A method for preparing peat moss fermentation filtrate, its products and applications

Abstract

This invention provides a method for preparing peat moss fermentation filtrate, its products, and applications, belonging to the field of plant extract preparation technology. The method for preparing peat moss fermentation filtrate provided by this invention includes the following steps: (1) peat moss is crushed, sieved, and then extracted with an extract to obtain peat moss powder; (2) the peat moss powder is dissolved and inoculated with a bacterial strain, followed by fermentation; (3) after fermentation, the bacterial strain is centrifuged, and the supernatant is the peat moss fermentation filtrate; wherein, the bacterial strain mentioned in step (2) is any one of *Lactobacillus plantarum* and *Lactobacillus acidophilus*, or a mixture of both. This invention not only applies fermentation technology to the preparation of peat moss for the first time, but also allows the prepared peat moss fermentation filtrate to be used in the preparation of cosmetics that increase cell vitality, repair skin barrier function, have anti-aging properties, and whiten the skin.

Description

Technical Field

[0001] This invention belongs to the field of plant extract preparation technology, specifically relating to a method for preparing peat moss fermentation filtrate, its products, and applications. Background Technology

[0002] Sphagnum is a genus of bryophytes belonging to the family Sphagnaceae. It is a unique type of bryophyte that lives in peatlands and swamps. Sphagnum contains polyphenols, terpenoids, amino acids, and organic acids. These substances not only inhibit microbial activity and scavenge free radicals, possessing potential medicinal value, but also exhibit ecological functions such as heavy metal adsorption, carbon storage, and hydrological regulation.

[0003] Currently, in traditional Chinese medicine, peat moss is widely used to treat various diseases, possessing effects such as clearing heat and detoxifying, improving circulation, anti-tumor activity, and relieving inflammation. It can be used to treat skin diseases and itching caused by insect bites. Other studies have found that peat moss wound dressings have antibacterial and wound-healing properties. A patent application with publication number CN118662401A discloses a peat moss extract, its preparation method, and its application in repairing the skin barrier. This patent application describes a method for preparing and extracting peat moss extract using artificially cultivated peat moss in a laboratory. The extract involves drying, crushing, sieving, extraction with 95% ethanol, vacuum concentration at 50°C, and vacuum drying to constant weight to obtain peat moss extract powder, which is used to prepare cosmetics that increase cell vitality, improve skin health, repair skin barrier function, and increase skin elasticity and firmness. In summary, we can see that peat moss is widely distributed, abundant, and possesses various medicinal functions; however, research on its application in the cosmetic field is limited, and there are no reports of fermentation technology being used to develop new cosmetic raw materials from peat moss. Summary of the Invention

[0004] Based on the problems and deficiencies in the existing technology, this invention aims to provide a method for preparing peat moss fermentation filtrate, its products, and applications. The method for preparing peat moss fermentation filtrate provided by this invention includes the following steps: (1) peat moss is crushed, sieved, and then extracted with an extract to obtain peat moss powder; (2) the peat moss powder is dissolved and inoculated with a bacterial strain, followed by fermentation; (3) after fermentation, the bacterial strain is centrifuged, and the supernatant is the peat moss fermentation filtrate; wherein, the bacterial strain mentioned in step (2) is any one of *Lactobacillus plantarum* and *Lactobacillus acidophilus*, or a mixture of both. This invention not only applies fermentation technology to the preparation of peat moss for the first time, but also allows the prepared peat moss fermentation filtrate to be used in the preparation of cosmetics that increase cell vitality, repair skin barrier function, have anti-aging properties, and whiten the skin.

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

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

[0007] (1) After the peat moss is crushed and sieved, it is extracted with an extract and filtered to obtain peat moss powder.

[0008] (2) Dissolve the peat moss powder, inoculate with fungi, and then ferment.

[0009] (3) After fermentation, centrifuge the supernatant, which is the peat moss fermentation filtrate.

[0010] The bacterial strain mentioned in step (2) is any one of Lactobacillus plantarum and Lactobacillus acidophilus or a mixture of both.

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

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

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

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

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

[0016] On the other hand, the present invention provides the application of the peat moss fermentation filtrate prepared by the aforementioned 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 product is selected from any one of the following: essence water, essence lotion, gel, foundation, serum, concealer, face cream, and face mask.

[0019] In another aspect, the present invention provides a cosmetic with whitening, antioxidant and anti-aging properties, wherein the cosmetic comprises peat moss fermentation filtrate prepared by any of the aforementioned preparation methods.

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

[0021] This invention provides a peat moss fermentation filtrate by developing and optimizing the preparation process of peat moss. In vitro and cell experiments have verified that the peat moss water extract and fermentation filtrate possess antioxidant, skin barrier repair, anti-aging, and whitening effects, bringing innovation to the cosmetics field. The peat moss fermentation filtrate of this invention is prepared without using any chemicals toxic or harmful to humans or the environment, making it green, safe, and pollution-free, meeting the requirements of sustainable development. Attached Figure Description

[0022] Figure 1 The results show the OD values ​​of the peat moss fermentation filtrate under the conditions of "4% inoculum and 37°C" in Example 1.

[0023] Figure 2 The results show the OD values ​​of the peat moss fermentation filtrate under the conditions of "2% inoculum and 37°C fermentation temperature" in Example 2.

[0024] Figure 3 The results show the OD values ​​of the peat moss fermentation filtrate under the conditions of "6% inoculum and 34°C fermentation temperature" in Example 3.

[0025] Figure 4 The results show the OD values ​​of the peat moss fermentation filtrate under the condition of "the ratio of the two mixed bacterial strains is 1:5" in Example 4.

[0026] Figure 5 The results show the effect of peat moss fermentation filtrate on HaCaT cell viability.

[0027] Figure 6 The results show the antioxidant effects of peat moss fermentation filtrate on HaCaT cells.

[0028] Figure 7 The results of mitochondrial oxidative damage membrane potential assays on HaCaT cells from different samples.

[0029] Figure 8 The results show the DPPH free radical scavenging rates of samples from different groups.

[0030] Figure 9 The results show the ABTS radical scavenging rates of different sample groups.

[0031] Figure 10 The results show the hydroxyl radical scavenging rates of different sample groups.

[0032] Figure 11 The results show the superoxide anion radical scavenging rates of different sample groups.

[0033] Figure 12 The results of the pre-protection test of HaCaT cells against ultraviolet damage by peat moss fermentation filtrate.

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

[0035] Figure 14 Figure A shows the sampling and photography results of different groups at 0h and 24h, and Figure B shows the healing rate of UV scratches in different groups.

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

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

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

[0039] Figure 18 Results of tyrosinase activity assays in B16 cells treated with different samples.

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

[0041] Figure 20 Results of tyrosinase activity inhibition rates in B16 cells treated with different samples.

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

[0043] Figure 22 The results show the effect of ethanol-extracted peat moss fermentation filtrate on HaCaT cell viability.

[0044] The significance analysis in the attached figure uses t-tests. "*" indicates significance at the P<0.05 level; "**" indicates significance at the P<0.01 level; "***" indicates high significance at the P<0.001 level; "****" indicates extremely significant at the P<0.0001 level; and "ns" indicates no significance. Detailed Implementation

[0045] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0046] The sources of the materials and reagents used 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 Biotechnology Co., Ltd. Lactobacillus acidophilus Xi'an Mixian 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 of Guangdong Pharmaceutical University DCFH-DA Shanghai Beyotime Biotechnology Co., Ltd. β-galactosidase Shanghai Beyotime Biotechnology Co., Ltd. B16 cells New Drug R&D Center of Guangdong Pharmaceutical University DMEM medium Beijing Wokawei Biotechnology Co., Ltd. 1640 culture medium Beijing Wokawei Biotechnology Co., Ltd. Arbutin Shanghai McLean Biochemical Technology Co., Ltd.

[0049] In the following examples, "W" represents peat moss extract that has not undergone microbial fermentation; "Z" represents peat moss filtrate fermented with Lactobacillus plantarum; "S" represents peat moss filtrate fermented with Lactobacillus acidophilus; and "H" represents peat moss filtrate fermented with a mixture of Lactobacillus plantarum and Lactobacillus acidophilus.

[0050] Example 1: Preparation method of peat moss fermentation filtrate

[0051] (1) The sterile peat moss grown 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) is filtered to obtain peat moss powder;

[0053] (3) Prepare a 20 mg / mL solution of peat moss powder and sterilize it at 121℃ for 20 min;

[0054] (4) Directly inoculate the peat moss solution obtained in step (3) with *Lactobacillus plantarum* powder (Z), *Lactobacillus acidophilus* powder (S), or a mixture of the two bacteria (H), with a total inoculation amount of 4% (w / v), a fermentation temperature of 37℃, a fermentation time of 50 h, and a 600 nm OD value. In the mixed bacteria strain, the mass ratio of *Lactobacillus plantarum* powder (purchased from Xi'an Mixianer Biotechnology Co., Ltd., catalog number YQ20250105) to *Lactobacillus acidophilus* powder (purchased from Xi'an Mixianer Biotechnology Co., Ltd., catalog number MXE20250210) is 1:1. If step (4) is omitted and the supernatant is directly obtained by centrifugation in step (5), the resulting product is peat moss extract (W) without bacterial fermentation.

[0055] (5) Sterilize the fermentation broth, centrifuge at 4000 rpm for 20 min, and the supernatant is the peat moss fermentation filtrate. OD value test results are shown below. Figure 1 It can be seen that the growth abilities of Z, S and H bacteria are similar.

[0056] Example 2: Preparation method of peat moss fermentation filtrate

[0057] The difference from Example 1 is that the inoculum size in step (4) is 2%, and the fermentation temperature is 37°C. Other steps are the same as in Example 1; the OD value results are shown below. Figure 2 .

[0058] Example 3: Preparation method of peat moss fermentation filtrate

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

[0060] Example 4: Preparation method of peat moss fermentation filtrate

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

[0062] from Figure 2-4 The results show that, within a certain range, the inoculum size, fermentation temperature, and mixed strain ratio have a similar effect on the growth of peat moss fermentation filtrate.

[0063] In Examples 5-11 below, the samples in groups “W”, “Z”, “S” and “H” are the corresponding solutions obtained by the preparation method in Example 1.

[0064] Example 5: Effect of peat moss fermentation filtrate on HaCaT cell viability

[0065] (1) With 1.0×10 5 HaCaT cells in logarithmic growth phase were inoculated into 96-well plates (100 μL per well) at a cell density of 100 cells / mL and cultured in an incubator at 37°C and 5% CO2 for 24 h.

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

[0067] (3) Then, add 150 μL of DMSO solution to each well, and finally use an ELISA reader to detect the absorbance value at 490 nm.

[0068] This experiment was divided into 5 groups: "Control" represents blank control; "W" represents peat moss extract without bacterial fermentation; "Z" represents peat moss filtrate fermented with Lactobacillus plantarum; "S" represents peat moss filtrate fermented with Lactobacillus acidophilus; and "H" represents peat moss filtrate fermented with a mixture of Lactobacillus plantarum and Lactobacillus acidophilus. The samples of each group were prepared according to the method in Example 1.

[0069] The preparation method of the peat moss extract (“W”) without microbial fermentation is as follows:

[0070] The method in Example 1 was followed. The sterile peat moss grown in the laboratory was dried, crushed, and sieved through a 100-mesh sieve. Ethyl acetate was added for extraction. The extract obtained in step (1) was filtered to obtain peat moss powder. The peat moss powder was prepared into a 20 mg / mL solution and sterilized at 121°C for 20 min. The sterilized solution was the peat moss extract. The subsequent inoculation step was omitted.

[0071] HaCaT cell survival rate: 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 of the ELISA reader test are as follows Figure 5 (The left, middle, and right images show the test results for samples of 50, 100, and 150 μg / mL, respectively.) As shown, the W, H, Z, and S groups had virtually no effect on the proliferation rate of HaCaT cells, and the cell survival rate was greater than 90%, indicating no toxicity and high safety.

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

[0074] The groups in this experiment are as follows: "dz" represents the blank control group; "mx" represents the H2O2 oxidative damage model group; "UVB" represents the ultraviolet irradiation group; "W" represents the peat moss extract group without bacterial fermentation; "H" represents the peat moss filtrate group fermented by mixed fermentation of Lactobacillus plantarum and Lactobacillus acidophilus; "Z" represents the peat moss filtrate group fermented by Lactobacillus plantarum; and "S" represents the peat moss filtrate group fermented by Lactobacillus acidophilus.

[0075] 6.1 H2O2 Oxidative Damage Model

[0076] Establishing an H2O2 oxidative damage model using HaCaT cells: The optimal H2O2 concentration for cell damage is 900 μM.

[0077] (1) With 1.0×10 5 HaCaT cells in logarithmic growth phase were inoculated into 96-well plates (100 μL per well) at a cell density of 100 cells / mL and cultured in an incubator at 37°C and 5% CO2 for 24 h.

[0078] (2) Add 100 μL of medium containing 900 μM H2O2 and incubate for 2 h; replace with 100 μL of medium containing different concentrations (50, 100 and 150 μg / mL) of samples and incubate for 2 h; discard the old medium and add 100 μL of new medium containing 0.5 mg / mL MTT to each well and continue incubating for 4 h.

[0079] (3) Then, add 150 μL of DMSO solution to each well, and finally use an ELISA reader to detect the absorbance value at 490 nm.

[0080] HaCaT cell viability: Cell viability = A1 / A2 × 100%; A1 represents the absorbance value of the sample and H2O2 group; A2 represents the absorbance value of the control group;

[0081] The results of the ELISA reader test are as follows Figure 6 As shown, the peat moss extract without microbial fermentation has virtually no antioxidant properties, while the peat moss filtrate from microbial fermentation has significant antioxidant properties, and the higher the concentration of the peat moss filtrate, the better the antioxidant properties.

[0082] 6.2 Testing of mitochondrial membrane potential in a UV oxidative damage model

[0083] (1) With 1.0×10 4 HaCaT cells in logarithmic growth phase were inoculated into 96-well plates (100 μL per well) at a cell density of 100 cells / well and cultured in an incubator at 37°C and 5% CO2 for 24 h. The culture medium was then discarded.

[0084] (2) Add 100 μL of culture medium containing samples of different concentrations (100 μg / mL and 150 μg / mL), with 3 parallel wells for each concentration. Incubate for 24 h, discard the old culture medium, and wash each well three times with 100 μL of PBS.

[0085] (3) Add 5 μL of PBS and irradiate with UVB mode using a UV crosslinker to cause damage. Then, incubate the cells in a cell culture incubator at 37°C and 5% CO2 for 4 hours and discard the culture medium.

[0086] (4) After washing three times with PBS, add JC-1 staining solution to each well and incubate in a cell culture incubator at 37°C and 5% CO2 for 20 min, then discard the staining solution. Add 100 μL of pre-cooled staining buffer to each well and wash three times, then add 50 μL of culture medium for quantitative detection using a multi-functional microplate reader.

[0087] Results of mitochondrial membrane potential testing in a cell UV oxidative damage model are as follows: Figure 7 As shown, samples W, H, Z, and S have a certain effect on increasing the mitochondrial membrane potential of HaCaT cells after UV damage, indicating that peat moss extract and fermentation filtrate can effectively enhance the antioxidant capacity of cells. Furthermore, samples H, Z, and S have a significantly greater effect on increasing the mitochondrial membrane potential of HaCaT cells after UV damage compared to sample W.

[0088] 6.3 DPPH Free Radical Scavenging Experiment

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

[0090] Table 2. Composition of the reaction system for the DPPH method

[0091]

[0092]

[0093] At room temperature, the components were mixed evenly and reacted in a dark room for 30 minutes. The absorbance of the sample was measured at a wavelength of 517 nm, and the DPPH free radical scavenging rate was calculated according to the formula.

[0094] DPPH clearance rate (%) = {1 - (A1 - A2) / A0} × 100%;

[0095] Where A1 represents the absorbance measured after adding antioxidant and DPPH solution; A2 represents the absorbance measured after adding antioxidant and water; and A0 represents the absorbance measured after adding DPPH solution and water.

[0096] Experimental results are as follows Figure 8 As shown, the scavenging rate of ascorbic acid for free radicals tends to stabilize. With increasing concentration, the sample groups exhibited stronger DPPH free radical scavenging capabilities. Group H had the highest average free radical scavenging rate at 98.84%, followed by Group S at 94.77%, while Group W had the lowest DPPH free radical scavenging rate at only 73.57%. Overall, Group H (the filtrate from peat moss fermented with a mixture of Lactobacillus plantarum and Lactobacillus acidophilus) showed the best free radical scavenging effect among all concentrations.

[0097] 6.4 ABST Free Radical Scavenging Experiment

[0098] Prepare a 7.0 mmol / L ABTS and 2.6 mmol / L potassium persulfate solution at a volume ratio of 1:1. Incubate at room temperature in the dark for 12-16 hours to obtain the ABTS stock solution. Take 2 mL of the stock solution and dilute it 50 times with anhydrous ethanol. Measure the wavelength at 734 nm = 0.7 ± 0.02 to obtain the ABTS working solution for later use. The sample groups consist of W, H, S, and Z solutions with mass concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 mg / mL. The positive control is ascorbic acid at the same concentration gradient as the sample groups; add samples to several test tubes as shown in Table 3.

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

[0100]

[0101] After the components are mixed evenly, the reaction is carried out in the dark for 10 minutes. The absorbance of the sample is measured at a wavelength of 734 nm, and the ABTS free radical scavenging rate is calculated according to the formula.

[0102] ABTS clearance rate (%) = {1 - (A1 - A2) / A0} × 100%;

[0103] Where A1 represents the absorbance measured with the addition of antioxidant and ABTS working solution; A2 represents the absorbance measured with the addition of antioxidant and anhydrous ethanol; and A0 represents the absorbance measured with the addition of ABTS working solution and water.

[0104] Experimental results are as follows Figure 9 The scavenging rate of ascorbic acid for free radicals tended to stabilize. As the concentration increased, the scavenging ability of different groups for ABTS free radicals became stronger. At the maximum concentration, the scavenging effects of groups H, S, and Z on ABTS free radicals were similar, at 88.48%, 90.63%, and 85.88%, respectively, while the scavenging rate of group W on ABTS free radicals was only 58.24%. Considering the overall situation, the scavenging effect of peat moss filtrate after microbial fermentation on ABTS free radicals was significantly higher than that of peat moss filtrate without microbial fermentation at all concentrations (P<0.05).

[0105] 6.5 Hydroxyl radical scavenging experiment

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

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

[0108]

[0109] After mixing all components, the sample was heated in a water bath at 37°C for 15 minutes, and the absorbance was measured at a wavelength of 510 nm. The hydroxyl radical scavenging rate was calculated according to the formula.

[0110] Hydroxyl radical scavenging rate (%) = {1 - (A1 - A2) / A0} × 100%;

[0111] Where A1 represents the absorbance of the sample group measured at a wavelength of 510 nm; A2 represents the absorbance of the blank group measured at a wavelength of 510 nm; and A0 represents the absorbance of the control group measured at a wavelength of 510 nm.

[0112] Experimental results are as follows Figure 10 The scavenging rate of ascorbic acid for free radicals tended to stabilize. At a concentration of 4 mg / mL, the scavenging effects of groups Z, H, and S on hydroxyl radicals were similar to those of group W. At the maximum concentration, the average scavenging rates of hydroxyl radicals for groups H, S, and Z were 22.02%, 26.37%, and 22.87%, respectively, while the average scavenging rate for group W was 16.80%. Overall, the scavenging effect of peat moss filtrate after microbial fermentation on hydroxyl radicals was significantly higher than that of peat moss filtrate without microbial fermentation (P<0.05).

[0113] 6.6 Superoxide anion free radical scavenging experiment

[0114] Prepare 0.1 mol / L Tris solution, 0.1 mol / L HCl solution, and 0.05 mol / L Tris-HCl buffer solution (pH 7.4). Weigh 0.0189 g of pyrogallol powder and dilute to 50 mL with 10 mmol / L HCl to prepare the pyrogallol working solution; prepare fresh before use. The sample groups consist of W, H, S, and Z solutions with mass concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 mg / mL. The positive control is ascorbic acid at the same concentration gradient as the sample groups; add samples to several test tubes as shown in Table 5.

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

[0116]

[0117] First, add Tris-HCl buffer to the cuvette, then add the sample, and finally quickly add pyrogallol. Mix quickly with a pipette. Select the dynamic measurement mode and measure the A value every 30 seconds at a wavelength of 325 nm until 300 seconds have elapsed.

[0118] ΔA0=A0(325nm) 300s -A0(325nm) 30s ;

[0119] ΔA1=A1(325nm) 300s -A1 (325nm) 30s ;

[0120] Superoxide anion free radical scavenging rate (%) = (A1-A2) / A0 × 100%.

[0121] Experimental results are as follows Figure 11At concentrations of 2-5 mg / mL, the scavenging rate of ascorbic acid for free radicals tended to stabilize. At the maximum concentration, the average scavenging rates of superoxide anion free radicals in groups H, S, and Z were 86.38%, 88.21%, and 89.27%, respectively, while the average scavenging rate in group W was 55.74%. At concentrations of 1 mg / mL and 3 mg / mL, the scavenging effect of peat moss filtrate without fermentation was slightly higher than that of peat moss filtrate after fermentation, possibly because the effective components after fermentation were inactivated due to prolonged sample storage. Overall, the scavenging effect of peat moss filtrate after fermentation was significantly higher than that of peat moss filtrate without fermentation.

[0122] Example 7: Test on the remediation efficacy of peat moss fermentation filtrate

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

[0124] 7.1 UV Damage Pre-protection Test

[0125] (1) With 1.0×10 4 HaCaT cells in logarithmic growth phase were inoculated into 96-well plates (100 μL per well) at a cell density of 100 cells / well and cultured in an incubator at 37°C and 5% CO2 for 24 h.

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

[0127] (3) Add 50 μL of PBS and irradiate the cells in UVB mode using a UV crosslinker. Then, incubate the cells in a cell culture incubator at 37°C and 5% CO2 for 24 hours and discard the culture medium. The cell viability was measured by the MTT assay.

[0128] The results are as follows Figure 12 As shown, it can be seen that the peat moss extract without microbial fermentation has no pre-protective effect against ultraviolet damage, while the peat moss filtrate with microbial fermentation has a significant pre-protective effect against ultraviolet damage.

[0129] 7.2 Cell Scratch Repair Test

[0130] (1) HaCaT cells in the logarithmic growth phase were digested with trypsin into a single-cell suspension and seeded into a 6-well culture plate (30w / well). The total amount of culture medium in each well was 2mL and cultured at 37℃ in a 5% CO2 incubator for 24h.

[0131] (2) Use a 200μL pipette tip to make a horizontal line on the back of the cell, either parallel or perpendicular to the back of the cell, and then rinse the cells with PBS three times to remove the cells that have been drawn.

[0132] (3) The blank control group was added with serum-free culture medium, and the sample group was added with serum-free culture medium containing W, H, Z and S. Samples were taken at 0h and 24h of culture time and photographed.

[0133] The scratch area was analyzed using ImageJ software. The cell scratch healing rate was calculated as (initial scratch area at 0h - scratch area at 24h) / initial scratch area at 0h.

[0134] The results are as follows Figure 13 As shown, the cell scratch healing rate of the blank control group was only about 25%, while the cell scratch healing rate of fermentation filtrate prepared by different strains reached about 44%-50%.

[0135] 7.3 UV-induced scratch repair test

[0136] (1) HaCaT cells in the logarithmic growth phase were digested with trypsin into a single-cell suspension and seeded into a 6-well culture plate (30w / well). The total amount of culture medium in each well was 2mL and cultured at 37℃ in a 5% CO2 incubator for 24h.

[0137] (2) Using 15mj / cm 2 The cells were irradiated with ultraviolet light, and then a 200μL pipette tip was used to make horizontal lines parallel or perpendicular to the back of the cell. The cells were then washed three times with PBS to remove the cells that had been drawn.

[0138] (3) The blank control group was added with serum-free culture medium, and the sample group was added with serum-free culture medium containing W, H, Z and S. Samples were taken at 0h and 24h of culture time and photographed.

[0139] The scratch area was analyzed using ImageJ software. The cell scratch healing rate was calculated as (initial scratch area at 0h - scratch area at 24h) / initial scratch area at 0h.

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

[0141] Example 8: Detection of the anti-aging effects of peat moss fermentation filtrate

[0142] In this experiment, "dz" represents the blank control group; "mx" represents the H2O2 oxidative damage model group; "UVB" represents the ultraviolet irradiation group; "W" represents the peat moss extract group without bacterial fermentation; "H" represents the peat moss filtrate group fermented by mixed fermentation of Lactobacillus plantarum and Lactobacillus acidophilus; "Z" represents the peat moss filtrate group fermented by Lactobacillus plantarum; and "S" represents the peat moss filtrate group fermented by Lactobacillus acidophilus.

[0143] 8.1 Fluorescence detection of ROS levels

[0144] (1) With 1.0×10 6 HaCaT cells in logarithmic growth phase were inoculated into 6-well culture plates (2 mL per well) at a cell density of 1 cell / well and cultured in an incubator at 37°C and 5% CO2 for 24 h. The culture medium was then discarded.

[0145] (2) Add 2 mL of culture medium of a certain concentration of sample, and set up 3 parallel wells for each concentration. Incubate for 24 h and then irradiate with ultraviolet light under an ultraviolet crosslinker.

[0146] (3) Discard the old culture medium, wash each well three times with PBS, then add 1 mL of DCFH-DA working solution, and continue incubation in an incubator for 30 min, shaking once every 5 min to ensure full contact between the probe and the cells. The entire process should be carried out in the dark. Take pictures under a fluorescence microscope.

[0147] Fluorescence microscopy results as follows Figure 15 As shown, the UVB ultraviolet irradiation group had high ROS levels, while the W, H, Z, and S groups had low ROS levels. Cell senescence is accompanied by increased ROS levels, revealing that peat moss filtrate has anti-aging effects.

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

[0149] (1) With 1.0×10 6 The cell density was 1 cell / well. Logarithmic growth phase HDF cells were inoculated into 6-well culture plates (2 mL per well) and cultured in an incubator at 37°C and 5% CO2 for 24 h. The culture medium was then discarded.

[0150] (2) Add 2 mL of culture medium for a certain concentration of sample, set up 3 parallel wells for each concentration, and let stand at room temperature for 2 h;

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

[0152] Photo results as follows Figure 16 As shown, groups W, H, Z and S can alleviate the aging of HDF cells, especially group Z, which has a stronger anti-aging effect.

[0153] HDF cells were seeded into 96-well plates. Old culture medium was aspirated, and 100 μL of culture medium containing different concentrations of the sample was added, with three replicates for each concentration. After incubation for 24 h, the cell supernatant was collected, and the levels of MMP-1, MMP-3, and MMP-9 in the cell supernatant were detected by ELISA (the levels of MMP-1, MMP-3, and MMP-9 increase during cell senescence). The results are shown below. Figure 17 As shown, this also indicates that peat moss fermentation filtrate has anti-aging effects.

[0154] Example 9: Whitening Efficacy Test of Peat Sphagnum Fermentation Filtrate

[0155] In this experiment, "dz" represents the blank control group; "bear" represents the arbutin positive control group; "W" represents the peat moss extract group without bacterial fermentation; "H" represents the peat moss filtrate group fermented by mixed fermentation of Lactobacillus plantarum and Lactobacillus acidophilus; "Z" represents the peat moss filtrate group fermented by Lactobacillus plantarum; and "S" represents the peat moss filtrate group fermented by Lactobacillus acidophilus.

[0156] 9.1 Cellular Tyrosinase Activity Experiment

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

[0158] (2) B16 cells were inoculated at 1.0 × 10⁻⁶. 4 Cells were seeded at a density of 100 μL per well in a 96-well plate and incubated for 24 hours, after which the culture medium was discarded.

[0159] (3) Add 100 μL of DMEM medium containing different samples, set three replicates for each concentration, and incubate in a 37℃, 5% CO2 incubator for 24 h before discarding the medium.

[0160] (4) Wash twice with PBS, add 50 μL of 1% Triton X-100 solution, place in a -80℃ freezer for 1 h, thaw at room temperature, add 100 μL of 0.1% levodopa solution to each well and shake at room temperature for 2 h. Measure absorbance at a wavelength of 475 nm. Tyrosinase activity = (sample well / blank well) × 100%.

[0161] Experimental results are as follows Figure 18 As shown, the tyrosinase activity of cells in different sample groups was high.

[0162] 9.2 Determination of melanin content in cells

[0163] The experimental procedure differs from 8.1 in the following ways:

[0164] Step (4) is different. The sample was washed twice with PBS, and 50 μL of a 1 mol / L NaOH solution containing 10% DMSO was added. The sample was then placed in a 90℃ water bath for 1 hour, and the absorbance was measured at a wavelength of 405 nm. The results are as follows: Figure 19 As shown, the melanin content of cells in different sample groups was higher.

[0165] 9.3 Determination of Tyrosinase Activity Inhibition

[0166] Add 1 mL of peat moss fermentation filtrate and 1 mL of L-tyrosine solution to 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 measure the absorbance of each group at 475 nm using a microplate reader. Use 0.1% vitamin C (VC) as a positive control.

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

[0168] Tyrosinase activity inhibition rate = [1 - (T - T0) / (C - CO)] × 100%;

[0169] T represents the absorbance of the peat moss fermentation filtrate sample with tyrosinase and L-tyrosine; T0 represents the absorbance of the peat moss fermentation filtrate sample with L-tyrosine but without tyrosinase; C represents the absorbance of the tyrosinase and L-tyrosine sample without the sample; and C0 represents the absorbance of the sample with only L-tyrosine, without the sample and tyrosinase.

[0170] The results are as follows Figure 20 As shown, the tyrosinase activity inhibition rate was low in the peat moss fermentation filtrate sample group. In conclusion, the peat moss 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 and solid culture media and autoclave them at 121°C for 20 min.

[0173] (2) Take Staphylococcus aureus and Escherichia coli out of the refrigerator and thaw them. Activate them continuously to the third generation with an inoculation ratio of bacterial culture to LB liquid medium of 1:6. The activation conditions for each generation are 37℃ for 24h and vibration culture.

[0174] (3) Take the third-generation activated bacterial strains and dilute each bacterial concentration to 0.5 MCF (McFländer turbidity units) as measured by a bacterial turbidimeter. Take 1 mL of the diluted bacterial solution and slowly inject it along the tube wall into a test tube containing 9 mL of sterile physiological saline. Mix well and take 10 mL of the solution. 6 10 710 8 Preliminary dilution and plating experiments were conducted using bacterial suspensions diluted at multiples of 10, with 10 being the final dilution factor selected. 7 Plate count method was used as the dilution factor;

[0175] (4) Take the filtrate of the mixed fermentation of 20 mg / mL peat moss culture and the filtrate of unfermented peat moss, filter them through a 0.22 μm microporous membrane, and set aside. Mix the diluted bacterial solution with the peat moss sample at a 1:1 ratio, and spread 100 μL of the mixture. Control group: physiological saline instead of bacterial solution. Incubate the culture medium in a 37℃ incubator for 16 h, observe the colony distribution and morphology of each group, and count the total number of colonies. Repeat the experiment 3 times for each group, take the average value, and calculate the inhibition rate.

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

[0177] Experimental results are as follows Figure 21 , Figure 21 As can be seen from Figure A, the average colony count in the saline control group was 10. 6 The average colony count in the mixed fermentation sample group of peat moss was 43, while the average colony count in the unfermented peat moss sample group was 60. The inhibition rate of the mixed fermentation sample group against Escherichia coli was 59.43%, while that of the unfermented sample group was 43.40%. This indicates that peat moss extract has a certain inhibitory effect on Escherichia coli, and the inhibitory effect of the mixed fermentation sample was better than that of the unfermented sample. Figure 21 As can be seen from B, the average colony count in the saline control group was 10. 7 The average colony count in the mixed fermentation sample group of peat moss was 35, while the average colony count in the unfermented peat moss sample group was 39. The inhibition rate of the mixed fermentation sample group against Staphylococcus aureus was 67.29%, while that of the unfermented sample group was 63.55%. This indicates that peat moss extract has a certain inhibitory effect on Staphylococcus aureus, and the inhibitory effect of peat moss before and after fermentation is similar.

[0178] Example 11: Irritation test of peat moss fermentation filtrate

[0179] By directly contacting the test sample with the chorioallantoic membrane (CAM) of chicken embryos, the CAM's response was observed through indicators such as bleeding, coagulation, and angiogenesis, and a score was given to objectively assess the eye irritation of the test sample.

[0180] (1) Purchase 0-day-old chicken embryos, use a smart incubator with a temperature of 37℃ and a humidity of 70%, and after 9 days of incubation, check and discard defective chicken embryos. Mark the air cell position on normal chicken embryos, use tweezers to knock open the shell of the air cell part to expose the white membrane, moisten it with 0.9% physiological saline, pour out the excess liquid and remove the inner membrane to ensure that the blood vessels are not damaged.

[0181] (2) Divided into 6 groups: positive control group (0.1 mol / L NaOH solution), negative control group (0.9% NaCl solution), and sample group (sphagnum moss fermentation filtrate with a crude drug concentration of 20 mg / mL). Each group had 6 chicken embryos and 3 parallel groups.

[0182] (3) Take 0.3 mL of each group solution and add it directly to the CAM surface. Record the time of occurrence of the three reactions of hemorrhage, coagulation and vascular dissolution within 300 s after addition, accurate to the second, and record the degree of reaction. Calculate the irritation score (IS) using the following formula, and evaluate the irritation of the samples before and after peat moss fermentation according to Table 6.

[0183]

[0184] In the formula: secH represents the average time (s) from the start of sample addition to the occurrence of bleeding; secL represents the average time (s) from the start of sample addition to the occurrence of clotting; secC represents the average time (s) from the start of sample addition to the occurrence of dissolution.

[0185] Table 6 Evaluation of Stimulus Rating Method Results

[0186] Stimulus Score / IS Predictive classification IS<1 Non-irritating 1<IS<5 Mild irritation 5<IS<9 moderate irritation IS>10 Strongly irritating / corrosive

[0187] Table 7 Evaluation of CAM irritant effects in each group

[0188]

[0189]

[0190] The irritation results are shown in Table 7. No bleeding, coagulation, or vascularization was observed in the three parallel chicken embryos of the negative control group and the sample group. The average IS value was 0, which means that the peat moss fermentation filtrate with a crude drug concentration of 20 mg / mL was not irritating to CAM. All three parallel chicken embryos of the positive control group showed strong irritation reactions.

[0191] Comparative Example 1: A method for preparing peat moss fermentation filtrate

[0192] The difference from Example 1 is that the extract is 95% ethanol, 40 mesh sieve, and other experimental steps are the same as in Example 1 for the preparation of peat moss fermentation filtrate.

[0193] The prepared peat moss fermentation filtrate (“W’” represents ethanol extraction of peat moss extract without bacterial fermentation; “Z’” represents ethanol extraction of peat moss filtrate fermented with Lactobacillus plantarum; “S’” represents ethanol extraction of peat moss filtrate fermented with Lactobacillus acidophilus; “H’” represents ethanol extraction of peat moss filtrate fermented with a mixture of Lactobacillus plantarum and Lactobacillus acidophilus) was used to verify the effect on HaCaT cell viability according to the experimental steps in Example 5.

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

Claims

1. A method for the preparation of a Sphagnum moss fermentation filtrate, characterized by, The method comprises the following steps: (1) extracting the broken and sieved sphagnum moss with an extraction solution to obtain sphagnum moss powder after filtration; (2) dissolving the sphagnum moss powder and inoculating bacteria to ferment; (3) centrifuging after fermentation, and the supernatant is the sphagnum moss fermentation filtrate; In the step (2), the bacteria are any one of lactobacillus plantarum and lactobacillus acidophilus or a mixed bacteria of the two, the mass ratio of lactobacillus plantarum and lactobacillus acidophilus in the mixed bacteria is 1:1-1:5, the total inoculation amount of the bacteria in step (2) is 2%-6% in mass fraction, the fermentation temperature in step (3) is 34-37℃, the fermentation time in step (3) is 50h, the extraction solution in step (1) is ethyl acetate, and the sieving in step (1) is 100 mesh sieve.

2. The application of the sphagnum moss fermentation filtrate prepared by the preparation method of claim 1 in the preparation of cosmetic products.

3. Use according to claim 2, characterized in that, The cosmetic products include whitening cosmetic products, antioxidant cosmetic products or anti-aging cosmetic products.

4. Use according to claim 3, characterized in that, The cosmetic is any one of essence, essence cream, serum, foundation, primer, concealer, cream and mask.

5. A cosmetic having whitening, antioxidant and anti-aging effects, characterized by, The cosmetic includes the sphagnum moss fermentation filtrate prepared by the preparation method of claim 1.

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