Preparation method of sphagnum fermentation filtrate as well as product and application of sphagnum fermentation filtrate

Abstract

The invention provides a preparation method of sphagnum fermentation filtrate as well as a product and application of the sphagnum fermentation filtrate, and belongs to the technical field of plant extract preparation. The preparation method of the sphagnum fermentation filtrate provided by the invention comprises the following steps: (1) crushing and sieving sphagnum, and extracting a leaching solution to obtain sphagnum powder; (2) dissolving sphagnum moss powder, inoculating a strain, and fermenting; (3) after strain fermentation, centrifuging, wherein supernate is sphagnum palustre fermentation filtrate; wherein the strain in the step (2) is any one of lactobacillus plantarum and lactobacillus acidophilus or a mixed strain of the lactobacillus plantarum and the lactobacillus acidophilus. The fermentation technology is applied to preparation of sphagnum moss for the first time, and meanwhile, the prepared sphagnum moss fermentation filtrate can also be used for preparing cosmetics with the functions of increasing cell viability, repairing skin barrier, resisting aging and whitening.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant extract preparation, and particularly relates to a preparation method, product and application of a Sphagnum fermentation filtrate. Background Art

[0002] Sphagnum is a genus in the Bryophyta, belonging to the Sphagnaceae, and is a special type of bryophyte living in peatlands and swamps. Sphagnum contains polyphenols, terpenoids, amino acids and organic acids. These substances not only inhibit microbial activities, scavenge free radicals, have potential medicinal value, but also have ecological functions such as heavy metal adsorption, carbon storage, and hydrological regulation.

[0003] Currently in traditional Chinese medicine, Sphagnum is widely used to treat various diseases, with effects such as clearing heat and detoxifying, improving blood circulation, anti-tumor, and relieving inflammation, and can be used to treat skin diseases and itching caused by insect bites. Another study found that Sphagnum wound dressings have antibacterial and wound healing promoting functions. The patent application with the publication number CN118662401A discloses a Sphagnum extract, its preparation method and application in repairing skin barriers. This patent application provides a method for preparing a Sphagnum extract by using Sphagnum artificially cultured in the laboratory, which is dried, crushed, sieved, extracted with 95% ethanol, vacuum concentrated at 50°C, and vacuum dried to constant weight to obtain a Sphagnum extract powder, which is used to prepare cosmetics with functions of increasing cell viability, improving skin health status, repairing skin barrier function, and increasing skin elasticity and firmness. In summary, we can see that Sphagnum is widely distributed, rich in resources, and has various medicinal functions, but its research in the cosmetics field is less, and there is no report on using fermentation technology to develop new raw materials for cosmetics from Sphagnum. Summary of the Invention

[0004] Based on the problems and deficiencies in the prior art, the present invention aims to provide a preparation method, product and application of a Sphagnum fermentation filtrate. The preparation method of the Sphagnum fermentation filtrate provided by the present invention includes the following steps: (1) extracting an extract from Sphagnum after crushing and sieving to obtain Sphagnum powder; (2) dissolving the Sphagnum powder, inoculating the strain, and then fermenting; (3) centrifuging after the strain ferments, and the supernatant is the Sphagnum fermentation filtrate; wherein, the strain in step (2) is any one or a mixed strain of Lactobacillus plantarum and Lactobacillus acidophilus. The present invention not only applies fermentation technology to the preparation of Sphagnum for the first time, but also the prepared Sphagnum fermentation filtrate can be used to prepare cosmetics with functions of increasing cell viability, repairing skin barrier function, anti-aging, and whitening.

[0005] The technical solution of the present invention is as follows:

[0006] On the one hand, the present invention provides a method for preparing a Sphagnum fermentation filtrate, comprising the following steps:

[0007] (1) After Sphagnum is crushed and sieved, it is extracted with an extraction solution, and Sphagnum powder is obtained by filtration;

[0008] (2) The Sphagnum powder is dissolved, inoculated with a strain, and then fermented;

[0009] (3) After fermentation, centrifugation is carried out, and the supernatant is the Sphagnum fermentation filtrate;

[0010] Wherein, the strain in step (2) is any one or a mixed strain of Lactobacillus plantarum and Lactobacillus acidophilus.

[0011] Preferably, the mass ratio of Lactobacillus plantarum to Lactobacillus acidophilus in the mixed strain is 1:1 - 1:5.

[0012] Preferably, the total inoculation amount mass fraction of the strain in step (2) is 2% - 6%.

[0013] Preferably, the temperature of the fermentation in step (3) is 34°C - 37°C, and the time of the fermentation is 20 - 50 h.

[0014] Specifically, the extraction solution in step (1) is ethyl acetate.

[0015] Specifically, the sieving in step (1) is through a 100-mesh sieve.

[0016] On the other hand, the present invention provides an application of the Sphagnum fermentation filtrate prepared by the foregoing preparation method in cosmetic products.

[0017] Specifically, the cosmetic products include: whitening cosmetic products, antioxidant cosmetic products, and anti-aging cosmetic products.

[0018] More specifically, the cosmetic is selected from any one of essence water, essence milk, gel, liquid foundation, essence lotion, concealer, cream, and facial mask.

[0019] On yet another hand, the present invention provides a cosmetic having whitening, antioxidant, and anti-aging effects, and the cosmetic comprises the Sphagnum fermentation filtrate prepared by the preparation method described in any one of the foregoing items.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] By developing and optimizing the preparation process of Sphagnum moss, the present invention provides a Sphagnum moss fermentation filtrate. Through in vitro and cell experiments, it is verified that the water extract and fermentation filtrate of Sphagnum moss have effects such as antioxidant, skin barrier repair, anti-aging, and whitening, which can bring innovation to the application in the cosmetics field. In the preparation process of the Sphagnum moss fermentation filtrate described in the present invention, no chemicals toxic and harmful to humans or the environment are used, which is green, safe, pollution-free, and meets the requirements of sustainable development. Description of the Drawings

[0022] Figure 1 OD value results of the Sphagnum moss fermentation filtrate under the conditions of Example 1 "inoculum size 4%, fermentation temperature 37°C".

[0023] Figure 2 OD value results of the Sphagnum moss fermentation filtrate under the conditions of Example 2 "inoculum size 2%, fermentation temperature 37°C".

[0024] Figure 3 OD value results of the Sphagnum moss fermentation filtrate under the conditions of Example 3 "inoculum size 6%, fermentation temperature 34°C".

[0025] Figure 4 OD value results of the Sphagnum moss fermentation filtrate under the conditions of Example 4 "the ratio of the two mixed strains is 1:5".

[0026] Figure 5 Results of the effect of the Sphagnum moss fermentation filtrate on the viability of HaCaT cells.

[0027] Figure 6 Results of the antioxidant detection of the Sphagnum moss fermentation filtrate on HaCaT cells.

[0028] Figure 7 Results of the mitochondrial oxidative damage membrane potential test of different samples on HaCaT cells.

[0029] Figure 8 Results of the DPPH radical scavenging rate of different groups of samples.

[0030] Figure 9 Results of the ABTS radical scavenging rate of different groups of samples.

[0031] Figure 10 Results of the hydroxyl radical scavenging rate of different groups of samples.

[0032] Figure 11 Results of the superoxide anion radical scavenging rate of different groups of samples.

[0033] Figure 12 Results of the pre-protection test of the Sphagnum moss fermentation filtrate on the ultraviolet damage of HaCaT cells.

[0034] Figure 13 The results of the effect of Sphagnum fermentation filtrate on the cell scratch healing rate.

[0035] Figure 14 In it, A is the results of sampling and photographing of different groups at 0 h and 24 h, and B is the results of the healing rate of ultraviolet scratches of different groups.

[0036] Figure 15 The results of the detection of ROS levels in HaCaT cells treated with different samples.

[0037] Figure 16 The results of the effect of β-galactosidase staining on the senescence of HDF cells.

[0038] Figure 17 The results of the detection of the contents of MMP-1, MMP-3 and MMP-9 in HDF cells treated with different samples.

[0039] Figure 18 The results of the detection of tyrosinase activity in B16 cells treated with different samples.

[0040] Figure 19 The results of the detection of melanin content in B16 cells treated with different samples.

[0041] Figure 20 The results of the inhibition rate of tyrosinase activity in B16 cells treated with different samples.

[0042] Figure 21 The results of the inhibition of bacterial activity by different samples; among them, A is the results of the inhibition of the activity of Escherichia coli by different samples, and B is the results of the inhibition of the activity of Staphylococcus aureus by different samples.

[0043] Figure 22 The results of the effect of ethanol-extracted Sphagnum fermentation filtrate on the viability of HaCaT cells.

[0044] In the accompanying drawings, the significance analysis uses a t-test, "*" represents significant at the P<0.05 level; "**" represents significant at the P<0.01 level; "***" represents highly significant at the P<0.001 level; "****" represents extremely significant at the P<0.0001 level; "ns" represents no significance. Detailed implementation manners

[0045] The following combines specific embodiments to further elaborate on the present invention. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments, unless otherwise specified, and the experimental methods without specific conditions noted in the embodiments are usually in accordance with conventional conditions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0046] The sources of the materials and reagents involved in the following examples are shown in the table below:

[0047] Table 1 Sources of Materials and Reagents

[0048] Materials / Reagents Purchase Source Lactobacillus plantarum Xi'an Mixian'er Biotechnology Co., Ltd. Lactobacillus acidophilus Xi'an Mixian'er Biotechnology Co., Ltd. HaCaT cells School of Life Sciences, Guangdong Pharmaceutical University MTT Medium Shanghai Beyotime Biotechnology Co., Ltd. HDF cells New Drug R & D Center, Guangdong Pharmaceutical University DCFH-DA Shanghai Beyotime Biotechnology Co., Ltd. β-galactosidase Shanghai Beyotime Biotechnology Co., Ltd. B16 cells New Drug R & D Center, Guangdong Pharmaceutical University DMEM Medium Beijing Wokaway Biotechnology Co., Ltd. 1640 Medium Beijing Wokaway Biotechnology Co., Ltd. Arbutin Shanghai Macklin Biochemical Co., Ltd.

[0049] In the following examples, "W" represents Sphagnum extract without strain fermentation; "Z" represents the filtrate of Sphagnum fermented by Lactobacillus plantarum; "S" represents the filtrate of Sphagnum fermented by Lactobacillus acidophilus; "H" represents the filtrate of Sphagnum fermented by the mixed fermentation of Lactobacillus plantarum and Lactobacillus acidophilus.

[0050] Example 1: Preparation Method of Sphagnum Fermentation Filtrate

[0051] (1) The aseptic Sphagnum moss cultured in the laboratory was dried, crushed, sieved through a 100-mesh sieve, and extracted with ethyl acetate.

[0052] (2) The extract obtained in step (1) was filtered by suction to obtain Sphagnum moss powder.

[0053] (3) The Sphagnum moss powder was formulated into a 20 mg / mL solution and sterilized at 121 °C for 20 min.

[0054] (4) The direct inoculation of Lactobacillus plantarum powder (Z), or Lactobacillus acidophilus powder (S), or the mixed strain of the two strains (H) was added to the Sphagnum moss solution obtained in step (3). The total inoculation amount of the strain was 4% (w / v), the fermentation temperature was 37 °C, and the fermentation time was 50 h. The OD value was measured at 600 nm. Among them, the mass ratio of Lactobacillus plantarum powder (purchased from Xi'an Mixian'er Biotechnology Co., Ltd., product number YQ20250105) and Lactobacillus acidophilus powder (purchased from Xi'an Mixian'er Biotechnology Co., Ltd., product number MXE20250210) in the mixed strain of the two strains was 1:1. If step (4) was omitted and step (5) of centrifuging to obtain the supernatant was directly carried out, then the Sphagnum moss extract without strain fermentation (W) was obtained.

[0055] (5) The fermentation broth was sterilized, centrifuged at 4000 rpm for 20 min, and the supernatant was the Sphagnum moss fermentation filtrate. The OD value test results are shown in Figure 1 , and it can be seen that the growth abilities of strains Z, S, and H are similar.

[0056] Example 2: Preparation Method of Sphagnum Fermentation Filtrate

[0057] The difference from Example 1 was that: in step (4), the inoculation amount of the strain was 2%, and the fermentation temperature was 37 °C. Other steps refer to Example 1, and the OD value results are shown in Figure 2 .

[0058] Example 3: Preparation Method of Sphagnum Fermentation Filtrate

[0059] The difference from Example 1 is that: in step (4), the inoculum size of the strain is 6%, and the fermentation temperature is 34°C. Other steps refer to Example 1, and the OD value results are shown in Figure 3 .

[0060] Example 4: Preparation method of Sphagnum fermentation filtrate

[0061] The difference from Example 1 is that: in step (4), the ratio of the two mixed strains is 1:5. Other steps refer to Example 1, and the OD value results are shown in Figure 4 .

[0062] From Figures 2 - 4 the results, it can be seen that within a certain range, the inoculum size of the strain, the fermentation temperature, and the ratio of the mixed strains have basically the same effect on the growth of the Sphagnum fermentation filtrate.

[0063] In the following Examples 5-11, the samples of "W", "Z", "S", and "H" groups are all the corresponding solutions obtained by the preparation method of Example 1.

[0064] Example 5: Effect of Sphagnum fermentation filtrate on the viability of HaCaT cells

[0065] (1) Inoculate HaCaT cells in the logarithmic growth phase into a 96-well plate (100 μL per well) at a cell density of 1.0×10 5 cells / mL, and culture them adherently in an incubator at 37°C with 5% CO 2 for 24 h.

[0066] (2) Add 100 μL of medium containing samples with different concentrations (50, 100, and 150 μg / mL), and set 3 parallel experiments for each concentration. Incubate for 24 h, discard the old medium, add 100 μL of 0.5 mg / mL MTT new medium to each well, and continue to incubate for 4 h.

[0067] (3) Subsequently, add 150 μL of DMSO solution to each well, and finally measure the absorbance value at 490 nm with an enzyme-linked immunosorbent assay instrument.

[0068] This experiment is divided into 5 groups: "Control" represents the blank control; "W" represents the Sphagnum extract without strain fermentation; "Z" represents the Sphagnum filtrate fermented by Lactobacillus plantarum; "S" represents the Sphagnum filtrate fermented by Lactobacillus acidophilus; "H" represents the Sphagnum filtrate fermented by the mixed fermentation of Lactobacillus plantarum and Lactobacillus acidophilus; the samples of each group are prepared according to the method of Example 1.

[0069] Among them, the preparation method of the Sphagnum extract without strain fermentation ("W") is:

[0070] It was carried out according to the method in Example 1. The aseptic Sphagnum moss for hydroponics in the laboratory was dried, crushed, sieved through a 100-mesh sieve, and extracted with ethyl acetate; the extract obtained in step (1) was filtered by suction to obtain Sphagnum moss powder; the Sphagnum moss powder was formulated into a 20 mg / mL solution and sterilized at 121 °C for 20 min, and the sterilized solution was the Sphagnum moss extract, and the subsequent steps of inoculating bacteria were omitted.

[0071] The survival rate of HaCaT cells: cell survival rate = A1 / A2 × 100%; A1 represents the absorbance value of the sample group; A2 represents the absorbance value of the Control group;

[0072] The results measured by the microplate reader are as Figure 5 (where the left, middle, and right figures are the test results corresponding to the samples of 50, 100, and 150 μg / mL respectively). The W group, H group, Z group, and S group had basically no effect on the proliferation rate of HaCaT cells, the cell survival rate was greater than 90%, there was no toxicity, and the safety was high.

[0073] Example 6: Detection of the antioxidant effect of the Sphagnum moss fermentation filtrate

[0074] The groups in this experiment: "dz" represents the blank control group; "mx" represents the H 2 O 2 oxidative damage model group; "UVB" represents the ultraviolet irradiation group; "W" represents the Sphagnum moss extract group without bacterial fermentation; "H" represents the Sphagnum moss filtrate group fermented by the mixed fermentation of Lactobacillus plantarum and Lactobacillus acidophilus; "Z" represents the Sphagnum moss filtrate group fermented by Lactobacillus plantarum; "S" represents the Sphagnum moss filtrate group fermented by Lactobacillus acidophilus.

[0075] 6.1. H 2 O 2 oxidative damage model

[0076] Establish an H 2 O 2 oxidative damage model for HaCaT cells: The optimal H 2 O 2 concentration is 900 μM.

[0077] (1) Inoculate HaCaT cells in the logarithmic growth phase into a 96-well plate (100 μL per well) at a cell density of 1.0×10 5 cells / mL, and incubate them in a 37 °C, 5% CO 2 incubator for 24 h for adherent culture.

[0078] (2) Add 100 μL of a solution containing 900 μM H 2 O 2The culture medium was incubated for 2 h; 100 μL of the culture medium containing samples at different concentrations (50, 100, and 150 μg / mL) was replaced and incubated for 2 h; the old culture medium was discarded, and 100 μL of the new culture medium containing 0.5 mg / mL MTT was added to each well and incubated for another 4 h.

[0079] (3) Subsequently, 150 μL of DMSO solution was added to each well, and finally the absorbance value at 490 nm was measured using a microplate reader.

[0080] Survival rate of HaCaT cells: Cell survival rate = A1 / A2 × 100%; A1 represents the absorbance value of the group with samples and H 2 O 2 ; A2 represents the absorbance value of the Control group;

[0081] The results measured by the microplate reader are as Figure 6 shown. It can be seen that the Sphagnum extract without strain fermentation basically has no antioxidant activity, while the filtrate of Sphagnum fermented by strains has significant antioxidant activity, and the higher the concentration of the Sphagnum filtrate, the better the antioxidant activity.

[0082] 6.2. Measurement of mitochondrial membrane potential in the ultraviolet oxidation damage model

[0083] (1) HaCaT cells in the logarithmic growth phase were seeded into 96-well plates (100 μL per well) at a cell density of 1.0×10 4 cells / well and cultured adherently in an incubator at 37 °C with 5% CO 2 for 24 h, and then the culture medium was discarded;

[0084] (2) 100 μL of the culture medium containing samples at different concentrations (100 μg / mL and 150 μg / mL) was added, and 3 parallel wells were set for each concentration. After incubation for 24 h, the old culture medium was discarded, and each well was washed three times with 100 μL of PBS;

[0085] (3) Then 5 μL of PBS was added, and damage was induced by irradiation with a UV crosslinker in the UVB mode. Subsequently, the cells were incubated in an incubator at 37 °C with 5% CO 2 for 4 h, and then the culture medium was discarded;

[0086] (4) After washing three times with PBS, JC-1 staining solution was added to each well and incubated in an incubator at 37 °C with 5% CO 2 for 20 min, and then the staining solution was discarded. Each well was washed three times with 100 μL of pre-cooled staining buffer, and then 50 μL of culture medium was added for quantitative detection using a multifunctional microplate reader.

[0087] The test results of mitochondrial membrane potential in the cell ultraviolet oxidation damage model are as Figure 7As shown, W, H, Z, and S have a certain effect on improving the mitochondrial membrane potential of HaCaT cells after ultraviolet damage, indicating that Sphagnum extract and fermentation filtrate can effectively enhance the antioxidant capacity of cells. Moreover, the three sample groups of H, Z, and S have a significant effect on improving the mitochondrial membrane potential of HaCaT cells after ultraviolet damage compared with the W group.

[0088] 6.3, DPPH Free Radical Scavenging Experiment

[0089] Weigh 7.88 mg of DPPH and dissolve it in 100 mL of absolute ethanol to obtain a 0.2 mmol / L DPPH ethanol solution, which is stored in the dark at 4°C for later use. The sample groups are W, H, S, and Z with mass concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 mg / mL. The positive control is ascorbic acid with the same concentration gradient as the sample groups. Take several test tubes and add samples as shown in Table 2.

[0090] Table 2 Composition of the Reaction System for the DPPH Method

[0091]

[0092]

[0093] At room temperature, mix each component evenly and place it in a dark room to react for 30 min. Measure the absorbance of the sample at a wavelength of 517 nm, and calculate the DPPH free radical scavenging rate according to the formula.

[0094] DPPH Scavenging Rate (%) = {1 - (A 1 - A 2 ) / A 0} × 100%;

[0095] Among them, A 1 represents the absorbance measured by adding an antioxidant and DPPH solution; A 2 represents the absorbance measured by adding an antioxidant and water; A 0 represents the absorbance measured by adding DPPH solution and water.

[0096] The experimental results are as Figure 8 shown. The scavenging rate of ascorbic acid for free radicals tends to be stable; as the concentration increases, the ability of the sample group to scavenge DPPH free radicals becomes stronger. The average free radical scavenging rate of the H group is the highest, up to 98.84%, followed by the S group with an average free radical scavenging rate of 94.77%. The DPPH free radical scavenging rate of the W group is the lowest, only reaching 73.57%. Considering the overall situation, the H group (Sphagnum filtrate fermented by a mixture of Lactobacillus plantarum and Lactobacillus acidophilus) has the best free radical scavenging effect at each concentration.

[0097] 6.4, ABST Free Radical Scavenging Experiment

[0098] Prepare 7 mmol / L ABTS and 2.6 mmol / L potassium persulfate, mix them in a volume ratio of 1:1, and react in the dark at room temperature for 12 - 16 h to obtain the ABTS stock solution. Take 2 mL of the stock solution, dilute it 50 times with absolute ethanol, measure the absorbance at a wavelength of 734 nm to be 0.7 ± 0.02 to obtain the ABTS working solution for standby. The sample groups are W, H, S, and Z with mass concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 mg / mL. The positive control is ascorbic acid with the same concentration gradient as the sample groups. Take several test tubes and add samples according to Table 3.

[0099] Table 3 Composition of the reaction system for the ABTS method

[0100]

[0101] After mixing each component evenly, react in the dark for 10 min, measure the absorbance of the sample at a wavelength of 734 nm, and calculate the ABTS radical scavenging rate according to the formula.

[0102] ABTS scavenging rate (%) = {1 - (A 1 - A 2 ) / A 0} × 100%;

[0103] Among them, A 1 represents the absorbance measured by adding the antioxidant and the ABTS working solution; A 2 represents the absorbance measured by adding the antioxidant and absolute ethanol; A 0 represents the absorbance measured by adding the ABTS working solution and water.

[0104] The experimental results are as Figure 9 , the scavenging rate of ascorbic acid on free radicals tends to be stable; as the concentration increases, the ability of different groups to scavenge ABTS free radicals becomes stronger; at the maximum concentration, the scavenging effects of groups H, S, and Z on ABTS free radicals are similar, being 88.48%, 90.63%, and 85.88% respectively, and the scavenging rate of group W on ABTS free radicals is only 58.24%; considering the overall situation, the scavenging effect of the Sphagnum filtrate after strain fermentation on ABTS free radicals is significantly higher than that of the Sphagnum filtrate without strain fermentation at each concentration (P < 0.05).

[0105] 6.5. Hydroxyl radical scavenging experiment

[0106] Prepare a 9 mmol / L ferrous sulfate solution, a 9 mmol / L ethanol salicylic acid solution, and an 8.8 mmol / L hydrogen peroxide solution. The sample groups are W, H, S, and Z with mass concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 mg / mL. The positive control is ascorbic acid with the same concentration gradient as the sample groups. Take several test tubes and add samples according to Table 4.

[0107] Table 4 Composition of the reaction system for the hydroxyl radical method

[0108]

[0109] After mixing each component, heat in a water bath at 37 °C for 15 min, measure the absorbance of the sample at a wavelength of 510 nm, and calculate the hydroxyl radical scavenging rate according to the formula.

[0110] Hydroxyl radical scavenging rate (%) = {1 - (A 1 - A 2 ) / A 0} × 100%;

[0111] Among them, A 1 represents the absorbance measured for the sample group at a wavelength of 510 nm; A 2 represents the absorbance measured for the blank group at a wavelength of 510 nm; A 0 represents the absorbance measured for the control group at a wavelength of 510 nm.

[0112] The experimental results are as Figure 10 , the scavenging rate of ascorbic acid on free radicals tends to be stable; at a concentration of 4 mg / mL, the effects of groups Z, H, and S on scavenging hydroxyl radicals are similar to those of group W; at the maximum concentration, the average scavenging rates of groups H, S, and Z on hydroxyl radicals are 22.02%, 26.37%, and 22.87% respectively, and the average scavenging rate of group W is 16.80%. Considering the overall situation, the peat moss filtrate after strain fermentation has a significantly higher hydroxyl radical scavenging effect than the peat moss filtrate without strain fermentation (P < 0.05).

[0113] 6.6. Superoxide anion radical scavenging experiment

[0114] Prepare a 0.1 mol / L Tris solution, a 0.1 mol / L HCl solution, and a 0.05 mol / L Tris-HCl buffer solution with pH 7.4. Weigh 0.0189 g of pyrogallol powder, dilute it to 50 mL with 10 mmol / L HCl to prepare a pyrogallol working solution, and use it immediately after preparation. The sample groups are W, H, S, and Z with mass concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 mg / mL. The positive control is ascorbic acid with the same concentration gradient as the sample groups. Take several test tubes and add samples according to Table 5.

[0115] Table 5 Composition of the reaction system for the superoxide anion radical method

[0116]

[0117] First add Tris-HCl buffer solution to the cuvette, then add the sample, and finally quickly add pyrogallol. Immediately pipette and mix well. Select the dynamic measurement mode. Measure the absorbance value (A) every 30 seconds at a wavelength of 325 nm until 300 seconds.

[0118] ΔA 0 = A 0 (325 nm) 300s - A 0 (325 nm) 30s ;

[0119] ΔA 1 = A 1 (325 nm) 300s - A 1 (325 nm) 30s ;

[0120] Superoxide anion radical scavenging rate (%) = (A 1 - A 2 ) / A 0 × 100%.

[0121] The experimental results are as Figure 11 , when the mass concentration is 2 - 5 mg / mL, the scavenging rate of ascorbic acid on free radicals tends to be stable; at the maximum concentration, the average scavenging rates of the superoxide anion radicals in groups H, S, and Z are 86.38%, 88.21%, and 89.27% respectively, and the average scavenging rate of group W is 55.74%; at the concentrations of 1 mg / mL and 3 mg / mL, the scavenging effect of the Sphagnum filtrate without strain fermentation is slightly higher than that of the Sphagnum filtrate after strain fermentation, probably because the active ingredients in the fermented sample are inactivated due to long-term storage; considering the overall situation, the Sphagnum filtrate after strain fermentation has a significantly higher scavenging effect on the superoxide anion radical than the Sphagnum filtrate without strain fermentation.

[0122] Example 7: Detection of the repair efficacy of the Sphagnum fermented filtrate

[0123] In this experiment, "dz" represents the blank control group; "UVB" represents the ultraviolet irradiation group; "W" represents the group of Sphagnum extract without strain fermentation; "H" represents the group of Sphagnum filtrate fermented by the mixed fermentation of Lactobacillus plantarum and Lactobacillus acidophilus; "Z" represents the group of Sphagnum filtrate fermented by Lactobacillus plantarum; "S" represents the group of Sphagnum filtrate fermented by Lactobacillus acidophilus.

[0124] 7.1. Pre-protection test for ultraviolet damage

[0125] (1) Seed HaCaT cells in the logarithmic growth phase into a 96-well plate (100 μL per well) at a cell density of 1.0×10 4 cells / well, and incubate them adherently in an incubator at 37°C with 5% CO 2 for 24 h;

[0126] (2) Add 100 μL of medium containing samples at different concentrations (50, 100, and 150 μg / mL), and set 3 parallel wells for each concentration. Incubate for 24 h, discard the old medium, and add 100 μL of PBS to each well and wash three times;

[0127] (3) Then add 50 μL of PBS, irradiate with a UV crosslinker in the UVB mode for damage, and then incubate in a cell incubator at 37°C with 5% CO 2 for 24 h, then discard the medium, and measure the cell viability by the MTT method.

[0128] The results are as Figure 12 shown. It can be seen that the Sphagnum extract without strain fermentation has no pre-protective effect on UV damage, while the filtrate of Sphagnum fermented by strains has a significant pre-protective effect on UV damage.

[0129] 7.2. Cell scratch repair test

[0130] (1) Digest HaCaT cells in the logarithmic growth phase with trypsin into a single-cell suspension, inoculate it into a 6-well culture plate (30w / well), with a final total volume of 2 mL of medium in each well, and incubate in an incubator at 37°C with 5% CO 2 for 24 h;

[0131] (2) Scratch parallel or perpendicular to the horizontal line behind with a 200 μL pipette tip, and then wash the cells 3 times with PBS to remove the scratched cells;

[0132] (3) Add serum-free medium to the blank control group, and add serum-free medium containing W, H, Z, and S to the sample group. Sampling and photographing are performed at the time points of 0 h and 24 h of culture.

[0133] Use Image J software to analyze the scratch area. The cell scratch healing rate = (initial scratch area at 0 h - scratch area at 24 h) / initial scratch area at 0 h.

[0134] The results are as Figure 13 shown. The cell scratch healing rate of the blank control group is only about 25%, while the healing rate of the fermented filtrates prepared from different strains for cell scratches reaches about 44% - 50%.

[0135] 7.3. UV-induced scratch repair test

[0136] (1) Trypsinize HaCaT cells in the logarithmic growth phase into single-cell suspensions, and inoculate them into 6-well culture plates (30w / well). The total volume of the medium in each well is 2 mL, and culture in an incubator at 37 °C and 5% CO 2 for 24 h;

[0137] (2) Perform ultraviolet irradiation with an ultraviolet dose of 15 mj / cm 2 . Then, use a 200 μL pipette tip to scratch parallel or perpendicular to the horizontal line on the back, and rinse the cells 3 times with PBS to remove the scratched cells;

[0138] (3) Add serum-free medium to the blank control group, and add serum-free medium containing W, H, Z, and S to the sample group. Sampling and photographing are performed at the time points of 0 h and 24 h of culture.

[0139] Use Image J software to analyze the scratch area. The cell scratch healing rate = (initial scratch area at 0 h - scratch area at 24 h) / initial scratch area at 0 h.

[0140] The results are as Figure 14 shown. The H, Z, and S groups have significant scratch repair effects.

[0141] Example 8: Detection of the anti-aging effect of Sphagnum fermentation filtrate

[0142] In this experiment, "dz" represents the blank control group; "mx" represents the H 2 O 2 oxidative damage model group; "UVB" represents the ultraviolet irradiation group; "W" represents the Sphagnum extract group without strain fermentation; "H" represents the Sphagnum filtrate group fermented by the mixed fermentation of Lactobacillus plantarum and Lactobacillus acidophilus; "Z" represents the Sphagnum filtrate group fermented by Lactobacillus plantarum; "S" represents the Sphagnum filtrate group fermented by Lactobacillus acidophilus.

[0143] 8.1. Fluorescence detection of ROS level

[0144] (1) Inoculate HaCaT cells in the logarithmic growth phase into 6-well culture plates (2 mL per well) at a cell density of 1.0×10 6 cells / well, and culture them adherently in an incubator at 37 °C and 5% CO 2 for 24 h, then discard the medium;

[0145] (2) Add 2 mL of medium containing a certain concentration of the sample, and set 3 parallel wells for each concentration. Incubate for 24 h and perform ultraviolet irradiation under a UV crosslinker.

[0146] (3) Discard the old culture medium, add PBS to each well and wash three times, then add 1 mL of DCFH-DA working solution, and continue to incubate in the incubator for 30 min. Shake once every 5 min during this period to ensure full contact between the probe and the cells. The operation should be carried out in the dark throughout the process. Place it under a fluorescence microscope for photography.

[0147] The results of the fluorescence microscope are as Figure 15 shown. It can be seen that the ROS level in the UVB ultraviolet irradiation group is high, while the ROS levels in the W, H, Z, and S groups are relatively low. Cell senescence is accompanied by an increase in the ROS level, revealing that the Sphagnum filtrate has anti-aging effects.

[0148] 8.2. Effects of β-galactosidase staining on HDF cell senescence

[0149] (1) Inoculate HDF cells in the logarithmic growth phase into a 6-well culture plate (2 mL per well) at a cell density of 1.0×10 6 cells / well, and culture them adherently in an incubator at 37°C with 5% CO 2 for 24 h. Discard the culture medium;

[0150] (2) Add 2 mL of culture medium containing a certain concentration of the sample. Set 3 parallel wells for each concentration and place them at room temperature for 2 h;

[0151] (3) Discard the old culture medium, add PBS to each well and wash three times, then add 1 mL of staining fixative, incubate in the incubator for 48 h, and place it under an inverted microscope for photography.

[0152] The photography results are as Figure 16 shown. It can be seen that the W, H, Z, and S groups can alleviate the senescence of HDF cells, especially the Z group has a stronger anti-aging effect.

[0153] Seed HDF cells into a 96-well plate. Aspirate the old culture medium, add 100 μL of culture medium containing different concentrations of the sample. Set 3 replicate wells for each concentration. After incubating for 24 h, collect the cell supernatant, and detect the contents of MMP-1, MMP-3, and MMP-9 in the cell supernatant by ELISA method (the contents of MMP-1, MMP-3, and MMP-9 increase during cell senescence). The results are as Figure 17 shown, which also indicates that the Sphagnum fermentation filtrate has anti-aging effects.

[0154] Example 9: Detection of the whitening effect of the Sphagnum fermentation filtrate

[0155] In this experiment, "dz" represents the blank control group; "xiong" represents the arbutin positive control group; "W" represents the Sphagnum extract group without strain fermentation; "H" represents the Sphagnum filtrate group fermented by a mixture of Lactobacillus plantarum and Lactobacillus acidophilus; "Z" represents the Sphagnum filtrate group fermented by Lactobacillus plantarum; "S" represents the Sphagnum filtrate group fermented by Lactobacillus acidophilus.

[0156] 9.1 Cell Tyrosinase Activity Experiment

[0157] (1) Culture B16 cells in DMEM medium:1640 medium at a ratio of 1:1 for 24 h until the B16 cells turn gray;

[0158] (2) Inoculate B16 cells at a cell density of 1.0×10 4 cells / well in a 96-well plate (100 μL per well) and incubate in an incubator for 24 h, then discard the medium;

[0159] (3) Add 100 μL of DMEM medium containing different samples, set three replicates for each concentration, and incubate adherently in an incubator at 37 °C and 5% CO 2 for 24 h, then discard the medium.

[0160] (4) Wash twice with PBS, add 50 μL of 1% Triton X-100 solution, place in a -80 °C refrigerator for 1 h, melt at room temperature, then add 100 μL of 0.1% L-DOPA solution to each well and react with shaking at room temperature for 2 h. Measure the absorbance at a wavelength of 475 nm. Tyrosinase activity = absorbance of sample well / absorbance of blank well × 100%.

[0161] The experimental results are as Figure 18 shown, and the tyrosinase activities of cells in different sample groups are higher.

[0162] 9.2 Determination of Cellular Melanin Content

[0163] The difference in the experimental procedure from 8.1 is as follows:

[0164] Step (4) is different. Wash twice with PBS, add 50 μL of 1 mol / L NaOH solution containing 10% DMSO, place in a 90 °C water bath for 1 h, and measure the absorbance value at a wavelength of 405 nm. The results are as Figure 19 shown, and the melanin contents of cells in different sample groups are higher.

[0165] 9.3 Determination of Tyrosinase Activity Inhibition

[0166] Pipette 1 mL of Sphagnum fermentation filtrate sample and 1 mL of L-tyrosine solution into a 5 mL centrifuge tube, mix well and incubate at 37 °C for 10 min. Add 0.5 mL of tyrosinase solution and incubate for 20 min. Then pipette 200 μL into a 96-well plate and detect the absorbance values of each group with an enzyme-labeling instrument at a wavelength of 475 nm. Use 0.1% vitamin C (VC) as the positive control.

[0167] The calculation formula for the tyrosinase activity inhibition rate is:

[0168] Tyrosinase activity inhibition rate = [1-(TT 0 ) / (CC 0 )]×100%;

[0169] T is the absorbance value of peat moss fermentation filtrate sample + tyrosinase + L-tyrosine; T 0 is the absorbance value of peat moss fermentation filtrate sample + L-tyrosine without tyrosinase; C is the absorbance value of tyrosinase + L-tyrosine without sample; C 0 It is the absorbance value when only L-tyrosine is added without adding sample and tyrosinase.

[0170] The results are as follows Figure 20 As shown, the sphagnum fermentation filtrate sample group has a lower tyrosinase activity inhibition rate. In summary, the sphagnum fermentation filtrate sample can inhibit tyrosinase activity and has a whitening effect.

[0171] Example 10: Antibacterial activity detection of peat moss fermentation filtrate

[0172] (1) Prepare LB liquid medium and solid medium, and sterilize by autoclave at 121°C for 20 min;

[0173] (2) Staphylococcus aureus and Escherichia coli were taken out of the refrigerator and thawed, and activated continuously to the third generation with an inoculum ratio of bacterial liquid:LB liquid medium = 1:6. The activation conditions for each generation were 37°C for 24 hours with shaking culture;

[0174] (3) Take the third generation activated bacteria and dilute the concentration of each bacteria to 0.5 MCF (McFarland turbidity unit) measured by the bacterial turbidity meter. Take 1 mL of the diluted bacterial solution and slowly inject it into a test tube containing 9 mL of sterile saline along the tube wall. Mix well and take 10 6 , 10 7 , 10 8 The dilution multiples of bacterial solution were used for dilution and coating pre-experiment, and 10 7 As the dilution factor, plate count method was performed;

[0175] (4) Take the filtrate of the mixed fermentation of sphagnum moss strains with a concentration of 20 mg / mL and the filtrate of unfermented sphagnum moss, filter through a 0.22 μm microporous filter membrane and set aside. Mix the diluted bacterial solution with the sphagnum moss sample in a ratio of 1:1, and apply 100 μL of the mixed solution. Control group: replace the bacterial solution with physiological saline. Incubate the culture medium in a 37°C incubator for 16 hours, observe the distribution and morphology of the colonies in each group, and count the total number of colonies. Repeat the experiment 3 times for each group, take the average value, and calculate the antibacterial rate.

[0176] Inhibition rate = [1-(number of colonies on the sample plate / number of colonies on the blank control plate)] × 100%.

[0177] The experimental results are as follows Figure 21 , Figure 21 in Figure 21 , it can be seen that the average number of colonies in the normal saline control group is 10 6 , the average number of colonies in the Sphagnum moss strain mixed fermentation sample group is 43, the average number of colonies in the unfermented Sphagnum moss sample group is 60, the antibacterial rate of the Sphagnum moss strain mixed fermentation sample group against Escherichia coli is 59.43%, and the antibacterial rate of the unfermented sample group is 43.40%. Thus, it can be seen that the Sphagnum moss extract has a certain antibacterial effect on Escherichia coli, and the antibacterial effect of the sample after strain mixed fermentation is better than that of the unfermented sample. Figure 21 in Figure 21 , it can be seen that the average number of colonies in the normal saline control group is 10 7 , the average number of colonies in the Sphagnum moss strain mixed fermentation sample group is 35, the average number of colonies in the unfermented Sphagnum moss sample group is 39, the antibacterial rate of the Sphagnum moss strain mixed fermentation sample group against Staphylococcus aureus is 67.29%, and the antibacterial rate of the unfermented sample group is 63.55%. Thus, it can be seen that the Sphagnum moss extract has a certain antibacterial effect on Staphylococcus aureus, and the antibacterial effect before and after Sphagnum moss fermentation is similar.

[0178] Example 11: Irritation detection of Sphagnum moss fermentation filtrate

[0179] By directly contacting the test sample with the chorioallantoic membrane (CAM) of chicken embryos, observing the reaction of CAM through indicators such as bleeding, blood coagulation, and vascular lysis, and giving a score to objectively evaluate the eye irritation of the test sample.

[0180] (1) Purchase 0-day-old chicken embryos. The temperature of the intelligent egg incubator is 37 °C and the humidity is 70%. After 9 days of incubation, check and discard the defective chicken embryos. Mark the position of the air chamber on the normal chicken embryos. Use forceps to knock open the eggshell of the air chamber part to expose the white membrane. Moisten it with 0.9% normal saline. Pour out the excess liquid and then remove the inner membrane to ensure that the blood vessels are not damaged;

[0181] (2) Divide into 6 groups: positive control group (0.1 mol / L NaOH solution), negative control (0.9% NaCl solution), sample group (Sphagnum moss fermentation filtrate with crude drug concentration of 20 mg / mL), with 6 chicken embryos in each group and 3 parallel groups;

[0182] (3) Take 0.3 mL of the solution of each group and directly drop it on the surface of CAM. Record the time when the three reactions of bleeding, blood coagulation, and vascular lysis occur within 300 s after dropping, accurate to seconds, and record the reaction degree. Use the following formula to calculate the irritation score (IS), and evaluate the irritation of the sample before and after Sphagnum moss fermentation according to Table 6.

[0183]

[0184] Where: secH represents the average time in seconds from the start of sample addition to bleeding; secL represents the average time in seconds from the start of sample addition to coagulation; secC represents the average time in seconds from the start of sample addition to lysis;

[0185] Table 6 Result Evaluation of Stimulation Scoring Method

[0186] Stimulation Score / IS Predicted Classification IS<1 Non-irritant 1 < IS < 5 Mildly irritant 5 < IS < 9 Moderately irritant IS > 10 Severely irritant / Corrosive

[0187] Table 7 Evaluation of CAM Stimulation Results for Each Group

[0188]

[0189]

[0190] The stimulation results are shown in Table 7. No bleeding, coagulation, or vascular lysis occurred in the 3 parallel chicken embryos corresponding to the negative control group and the sample group. The average IS value was 0, indicating that the peat moss fermentation filtrate with a crude drug concentration of 20 mg / mL had no irritation to the CAM. Strong irritation reactions occurred in the 3 parallel groups of chicken embryos in the positive control group.

[0191] Comparative Example 1: A Preparation Method of Peat Moss Fermentation Filtrate

[0192] The difference from Example 1 is that the extraction solution is 95% ethanol and a 40-mesh sieve. Other experimental steps are the same as those in Example 1 for the preparation of peat moss fermentation filtrate.

[0193] The prepared peat moss fermentation filtrate (“W'” represents the peat moss extract extracted with ethanol without strain fermentation; “Z'” represents the peat moss filtrate fermented by Lactobacillus plantarum extracted with ethanol; “S'” represents the peat moss filtrate fermented by Lactobacillus acidophilus extracted with ethanol; “H'” represents the peat moss filtrate fermented by the mixed fermentation of Lactobacillus plantarum and Lactobacillus acidophilus extracted with ethanol). Refer to the experimental steps in Example 5 to verify the effect on HaCaT cell viability.

[0194] The experimental results show that Figure 22 , the cell survival rate of the peat moss filtrate prepared by the preparation method of Comparative Example 1 is less than 80%, indicating that the preparation method of the present invention can achieve better technical effects.

Claims

1. A method for preparing peat moss fermentation filtrate, characterized in that: The following steps are involved: (1) crushing and sieving the peat moss, extracting it with an extract, and filtering it to obtain peat moss powder; (2) dissolving peat moss powder, inoculating bacteria and fermenting; (3) After fermentation, centrifuge and the supernatant is the peat moss fermentation filtrate; Wherein, the bacterial strain in step (2) is any one of Lactobacillus plantarum and Lactobacillus acidophilus or a mixture of the two.

2. The preparation method according to claim 1, characterized in that: The mass ratio of Lactobacillus plantarum to Lactobacillus acidophilus in the mixed strains is 1:1-1:

5.

3. The preparation method according to claim 1, characterized in that: The total inoculation amount of the bacterial strain in step (2) is 2%-6% by mass.

4. The preparation method according to claim 1, characterized in that: The fermentation temperature of step (3) is 34°C-37°C, and the fermentation time of step (3) is 20-50h.

5. The preparation method according to claim 1, characterized in that: The extract in step (1) is ethyl acetate.

6. The preparation method according to claim 1, characterized in that: The sieving in step (1) is a 100-mesh sieve.

7. Use of the peat moss fermentation filtrate prepared by the preparation method according to any one of claims 1 to 6 in cosmetic products.

8. The use according to claim 7, characterized in that: The cosmetic products include whitening cosmetic products, antioxidant cosmetic products or anti-aging cosmetic products.

9. The use according to claim 8, characterized in that: The cosmetic is selected from any one of essence water, essence milk, gel, liquid foundation, essence lotion, concealer, face cream and facial mask.

10. A cosmetic with whitening, anti-oxidation and anti-aging effects, characterized in that: The cosmetic comprises peat moss fermentation filtrate prepared by the preparation method according to any one of claims 1 to 6.

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