactobacillus to extract the active ingredients in mother-of-pearl through a composite fermentation method, and effectively combines it with phosphatase during the fermentation process, so that the prepared fermentation product has ideal antioxidant, anti-aging, anti-wrinkle and other effects.

[0006] This application adopts the following technical solutions to solve the above technical problems:

[0007] The present application provides a method for preparing a pearl mother-of-pearl fermentation product, which comprises the following steps: inoculating lactobacillus into a fermentation substrate, culturing by fermentation, and sterilizing;

[0008] The fermentation substrate comprises mother of pearl, bosogenin, a carbon source, a nitrogen source and water, and the lactobacillus consists of fermentative mucinous bacteria and helveticus lactobacillus.

[0009] In some embodiments, the mother-of-pearl comprises the shell of the cuming oyster, the pleated crown oyster, or the pearl oyster of the nacre family, preferably the shell of the Australian white pearl;

[0010] In some embodiments, the mother-of-pearl is mother-of-pearl powder, and the preparation method of the mother-of-pearl powder includes: crushing and sieving the mother-of-pearl.

[0011] Wherein, the particle size of the crushed particles after sieving is 50 to 200 meshes.

[0012] In some embodiments, the mass ratio of the mother-of-pearl to the water is 1:(20-200), preferably 1:(50-200), and more preferably 1:100.

[0013] In some embodiments, the mass ratio of the mother-of-pearl to the bosogen is (5-20):1, preferably (5-10):1.

[0014] In some embodiments, the carbon source includes one of corn flour, rice flour or glucose.

[0015] In some embodiments, the nitrogen source includes one of soybean flour, soybean meal or soybean peptide.

[0016] In some embodiments, the water includes deionized water or distilled water.

[0017] In some embodiments, the mass ratio of the carbon source to the water is 1:(100-1000), preferably 1:(200-500).

[0018] In some embodiments, the mass ratio of the nitrogen source to the water is 1:(100-1000), preferably 1:(200-500).

[0019] In some embodiments, the method for preparing the fermentation substrate comprises: mixing the components in the fermentation substrate until the components are completely dissolved.

[0020] In some embodiments, the fermentation substrate may be further sterilized before use.

[0021] Wherein, the method for sterilizing the fermentation substrate can generally be a high temperature sterilization method.

[0022] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature may be a conventional temperature for such operations in the art, preferably 110°C to 125°C, more preferably 115°C to 121°C, for example 121°C.

[0023] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization pressure may be a conventional pressure for such operations in the art, preferably 0.1-0.14 MPa, more preferably 0.1-0.13 MPa, such as 0.13 MPa.

[0024] When the high temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time can be the conventional time for such operation in the art, preferably 15 to 35 minutes, more preferably 15 to 25 minutes, for example 20 minutes.

[0025] In some embodiments, the fermented mucus lactobacillus is deposited in the China Industrial Microbiological Culture Collection Center with a deposit number of CICC 24575.

[0026] In some embodiments, the Lactobacillus helveticus is deposited in the China Industrial Microbiological Culture Collection Center with a deposit number of CICC 6261.

[0027] In some embodiments, the ratio of the viable cell count of the fermented Lactobacillus mucis to that of the Lactobacillus helveticus is 1:(0.2-5), preferably 1:1.

[0028] In some embodiments, the fermented Lactobacillus mucilaginosus is added in the form of a fermented Lactobacillus mucilaginosus bacterial solution, and the live bacteria concentration of the fermented Lactobacillus mucilaginosus bacterial solution is 10 9 ~10 13 CFU / mL, preferably 10 9 ~10 11 CFU / mL.

[0029] The method for preparing the fermented mucus lactobacillus liquid comprises: inoculating the fermented mucus lactobacillus into an MRS culture medium and culturing the culture medium at 37° C. for 24 hours.

[0030] In some embodiments, the Lactobacillus helveticus is added in the form of a Lactobacillus helveticus bacterial solution. The live bacteria concentration of the Lactobacillus helveticus bacterial solution is 10 9 ~10 13 CFU / mL, preferably 10 9 ~10 11 CFU / mL.

[0031] The method for preparing the Lactobacillus helveticus bacterial liquid comprises: inoculating Lactobacillus helveticus into MRS culture medium and culturing at 37° C. for 24 hours.

[0032] In some embodiments, based on the water in the fermentation substrate, the number of viable lactobacilli inoculated per unit mass of the water is 10 9 ~10 13 CFU / mL, preferably 109 ~10 11 CFU / mL.

[0033] In some embodiments, the conditions and methods of the fermentation culture may be conventional in the art, and may generally be static fermentation.

[0034] In some embodiments, the fermentation culture temperature is 30-45°C, preferably 35-40°C, such as 37°C.

[0035] In some embodiments, the fermentation culture time is 12 to 48 hours, preferably 12 to 24 hours, such as 15 hours.

[0036] In some embodiments, the sterilization method may generally be high temperature sterilization.

[0037] The sterilization temperature may be a conventional temperature for such operations in the art, preferably 110°C to 125°C, more preferably 115°C to 121°C, for example 121°C.

[0038] The sterilization pressure may be a conventional pressure for such operations in the art, preferably 0.1-0.14 MPa, more preferably 0.1-0.13 MPa, for example 0.13 MPa.

[0039] The sterilization time may be the conventional time for such operation in the art, preferably 15 to 35 minutes, more preferably 20 to 35 minutes, for example 30 minutes.

[0040] In some embodiments, after the sterilization operation, the sterilization may further include at least one of cooling, centrifuging and collecting the supernatant, secondary sterilization, or mixing with a preservative.

[0041] Wherein, according to the common practice in the art, the cooling may be cooling to room temperature.

[0042] The centrifugal rotation speed is a conventional rotation speed for this type of operation in the art, preferably 3000-9000 rpm, more preferably 4000-6000 rpm, for example 5000 rpm.

[0043] Wherein, the radius of the centrifuge is 8 to 15 cm.

[0044] The centrifugation time is conventional in the art for this type of operation, preferably 10 to 40 min, more preferably 20 to 40 min, for example 30 min.

[0045] The secondary sterilization method is generally a high temperature sterilization method.

[0046] The temperature of the secondary sterilization is a conventional temperature for such operations in the art, preferably 110°C to 125°C, more preferably 115°C to 121°C, for example 121°C.

[0047] The pressure of the secondary sterilization is a conventional pressure for such operations in the art, preferably 0.1-0.14 MPa, more preferably 0.1-0.13 MPa, for example 0.13 MPa.

[0048] The time for the secondary sterilization is the conventional time for such operations in the art, preferably 15 to 35 minutes, more preferably 20 to 35 minutes, for example 30 minutes.

[0049] Wherein, during the mixing process with the preservative, the mixing temperature is a conventional temperature for such operation in the art, preferably 50 to 80°C, more preferably 70 to 80°C.

[0050] Wherein, in the process of mixing with the preservative, the preservative comprises p-hydroxyacetophenone and / or 1,2-hexanediol according to the conventional practice in the art.

[0051] When the preservative includes p-hydroxyacetophenone, the mass percentage of p-hydroxyacetophenone in the supernatant of the material obtained after sterilization may be 0.5% to 2%, preferably 0.1% to 0.6%, and more preferably 0.5%.

[0052] When the preservative includes 1,2-hexanediol, the mass fraction of 1,2-hexanediol in the supernatant of the material obtained after sterilization is 0.5% to 2%, preferably 0.5% to 1%, and more preferably 0.5%.

[0053] In some embodiments, the room temperature generally refers to 15-40°C.

[0054] The present application also provides a pearl mother-of-pearl fermented product, which is prepared by the preparation method of the pearl mother-of-pearl fermented product as described above.

[0055] The present application also provides a pearl mother-of-pearl fermentation product, which can be used directly as a product, as an additive or as a base in the preparation of a skin external preparation.

[0056] In some embodiments, the pearl mother-of-pearl fermentation product is used as at least one of the antioxidant active ingredient, anti-aging active ingredient, and anti-wrinkle active ingredient in the skin topical preparation.

[0057] The present application also provides a skin external preparation, which comprises the pearl mother-of-pearl fermentation product as described above.

[0058] In some embodiments, the skin topical preparation further includes active ingredients conventionally used in the art, which may generally include at least one of moisturizing active ingredients, whitening active ingredients, anti-inflammatory active ingredients, anti-allergic active ingredients and antioxidant active ingredients.

[0059] In some embodiments, the topical skin preparation may include but is not limited to a facial mask, an essence or a toner according to conventional methods in the art.

[0060] In some embodiments, the mass percentage of the pearl mother-of-pearl fermentation product in the skin external preparation may be 5% to 99%, preferably 60% to 99%.

[0061] On the basis of conforming to the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present application.

[0062] The reagents and raw materials used in this application are commercially available.

[0063] The positive progressive effect of the present application is that the present application ferments Australian white pearl mother-of-pearl and bosera by lactic acid bacteria, which can not only exert the efficacy of Australian white pearl mother-of-pearl and bosera themselves, but also produce probiotic metabolites such as amino acids, proteins, polysaccharides, etc., to better exert their skin care effects. At the same time, the prepared products can be used directly as cosmetics or added to the basic formula of cosmetics, and have good firming, anti-oxidation, and cell proliferation promoting effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] The present application can be better understood by referring to the description given below in conjunction with the accompanying drawings. The accompanying drawings, together with the following detailed description, are included in this specification and form a part of this specification, and are used to further illustrate the preferred embodiments of the present application and explain the principles and advantages of the present application.

[0065] in:

[0066] Figure 1 This is a comparison chart of the DPPH free radical scavenging ability of the products obtained in Examples 1 to 4 and Comparative Examples 1 to 4;

[0067] Figure 2 A comparison chart of the hydroxyl radical scavenging abilities of the products obtained in Examples 1 to 4 and Comparative Examples 1 to 4;

[0068] Figure 3 This is a comparison chart of cell viability after cells were treated with products obtained in Examples 1 to 4 and Comparative Examples 1 to 4;

[0069] Figure 4 This is a comparison chart of intracellular COL-I content after cells were treated with the products obtained in Examples 1 to 4 and Comparative Examples 1 to 4. DETAILED DESCRIPTION

[0070] The present application is further described below by way of examples, but the present application is not limited to the scope of the examples. The experimental methods without specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product instructions.

[0071] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0072] The raw materials in the following examples are all commercially available.

[0073] In the following examples and comparative examples, Limosilactobacillus fermentum was purchased from China Industrial Microbiological Culture Collection Center with a collection number of CICC 24575.

[0074] In the following examples and comparative examples, Lactobacillus helveticus was purchased from China Industrial Microbiological Culture Collection Center with a collection number of CICC 6261.

[0075] In the following comparative examples, Saccharomyces cerevisiae was purchased from China Industrial Microbiological Culture Collection Center with a collection number of CICC 1538.

[0076] Example 1

[0077] 5 g of Australian white mother-of-pearl powder, 1 g of bosaicin, 1.5 g of glucose, 1.5 g of soybean peptide and 300 mL of deionized water were mixed evenly, and then sterilized in an autoclave at a temperature of 121° C. and a pressure of 0.12 MPa for 30 min, and then cooled to room temperature after sterilization to obtain a fermentation substrate;

[0078] The preparation method of the fermented mucus lactobacillus liquid comprises: inoculating the fermented mucus lactobacillus into an MRS medium and culturing it at 37° C. for 24 hours, wherein the number of viable bacteria in the fermented mucus lactobacillus liquid is 10 10 CFU / mL.

[0079] The preparation method of the Lactobacillus helveticus liquid comprises: inoculating Lactobacillus helveticus in an MRS medium and culturing at 37° C. for 24 hours, wherein the number of viable bacteria in the Lactobacillus helveticus liquid is 10 10 CFU / mL.

[0080] 1.5 mL of fermented Lactobacillus culture and 1.5 mL of Lactobacillus helveticus culture were inoculated into the fermentation substrate prepared above, and the mixture was shaken and cultured in a constant temperature incubator at 37° C. for 15 h. After the fermentation culture was completed, the mixture was sterilized in an autoclave at a temperature of 121° C. and a pressure of 0.12 MPa for 30 min. After the sterilization was completed, the mixture was cooled to room temperature and centrifuged, wherein the centrifugal speed was 4800 r / min, the centrifugal time was 30 min, and the centrifugal radius was 15 cm. After the centrifugation was completed, secondary sterilization was performed, and the secondary sterilization conditions were 121° C., a pressure of 0.13 MPa, and a time of 30 min. After the sterilization was completed, a preservative needed to be added to the fermentation liquid, and during the mixing process with the preservative, the mixing temperature was 75° C. Based on the mass of the fermentation stock solution, 0.5% of p-hydroxyacetophenone and 0.5% of 1,2-hexanediol were added, and the mixture was evenly mixed to obtain mother-of-pearl Bose fermentation product.

[0081] Example 2

[0082] Compared with Example 1, the only difference is that the fermentation time is 18 hours, and the other condition parameters are the same as Example 1.

[0083] Example 3

[0084] Compared with Example 1, the only difference is that 1 ml of fermented mucus Lactobacillus liquid and 2 ml of Lactobacillus helveticus liquid are added, and other condition parameters are the same as Example 1.

[0085] Example 4

[0086] Compared with Example 1, the only difference is that 2 ml of fermented mucus Lactobacillus liquid and 1 ml of Lactobacillus helveticus liquid are added, and other condition parameters are the same as Example 1.

[0087] Comparative Example 1

[0088] The preparation method is the same as in Example 1, except that fermentation is not performed. Other conditions and parameters are the same as in Example 1. The specific operations are as follows:

[0089] Mix 5g of Australian white mother-of-pearl powder, 1g of bosacein, 1.5g of glucose, 1.5g of soybean peptide and 300mL of deionized water, and then sterilize in an autoclave at a temperature of 121°C and a pressure of 0.12MPa for 20min. After sterilization, cool to room temperature.

[0090] Comparative Example 2

[0091] The preparation method is the same as that in Example 1, except that only 3 mL of fermented mucus Lactobacillus is inoculated, and other condition parameters are the same as those in Example 1.

[0092] Comparative Example 3

[0093] The preparation method is the same as that in Example 1, except that Saccharomyces cerevisiae (CICC 1538) is used for fermentation, and other conditions and parameters are the same as those in Example 1.

[0094] Comparative Example 4

[0095] The preparation method is the same as that in Example 1, except that no carbon source and nitrogen source are added, and other conditions and parameters are the same as those in Example 1.

[0096] Effect Example 1

[0097] DPPH free radical scavenging experiment

[0098] DPPH is an early synthesized organic free radical, which is often used to evaluate the hydrogen-donating capacity of antioxidants. It is very stable in organic solvents, purple, and has a characteristic absorption peak at 517nm. When encountering free radical scavengers, the lone pair of electrons of DPPH is paired and it fades, that is, the absorbance at the maximum absorption wavelength becomes smaller. Therefore, the scavenging effect of the sample on DPPH free radicals can be evaluated by measuring the change in absorbance.

[0099] The specific experimental steps of the DPPH free radical scavenging experiment are as follows: (sample diluted 2 times)

[0100] (1) Take an equal volume (1 mL) of the test solution and 2 × 10 -4 mol / L DPPH solution and mix well (tube A1);

[0101] (2) Take an equal volume (1 mL) of anhydrous ethanol (solvent for the test substance) and 2×10 -4 mol / L DPPH solution and mix well (tube A2);

[0102] (3) Take an equal volume (1 mL) of anhydrous ethanol and mix it with the test solution (tube A3);

[0103] (4) After 30 minutes of reaction in the dark, measure the absorbance of tubes A1, A2, and A3 at 517 nm. The clearance rate is calculated as follows: clearance rate = [(A2 + A3) - A1] / A2 × 100%.

[0104] This experiment tested the DPPH free radical scavenging experiment on the embodiments and comparative examples. The results are shown in Tables 1 and Figure 1 .

[0105] Table 1

[0106] serial number DPPH free radical scavenging rate (%) Example 1 65.41±1.85 Example 2 58.67±1.35 Example 3 54.16±1.35 Example 4 57.76±0.77 Comparative Example 1 35.88±2.2 Comparative Example 2 42.08±1.2 Comparative Example 3 41.92±0.96 Comparative Example 4 41.18±0.71

[0107] The results show that the DPPH radical scavenging rates of the products prepared in Examples 1 to 4 are much higher than those of the products prepared in Comparative Examples 1 to 4. It can be seen that the products prepared in the examples of the present application have ideal antioxidant effects, and the fermentation culture time and the fermentation substrate have a great influence on the antioxidant properties of the final product.

[0108] Effect Example 2

[0109] Hydroxyl free radical scavenging assay

[0110] The Fenton reaction is used to generate hydroxyl radicals (·OH), and salicylic acid is added to the reaction system. ·OH reacts with salicylic acid to generate a colored compound 2,3-dihydroxybenzoic acid with characteristic absorption at 510nm. The fixed reaction time method is used to measure the absorbance of the reaction solution containing the analyte at 510nm, and compared with the blank solution to determine the scavenging effect of the analyte on ·OH.

[0111] The test solution is the product obtained in the above-mentioned embodiment and comparative example (diluted 2 times);

[0112] Add reagents to the test tube according to Table 1, and add 9mmol / L FeSO4, 9mmol / L salicylic acid ethanol solution, test solution, appropriate amount of deionized water and 8.8mmol / L H2O2 solution in sequence. Shake well, heat in a 37℃ water bath for 15 minutes, then take out and measure the absorbance A0 of the blank control and the absorbance A0 of the sample group. x and the background absorbance of the solution without H2O2 x0 When determining A0, the reference solution was a system without hydrogen peroxide. Three parallel experiments were performed in each group, and the absorbance values ​​were tested and the average values ​​were taken. The results are shown in Table 2. The hydroxyl radical scavenging rate of each group was calculated according to the following formula. The results are shown in Tables 3 and Figure 2 .

[0113] Hydroxyl radical scavenging rate = (A0-(A x -A x0 )) / A0×100%.

[0114] Table 2

[0115]

[0116] Table 3

[0117] serial number Hydroxyl free radical scavenging rate (%) Example 1 83.85±0.71 Example 2 78.88±1.98 Example 3 75.31±1.48 Example 4 76.76±2.52 Comparative Example 1 52.23±1.44 Comparative Example 2 62.42±2.44 Comparative Example 3 56.21±1.95 Comparative Example 4 59.39±1.17

[0118] The results show that the hydroxyl radical scavenging rates of the products prepared in Examples 1 to 4 are much higher than those of the products prepared in Comparative Examples 1 to 4. It can be seen that the products prepared in the examples of the present application have ideal antioxidant effects, and the fermentation culture time and the fermentation substrate have a great influence on the antioxidant properties of the final product.

[0119] Effect Example 3

[0120] Cell survival rate (promote cell proliferation)

[0121] This experiment uses human skin fibroblasts from the Chinese Science Cell Bank to verify the cytotoxicity of the products obtained in the above examples and comparative examples.

[0122] Reagents: 0.25% (containing EDTA) trypsin was produced by GIBCO, USA; DMEM culture medium was produced by GIBCO, USA; double antibody was produced by Corning, USA; CCK-8 was produced by Beijing Biode Biotech Co., Ltd.; fetal bovine serum was produced by GIBCO, USA; phosphate buffer was produced by Beijing Bioregi Biotech Co., Ltd.

[0123] Equipment: The manufacturer of WJ-80A-Ⅱ CO2 constant temperature incubator is Shanghai Shengke Instrument Equipment Co., Ltd.; the manufacturer of Sunrise microplate reader is Tecan Trading Co., Ltd.; the manufacturer of TL80-2 medical centrifuge is Jiangsu Tianli Medical Equipment Co., Ltd.; the manufacturer of NUNC 96-well cell culture plate is Thermo Fisher Scientific.

[0124] 1. Experimental steps:

[0125] The products prepared in the above examples and comparative examples were respectively prepared into 2.5% of the test solution in the experimental group using serum-free DMEM medium. Human skin fibroblasts were cultured in a medium containing 10% fetal bovine serum and 1% double antibody (1×10 5 U / L penicillin, 100 mg / L streptomycin) in DMEM medium. The cells were grown in an incubator at 37°C and 5% CO2 saturated humidity. When the cell fusion reached more than 85%, the cells in the logarithmic growth phase were digested with 0.05% trypsin and the digestion reaction was terminated with serum-containing DMEM. The cell count was performed on a cell counting plate and the cell suspension concentration was adjusted to 7×10 4 / mL, inoculate the cell suspension into a 96-well plate at a ratio of 100μL per well, and incubate at 37℃, 5% CO2 for 12h. Remove the old culture medium and wash the cells twice with phosphate buffer. Add 100μL of the above-configured, filtered and sterilized test solution of different concentrations to each well in the experimental group, and make 3 replicate wells for each test solution; the control group contains cells, and serum-free DMEM culture medium is added; the blank control group has no cells, and 100μL of PBS is added. Incubate for another 24h at 37℃, 5% CO2. Then add 10μL of CCK-8 solution to each well, incubate for another 3h, measure the absorbance at a wavelength of 450nm, and calculate the cell survival rate of each group. The results are shown in Tables 4 and Figure 3 .

[0126] The cell viability was calculated as follows:

[0127] Cell survival rate (%) = (A experimental group - A blank control group) / (A control group - A blank control group) × 100%.

[0128] Table 4

[0129] serial number Cell survival rate (%) Example 1 109.88±2.04 Example 2 107.45±1.31 Example 3 104.61±1.19 Example 4 108.64±3.63 Comparative Example 1 89.99±1.08 Comparative Example 2 95.98±3.39 Comparative Example 3 93.45±1.6 Comparative Example 4 92.02±1.87

[0130] The results show that the cell survival rates of cells treated with the products prepared in Examples 1 to 4 are much higher than those of cells treated with the products prepared in Comparative Examples 1 to 4. It can be seen that the products prepared in the examples of the present application have a certain effect of promoting cell proliferation, and the fermentation culture time and fermentation substrate have a great influence on the cell proliferation effect of the final product.

[0131] Effect Example 4

[0132] Intracellular collagen content determination (COL-I content determination)

[0133] This experiment verifies the ability of the products prepared in the above examples and comparative examples to repair damaged skin by measuring the COL-I content in human skin fibroblasts.

[0134] Human skin fibroblasts in logarithmic phase were taken and the cell suspension was inoculated on a 6-well cell culture plate at a density of 250,000 / mL. 2 mL of cell suspension was added to each well and cultured for 12 hours. Experimental group, blank group and model group were set up. Among them, the experimental group and model group were treated with 18mJ / cm 2 The cells were irradiated with UVA for 40 min. After irradiation, the supernatant in the experimental group, blank group and model group was discarded, 2 mL of DMEM solution was added to the blank group and model group, and 2 mL of the test solution was added to the experimental group (the product prepared in the above embodiment or comparative example was configured into a test solution with a volume percentage of 2.5% using DMEM, and the test solution needed to be filtered through a 0.22 μm sterile filter membrane before use), and the cells were treated for 24 h after addition, the supernatant was discarded, the cells were washed 2 to 3 times with PBS, and then the cells were treated with cell lysis solution, transferred to a centrifuge tube, centrifuged at 10000 r / min and 4°C for 10 min, and the supernatant was taken to obtain the cell lysate. Take 20 μL of cell lysate and use the BCA kit to detect the total protein content in the sample; the collagen content was determined according to the ELISA kit instructions, and the OD values ​​were measured at 450 nm. The expression of collagen x was calculated according to the standard curve, and the total protein content b was used for correction to obtain A=x / b, and then the relative expression of collagen I was calculated, and the relative expression of collagen I=A 实验组 / 模型组 / A 空白组 The results are shown in Table 5 and Figure 4 (In the figure,### p<0.001, indicating an extremely significant decrease compared with the blank group; p>0.05, ns, indicating no significant increase compared with the model group; *p<0.05, indicating an increase compared with the model group, **p<0.01, indicating a significant increase compared with the model group, ***p<0.001, indicating an extremely significant increase compared with the model group).

[0135] Table 5

[0136] serial number COL-I content (ng / mg) Blank Group 0.476±0.023 Model Group 0.29±0.015 Example 1 0.552±0.009 Example 2 0.532±0.011 Example 3 0.499±0.008 Example 4 0.507±0.017 Comparative Example 1 0.392±0.014 Comparative Example 2 0.439±0.01 Comparative Example 3 0.412±0.008 Comparative Example 4 0.408±0.014

[0137] The results show that after the cells were treated with the products obtained in Examples 1 to 4, the COL-I content was higher than the COL-I content after the cells were treated with the products obtained in Comparative Examples 1 to 4. It can be seen that the products obtained in the examples of the present application have a certain ability to repair damaged skin, and the fermentation culture time and the fermentation substrate have a great influence on the ability of the final product to repair damaged skin. Finally, it should be noted that in this application, the terms "comprises", "comprising" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device that includes a series of elements includes not only those elements, but also includes other elements that are not explicitly listed, or also includes elements that are inherent to such process, method, article or device.

[0138] Although the present application has been disclosed above through the description of the specific embodiments of the present application, it should be understood that those skilled in the art can design various modifications, improvements or equivalents to the present application within the spirit and scope of the attached scheme. These modifications, improvements or equivalents should also be considered to be included in the scope of protection claimed in the present application.

Claims

1. A method for preparing a fermented product of mother-of-pearl, characterized in that: The specific steps include: inoculating lactobacillus into a fermentation substrate, culturing by fermentation, and sterilizing; The fermentation substrate comprises mother of pearl, bosogenin, a carbon source, a nitrogen source and water, and the lactobacillus consists of fermentative mucinous bacteria and helveticus lactobacillus.

2. The method for preparing the mother-of-pearl fermented product according to claim 1, characterized in that: The method for preparing the pearl mother-of-pearl fermentation product satisfies at least one of the following conditions: The mother-of-pearl comprises the shell of the mullet Hyriopsis cumingii, Hyriopsis plicata or the pearl oyster of the family Pectinidae, preferably the shell of the Australian white pearl; The mother-of-pearl is mother-of-pearl powder. Preferably, the preparation method of the mother-of-pearl powder comprises: crushing and sieving the mother-of-pearl, more preferably, the crushed particles are sieved with a particle size of 50 to 200 meshes; The mass ratio of the mother-of-pearl to the water is 1:(20-200), preferably 1:(50-200), and more preferably 1:100; The mass ratio of the mother-of-pearl to the bosacene is (5-20):1, preferably (5-10):

1.

3. The method for preparing the mother-of-pearl fermented product according to claim 1, characterized in that: The method for preparing the pearl mother-of-pearl fermentation product satisfies at least one of the following conditions: The carbon source includes one of corn flour, rice flour or glucose; The nitrogen source includes one of soybean powder, soybean meal or soybean peptide; The water includes deionized water or distilled water; The mass ratio of the carbon source to the water is 1:(100-1000), preferably 1:(200-500); The mass ratio of the nitrogen source to the water is 1:(100-1000), preferably 1:(200-500); The method for preparing the fermentation substrate comprises: mixing the components in the fermentation substrate until the components are completely dissolved.

4. The method for preparing the mother-of-pearl fermented product according to claim 1, characterized in that: The method for preparing the pearl mother-of-pearl fermentation product satisfies at least one of the following conditions: The fermented mucus lactobacillus (Limosilactobacillus fermentum) is deposited in the China Industrial Microbiological Culture Collection Center with a deposit number of CICC 24575; The Lactobacillus helveticus is deposited in the China Industrial Microbiological Culture Collection Center with a deposit number of CICC 6261; The ratio of the viable bacteria count of the fermented mucus lactobacillus to the viable bacteria count of the helvetic acid lactobacillus is 1:(0.2-5), preferably 1:1; The fermented mucus lactobacillus is added in the form of a fermented mucus lactobacillus bacterial liquid. Preferably, the live bacteria concentration of the fermented mucus lactobacillus bacterial liquid is 10 9 ~10 13 CFU / mL, preferably 10 9 ~10 11 CFU / mL; The method for preparing the fermented mucus lactobacillus liquid comprises: inoculating the fermented mucus lactobacillus into an MRS medium and culturing at 37° C. for 24 hours; The Lactobacillus helveticus is added in the form of a Lactobacillus helveticus bacterial liquid. Preferably, the live bacteria concentration of the Lactobacillus helveticus bacterial liquid is 10 9 ~10 13 CFU / mL, preferably 10 9 ~10 11 CFU / mL; The preparation method of the Lactobacillus helveticus bacterial liquid comprises: inoculating Lactobacillus helveticus into MRS medium and culturing at 37° C. for 24 hours; Based on the water in the fermentation substrate, the number of viable lactobacilli inoculated per unit mass of the water is 10 9 ~10 13 CFU / mL, preferably 10 9 ~10 11 CFU / mL; The fermentation culture is static fermentation; The fermentation culture temperature is 30-45°C, preferably 35-40°C, for example 37°C; The fermentation culture time is 12 to 48 hours, preferably 12 to 24 hours, for example 15 hours; The sterilization method is a high-temperature sterilization method, the sterilization temperature is 110°C to 125°C, more preferably 115°C to 121°C, for example 121°C, the sterilization pressure is 0.1 to 0.14MPa, more preferably 0.1 to 0.13MPa, for example 0.13MPa, and the sterilization time is 15 to 35min, more preferably 20 to 35min, for example 30min.

5. The method for preparing the mother-of-pearl fermented product according to claim 1, characterized in that: After the sterilization operation, the method further includes at least one of cooling, centrifuging and collecting the supernatant, secondary sterilization or mixing with a preservative.

6. The method for preparing the fermented product of mother-of-pearl Bose as claimed in claim 5, characterized in that: The method for preparing the pearl mother-of-pearl fermentation product satisfies at least one of the following conditions: The cooling is cooling to room temperature; The centrifugal speed is 3000-9000 rpm, more preferably 4000-6000 rpm, for example 5000 rpm; The centrifugal radius is 8 to 15 cm; The centrifugation time is 10 to 40 minutes, preferably 20 to 40 minutes, for example 30 minutes; The secondary sterilization method is a high temperature sterilization method. Preferably, the temperature of the secondary sterilization is 110°C to 125°C, more preferably 115°C to 121°C, for example 121°C; the pressure of the secondary sterilization is 0.1 to 0.14MPa, more preferably 0.1 to 0.13MPa, for example 0.13MPa; the time of the secondary sterilization is 15 to 35min, more preferably 15 to 25min, for example 20min; During the mixing process with the preservative, the mixing temperature is 50-80°C, more preferably 70-80°C; In the process of mixing with the preservative, the preservative includes p-hydroxyacetophenone and / or 1,2-hexanediol; when the preservative includes p-hydroxyacetophenone, the mass percentage of p-hydroxyacetophenone in the supernatant of the material obtained after sterilization is 0.5% to 2%, preferably 0.1% to 0.6%, and more preferably 0.5%; when the preservative includes 1,2-hexanediol, the mass percentage of 1,2-hexanediol in the supernatant of the material obtained after sterilization is 0.5% to 2%, preferably 0.5% to 1%, and more preferably 0.5%.

7. A pearl mother-of-pearl fermentation product, characterized in that: The invention is prepared by the method for preparing the mother-of-pearl Bose fermentation product according to any one of claims 1 to 5.

8. A fermented product of mother-of-pearl, used directly as a product, as an additive or as a base in the preparation of a skin external preparation; Preferably, the pearl mother-of-pearl fermentation product is used as at least one of the antioxidant active ingredient, anti-aging active ingredient, and anti-allergic active ingredient in the skin external preparation; More preferably, the antioxidant active ingredient is an antioxidant active ingredient having a DPPH free radical scavenging effect or an antioxidant active ingredient having a hydroxyl free radical scavenging effect; More preferably, the anti-aging active ingredient is an anti-aging active ingredient having a cell proliferation effect; More preferably, the anti-allergic active ingredient is an anti-allergic active ingredient having the effect of inhibiting hyaluronidase.

9. A skin external preparation, characterized in that: The invention comprises the mother-of-pearl Bose fermentation product as claimed in claim 7.

10. The external skin preparation according to claim 9, wherein The skin external preparation satisfies at least one of the following conditions: The skin external preparation further comprises at least one of a moisturizing active ingredient, a whitening active ingredient, an anti-inflammatory active ingredient, an anti-allergic active ingredient and an antioxidant active ingredient; The skin external preparation includes a facial mask, essence or toner; The mass percentage of the pearl mother-of-pearl fermented product in the skin external preparation is 5% to 99%, preferably 60% to 99%.

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