Enzymatic soybean lecithin with whitening and anti-aging effects as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of soybean phospholipids, and particularly relates to enzymolysis soybean lecithin with whitening and anti-aging effects as well as a preparation method and application of the enzymolysis soybean lecithin. The preparation method comprises the following steps: (1) taking soybean powder phospholipid, adding a solution A, stirring and centrifuging to obtain a precipitate; (2) adding absolute ethyl alcohol into the precipitate, standing, and concentrating the supernate to obtain a concentrated solution; (3) adding a compound enzyme and a buffer solution into the concentrated solution for enzymolysis, and drying to obtain crude phospholipid; and (4) eluting the obtained crude phospholipid through a silica gel-alumina column, collecting concentrated effluent, and drying to obtain the enzymolysis soybean lecithin. Wherein the solution A in the step (1) is a mixed solution of acetone, methanol and a citric acid aqueous solution. The prepared enzymolysis soybean lecithin is high in purity, good in activity and high in hydrophilicity, and has remarkable whitening and anti-aging effects.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of soybean phospholipids, and particularly relates to an enzymatic soybean lecithin with whitening and anti-aging effects, a preparation method thereof and application thereof. BACKGROUND

[0002] Wrinkles, aging, and melanin deposition are common problems that affect the appearance of the skin. With age, or long-term exposure of the skin to harmful environments (such as ultraviolet radiation), it is easy to cause the skin to become dull, pigmented, and lose luster; in addition, active oxygen produced by human metabolism can destroy cell structure, causing collagen and elastin to be unable to be normally secreted, resulting in skin relaxation and the generation of wrinkles.

[0003] The mainstream anti-aging ingredients of current cosmetics include vitamin C, retinol, bovine growth factor, and polypeptide ingredients, and the mechanisms of action are mainly to promote collagen synthesis, inhibit the degradation of collagen by matrix metalloproteinase, or remove free radicals through antioxidant to delay skin aging. The mainstream whitening ingredients are mainly niacinamide, ursolic acid, glabridin, and histidine, and the core raw materials are to inhibit the activity of tyrosinase, block the melanin generation pathway, or accelerate the metabolism and excretion of melanin. However, there are mainly two problems in current cosmetic products: 1. poor synergistic effect of functional ingredients: most products only add single anti-aging or whitening ingredients, or simply mix multiple ingredients, but do not solve the conflict of the mechanisms of action of different ingredients; 2. insufficient skin permeability: anti-aging and whitening ingredients are mostly macromolecular active substances, which are difficult to penetrate the stratum corneum of the skin by relying on the ingredients themselves alone, resulting in weak actual efficacy.

[0004] Emulsifiers are key auxiliary materials in cosmetics, and can be divided into synthetic and natural emulsifiers according to their sources. Chemically synthesized emulsifiers have stronger emulsifying performance and stability, and are cost-effective. However, chemically synthesized emulsifiers have certain irritation to sensitive skin and high sensitization risk. The natural emulsifiers that are currently studied more include lecithin, sugar esters, glycosides, and glycerol esters.

[0005] Among them, soybean lecithin (Lecithin High Potency), also known as soybean yolk, is mainly extracted from soybean foot oil, crude soybean phospholipid powder, etc., and contains choline, vitamins, minerals, linolenic acid, and secondary linolenic acid, etc. Choline is the main component of soybean lecithin and is also an essential nutrient for the human body, and has the functions of emulsification, antioxidant, cholesterol reduction, blood lipid regulation, anti-aging, memory enhancement, and prevention of cardiovascular and cerebrovascular diseases, and has been widely used in food, cosmetics, and feed industries.

[0006] The traditional extraction method is generally through organic solvent extraction, supercritical fluid extraction, ion exchange resin, ultrasonic extraction, etc., and then through centrifugation, alcohol washing to obtain lecithin. However, the soybean lecithin extracted in the prior art has low purity, poor activity and low hydrophilicity.

[0007] Therefore, how to provide an enzymatic soybean lecithin extraction method with high purity, high activity and high hydrophilicity has become a technical problem to be solved by those skilled in the art. SUMMARY

[0008] In view of the deficiencies of the prior art, the present application provides an enzymatic soybean lecithin with whitening and anti-aging effects, a preparation method and application thereof.

[0009] The present application is realized by the following technical solutions: A preparation method of enzymatic soybean lecithin with whitening and anti-aging effects, comprising the following steps: (1) Take soybean powder phospholipid, add solution A, stir, centrifuge, and obtain precipitate; (2) Add anhydrous ethanol to the precipitate, stand, concentrate the supernatant, and obtain a concentrated solution; (3) Add a complex enzyme and a buffer solution to the concentrated solution for enzymatic hydrolysis, dry, and obtain crude phospholipid; (4) Elute the obtained crude phospholipid through a silica gel-aluminum oxide column, concentrate the effluent, and dry to obtain the enzymatic soybean lecithin; In step (1), the solution A is a mixed solution of acetone, methanol and citric acid aqueous solution.

[0010] Preferably, the mass concentration of the citric acid aqueous solution is 8%-12%, and the volume ratio of the acetone, methanol and citric acid aqueous solution is 70:20-30:3-7.

[0011] Preferably, in step (1), the ratio of the feed liquid is 1g:8-10mL, the stirring temperature is 40-50℃, the stirring time is 1-2h, and the stirring speed is 250-350rpm.

[0012] Preferably, in step (2), the temperature of the anhydrous ethanol is -15℃~-25℃, the amount of anhydrous ethanol added is 5-10 times the volume of the precipitate, the standing temperature is 0-5℃, and the standing time is 10-12h.

[0013] Preferably, in step (3), the complex enzyme is a mixture of phospholipase A2, alkaline protease and laccase, and the mass ratio of the phospholipase A2, alkaline protease and laccase is 8:2-3:0.5-1.

[0014] Preferably, in step (3), the buffer solution is 0.2M sodium phosphate dibasic solution, the volume ratio of the concentrated solution to the buffer solution is 1:20-25, the added amount of the complex enzyme is 0.2%-0.5% of the mass of the concentrated solution, the enzymolysis temperature is 30-50℃, the enzymolysis time is 8-12h, and the drying temperature is 45-55℃.

[0015] Preferably, in step (4), the elution includes: eluent A: a mixture of n-hexane and ethyl acetate at a volume ratio of 4:1, eluted with 3-5 column volumes; eluent B: a mixture of ethyl acetate and anhydrous ethanol at a volume ratio of 1:1, eluted with 5-7 column volumes; eluent C: a mixture of anhydrous ethanol, water and glacial acetic acid at a volume ratio of 85:15:0.5, eluted with 8-10 column volumes; the flow rate of the eluent is 1.5-2mL / min, the mass ratio of the crude phospholipid to the silica-alumina column is 1:8-10; in the silica-alumina column, the column height ratio of silica gel to alumina is 1.5-2.5:1, and the effluent of the eluent C part is collected; and the drying temperature is 50-60℃.

[0016] The present application also relates to the enzymatic soybean lecithin prepared by the preparation method.

[0017] The present application also provides an emulsifier, which comprises glycerol and the enzymatic soybean lecithin prepared by the preparation method.

[0018] The present application also relates to the application of the enzymatic soybean lecithin prepared by the preparation method or the emulsifier in cosmetics.

[0019] Compared with the prior art, the present application has the following beneficial effects: (1) The present application uses an acetone-methanol-citric acid system as the extraction solution, which can effectively improve the soybean phospholipid yield. Methanol can break the hydrogen bond between phospholipid and protein, promote the dissolution of lecithin, and citric acid can provide an acidic environment, which can prevent phospholipid degradation during extraction and reduce oxidation. The synergistic effect of the ternary solvent can not only improve the extraction yield, but also effectively reduce the peroxide value.

[0020] (2) In the enzymolysis process, the present application uses specific complex enzymes for enzymolysis. Alkaline protease can effectively decompose the bound protein, solve the steric hindrance, and laccase can eliminate the interference of antioxidant components. The three enzymes work together to further improve the enzymolysis efficiency and the hydrophilicity and activity of lecithin. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is the effect of the emulsifier obtained by compounding the enzymatic soybean lecithin in Example 1 with glycerol at different concentrations on the activity of B16 cells, wherein, compared with the negative control group, P<0.01, P < 0.001. DETAILED DESCRIPTION

[0022] The application will be further described in conjunction with specific examples. The following examples are not intended to limit the application, but merely to illustrate the application. Unless otherwise specified, the experimental methods used in the following examples are generally performed according to conventional conditions. Unless otherwise specified, the materials, reagents, etc. used in the following examples are commercially available.

[0023] The application will be further described in conjunction with specific examples. The various instruments, devices, equipment, reagents, products, etc. used in the examples of the application are obtained through conventional commercial channels unless otherwise specified.

[0024] Phospholipase A2 (product number: S23892), laccase (product number: S10188) and alkaline protease (product number: S10154) are all purchased from Shanghai Yuan Ye Biotechnology Co., Ltd.

[0025] Example 1 A preparation method of an enzymatic soy lecithin with whitening and anti-aging effects, the steps are as follows: (1) Take soybean powder phospholipid, add a mixed solution of acetone, methanol and citric acid aqueous solution, the solid-liquid ratio is 1 g:9 mL, stir at 45°C, 300 rpm for 1.5 h, centrifuge to obtain a precipitate; The mass concentration of the citric acid aqueous solution is 10%, and the volume ratio of acetone, methanol and citric acid aqueous solution is 70:25:5.

[0026] (2) Add 7 times the amount of anhydrous ethanol at -20°C to the precipitate, stand at 4°C for 10 h, concentrate the supernatant to obtain a concentrated solution.

[0027] (3) Add a complex enzyme (phospholipase A2, alkaline protease and laccase) and 0.2M sodium phosphate dibasic solution to the concentrated solution, and enzymatically hydrolyze at 45°C for 10 h, and dry at 50°C to obtain a crude phospholipid; The mass ratio of phospholipase A2, alkaline protease and laccase is 8:2.4:0.6, the volume ratio of the concentrated solution and the buffer solution is 1:20, and the addition amount of the complex enzyme is 0.3% of the mass of the concentrated solution.

[0028] (4) The crude phospholipid is eluted through a silica gel-alumina column (the mass ratio of crude phospholipid to silica gel-alumina column is 1:9, and the column height ratio of silica gel to alumina is 2:1), and the following eluent systems are used for elution in turn, and the flow rate is controlled at 1.5 mL / min; Eluent A: mixture of n-hexane and ethyl acetate with volume ratio of 4:1, eluted with 4 column volumes; Eluent B: mixture of ethyl acetate and absolute ethanol with volume ratio of 1:1, eluted with 6 column volumes; Eluent C: mixture of absolute ethanol, water and glacial acetic acid with volume ratio of 85:15:0.5, eluted with 9 column volumes.

[0029] The effluent of the eluent C part was collected, concentrated, and dried at 55℃ to obtain the enzymatic soy lecithin.

[0030] Example 2 A preparation method of an enzymatic soy lecithin with whitening and anti-aging effects, comprising the following steps: (1) Soybean powder phospholipid was taken and added into a mixed solution of acetone, methanol and citric acid aqueous solution, with a material-to-liquid ratio of 1g:8mL, stirred at 40℃ and 250rpm for 2h, and centrifuged to obtain a precipitate; The mass concentration of the citric acid aqueous solution was 8%, and the volume ratio of acetone, methanol and citric acid aqueous solution was 70:23:7.

[0031] (2) 5 times of absolute ethanol at -25℃ was added to the precipitate, and the mixture was placed at 4℃ for 12h, and the supernatant was concentrated to obtain a concentrated solution.

[0032] (3) Compound enzymes (phospholipase A2, alkaline protease and laccase) and 0.2M sodium phosphate dibasic solution were added to the concentrated solution, and the mixture was enzymatically hydrolyzed at 40℃ for 12h, and then dried at 45℃ to obtain a crude phospholipid; The mass ratio of phospholipase A2, alkaline protease and laccase was 8:2:0.5, the volume ratio of the concentrated solution and the buffer solution was 1:25, and the addition amount of the compound enzymes was 0.2% of the mass of the concentrated solution.

[0033] (4) The crude phospholipid was eluted through a silica gel-alumina column (the mass ratio of the crude phospholipid to the silica gel-alumina column was 1:9, and the column height ratio of silica gel to alumina was 2:1), and the following eluent systems were used for elution in sequence, with a flow rate controlled at 1.5mL / min; Eluent A: mixture of n-hexane and ethyl acetate with volume ratio of 4:1, eluted with 4 column volumes; Eluent B: mixture of ethyl acetate and absolute ethanol with volume ratio of 1:1, eluted with 6 column volumes; Eluent C: mixture of absolute ethanol, water and glacial acetic acid with volume ratio of 85:15:0.5, eluted with 9 column volumes.

[0034] The effluent of the eluent C part was collected, concentrated, and dried at 50℃ to obtain the enzymatic soy lecithin.

[0035] Example 3 A preparation method of an enzymatic soy lecithin with whitening and anti-aging effects, comprising the following steps: (1) Take soybean powder phospholipid, add a mixed solution of acetone, methanol and citric acid aqueous solution, the solid-liquid ratio is 1 g:10 mL, stir at 50℃, 350 rpm for 1 h, centrifuge, and obtain the precipitate; The mass concentration of the citric acid aqueous solution is 12%, and the volume ratio of acetone, methanol and citric acid aqueous solution is 70:27:3.

[0036] (2) Add 10 times the amount of anhydrous ethanol at -15℃ to the precipitate, stand at 4℃ for 10 h, concentrate the supernatant, and obtain the concentrated solution.

[0037] (3) Add a composite enzyme (phospholipase A2, alkaline protease and laccase) and 0.2M sodium phosphate dibasic solution to the concentrated solution, enzymatically hydrolyze at 50℃ for 12 h, dry at 55℃, and obtain the crude phospholipid; The mass ratio of phospholipase A2, alkaline protease and laccase is 8:3:1, the volume ratio of the concentrated solution and the buffer solution is 1:25, and the addition amount of the composite enzyme is 0.5% of the mass of the concentrated solution.

[0038] (4) Elute the crude phospholipid through a silica gel-alumina column (the mass ratio of the crude phospholipid to the silica gel-alumina column is 1:9, and the column height ratio of silica gel to alumina is 2:1), and sequentially use the following eluent systems for elution, with a flow rate controlled at 2.0 mL / min; Eluent A: a mixture of n-hexane and ethyl acetate with a volume ratio of 4:1, eluted with 5 column volumes; Eluent B: a mixture of ethyl acetate and anhydrous ethanol with a volume ratio of 1:1, eluted with 7 column volumes; Eluent C: a mixture of anhydrous ethanol, water and glacial acetic acid with a volume ratio of 85:15:0.5, eluted with 10 column volumes.

[0039] Collect the effluent of the Eluent C part, concentrate, and dry at 55℃ to obtain the enzymatically hydrolyzed soy lecithin.

[0040] Comparative Example 1 The difference between this comparative example and Example 1 is only that the mixed solution in step (1) is replaced by acetone only.

[0041] Comparative Example 2 The difference between this comparative example and Example 1 is only that the mixed solution in step (1) is replaced by acetone and methanol with a volume ratio of 75:25.

[0042] Comparative Example 3 The composite enzyme in step (3) is replaced by phospholipase A2 only.

[0043] Comparative Example 4 The composite enzyme in step (3) is replaced by alkaline protease and laccase with a mass ratio of 8.8:2.2.

[0044] Comparative Example 5 The complex enzyme in step (3) is replaced by phospholipase A2 and alkaline protease at a mass ratio of 8:3.

[0045] Comparative Example 6 The complex enzyme in step (3) is replaced by phospholipase A2 and laccase at a mass ratio of 8:3.

[0046] Effect Test The enzymatic soybean lecithin prepared in Examples 1-3 and Comparative Examples 1-6 is mixed with glycerol (mass ratio, enzymatic soybean lecithin: glycerol = 1:4) to obtain respective emulsifiers, which are subjected to the following effect test.

[0047] Test Example 1 Whitening Effect Test 1. Inhibition of B16 Cell Melanin Synthesis Effect Test Instrument: RT-6100 enzyme label analyzer.

[0048] Test Cell: B16 mouse melanocyte.

[0049] Test Method: (1) Sample Treatment Each emulsifier sample is diluted with pure water to a volume fraction of 4%, and then filtered with a 0.22 μm filter to collect the filtrate as a sample mother liquor.

[0050] Negative Control: Base medium.

[0051] (2) Operation Steps Cell Activity Test: B16 cells are taken and plated in a 96-well plate, and after 24 h, the culture medium is aspirated and replaced with a base medium containing different concentrations of the emulsifier of Example 1. After 24 h, the OD is detected by MTT method. 490nm The effect of the test sample on B16 cell activity is analyzed by t-test.

[0052] B16 Cell Melanin Synthesis Relative Content Determination: B16 cells are taken and plated in a 6-well plate, and after 24 h, the culture medium is replaced with a base medium containing different test samples (Examples 1-3, Comparative Examples 1-6). After two more medium changes, the cells are washed twice with PBS, 200 μL of 0.25% trypsin is added to each well to digest the cells, and the cells are collected in a centrifuge tube and centrifuged for 5 min. 200 μL of melanin extraction solution is added to each tube, shaken uniformly, and transferred to a 96-well plate. The absorbance at 405 nm is detected by an enzyme label analyzer.

[0053] (3) Calculation Formula Melanin Synthesis Relative Content (%) = (T-C) / C × 100%; In the formula, T is the absorbance of the test sample well, and C is the average of three absorbance values of the negative control group.

[0054] (4) Data analysis Statistical analysis software was SPSS, and the comparison between the test sample and the negative control was performed by independent sample t test. The above statistical analysis was a two-tailed test, and the significance level was a = 0.05. P > 0.05 indicated that there was no significant difference between the two groups; P < 0.05 indicated that there was a significant difference between the two groups.

[0055] (5) Test results The cell activity test results are shown in Table 1. Figure 1 As shown in Table 1, the cell activity of the sample of Example 1 was > 90% when the concentration was 0.25%.

[0056] In the relative content test of cell melanin synthesis, the sample concentration of Example 1-3 and Comparative Example 1-6 was 0.25%, and the test results are shown in Table 1.

[0057]

[0058] Note: compared with the negative control group, # P < 0.01, ## P < 0.001; compared with Example 1 group, & P < 0.05.

[0059] 2. Tyrosinase inhibition rate test Instrument equipment: BSA224S analytical balance, RT-6100 enzyme label analyzer.

[0060] Reagent: polyphenol oxidase (mushroom), BR; levodopa, BR.

[0061] Test method: (1) Control and test sample treatment The samples of Example 1-3 and Comparative Example 1-6 were diluted with pure water to a sample concentration of 5% respectively; Positive control (kojic acid, purity ≥ 96%): diluted with pure water to a positive control concentration of 0.1%; Negative control: pure water.

[0062] (2) Test operation steps Set up sample tubes, sample background tubes, enzyme reaction tubes and solvent background tubes, and set up 3 parallel tubes for each group. Different reagent solutions were added, shaken gently, and left to stand at room temperature for 5 minutes. The reaction solutions of each group were moved into 1 cm cuvettes, and the absorbance value was measured at 475 nm.

[0063] (3) Calculation formula Tyrosinase inhibition rate (%) = (A0- As) / A0x 100%; ​In the formula: T—sample tube absorbance, i.e. absorbance of the solution after the sample reacts with tyrosinase; T0—background absorbance of the sample; C—average absorbance of the enzyme reaction tube for three times, i.e. absorbance of the reaction of tyrosinase and dopa without sample; C0—background absorbance of the solvent.

[0064] (4) Data analysis The statistical analysis software is SPSS, and the comparison between the test sample, the positive control and the negative control in terms of the tyrosinase inhibition rate is performed by independent sample t test. The above statistical analysis is a two-tailed test, and the significance level is α=0.05. P>0.05 indicates that there is no significant difference between the two groups; P<0.05 indicates that there is a significant difference between the two groups.

[0065] (5) Test results The tyrosinase inhibition rate of the positive control group is >50%, and the reaction system is effective. When the test samples are diluted into 5% aqueous solutions, Examples 1-3 and Comparative Examples 1-6 all have a significant difference (P<0.05) compared with the negative control group, and Comparative Example 1-6 also has a significant difference (P<0.05) compared with Example 1. The specific results are shown in Table 2.

[0066]

[0067] Note: Compared with the negative control group, # P<0.05; compared with the Example 1 group, & P<0.05.

[0068] Test Example 2 Anti-aging effect test Instrument equipment: BSA224S analytical balance, RT-6100 enzyme label analyzer.

[0069] Reagent: elastase (pig pancreas), BR; N-succinyl-L-alanyl-L-alanyl-L-alanine, 98%; epigallocatechin gallate (EGCG), 98%.

[0070] Test method: (1) Treatment of controls and test samples Sample group: diluted with pure water to a sample concentration of 0.5%; Positive control (EGCG): diluted with water to a positive control concentration of 0.1%; Negative control: pure water.

[0071] (2) Test operation steps Set up sample group, sample background group, solvent group and solvent background group, each group needs to set up 3 parallel, add different reagent solution in 96 well plate, shake gently, incubate at 25℃ for 15 min, then put it in the enzyme label instrument, measure the absorbance at 410 nm.

[0072] (3) Calculation formula Elastase inhibition rate (%) = (A - D) / (C - B) x 100%; In the formula: A is the absorbance of the reaction solution without sample; B is the absorbance of the reaction solution without sample and enzyme; C is the absorbance of the reaction solution containing sample and enzyme; D is the absorbance of the reaction solution containing sample and without enzyme.

[0073] (4) Data analysis The statistical analysis software is SPSS, and the comparison of elastase inhibition rates between the test sample, positive control and negative control uses independent sample t test. The above statistical analysis is two-tailed test, and the significance level is a=0.05. P>0.05 indicates that there is no significant difference between the two groups; P<0.05 indicates that there is significant difference between the two groups.

[0074] (5) Test results The positive control elastase inhibition rate is >50%, the reaction system is effective, when the sample concentration is 0.5%, the average elastase inhibition rate is 43.02%, which has significant difference compared with the negative control (P<0.05), indicating that the sample has certain anti-wrinkle and firming effect. The specific is shown in Table 3.

[0075]

[0076] Note: compared with the negative control group, # P<0.05; compared with the example 1 group, & P<0.05.

[0077] Test example 3 antioxidant capacity test 1. Reagent: DPPH (1,1-diphenyl-2-trinitrobenzene hydrazine), anhydrous ethanol (analytical pure).

[0078] 2. Test sample: Experimental group: enzyme hydrolyzed soy lecithin prepared in examples 1-3 and comparative examples 1-6, mixed with glycerol (mass ratio, enzyme hydrolyzed soy lecithin: glycerol = 1:4) to obtain respective emulsifiers.

[0079] Control group: 2,6-di-tert-butyl-p-cresol (BHT).

[0080] 3. Solution preparation: ​DPPH solution: Take 4 mL of DPPH solution, and take 6 mL of 75% ethanol to prepare 10 mL of stock solution, and store in dark for standby.

[0081] Sample solution: Mix 1 mL of sample, 4 mL of DPPH solution and 5 mL of 75% ethanol respectively.

[0082] 4. Test method: Determine the absorbance value of each solution at 517 nm wavelength, and the formula of DPPH clearance rate is as follows: DPPH clearance rate (%) = [1-(A1-A2) / A0]x100%; In the formula: A0 is the absorbance value of DPPH solution; A1 is the absorbance value of sample solution; A2 is the absorbance value of 75% ethanol solution.

[0083] The test results are shown in Table 4.

[0084]

[0085] The above detailed description is for one of the possible embodiments of the present application, which is not intended to limit the patent scope of the present application, and any equivalent implementation or change without departing from the present application shall be included in the scope of the technical scheme of the present application.

Claims

1. A method for preparing an enzymatic soy lecithin with whitening and anti-aging effects, characterized in that, The preparation method comprises the following steps: (1) taking soybean powder phospholipid, adding solution A, stirring, centrifuging, and obtaining precipitate; (2) adding anhydrous ethanol to the precipitate, standing, concentrating the supernatant, and obtaining concentrated solution; (3) adding complex enzyme and buffer solution to the concentrated solution for enzymolysis, and drying to obtain crude phospholipid; (4) eluting the crude phospholipid through a silica gel-alumina column, collecting and concentrating the eluate, and drying to obtain the enzymatic soybean lecithin. In step (1), the solution A is a mixed solution of acetone, methanol and citric acid aqueous solution.

2. The production method according to claim 1, characterized by, The mass concentration of the citric acid aqueous solution is 8%-12%, and the volume ratio of the acetone, methanol and citric acid aqueous solution is 70:20-30:3-7.

3. The preparation method according to claim 1, characterized in that, In step (1), the ratio of the liquid to the solid is 1g:8-10mL, the stirring temperature is 40-50℃, the stirring time is 1-2h, and the stirring speed is 250-350rpm.

4. The method of claim 1, wherein, In step (2), the temperature of the anhydrous ethanol is-15℃~-25℃, the amount of the anhydrous ethanol added is 5-10 times the volume of the precipitate, the standing temperature is 0-5℃, and the standing time is 10-12h.

5. The preparation method according to claim 1, characterized in that, In step (3), the complex enzyme is a mixture of phospholipase A2, alkaline protease and laccase, and the mass ratio of the phospholipase A2, alkaline protease and laccase is 8:2-3:0.5-1.

6. The method of claim 1, wherein, In step (3), the buffer solution is 0.2M sodium phosphate dibasic solution, the volume ratio of the concentrated solution to the buffer solution is 1:20-25, the amount of the complex enzyme added is 0.2%-0.5% of the mass of the concentrated solution, the enzymolysis temperature is 30-50℃, the enzymolysis time is 8-12h, and the drying temperature is 45-55℃.

7. The method of any one of claims 1-6, wherein, In step (4), the elution includes: eluent A: a mixture of n-hexane and ethyl acetate with a volume ratio of 4:1, eluted with 3-5 column volumes; eluent B: a mixture of ethyl acetate and anhydrous ethanol with a volume ratio of 1:1, eluted with 5-7 column volumes; eluent C: a mixture of anhydrous ethanol, water and glacial acetic acid with a volume ratio of 85:15:0.5, eluted with 8-10 column volumes; the flow rate of the eluent is 1.5-2mL / min, the mass ratio of the crude phospholipid to the silica gel-alumina column is 1:8-10, the column height ratio of silica gel to alumina in the silica gel-alumina column is 1.5-2.5:1, the flow of the eluent C part is collected, and the drying temperature is 50-60℃.

8. The enzymatic soybean lecithin prepared by the preparation method of any one of claims 1-7.

9. An emulsifier characterized by, The emulsifier comprises glycerol and the enzymatic soybean lecithin prepared by the preparation method of any one of claims 1-7.

10. The enzymatic soybean lecithin prepared by the preparation method of any one of claims 1-7 or the emulsifier of claim 9 is applied to cosmetics.

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