Enzymatically hydrolyzed soy lecithin with whitening and anti-aging effects, and preparation method and application thereof

Abstract

The application belongs to the technical field of soybean phospholipid, and particularly relates to an enzymatic soybean lecithin with whitening and anti-aging effects, a preparation method and application thereof. The preparation method comprises the following steps: (1) taking soybean powder phospholipid, adding solution A, stirring, centrifuging, and obtaining a precipitate; (2) adding anhydrous ethanol to the precipitate, standing, concentrating the supernatant, and obtaining a concentrated solution; (3) adding a composite enzyme and a buffer solution to the concentrated solution for enzymolysis, drying, and obtaining crude phospholipid; (4) eluting the crude phospholipid through a silica gel-aluminum oxide column, collecting and concentrating the eluent, and drying, to obtain the enzymatic soybean lecithin; wherein the solution A in step (1) is a mixed solution of acetone, methanol and a citric acid aqueous solution. The prepared enzymatic soybean lecithin has high purity, good activity and high hydrophilicity, and has significant whitening and anti-aging effects.

Description

Technical Field

[0001] This invention belongs to the field of soybean lecithin technology, specifically relating to an enzymatically hydrolyzed soybean lecithin with whitening and anti-aging effects, its preparation method, and its application. Background Technology

[0002] Wrinkles, aging, and melanin deposition are common problems affecting the appearance of the skin. As we age, or when the skin is exposed to harmful environments (such as ultraviolet radiation) for a long time, it is prone to dull skin tone, pigmentation, and loss of radiance. In addition, reactive oxygen species produced by the body's metabolism can damage cell structure, causing collagen and elastin to be unable to be secreted normally, resulting in loose skin and wrinkles.

[0003] Currently, mainstream anti-aging ingredients in cosmetics include vitamin C, retinol, pro-xylane, and peptides. Their mechanisms of action primarily involve promoting collagen synthesis, inhibiting the degradation of collagen by matrix metalloproteinases, or delaying skin aging by scavenging free radicals through antioxidation. Whitening ingredients are mainly composed of niacinamide, ursolic acid, glycyrrhizin, and tranexamic acid. Their core ingredients are inhibiting tyrosinase activity, blocking melanin production pathways, or accelerating melanin metabolism and excretion. However, current cosmetic products mainly suffer from two major problems: First, poor synergy of active ingredients: most products only add a single anti-aging or whitening ingredient, or simply mix multiple ingredients, without resolving the conflicting mechanisms of action between different ingredients; second, insufficient skin penetration: anti-aging and whitening ingredients are mostly large-molecule active substances, which are difficult to penetrate the stratum corneum on their own, resulting in weak actual efficacy.

[0004] Emulsifiers are key excipients in cosmetics, and can be divided into synthetic and natural types based on their source. Chemically synthesized emulsifiers have stronger emulsifying properties and stability, and are cost-effective. However, chemically synthesized emulsifiers can irritate sensitive skin and pose a high risk of sensitization. Currently, the most researched natural emulsifiers include lecithin, glycolipids, glycosides, and glycerides.

[0005] Soy lecithin, also known as soy egg yolk lecithin, is mainly extracted from soybean foot oil and crude soybean lecithin powder. It contains choline, vitamins, minerals, linolenic acid, and alpha-linolenic acid. Choline is the main component of soy lecithin and is an essential nutrient for the human body. It has functions such as emulsification, anti-oxidation, lowering cholesterol, regulating blood lipids, delaying aging, enhancing memory, and preventing cardiovascular and cerebrovascular diseases. It is currently widely used in the food, cosmetics, and feed industries.

[0006] Traditional extraction methods typically involve organic solvent extraction, supercritical fluid extraction, ion exchange resin extraction, and ultrasonic extraction, followed by centrifugation and alcohol washing to obtain lecithin. However, the soybean lecithin extracted using existing technologies has low purity, poor activity, and low hydrophilicity.

[0007] Therefore, how to provide a method for extracting soybean lecithin with high purity, high activity and high hydrophilicity has become a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides an enzymatically hydrolyzed soybean lecithin with whitening and anti-aging effects, its preparation method, and its applications.

[0009] This invention is achieved through the following technical solution: A method for preparing enzymatically hydrolyzed soybean lecithin with whitening and anti-aging effects includes the following steps: (1) Take soybean powder phospholipids, add them to solution A, stir, centrifuge, and obtain the precipitate; (2) Add anhydrous ethanol to the precipitate, let stand, concentrate the supernatant to obtain the concentrated solution; (3) Add a compound enzyme and buffer solution to the concentrate for enzymatic hydrolysis, dry, and obtain crude phospholipids; (4) The crude phospholipid obtained was eluted through a silica gel-alumina column, the eluent was concentrated and dried to obtain the enzymatically hydrolyzed soybean lecithin; In step (1), solution A is a mixture 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 material-to-liquid ratio 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, the temperature of the anhydrous ethanol in step (2) is -15℃ to -25℃, the amount of anhydrous ethanol added is 5-10 times the volume of the precipitate, the temperature of the settling is 0-5℃, and the settling time is 10-12h.

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

[0014] Preferably, the buffer solution in step (3) is a 0.2M disodium hydrogen phosphate solution, the volume ratio of the concentrate to the buffer solution is 1:20-25, the amount of the compound enzyme added is 0.2%-0.5% of the mass of the concentrate, the enzymatic hydrolysis temperature is 30-50℃, the enzymatic hydrolysis time is 8-12h, and the drying temperature is 45-55℃.

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

[0016] This invention also relates to enzymatically hydrolyzed soybean lecithin prepared by the above-described preparation method.

[0017] The present invention also provides an emulsifier comprising glycerol and enzymatically hydrolyzed soybean lecithin prepared by the above preparation method.

[0018] This invention also relates to the application of enzymatically hydrolyzed soybean lecithin prepared by the above preparation method or the above emulsifier in cosmetics.

[0019] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention uses an acetone-methanol-citric acid system as the extraction solvent, which can effectively improve the yield of soybean phospholipids. Methanol provides a way to break the hydrogen bond between phospholipids and proteins, which can promote the dissolution of lecithin. Citric acid provides an acidic environment, which can prevent the degradation of phospholipids during the extraction process and reduce the oxidation reaction. The synergistic effect of the ternary solvent can not only improve the extraction rate, but also effectively reduce the peroxide value.

[0020] (2) In the enzymatic hydrolysis process, the present invention uses a specific complex enzyme for enzymatic hydrolysis. Alkaline protease can effectively decompose the bound protein and solve the steric hindrance. Laccase can eliminate the interference of antioxidant components. The three work together to further improve the enzymatic hydrolysis efficiency and the hydrophilicity and activity of lecithin. Attached Figure Description

[0021] Figure 1 This is a graph showing the effect of different concentrations of the emulsifier obtained from the enzymatic hydrolysis of soybean lecithin and glycerol in Example 1 on the activity of B16 cells. Compared with the negative control group, P<0.01, P<0.001. Detailed Implementation

[0022] 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.

[0023] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

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

[0025] Example 1 A method for preparing enzymatically hydrolyzed soybean lecithin with whitening and anti-aging effects, comprising the following steps: (1) Take soybean powder phospholipids and add them to a mixed solution of acetone, methanol and citric acid aqueous solution. The material-to-liquid ratio is 1g:9mL. Stir at 45℃ and 300rpm for 1.5h, then centrifuge to obtain the precipitate. The citric acid aqueous solution has a mass concentration of 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℃ to the precipitate, let it stand at 4℃ for 10h, concentrate the supernatant to obtain the concentrated solution.

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

[0028] (4) Elute the crude phospholipid through a silica-alumina column (the mass ratio of crude phospholipid to silica-alumina column is 1:9, and the column height ratio of silica to alumina is 2:1). Use the following elution system sequentially for elution, and control the flow rate at 1.5 mL / min. Elution buffer A: a mixture of n-hexane and ethyl acetate in a volume ratio of 4:1, eluted with 4 column volumes; Elution buffer B: a mixture of ethyl acetate and anhydrous ethanol in a volume ratio of 1:1, eluted with 6 column volumes; Elution buffer C: a mixture of anhydrous ethanol, water, and glacial acetic acid in a volume ratio of 85:15:0.5, eluted with 9 column volumes.

[0029] Collect the eluent from part C of the eluent, concentrate it, and dry it at 55°C to obtain the enzymatically hydrolyzed soybean lecithin.

[0030] Example 2 A method for preparing enzymatically hydrolyzed soybean lecithin with whitening and anti-aging effects, comprising the following steps: (1) Take soybean powder phospholipids and add them to a mixed solution of acetone, methanol and citric acid aqueous solution. The material-to-liquid ratio is 1g:8mL. Stir at 40℃ and 250rpm for 2h, then centrifuge to obtain the precipitate. The mass concentration of the citric acid aqueous solution is 8%, and the volume ratio of acetone, methanol and citric acid aqueous solution is 70:23:7.

[0031] (2) Add 5 times the amount of anhydrous ethanol at -25℃ to the precipitate, let it stand at 4℃ for 12h, concentrate the supernatant to obtain the concentrated solution.

[0032] (3) Add a complex enzyme (phospholipase A2, alkaline protease and laccase) and 0.2M disodium hydrogen phosphate solution to the concentrate, enzymatically hydrolyze for 12 h at 40 °C, and dry at 45 °C to obtain crude phospholipids; The mass ratio of phospholipase A2, alkaline protease, and laccase is 8:2:0.5, the volume ratio of concentrate to buffer solution is 1:25, and the amount of the complex enzyme added is 0.2% of the mass of the concentrate.

[0033] (4) Elute the crude phospholipid through a silica-alumina column (the mass ratio of crude phospholipid to silica-alumina column is 1:9, and the column height ratio of silica to alumina is 2:1). Use the following elution system sequentially for elution, and control the flow rate at 1.5 mL / min. Elution buffer A: a mixture of n-hexane and ethyl acetate in a volume ratio of 4:1, eluted with 4 column volumes; Elution buffer B: a mixture of ethyl acetate and anhydrous ethanol in a volume ratio of 1:1, eluted with 6 column volumes; Elution buffer C: a mixture of anhydrous ethanol, water, and glacial acetic acid in a volume ratio of 85:15:0.5, eluted with 9 column volumes.

[0034] Collect the eluent from part C of the eluent, concentrate it, and dry it at 50°C to obtain the enzymatically hydrolyzed soybean lecithin.

[0035] Example 3 A method for preparing enzymatically hydrolyzed soybean lecithin with whitening and anti-aging effects, comprising the following steps: (1) Take soybean powder phospholipids and add them to a mixed solution of acetone, methanol and citric acid aqueous solution. The material-to-liquid ratio is 1g:10mL. Stir at 50℃ and 350rpm for 1h, then centrifuge to 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, let it stand at 4℃ for 10h, concentrate the supernatant to obtain the concentrate.

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

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

[0039] Collect the eluent from part C of the eluent, concentrate it, and dry it at 55°C to obtain the enzymatically hydrolyzed soybean lecithin.

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

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

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

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

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

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

[0046] Effect test The enzymatically hydrolyzed soybean lecithin prepared in Examples 1-3 and Comparative Examples 1-6 was mixed with glycerol (mass ratio: enzymatically hydrolyzed soybean lecithin: glycerol = 1:4) to obtain their respective emulsifiers, and the following effect tests were conducted.

[0047] Test Example 1: Whitening Effect Test 1. Effect test on inhibiting melanin synthesis in B16 cells Instrumentation: RT-6100 enzyme-linked immunosorbent assay (ELISA) analyzer.

[0048] Cells used in the experiment: B16 mouse melanocytes.

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

[0050] Negative control: basal culture medium.

[0051] (2) Operating steps Cell viability assay: B16 cells were seeded into 96-well plates. After 24 hours, the culture medium was discarded, and basal culture medium containing different concentrations of the emulsifier from Example 1 was added. After 24 hours, OD was measured by MTT assay. 490nm The effect of the experimental samples on the activity of B16 cells was analyzed by t-test.

[0052] Determination of relative melanin synthesis content in B16 cells: B16 cells were seeded into 6-well plates and cultured for 24 hours. The medium was then replaced with basal medium containing different experimental samples (Examples 1-3, Comparative Examples 1-6). After two medium changes, the cells were washed twice with PBS. 200 μL of 0.25% trypsin was added to each well to digest the cells. The cells were collected in centrifuge tubes and centrifuged for 5 minutes. 200 μL of melanin extraction solution was added to each tube, shaken well, and transferred to a 96-well plate. The absorbance at 405 nm was measured using a microplate reader.

[0053] (3) Calculation formula Relative melanin synthesis content (%) = ×100%; Where: T—absorbance of the test sample well; C—average of three absorbance measurements of the negative control group.

[0054] (4) Data Analysis Statistical analysis was performed using SPSS software. Independent samples t-tests were used to compare the experimental samples and the negative control. All statistical analyses were two-tailed tests, with a significance level of α = 0.05. P > 0.05 indicated no significant difference between the two groups; P < 0.05 indicated a significant difference between the two groups.

[0055] (5) Test results Cell viability assay results as follows Figure 1 As shown, in Example 1, when the sample concentration was 0.25%, the cell viability was >90%.

[0056] In the relative content test of cellular melanin synthesis, the concentration of samples in Examples 1-3 and Comparative Examples 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 Instruments and equipment: BSA224S analytical balance, RT-6100 enzyme-linked immunosorbent assay (ELISA) analyzer.

[0060] Reagents: Polyphenol oxidase (mushrooms), BR; Levodopa, BR.

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

[0062] (2) Experimental operation procedures Set up sample tubes, sample background tubes, enzyme reaction tubes, and solvent background tubes. Each group should have 3 parallel tubes. Add different reagent solutions to each tube, shake gently, and let stand at room temperature for 5 minutes. Transfer the reaction solutions of each group into a 1 cm cuvette and measure the absorbance at 475 nm.

[0063] (3) Calculation formula Tyrosinase inhibition rate (%) = ×100%; Where: T—absorbance of the sample tube, i.e., absorbance of the solution after the sample reacts with tyrosinase; T0—background absorbance of the sample; C—average of three absorbance values ​​of the enzyme reaction tube, i.e., absorbance of the reaction between tyrosinase and dopa without the addition of the sample; C0—background absorbance of the solvent.

[0064] (4) Data Analysis Statistical analysis was performed using SPSS software. Independent samples t-tests were used to compare the tyrosinase inhibition rates of the test samples, positive controls, and negative controls. All statistical analyses were two-tailed tests, with a significance level of α = 0.05. P > 0.05 indicated no significant difference between the two groups; P < 0.05 indicated a significant difference between the two groups.

[0065] (5) Test results The positive control group showed a tyrosinase inhibition rate >50%, indicating an effective reaction system. When the test samples were diluted to 5% aqueous solutions, Examples 1-3 and Comparative Examples 1-6 showed significant differences compared to the negative control group (P<0.05). Comparative Examples 1-6 also showed significant differences compared to Example 1 (P<0.05). See Table 2 for details.

[0066]

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

[0068] Test Example 2: Anti-aging Effect Test Instruments and equipment: BSA224S analytical balance, RT-6100 enzyme-linked immunosorbent assay (ELISA) analyzer.

[0069] Reagents: Elastase (porcine pancreas), BR; N-succinyl-L-alanyl-L-alanyl-L-alanine, 98%; Epigallocatechin gallate (EGCG), 98%.

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

[0071] (2) Experimental operation procedures Set up a sample group, a sample background group, a solvent group, and a solvent background group. Each group should be set up in 3 replicates. Add different reagent solutions to 96-well plates, shake gently, incubate at 25°C for 15 min, and then place them in an ELISA reader to measure the absorbance at 410 nm.

[0072] (3) Calculation formula elastase inhibition rate (%) = ×100%; In the formula: A—absorbance of the reaction solution without sample; B—absorbance of the reaction solution without sample and enzyme; C—absorbance of the reaction solution containing sample and enzyme; D—absorbance of the reaction solution containing sample and without enzyme.

[0073] (4) Data Analysis Statistical analysis was performed using SPSS software. Independent samples t-tests were used to compare the elastase inhibition rates of the test samples, positive controls, and negative controls. All statistical analyses were two-tailed tests, with a significance level of α = 0.05. P > 0.05 indicated no significant difference between the two groups; P < 0.05 indicated a significant difference between the two groups.

[0074] (5) Test results The positive control showed an elastase inhibition rate >50%, indicating an effective reaction system. When the sample concentration was 0.5%, the average elastase inhibition rate was 43.02%, which was significantly different from the negative control (P<0.05), suggesting that the sample has certain anti-wrinkle and firming effects. See Table 3 for details.

[0075]

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

[0077] Test Example 3 Antioxidant Capacity Test 1. Reagents: DPPH (1,1-diphenyl-2-trinitrophenylhydrazine), anhydrous ethanol (analytical grade).

[0078] 2. Sample to be tested: Experimental group: The enzymatically hydrolyzed soybean lecithin prepared in Examples 1-3 and Comparative Examples 1-6 was mixed with glycerol (mass ratio, enzymatically hydrolyzed soybean lecithin: glycerol = 1:4) to obtain their respective emulsifiers.

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

[0080] 3. Solution preparation: DPPH solution: Accurately take 4 mL of DPPH solution and prepare a 10 mL stock solution with 6 mL of 75% ethanol. Store in the dark for later use.

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

[0082] 4. Testing Method: The absorbance of each solution was measured at a wavelength of 517 nm. The formula for DPPH scavenging rate is as follows: DPPH clearance rate (%) = [1 - (A1 - A2) / A0] × 100%; In the formula: A0 is the absorbance value of the DPPH solution; A1 is the absorbance value of the sample solution; A2 is the absorbance value of the 75% ethanol solution.

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

[0084]

[0085] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. A method for preparing enzymatically hydrolyzed soybean lecithin with whitening and anti-aging effects, characterized in that, Includes the following steps: (1) Take soybean powder phospholipids, add them to solution A, stir, centrifuge, and obtain the precipitate; (2) Add anhydrous ethanol to the precipitate, let stand, concentrate the supernatant to obtain the concentrated solution; (3) Add a compound enzyme and buffer solution to the concentrate for enzymatic hydrolysis, dry, and obtain crude phospholipids; (4) The crude phospholipid obtained was eluted through a silica gel-alumina column, the concentrated effluent was collected and dried to obtain the enzymatically hydrolyzed soybean lecithin; Wherein, solution A in step (1) is a mixed solution of acetone, methanol and citric acid aqueous solution; the mass concentration of the citric acid aqueous solution is 8%-12%, and the volume ratio of acetone, methanol and citric acid aqueous solution is 70:20-30:3-7; The complex enzyme mentioned in step (3) is a mixture of phospholipase A2, alkaline protease and laccase, and the mass ratio of phospholipase A2, alkaline protease and laccase is 8:2-3:0.5-1.

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

3. The preparation method according to claim 1, characterized in that, In step (2), the temperature of the anhydrous ethanol is -15℃ to -25℃, the amount of anhydrous ethanol added is 5-10 times the volume of the precipitate, the temperature of the standing is 0-5℃, and the standing time is 10-12h.

4. The preparation method according to claim 1, characterized in that, The buffer solution in step (3) is a 0.2M disodium hydrogen phosphate solution, the volume ratio of the concentrate to the buffer solution is 1:20-25, the amount of the compound enzyme added is 0.2%-0.5% of the mass of the concentrate, the enzymatic hydrolysis temperature is 30-50℃, the enzymatic hydrolysis time is 8-12h, and the drying temperature is 45-55℃.

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

6. An enzymatically hydrolyzed soybean lecithin prepared by the preparation method according to any one of claims 1-5.

7. An emulsifier, characterized in that, The emulsifier comprises glycerol and enzymatically hydrolyzed soybean lecithin prepared by any one of claims 1-5.

8. The application of enzymatically hydrolyzed soybean lecithin prepared by any one of claims 1-5 or the emulsifier of claim 7 in cosmetics.

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