Preparation method of plant fermentation liquor as well as product and application of plant fermentation liquor

Abstract

The invention relates to a preparation method of plant fermentation liquor and a product and application thereof, the preparation method comprises the following steps: (1) breaking walls of an algae raw material, and crushing to obtain algae powder; crushing pomegranate seeds, and mixing the crushed pomegranate seeds with the algae powder to obtain mixed powder; (2) adding the mixed powder into a fermentation culture medium, inoculating a fermentation strain, and performing fermentation culture to obtain crude fermentation liquor; and (3) centrifuging and filtering the crude fermentation liquor, collecting filtrate, and concentrating the filtrate to obtain the plant fermentation liquor. The algae raw materials comprise chlorella vulgaris, spirulina maxima, codium fragile and haematococcus pluvialis. Compared with a traditional plant extract preparation method, the preparation method has the advantages that an organic solvent extraction process is omitted, loss of active ingredients in the extraction process is reduced, and the active ingredients in plant raw materials are extracted to the maximum extent. In addition, the preparation method provided by the invention has the advantages of simple steps, no use of an organic solvent, greenness and environmental protection, and is suitable for industrial expanded production.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and in particular relates to a method for preparing plant fermentation liquid, a product thereof and an application thereof. Background Art

[0002] With the improvement of people's living standards, in recent years, with the gradual improvement of people's skin care and beauty awareness, beauty and skin care products have also developed rapidly. At present, there are many chemical synthetic components in the beauty and skin care products on the market, and these components will have a certain degree of irritation to the skin. Compared with compounds, natural ingredients such as plant extracts are milder, safer for the skin, and have fewer side effects. Therefore, they are loved by consumers.

[0003] CN117598960A discloses a plant fermentation filtrate and a preparation method thereof. The invention prepares a plant fermentation filtrate through the steps of pretreatment, separation and purification of Eurotium cristatum, preparation of Eurotium cristatum spore inoculation liquid, inoculation of Eurotium cristatum spores, fermentation, extraction and the like. In the present invention, Eurotium cristatum and a plant composition are used for co-fermentation, the active substance components in the plant fermentation liquid are increased, and the anti-inflammatory, anti-oxidation and anti-UVB damage effects thereof are improved.

[0004] CN117180157A discloses an anti-aging plant extract and its application in cosmetics. The preparation method thereof comprises the following steps: mixing clove basil, lavender, thyme, scutellaria baicalensis, carrot seeds and hawthorn, crushing them, adding water and compound enzyme to stir, enzymolysis, sterilization to obtain enzymolysis material; adding compound bacteria to the enzymolysis material to stir, ferment, sterilize to obtain fermentation material; filtering and ultrafiltration of the fermentation material, concentrating and drying to obtain fermentation active substance; adding the fermentation active substance and sodium alginate to a buffer solution and mixing evenly to obtain an active liquid; adding the active liquid to a liposome liquid for homogenization and drying to obtain. The anti-aging plant extract of the invention can help skin tissue reconstruction, strengthen skin barrier, make skin elastic, and reduce wrinkles. At the same time, the plant extract of the invention has stable performance and can effectively avoid the reduction of its activity during storage and application.

[0005] At present, although there are many methods for preparing plant extracts, there are still problems such as complicated steps and difficulty in extracting active substances. Therefore, it is very meaningful to develop a method for preparing plant extracts with simple steps and more significant product efficacy. Summary of the invention

[0006] In view of the deficiencies of the prior art, the object of the present invention is to provide a method for preparing plant fermentation liquid and its product and application.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing a plant fermentation liquid, the preparation method comprising:

[0009] (1) breaking the algae raw material into pieces and then pulverizing them to obtain algae powder;

[0010] The pomegranate seeds are crushed and mixed with the algae powder to obtain a mixed powder;

[0011] (2) adding the mixed powder to a fermentation medium, inoculating the fermentation bacteria, and fermenting and culturing to obtain a crude fermentation liquid;

[0012] (3) centrifuging the crude fermentation liquid, filtering, collecting the filtrate, and concentrating the filtrate to obtain the plant fermentation liquid;

[0013] The algae raw materials include microscopic chlorella, giant spirulina, spinulosa and haematococcus pluvialis.

[0014] Preferably, the mass ratio of the microscopic chlorella, the giant spirulina, the spinulosa and the pluvialis haematococcus is (3-5):(1-2):(1-3):(3-4).

[0015] Wherein "3-5" can be 3.1, 3.3, 3.5, 3.7, 3.9, 4, 4.2, 4.5, 4.7 or 4.9, etc.;

[0016] "1-2" can be 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 or 1.9, etc.;

[0017] "1-3" can be 1.1, 1.3, 1.5, 1.7, 1.9, 2, 2.2, 2.5, 2.7 or 2.9, etc.;

[0018] “3-4” can be 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8 or 3.9, etc.

[0019] Preferably, the mass ratio of the pomegranate seeds to the algae powder is 1:(3-6), for example, it can be 1:3.2, 1:3.5, 1:3.8, 1:4, 1:4.2, 1:4.5, 1:4.8, 1:5, 1:5.2, 1:5.5 or 1:5.8, etc.

[0020] Preferably, the fermentation bacteria include yeast species and lactobacillus species.

[0021] Preferably, the total inoculation amount of the fermentation bacteria is 3-10% (v / v), for example, it can be 4% (v / v), 5% (v / v), 6% (v / v), 7% (v / v), 8% (v / v) or 9% (v / v), etc.

[0022] Preferably, the total inoculation amount of the fermentation bacteria is 5-8% (v / v).

[0023] Preferably, the fermentation medium comprises water, a carbon source, a nitrogen source and inorganic salts.

[0024] The carbon source includes any one of glucose, sucrose, maltose or glycerol, or a combination of at least two of them.

[0025] The nitrogen source includes any one of soy peptone, tryptone, yeast extract, and corn steep liquor powder, or a combination of at least two of them.

[0026] The inorganic salt is selected from any one of sodium chloride, potassium chloride, dipotassium hydrogen phosphate, ammonium sulfate or magnesium sulfate, or a combination of at least two thereof.

[0027] Preferably, the components of the fermentation medium include glucose 10-20 g / L (for example, 11 g / L, 13 g / L, 15 g / L, 17 g / L or 19 g / L, etc.), glycerol 1-10 g / L (for example, 2 g / L, 4 g / L, 6 g / L or 8 g / L, etc.), soy peptone 20-40 g / L (for example, 21 g / L, 23 g / L, 25 g / L, 27 g / L, 29 g / L, 30 g / L, 32 g / L, 35 g / L, 37 g / L or 39 g / L, etc.), yeast extract 1-5 g / L (for example, 1.5 g / L L, 2g / L, 2.5g / L, 3g / L, 3.5g / L, 4g / L or 4.5g / L, etc.), dipotassium hydrogen phosphate 0.5-2g / L (for example, 0.6g / L, 0.8g / L, 1g / L, 1.2g / L, 1.4g / L, 1.6g / L or 1.8g / L, etc.) and water; the addition amount of the mixed powder in the fermentation medium is 20-40g / L (for example, 21g / L, 23g / L, 25g / L, 27g / L, 29g / L, 30g / L, 32g / L, 35g / L, 37g / L or 39g / L, etc.).

[0028] Preferably, the fermentation conditions in step (2) are: dissolved oxygen is 30-40% (for example, it can be 31%, 33%, 35%, 37% or 39%, etc.), stirring speed is 100-300 rpm (for example, it can be 120 rpm, 150 rpm, 180 rpm, 200 rpm, 220 rpm, 250 rpm or 280 rpm, etc.), pH value is 7-8 (for example, it can be 7.1, 7.3, 7.5, 7.7 or 7.9, etc.), temperature is 35-40°C (for example, it can be 36°C, 37°C, 38°C or 39°C, etc.), time is 48-96h (for example, it can be 50h, 55h, 60h, 65h, 70h, 75h, 80h, 85h, 90h or 95h, etc.).

[0029] Preferably, the centrifugal speed in step (3) is 3000-5000 rpm, and the time is 5-20 min. For example, the speed can be 3200 rpm, 3500 rpm, 3800 rpm, 4000 rpm, 4200 rpm, 4500 rpm or 4800 rpm, and the time can be 6 min, 8 min, 10 min, 12 min, 14 min, 16 min or 18 min, etc.

[0030] Preferably, the concentration in step (3) is 10-20% of the volume of the filtrate, for example, 11%, 13%, 15%, 17% or 19%.

[0031] In a second aspect, the present invention provides a plant fermentation liquid, which is prepared by the method described in the first aspect.

[0032] In a third aspect, the present invention provides a use of the plant fermentation liquid as described in the second aspect in preparing cosmetics.

[0033] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) Compared with the traditional method for preparing plant extracts, the preparation method of the present invention does not require an organic solvent extraction process, thereby reducing the loss of active ingredients during the extraction process and maximizing the extraction of active ingredients from the plant raw materials.

[0036] (2) The plant extract prepared by the present invention has a high content of polyphenols, and thus has excellent antioxidant and anti-inflammatory effects.

[0037] (3) The preparation method of the present invention has simple steps and does not use organic solvents during the preparation process, which is more environmentally friendly and suitable for industrial production. DETAILED DESCRIPTION

[0038] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0039] The following materials and comparative examples are as follows:

[0040]

[0041] The rest of the raw materials can be used as long as they are purchased from regular dealers.

[0042] Preparation Example 1

[0043] The yeast strain was taken out from a -80°C refrigerator, inoculated into a liquid culture medium at an inoculation rate of 3% (v / v), cultured at 35°C for 24 hours for activation, and activated twice in succession to obtain an activated solution; the activated solution was inoculated into a liquid culture medium at an inoculation rate of 3% (v / v), and cultured at 37°C for 24 hours to obtain a yeast solution.

[0044] Preparation Example 2

[0045] The yeast strain was taken out from a -80°C refrigerator, inoculated into a liquid culture medium at an inoculation rate of 2% (v / v), cultured at 37°C for 36 hours for activation, and activated twice to obtain an activated solution; the activated solution was inoculated into a liquid culture medium at an inoculation rate of 2% (v / v), cultured at 37°C for 36 hours to obtain a lactobacillus culture solution.

[0046] Example 1

[0047] This embodiment provides a plant fermentation liquid, and the plant fermentation liquid is prepared by the following method:

[0048] (1) 40 parts of microscopic Chlorella, 15 parts of Spirulina, 20 parts of Spirulina, and 35 parts of Haematococcus pluvialis are crushed and mixed to obtain algae powder;

[0049] (2) crushing 25 portions of pomegranate seeds and mixing them with the algae powder obtained in step (1) to obtain a mixed powder;

[0050] (3) adding the mixed powder to a fermentation medium, wherein the composition of the fermentation medium is 15 g / L glucose, 5 g / L glycerol, 30 g / L soy peptone, 3 g / L yeast extract, 1 g / L potassium dihydrogen phosphate, 30 g / L mixed powder and 1 L water, and then inoculating the fermentation bacteria (yeast liquid and lactobacillus liquid in a volume ratio of 5:4), the inoculation amount is 6% (v / v), and the fermentation culture is carried out under the following fermentation conditions: dissolved oxygen is 35%, stirring speed is 200 rpm, pH value is 7.5, temperature is 38° C., and time is 72 h, to obtain a crude fermentation liquid;

[0051] (4) The crude fermentation liquid was centrifuged at 4000 rpm for 15 min, the filtrate was collected after filtration, and the filtrate was concentrated to 15% of the original volume to obtain the plant fermentation liquid.

[0052] Example 2

[0053] This embodiment provides a plant fermentation liquid, and the plant fermentation liquid is prepared by the following method:

[0054] (1) 50 parts of microscopic Chlorella, 10 parts of Spirulina, 30 parts of Spirulina, and 30 parts of Haematococcus pluvialis are crushed and mixed to obtain algae powder;

[0055] (2) crushing 40 portions of pomegranate seeds and mixing them with the algae powder obtained in step (1) to obtain a mixed powder;

[0056] (3) adding the mixed powder to a fermentation medium, wherein the composition of the fermentation medium is 20 g / L glucose, 3 g / L glycerol, 40 g / L soy peptone, 2 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, 20 g / L mixed powder and 1 L water, and then inoculating fermentation bacteria (yeast culture and lactobacillus culture in a volume ratio of 7:3) with an inoculation amount of 8% (v / v), and fermenting and culturing under the following fermentation conditions: dissolved oxygen of 30%, stirring speed of 150 rpm, pH of 7, temperature of 35° C., and time of 60 h to obtain a crude fermentation liquid;

[0057] (4) The crude fermentation liquid was centrifuged at 5000 rpm for 10 min, the filtrate was collected after filtration, and the filtrate was concentrated to 10% of the original volume to obtain the plant fermentation liquid.

[0058] Example 3

[0059] This embodiment provides a plant fermentation liquid, and the plant fermentation liquid is prepared by the following method:

[0060] (1) 30 parts of microscopic Chlorella, 20 parts of Spirulina, 10 parts of Spirulina serrata and 40 parts of Haematococcus pluvialis were crushed and mixed to obtain algae powder;

[0061] (2) crushing 20 portions of pomegranate seeds and mixing them with the algae powder obtained in step (1) to obtain a mixed powder;

[0062] (3) adding the mixed powder to a fermentation medium, wherein the composition of the fermentation medium is 10 g / L glucose, 5 g / L glycerol, 20 g / L soy peptone, 5 g / L yeast extract, 0.5 g / L potassium dihydrogen phosphate, 40 g / L mixed powder and 1 L water, and then inoculating fermentation bacteria (yeast culture and lactobacillus culture in a volume ratio of 4:5) with an inoculation amount of 5% (v / v), and fermenting and culturing under the following fermentation conditions: dissolved oxygen of 40%, stirring speed of 300 rpm, pH of 8, temperature of 40° C., and time of 96 h to obtain a crude fermentation liquid;

[0063] (4) The crude fermentation liquid was centrifuged at 3000 rpm for 20 min, filtered, and the filtrate was collected and concentrated to 18% of the original volume to obtain the plant fermentation liquid.

[0064] Example 4

[0065] This embodiment provides a plant fermentation liquid, which is different from that of Embodiment 1 only in that the amount of pomegranate seeds used in step (2) of the preparation method is 40 parts, and the remaining steps and raw material amounts are consistent with those of Embodiment 1.

[0066] Example 5

[0067] This embodiment provides a plant fermentation liquid, which is different from that of Embodiment 1 only in that the amount of pomegranate seeds used in step (2) of the preparation method is 15 parts, and the remaining steps and raw material amounts are consistent with those of Embodiment 1.

[0068] Example 6

[0069] This embodiment provides a plant fermentation liquid. The plant fermentation liquid is different from that in Embodiment 1 only in that the fermentation strain in the preparation method is only yeast liquid, and the inoculation amount is kept unchanged, and the remaining steps and raw material dosages are consistent with those in Embodiment 1.

[0070] Example 7

[0071] This embodiment provides a plant fermentation liquid. The plant fermentation liquid is different from that in Embodiment 1 only in that the fermentation bacteria in the preparation method are only lactobacillus liquid, and the inoculation amount is kept unchanged. The remaining steps and raw material dosages are consistent with those in Embodiment 1.

[0072] Comparative Example 1

[0073] This comparative example provides a plant fermentation liquid, which is different from Example 1 only in that step (1) in the preparation method is "23 parts of Spirulina maxima, 32 parts of Spirulina spinulosa and 55 parts of Haematococcus pluvialis are broken and crushed respectively, and then mixed to obtain algae powder", and the remaining steps and raw material amounts are consistent with Example 1.

[0074] Comparative Example 2

[0075] The present embodiment provides a plant fermentation liquid, which is different from the plant fermentation liquid in embodiment 1 only in that step (1) in the preparation method is "46.3 parts of microscopic Chlorella, 23.2 parts of Psoralea coccus and 40.5 parts of Haematococcus pluvialis are crushed and mixed to obtain algae powder", and the remaining steps and raw material amounts are the same as those in embodiment 1.

[0076] Comparative Example 3

[0077] The present embodiment provides a plant fermentation liquid, which is different from the plant fermentation liquid in embodiment 1 only in that step (1) in the preparation method is "48.8 parts of microscopic chlorella, 18.5 parts of spirulina and 42.7 parts of haematococcus pluvialis are broken and crushed respectively, and then mixed to obtain algae powder", and the remaining steps and raw material amounts are the same as those in embodiment 1.

[0078] Comparative Example 4

[0079] The present embodiment provides a plant fermentation liquid, which is different from the plant fermentation liquid in embodiment 1 only in that step (1) in the preparation method is "58.6 parts of microscopic Chlorella, 22 parts of Spirulina and 29.4 parts of Spirulina are broken and crushed respectively, and then mixed to obtain algae powder", and the remaining steps and raw material amounts are the same as those in embodiment 1.

[0080] Comparative Example 5

[0081] This embodiment provides a plant fermentation liquid, and the preparation method of the plant fermentation liquid is as follows:

[0082] (1) 40 parts of microscopic Chlorella, 15 parts of Spirulina, 20 parts of Spirulina, and 35 parts of Haematococcus pluvialis are crushed and mixed to obtain algae powder;

[0083] (2) adding algae powder to the fermentation medium, wherein the composition of the fermentation medium is 15 g / L glucose, 5 g / L glycerol, 30 g / L soy peptone, 3 g / L yeast extract, 1 g / L potassium dihydrogen phosphate, 30 g / L algae powder and 1 L water, and then inoculating the fermentation bacteria (yeast and lactobacillus in a volume ratio of 5:4), the inoculation amount is 6% (v / v), and the fermentation culture is carried out under the following fermentation conditions: dissolved oxygen is 35%, stirring speed is 200 rpm, pH value is 7.5, temperature is 38°C, time is 72 h, and crude fermentation liquid is obtained;

[0084] (3) The crude fermentation liquid was centrifuged at 4000 rpm for 15 min, filtered, and the filtrate was collected and concentrated to 15% of the original volume to obtain the plant fermentation liquid.

[0085] Comparative Example 6

[0086] The present invention provides a plant extract, and the preparation method of the plant extract is as follows:

[0087] 40 parts of microscopic Chlorella, 15 parts of Spirulina, 20 parts of Psoralea coccus and 35 parts of Haematococcus pluvialis are respectively broken and crushed, and then mixed to obtain algae powder, which is mixed with 10 times the mass of an ethanol aqueous solution with a volume fraction of 70%, heated to boiling, refluxed and extracted 3 times, each time for 30 minutes, and the extract is concentrated to 15% of the original volume to obtain the plant extract.

[0088] Test Example 1 Antioxidant Efficacy Study - DPPH Free Radical Scavenging Experiment

[0089] Test samples: samples provided in Examples 1-7 and Comparative Examples 1-6;

[0090] Test method:

[0091] Accurately weigh 47.7 mg of DPPH reagent, dilute to 100 ml brown volumetric flask, take 5 ml of it and dilute to 50 ml brown volumetric flask with anhydrous ethanol to obtain DPPH test solution (47.7 mg / L). Pipette 2 mL of DPPH test solution respectively, add 0.1 mL of plant fermentation liquid prepared in Examples 1-7 and Comparative Examples 1-4 respectively as shown in Table 2, and then add an appropriate amount of anhydrous ethanol to make the total mass equal. After fully mixing, let stand in the dark for 30 min, use anhydrous ethanol as a blank control, measure its absorbance at 517 nm, and set vitamin C as a control, and calculate the scavenging rate of each extract for DPPH according to the following formula:

[0092] DPPH removal rate (%) = [1-(As-Ar) / A0] × 100%

[0093] Where: As is the absorbance of the extract and DPPH reaction system; Ar is the absorbance of the extract-anhydrous ethanol; A0 is the absorbance of the blank control anhydrous ethanol.

[0094] Table 1 Reaction solution composition

[0095]

[0096] The DPPH free radical scavenging rate reflects its antioxidant capacity to a certain extent. The higher the scavenging rate, the better the antioxidant degree and the better the anti-aging effect. Therefore, the anti-aging effect of the essence can be judged by studying its ability to scavenge DPPH free radicals. Vitamin C is a highly effective antioxidant recognized by the cosmetics industry. This experiment used vitamin C as a control to examine its antioxidant capacity. The test results are shown in Table 1.

[0097] Table 1

[0098]

[0099]

[0100] As can be seen from Table 1, compared with the plant extract prepared by the conventional method in Comparative Example 6, the plant fermentation liquid prepared by the specific method of the present invention in Examples 1-3 has a better scavenging effect on DPPH, and the scavenging rate is above 89%. Among them, the scavenging rate of Examples 1-3 is higher than that of Comparative Examples 1-4, indicating that the algae raw materials of the present invention can be used in combination to make the antioxidant capacity of the final product better, verifying the rationality of the raw material selection. And compared with the positive control vitamin C, the scavenging rate of Examples 1-3 is only slightly lower than that of the positive control vitamin C and is very close, indicating that the plant fermentation liquid of the present invention has an antioxidant capacity similar to that of vitamin C and has a good antioxidant effect.

[0101] Test Example 2 Anti-inflammatory Efficacy Test

[0102] Test samples: samples provided in Examples 1-7 and Comparative Examples 1-6;

[0103] The test method is as follows:

[0104] Inhibition of TNF-α expression: Fresh blood was treated with EDTA as anticoagulant, blood cells were separated by Ficoll, and cells were resuspended in RIMP 1640 medium containing 10% fetal bovine serum. 100 mL of 1×10 5 cells / mL Freshly isolated cells, total number of cells per well: 10 4 3 wells for each sample; weigh the test sample, add sterile PBS solution to prepare the test sample stock solution (10 mg / mL), and then use sterile PBS to dilute the stock solution to the corresponding working concentration for use; add 10 mL of the test solution of the corresponding working concentration to the well of the culture plate. The culture plate is placed in an incubator at 37°C and 5% CO2 for 15 minutes; add 10 mL of LPS with a concentration of 100 mg / mL and incubate in an incubator at 37°C and 5% CO2 for 16 hours; the culture plate is centrifuged at 1000 rpm for 15 minutes, the supernatant is transferred to a new culture plate, and the TNF-α concentration in the culture supernatant is determined using an enzyme marker; or frozen at -20°C to avoid repeated freezing and thawing, and the inhibition rate is calculated.

[0105] The specific test results are shown in Table 2 below:

[0106] Table 2

[0107] Test samples Inhibition rate of TNF-α release / % Blank control 7.8 Example 1 92.6 Example 2 91.8 Example 3 92.3 Example 4 85.9 Example 5 84.2 Example 6 82.6 Example 7 80.8 Comparative Example 1 71.6 Comparative Example 2 73.3 Comparative Example 3 73.8 Comparative Example 4 70.9 Comparative Example 5 76.8 Comparative Example 6 82.9

[0108] It can be seen from the data in Table 2 that compared with the plant extract prepared by the conventional method in Comparative Example 6, the inhibition rate of TNF-α release by the plant fermentation liquid of the present invention is more than 90%, which fully demonstrates that the product prepared by the method of the present invention can extract the active ingredients better, and thus has a better anti-inflammatory and soothing effect, and can more effectively repair inflamed skin.

[0109] Test Example 3: Safety Evaluation Experiment

[0110] This test example conducts a safety test on the plant fermentation liquid prepared in Examples 1-7, and the test method is as follows:

[0111] The chorioallantoic membrane (CAM) is a respiratory membrane that surrounds the chicken embryo. Since the surface of the chicken embryo chorioallantoic membrane is rich in blood vessels and can be regarded as a complete organism, this test uses the characteristics of the complete, clear and transparent vascular system of the chorioallantoic membrane of the mid-stage incubated chicken embryo. A certain amount of the test substance is directly exposed to the chicken embryo chorioallantoic membrane. After the specified time, the changes in the toxic effect indicators of the chorioallantoic membrane (such as bleeding, coagulation and vascular melting) are observed, and scores are given. The mathematical mean value is calculated to evaluate the irritation of the test substance.

[0112] (1) Preparation of chicken embryos: Fertilized chicken embryos of varieties such as White Leghorn chicken, weighing 50g-60g, preferably SPF chicken embryos, are placed in an incubator for incubation, the temperature is controlled at 37.5°C ± 0.5°C, the relative humidity is 55%-70%, and the egg turning frequency is 3 times / h-6 times / h. On the 8th day of incubation, the eggs are checked with an egg candling device, unfertilized and inactive chicken embryos are discarded, and chicken embryos with well-developed blood vessels are selected, and the position of the air chamber is marked on the eggshell surface.

[0113] (2) Test process: There are at least 6 chicken embryos in each group, and negative and positive controls need to be set up for each test. The test started on the 9th day. Use dental curved forceps to carefully peel off the air chamber eggshell part, and drop a few drops of physiological saline on the surface of the eggshell membrane to fully moisten it. After pouring it out, use forceps to carefully remove the eggshell membrane to ensure that the exposed chorioallantoic membrane is intact and not damaged in any way. Take 0.3ml / 0.3g of the test substance and apply it directly to the CAM. Spread it as much as possible to ensure that the coverage area reaches at least 50%. After 3 minutes of action, gently rinse the surface of the CAM with double distilled water. The rinsing should be completed within 30s, pour out the liquid, and immediately observe the degree of change of each toxic effect under a stereomicroscope, and give a score (ES) according to the capillary bleeding, coagulation, and vascular melting.

[0114] (3) Observation results:

[0115] Bleeding: blood flows out of the blood vessels or capillaries of the CAM. No bleeding, mild bleeding, moderate bleeding, and severe bleeding are scored as 0, 1, 2, and 3 points, respectively.

[0116] Coagulation: refers to the denaturation of proteins inside and outside blood vessels, manifested as thrombus, swelling of the vessel wall, the appearance of coagulation points inside and outside blood vessels, milky turbidity outside blood vessels, etc. No coagulation, mild coagulation, moderate coagulation, and severe coagulation are scored as 0, 1, 2, and 3 points respectively.

[0117] Vascular ablation: refers to the ablation of blood vessels on the CAM membrane. No vascular ablation, mild vascular ablation, moderate vascular ablation, and severe vascular ablation were scored as 0, 1, 2, and 3 points, respectively.

[0118] (4) Result calculation and evaluation

[0119] Calculate the average ES score according to the following formula and determine the stimulus classification according to Table 3.

[0120]

[0121] Table 3 Results evaluation

[0122] End point score Irritation classification ES≤12 No / mild irritation 12<ES<16 Moderate irritation ES≥16 Severe irritation

[0123] The results are shown in Table 4:

[0124] Table 4

[0125]

[0126]

[0127] As can be seen from Table 4, the plant fermentation liquid of the present invention has no irritation to the skin and no positive reaction, which shows that the product of the present invention is safer and milder.

[0128] The applicant declares that the present invention illustrates the preparation method of a plant fermentation liquid and its product and application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0129] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0130] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for preparing a plant fermentation liquid, It is characterized in that The preparation method comprises: (1) breaking the algae raw material into pieces and then pulverizing them to obtain algae powder; The pomegranate seeds are crushed and mixed with the algae powder to obtain a mixed powder; (2) adding the mixed powder to a fermentation medium, inoculating the fermentation bacteria, and fermenting and culturing to obtain a crude fermentation liquid; (3) centrifuging the crude fermentation liquid, filtering, collecting the filtrate, and concentrating the filtrate to obtain the plant fermentation liquid; The algae raw materials include microscopic chlorella, giant spirulina, spinulosa and haematococcus pluvialis.

2. The method for preparing the plant fermentation liquid according to claim 1, It is characterized in that The mass ratio of the microscopic chlorella, the giant spirulina, the spinulosa and the pluvialis haematococcus is (3-5):(1-2):(1-3):(3-4).

3. The method for preparing the plant fermentation liquid according to claim 1 or 2, It is characterized in that The mass ratio of the pomegranate seeds to the algae powder is 1:(3-6).

4. The method for preparing a plant fermentation liquid according to any one of claims 1 to 3, It is characterized in that The fermentation strains include yeast strains and lactobacillus strains.

5. The method for preparing the plant fermentation liquid according to any one of claims 1 to 4, It is characterized in that The total amount of the fermentation bacteria inoculated is 3-10% (v / v); Preferably, the total inoculation amount of the fermentation bacteria is 5-8% (v / v).

6. The method for preparing the plant fermentation liquid according to any one of claims 1 to 5, It is characterized in that The fermentation medium comprises water, a carbon source, a nitrogen source and inorganic salts; The carbon source includes any one of glucose, sucrose, maltose or glycerol, or a combination of at least two thereof; The nitrogen source includes any one of soy peptone, tryptone, yeast extract or corn steep liquor powder, or a combination of at least two thereof; The inorganic salt is selected from any one of sodium chloride, potassium chloride, dipotassium hydrogen phosphate, ammonium sulfate or magnesium sulfate, or a combination of at least two thereof.

7. The method for preparing the plant fermentation liquid according to claim 6, It is characterized in that The components of the fermentation medium include 10-20 g / L of glucose, 1-10 g / L of glycerol, 20-40 g / L of soy peptone, 1-5 g / L of yeast extract, and 0.5-2 g / L of dipotassium hydrogen phosphate; the added amount of the mixed powder in the fermentation medium is 20-40 g / L.

8. The method for preparing the plant fermentation liquid according to any one of claims 1 to 7, It is characterized in that The fermentation conditions in step (2) are: dissolved oxygen 30-40%, stirring speed 100-300 rpm, pH 7-8, temperature 35-40° C., and time 48-96 h; Preferably, the centrifugation speed in step (3) is 3000-5000 rpm, and the time is 5-20 min; Preferably, the concentration in step (3) is to 10-20% of the volume of the filtrate.

9. A plant fermentation liquid, It is characterized in that The plant fermentation liquid is prepared by the method according to any one of claims 1 to 8.

10. Use of the plant fermentation liquid according to claim 9 in preparing cosmetics.

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