Stevia rebaudiana extract with whitening and freckle removing effects, preparation method of stevia rebaudiana extract and application of stevia rebaudiana extract in cosmetics
By employing citrate-disodium hydrogen phosphate buffer and ultrasonic-assisted extraction with a complex enzyme and macroporous resin column purification technology, combined with gradient elution with ethanol solution and glycosyltransferase catalysis, the problem of low stevioside extraction rate was solved, and the preparation of stevia extract with highly efficient enrichment of rebaudioside M2 was achieved, which has significant whitening and freckle-removing effects.
Patent Information
- Application Number
- CN202511345149.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-19
- Publication Date
- 2025-11-11
AI Technical Summary
Existing technologies have low stevioside extraction rates, making it difficult to enrich rare and high-value monomers such as rebaudioside M2. Furthermore, traditional hot water extraction methods are time-consuming, produce many impurities, and are difficult to separate and purify.
Stevia extract rich in rebaudioside M2 was prepared by ultrasonic-assisted extraction with a combination of citrate-disodium hydrogen phosphate buffer and a complex enzyme, followed by purification with macroporous resin column and gradient elution with ethanol solution. Rebaudioside was then converted to stevia extract by glycosyltransferase UGT76G1.
It significantly improved the dissolution rate of steviol glycosides and selectively enriched rebaudioside M2, shortened the extraction time, reduced the damage of heat-sensitive components, made it suitable for industrial production, and verified its whitening and spot-removing effects.
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Abstract
Description
Technical Field
[0001] This invention belongs to the biomedical engineering industry, and more specifically relates to the field of plant extract technology. Specifically, it relates to a stevia extract with whitening and freckle-removing effects, its preparation method, and its application in cosmetics. Background Technology
[0002] Stevia, also known as sweet grass, stevia, and sweet tea, is a perennial herbaceous plant native to South America, belonging to the genus Stevia in the family Asteraceae. It contains glycosides, flavonoids, chlorogenic acid, alkaloids, sterols, fatty acids, and polysaccharides, among which glycosides are the most numerous and abundant. Stevia glycosides, as extracts of stevia and also belonging to the biomedical engineering industry, are a class of tetracyclic diterpenoid mixtures composed of the same aglycone and different glycosidic groups. These include steviol glycosides, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, and rebaudioside M. These steviol glycosides are natural non-nutritive sweeteners formed by modifying the glycosyl backbone with different amounts of monosaccharides or oligosaccharides such as glucose and rhamnose, using steviol as the glycosyl backbone. Their sweetness is tens to hundreds of times that of sucrose. Currently, they are widely used in functional foods, health products, pharmaceuticals, and cosmetics.
[0003] Rebaudioside M2, a rare and high-value monomer, is present in low concentrations in stevia, making direct extraction from the plant difficult. Current extraction methods for steviol glycosides primarily involve hot water extraction, but this method suffers from low extraction rates, long processing times, and high impurity content in the crude extract, making subsequent separation and purification challenging and hindering the enrichment of the rare and high-value monomer rebaudioside M2. Therefore, developing a method for efficiently extracting steviol glycosides and enriching rebaudioside M2 from stevia raw materials has significant application value. Summary of the Invention
[0004] The purpose of this invention is to provide a stevia extract with whitening and freckle-removing effects, its preparation method, and its application in cosmetics, which solves the problems of low stevia glycoside extraction rate and difficulty in enriching active ingredients in the prior art.
[0005] The objective of this invention can be achieved through the following technical solutions:
[0006] The first aspect of this invention provides a method for preparing a stevia extract with whitening and freckle-removing effects, the preparation method specifically including the following steps:
[0007] S1. Pre-treat the stevia raw material to obtain stevia powder for later use;
[0008] S2. Take stevia powder, add citrate-disodium hydrogen phosphate buffer and compound enzyme, and extract with ultrasonic assistance to obtain crude extract.
[0009] S3. Purify the crude extract by passing it through a macroporous resin column and elute it with a gradient of ethanol solution to obtain the eluent.
[0010] S4. Distill the eluent under reduced pressure until no ethanol distills out, cool and centrifuge to obtain a precipitate, add deionized water to the precipitate to obtain mixture A, then add glycosyl donor UDPG and glycosyltransferase UGT76G1 to mixture A in sequence, and catalyze the enzyme to obtain mixture B. Concentrate under reduced pressure and cool to obtain stevia extract rich in rebaudioside M2.
[0011] As a preferred technical solution of the present invention, step S1 specifically includes:
[0012] Stevia leaves are dried, crushed, and sieved to obtain stevia powder, which is then set aside.
[0013] Furthermore, the sieving is performed through a 40-60 mesh sieve.
[0014] As a preferred technical solution of the present invention, step S2 specifically includes:
[0015] Stevia powder was mixed with citrate-disodium hydrogen phosphate buffer, and then a compound enzyme was added for enzymatic hydrolysis. After enzymatic hydrolysis, the enzyme was inactivated, cooled, and then extracted with ultrasound-assisted extraction followed by vacuum filtration to obtain the crude extract.
[0016] As a preferred embodiment of the present invention, the ratio of stevia powder to citrate-disodium hydrogen phosphate buffer in step S2 is 1g:80-90mL; the amount of compound enzyme added is 2-4% of the mass of the mixture.
[0017] Further, the complex enzyme in step S2 comprises cellulase, pectinase and papain in a mass ratio of 2-3:1-2:1.
[0018] Furthermore, the cellulase activity is 10,000-20,000 U / g, the pectinase activity is 8,000-12,000 U / g, and the papain activity is 1,600-2,000 U / g.
[0019] As a preferred embodiment of the present invention, the enzymatic hydrolysis time in step S2 is 2-3 hours and the enzymatic hydrolysis temperature is 45-55°C; the enzyme inactivation after enzymatic hydrolysis is performed by boiling water bath for 10 minutes; the cooling is performed by cooling to room temperature; the ultrasonic power of the ultrasonic-assisted extraction is 200-250W and the ultrasonic time is 30-60 minutes.
[0020] As a preferred technical solution of the present invention, step S3 specifically includes:
[0021] The crude extract was purified by adsorption through a macroporous resin column, followed by gradient elution with ethanol solution twice. The liquids from the two elutions were then mixed to obtain the eluent.
[0022] Furthermore, the resin in the macroporous resin column is D101 macroporous adsorption resin; the loading flow rate for both adsorption purification and elution is 1.0-2.5 mL / min; the gradient elution with ethanol solution is performed twice, first with a 50% ethanol solution and then with a 70% ethanol solution.
[0023] Furthermore, the amount of the 50% ethanol solution added is 1 times the volume of the crude extract. The amount of the 70% ethanol solution added is 2 times the volume of the crude extract.
[0024] As a preferred embodiment of the present invention, the amount of deionized water added in step S4 is 5-8 times the mass of the precipitate; the amount of glycosyl donor UDPG added is 0.5%-1.5% of the mass of mixture A; and the amount of glycosyltransferase UGT76G1 added is 3%-5% of the mass of mixture A.
[0025] As a preferred embodiment of the present invention, the enzyme catalysis temperature in step S4 is 45-55℃, and the enzyme catalysis time is 24-48h; the vacuum concentration is concentrated to 0.2-0.3 times the mass of mixture B; and the cooling is cooled to room temperature.
[0026] A second aspect of the present invention provides a stevia extract prepared by the above-described preparation method.
[0027] The third aspect of this invention provides the application of the stevia extract prepared as described above in cosmetics.
[0028] The beneficial effects of this invention are:
[0029] (1) This invention uses three enzymes for compound enzymatic hydrolysis. Pectinase degrades the pectin layer and destroys the adhesion structure between cells, so that steviol glycosides can be better separated. Cellulase decomposes the microfibril structural units, so that steviol glycosides can be better dissolved. Papain enzymatically hydrolyzes the proteins in the cell wall / intercellular layer, reducing the role of proteins and polysaccharides in maintaining cell wall integrity. Therefore, the compound enzymes used in this invention can synergistically degrade the cell wall structure and greatly improve the steviol glycoside dissolution rate.
[0030] (2) The present invention combines ultrasonic-assisted extraction and compound enzymatic hydrolysis, which shortens the extraction time, reduces the damage to heat-sensitive components in steviol glycosides caused by high-temperature extraction in traditional methods, and reduces energy consumption, making it suitable for industrial production.
[0031] (3) The present invention uses macroporous resin separation and purification and ethanol solution gradient elution, which can effectively adsorb rebaudine based on the difference in adsorption force between rebaudine and impurities on the resin, and elute in stages to avoid the problem of incomplete elution that may be caused by one-time elution. It selectively enriches rebaudine A, D, etc., and converts rebaudine into high-value-added rebaudine M2 through UGT76G1 enzyme catalysis, thus realizing the precise preparation of active ingredients.
[0032] (4) The stevia extract prepared by the method of the present invention contains more rebaudioside M2 than the extracts prepared by conventional methods on the market. Furthermore, the stevia extract prepared by the present invention has been tested and verified to have better whitening and spot-removing effects. Detailed Implementation
[0033] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0034] The UGT76G1 enzyme used in the embodiments, comparative examples and test examples of this invention was purchased from Mingcheng Huizhong (Jiangsu) Pharmaceutical Research Co., Ltd.; the glycosyl donor UDPG used was purchased from Beijing Bio-Lab Technology Co., Ltd.; the above will not be repeated hereafter.
[0035] Example 1
[0036] A method for preparing a stevia extract with whitening and freckle-removing effects, the preparation method specifically includes the following steps:
[0037] S1. Take stevia leaves, dry them, crush them, and pass them through a 40-mesh sieve to obtain stevia powder for later use;
[0038] S2. Take stevia powder and add it to citrate-disodium hydrogen phosphate buffer at a ratio of 1g:80mL. Stir to obtain a mixture, add compound enzyme, and hydrolyze at 50℃ for 2.5h. After hydrolysis, inactivate the enzyme in a boiling water bath for 10min, cool to room temperature, and extract with ultrasonic power of 225W for 45min. Then, vacuum filter to obtain crude extract.
[0039] The amount of the compound enzyme added is 3% of the mass of the mixture. The compound enzyme consists of cellulase, pectinase and papain in a mass ratio of 3:1:1.
[0040] The cellulase activity is 15000 U / g, the pectinase activity is 8000 U / g, and the papain activity is 2000 U / g.
[0041] S3. The crude extract is purified by adsorption through a D101 macroporous adsorption resin column. First, it is eluted with a 50% ethanol solution, and then with a 70% ethanol solution. The liquids after the two elutions are mixed to obtain the eluent.
[0042] The loading flow rate for both adsorption purification and elution was 1.8 mL / min;
[0043] The amount of 50% ethanol solution added is 1 times the volume of the crude extract, and the amount of 70% ethanol solution added is 2 times the volume of the crude extract.
[0044] S4. Distill the eluent under reduced pressure until no ethanol distills out, cool and centrifuge to obtain a precipitate, add deionized water to the precipitate to obtain mixture A, then add glycosyl donor UDPG and glycosyltransferase UGT76G1 to mixture A in sequence, catalyze with enzyme at 45℃ for 36 hours to obtain mixture B, concentrate under reduced pressure to 0.205 times the mass of mixture B, cool to room temperature to obtain stevia extract rich in rebaudioside M2;
[0045] The amount of deionized water added is 6.5 times the mass of the precipitate; the amount of glycosyl donor UDPG added is 1.0% of the mass of mixture A; and the amount of glycosyltransferase UGT76G1 added is 3% of the mass of mixture A.
[0046] Example 2
[0047] A method for preparing a stevia extract with whitening and freckle-removing effects, the preparation method specifically includes the following steps:
[0048] S1. Take stevia leaves, dry them, crush them, and pass them through a 60-mesh sieve to obtain stevia powder for later use;
[0049] S2. Take stevia powder and add it to citrate-disodium hydrogen phosphate buffer at a ratio of 1g:90mL. Stir to obtain a mixture, add compound enzyme, and enzymatically hydrolyze at 45℃ for 3 hours. After enzymatic hydrolysis, inactivate the enzyme in a boiling water bath for 10 minutes, cool to room temperature, and extract with ultrasonic assistance at a power of 200W for 60 minutes. Then, filter under vacuum to obtain crude extract.
[0050] The amount of the compound enzyme added is 4% of the mass of the mixture. The compound enzyme consists of cellulase, pectinase and papain in a mass ratio of 2:2:1.
[0051] The cellulase activity is 20,000 U / g, the pectinase activity is 10,000 U / g, and the papain activity is 1,600 U / g.
[0052] S3. The crude extract is purified by adsorption through a D101 macroporous adsorption resin column. First, it is eluted with a 50% ethanol solution, and then with a 70% ethanol solution. The liquids after the two elutions are mixed to obtain the eluent.
[0053] The loading flow rate for both adsorption purification and elution was 2.5 mL / min;
[0054] The amount of 50% ethanol solution added is 1 times the volume of the crude extract, and the amount of 70% ethanol solution added is 2 times the volume of the crude extract.
[0055] S4. Distill the eluent under reduced pressure until no ethanol distills out, cool and centrifuge to obtain a precipitate, add deionized water to the precipitate to obtain mixture A, then add glycosyl donor UDPG and glycosyltransferase UGT76G1 to mixture A in sequence, catalyze with enzyme at 55℃ for 24h to obtain mixture B, concentrate under reduced pressure to 0.208 times the mass of mixture B, cool to room temperature to obtain stevia extract rich in rebaudioside M2;
[0056] The amount of deionized water added is 8 times the mass of the precipitate; the amount of glycosyl donor UDPG added is 0.5% of the mass of mixture A; and the amount of glycosyltransferase UGT76G1 added is 4% of the mass of mixture A.
[0057] Example 3
[0058] A method for preparing a stevia extract with whitening and freckle-removing effects, the preparation method specifically includes the following steps:
[0059] S1. Take stevia leaves, dry them, crush them, and pass them through a 50-mesh sieve to obtain stevia powder for later use;
[0060] S2. Take stevia powder and add it to citrate-disodium hydrogen phosphate buffer at a ratio of 1g:85mL. Stir to obtain a mixture, add compound enzyme, and enzymatically hydrolyze at 55℃ for 2 hours. After enzymatic hydrolysis, inactivate the enzyme in a boiling water bath for 10 minutes, cool to room temperature, and extract with ultrasonic assistance at a power of 250W for 30 minutes. Then, filter under vacuum to obtain crude extract.
[0061] The amount of the compound enzyme added is 2% of the mass of the mixture, and the compound enzyme consists of cellulase, pectinase and papain in a mass ratio of 2.5:1.5:1.
[0062] The cellulase activity is 10000 U / g, the pectinase activity is 12000 U / g, and the papain activity is 1800 U / g.
[0063] S3. The crude extract is purified by adsorption through a D101 macroporous adsorption resin column. First, it is eluted with a 50% ethanol solution, and then with a 70% ethanol solution. The liquids after the two elutions are mixed to obtain the eluent.
[0064] The loading flow rate for both adsorption purification and elution was 1.0 mL / min;
[0065] The amount of 50% ethanol solution added is 1 times the volume of the crude extract, and the amount of 70% ethanol solution added is 2 times the volume of the crude extract.
[0066] S4. Distill the eluent under reduced pressure until no ethanol distills out, cool and centrifuge to obtain a precipitate, add deionized water to the precipitate to obtain mixture A, then add glycosyl donor UDPG and glycosyltransferase UGT76G1 to mixture A in sequence, catalyze with enzyme at 50℃ for 48h to obtain mixture B, concentrate under reduced pressure to 0.203 times the mass of mixture B, cool to room temperature to obtain stevia extract rich in rebaudioside M2;
[0067] The amount of deionized water added is 5 times the mass of the precipitate; the amount of glycosyl donor UDPG added is 1.5% of the mass of mixture A; and the amount of glycosyltransferase UGT76G1 added is 5% of the mass of mixture A.
[0068] Comparative Example 1
[0069] Compared with Example 2, the difference is that cellulase is not added to the complex enzyme in step S2 of Comparative Example 1, and the missing weight is made up of pectinase and papain in a mass ratio of 2:1. All other operation steps and parameters remain unchanged.
[0070] Comparative Example 2
[0071] Compared with Example 2, the difference is that pectinase is not added to the complex enzyme in step S2 of Comparative Example 2, and the missing weight is made up of cellulase and papain in a mass ratio of 2:1. All other operation steps and parameters remain unchanged.
[0072] Comparative Example 3
[0073] Compared with Example 2, the difference is that papain is not added to the complex enzyme in step S2 of Comparative Example 3, and the missing weight is made up of cellulase and pectinase in a mass ratio of 1:1. All other operation steps and parameters remain unchanged.
[0074] Comparative Example 4
[0075] Compared with Example 2, the difference is that no compound enzyme was added in step S2 of Comparative Example 4, while the other operation steps and parameters remained unchanged.
[0076] Comparative Example 5
[0077] Compared with Example 2, the difference is that ultrasound-assisted extraction is not used in step S2 of Comparative Example 5, while the other operation steps and parameters remain unchanged.
[0078] Comparative Example 6
[0079] Compared with Example 2, the difference is that in Comparative Example 6, step S3 does not use a 50% ethanol solution for elution, while the other operation steps and parameters remain unchanged.
[0080] Comparative Example 7
[0081] Compared with Example 2, the difference is that in Comparative Example 7, step S3 does not use a 70% ethanol solution for elution, while the other operation steps and parameters remain unchanged.
[0082] Test Example 1
[0083] (1) Determination of steviol glycoside concentration by anthrone-sulfuric acid method: 0.5 g of stevia extract prepared in Examples 1-3 and Comparative Examples 1-7 were placed in 100 mL volumetric flasks, diluted with distilled water, and shaken well to obtain the test solution; 2 mL of the test solution was placed in a 20 mL colorimetric tube, 10 mL of anthrone reagent was slowly added along the tube, shaken well, heated in a boiling water bath for 12 min, removed and rapidly cooled to room temperature under running water, and the absorbance was measured at 620 nm using a UV spectrophotometer. A standard curve was prepared using 0.5 g steviol glycoside standard (regression equation: y = 0.0047x - 0.0008, R...). 2 =0.9991) The concentration of steviol glycosides was calculated based on the standard curve; the results are shown in Table 1.
[0084] (2) Determination of rebaudioside M2 yield by UPLC: 0.5 g of stevia extract prepared in Examples 1-3 and Comparative Examples 1-7 were added to 100 mL of 75% methanol solution, centrifuged at 12000 r / min for 10 min, and the supernatant was collected and filtered through a 0.22 μm microporous membrane to obtain the test solution. A standard curve was plotted using a Waters Acquity UPLC system with the rebaudioside M2 standard solution (regression equation: y = 2382647.36992x + 1547.88625, R0). 2 =0.9955), the yield of rebaudioside M2 was determined according to the standard curve;
[0085] The Waters Acquisition UPLC system used a BEH C18 1.7 μM column (2.1 × 50 mm). The liquid phase conditions were: organic phase - acetonitrile, aqueous phase - ultrapure water; flow rate 0.3 mL / min; column temperature 40℃; UV detection wavelength 210 nm; detection program: 0-1 min 15% organic phase, 6 min 40% organic phase, 7-8 min 15% organic phase. The results are shown in Table 1.
[0086] Table 1
[0087] Stevioside concentration (%) Rebadinidin M2 yield (g / L) Example 1 78 1.65 Example 2 83 1.74 Example 3 76 1.62 Comparative Example 1 61 1.29 Comparative Example 2 65 1.36 Comparative Example 3 67 1.41 Comparative Example 4 27 0.55 Comparative Example 5 66 1.38 Comparative Example 6 55 1.15 Comparative Example 7 57 1.21
[0088] As shown in Table 1, the preparation process of this invention can effectively improve the dissolution rate of steviol glycosides and selectively enrich rebaudioside M2. Specifically, as can be seen from Comparative Examples 1-4, this invention uses three enzymes for compound enzymatic hydrolysis, which can synergistically degrade the cell wall structure and significantly improve the dissolution rate of steviol glycosides. It is not difficult to see from Comparative Example 5 that this invention combines ultrasonic-assisted extraction and compound enzymatic hydrolysis, which can reduce the damage to heat-sensitive components in steviol glycosides caused by high-temperature extraction in traditional methods. Comparative Examples 6-7 also show that this invention uses ethanol for staged elution, which can avoid the problem of incomplete elution that may be caused by one-time elution. Subsequently, rebaudioside is converted into high-value-added rebaudioside M2 by UGT76G1 enzyme catalysis, which can achieve precise preparation of active ingredients.
[0089] Test Example 2
[0090] Stevia extracts obtained in Examples 1-3 and Comparative Examples 1-7 were respectively formulated into toners. The toners, by mass percentage, comprised the following components: 3% stevia extract, 5% glycerin, 0.5% hydroxyacetophenone, and the balance deionized water. To verify the efficacy of the toners, the following tests were conducted, with the specific steps as follows:
[0091] One hundred female volunteers aged 35-45 with darker skin tone and age spots were selected and randomly divided into 10 groups of 10 each. Each group used one of the toners from Examples 1-3 and Comparative Examples 1-7, respectively. Each volunteer applied the toner to their face twice daily, morning and evening, and gently massaged it for 1 minute. The effects were assessed after two months of use. Evaluation criteria: Significant effect: After use, the area of age spots visibly decreased by more than 50%, and the skin tone became noticeably brighter; Improvement: After use, the area of age spots visibly decreased by more than 30% but less than 50%, and the skin tone became brighter; Ineffective: After use, the area of age spots visibly decreased by less than 30%, and the skin tone showed no significant change. The test results are shown in Table 2.
[0092] Table 2
[0093]
[0094] As can be seen from Table 2, the stevia extract prepared by the method of the present invention contains more rebaudioside M2 than the extracts prepared by conventional methods on the market. Furthermore, tests have verified that the stevia extract prepared by the present invention has better whitening and freckle-removing effects.
[0095] In the description of this specification, the references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.
[0096] The above description is merely an example and illustration of the concept of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described or use similar methods to replace them, as long as they do not deviate from the concept of the invention or exceed the scope defined in the claims, they should all fall within the protection scope of the present invention.
Claims
1. A method for preparing a stevia extract with whitening and freckle-removing effects, characterized in that, The preparation method specifically includes the following steps: S1. Pre-treat the stevia raw material to obtain stevia powder for later use; S2. Take stevia powder, add citrate-disodium hydrogen phosphate buffer and compound enzyme, and extract with ultrasonic assistance to obtain crude extract. S3. Purify the crude extract by passing it through a macroporous resin column and elute it with a gradient of ethanol solution to obtain the eluent. S4. Distill the eluent under reduced pressure until no ethanol distills out, cool and centrifuge to obtain a precipitate, add deionized water to the precipitate to obtain mixture A, then add glycosyl donor UDPG and glycosyltransferase UGT76G1 to mixture A in sequence, and catalyze the enzyme to obtain mixture B. Concentrate under reduced pressure and cool to obtain stevia extract rich in rebaudioside M2.
2. The method for preparing stevia extract with whitening and freckle-removing effects according to claim 1, characterized in that, Step S1 specifically includes: taking stevia leaves, drying, crushing, and sieving them to obtain stevia powder for later use.
3. The method for preparing stevia extract with whitening and freckle-removing effects according to claim 1, characterized in that, Step S2 specifically includes: taking stevia powder, adding citrate-disodium hydrogen phosphate buffer and stirring to obtain a mixture, adding a complex enzyme for enzymatic hydrolysis, inactivating the enzyme after enzymatic hydrolysis, cooling, ultrasonic-assisted extraction, and vacuum filtration to obtain a crude extract.
4. The method for preparing stevia extract with whitening and freckle-removing effects according to claim 3, characterized in that, The ratio of stevia powder to citrate-disodium hydrogen phosphate buffer is 1:80-90 g / mL; the amount of compound enzyme added is 2-4% of the mass of the mixture; the compound enzyme consists of cellulase, pectinase and papain in a mass ratio of 2-3:1-2:1; the activity of cellulase is 10000-20000 U / g, the activity of pectinase is 8000-12000 U / g and the activity of papain is 1600-2000 U / g.
5. The method for preparing stevia extract with whitening and freckle-removing effects according to claim 3, characterized in that, The enzymatic hydrolysis time is 2-3 hours, and the enzymatic hydrolysis temperature is 45-55℃; the enzyme inactivation after enzymatic hydrolysis is performed by boiling water bath for 10 minutes; the cooling is performed by cooling to room temperature; the ultrasonic power of the ultrasonic-assisted extraction is 200-250W, and the ultrasonic time is 30-60 minutes.
6. The method for preparing stevia extract with whitening and freckle-removing effects according to claim 1, characterized in that, Step S3 specifically includes: purifying the crude extract by adsorption through a macroporous resin column, then eluting twice with a gradient of ethanol solution, and mixing the liquids after the two elutions to obtain the eluent. The resin inside the macroporous resin column is D101 macroporous adsorption resin.
7. The method for preparing stevia extract with whitening and freckle-removing effects according to claim 6, characterized in that, The loading flow rate for both adsorption purification and elution is 1.0-2.5 mL / min; the gradient elution with ethanol solution is performed twice, first with a 50% ethanol solution and then with a 70% ethanol solution.
8. The method for preparing stevia extract with whitening and freckle-removing effects according to claim 1, characterized in that, In step S4, the amount of deionized water added is 5-8 times the mass of the precipitate; the amount of glycosyl donor UDPG added is 0.5%-1.5% of the mass of mixture A; the amount of glycosyltransferase UGT76G1 added is 3%-5% of the mass of mixture A; the enzyme catalysis temperature is 45-55℃, and the enzyme catalysis time is 24-48h; the vacuum concentration is concentrated to 0.2-0.3 times the mass of mixture B; and the cooling is cooled to room temperature.
9. A stevia extract prepared by the method described in any one of claims 1-8, which has whitening and freckle-removing effects.
10. The application of a stevia extract with whitening and freckle-removing effects prepared by any one of claims 1-8, or the stevia extract with whitening and freckle-removing effects prepared by the method of claim 9 in cosmetics.