Compound protease composition and application thereof

Abstract

The invention discloses a compound protease composition and application thereof. The compound protease composition comprises pancreatin, bromelain and papain, and each protease is wrapped by lipidosome to respectively obtain pancreatin lipidosome, bromelain lipidosome and papain lipidosome. The protease of the compound protease composition can stably exist, does not degrade each other, and can permeate into the skin to play a role, so that the compound protease composition has a wide application prospect in the fields of cosmetics and personal care.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of protease, in particular to a complex protease composition and application thereof. BACKGROUND

[0002] Human skin is composed of epidermis and dermis, and the outermost layer of the skin is the stratum corneum, which is mainly composed of keratin, silk polymer protein, keratin bridge protein (the action site of fruit acid), keratinization envelope protein, etc., and is the most important barrier, which plays a role in resisting external stimuli; abnormal keratin metabolism will lead to keratin accumulation, follicle orifice blockage, sebum accumulation, bacterial reproduction and inflammatory reaction, and finally lead to dull skin and acne; acne (acne) is a common problem that absolutely puzzles millions of people around the world, especially teenagers and young adults, which seriously affects people's quality of life.

[0003] Traditional exfoliation products were originally mainly physical abrasive preparations, and then developed into chemical peeling agents, mainly organic acids such as fruit acid; traditional exfoliation methods are irritating, and long-term use may damage the skin barrier function and cause problems such as sensitivity and stinging; later, enzyme preparations were gradually used for exfoliation, and biological enzyme exfoliation is more gentle, but also has the disadvantage of poor effect; the existing technology uses a single protease, especially keratinase, and other proteases such as papaya and pineapple proteases; the enzyme cleavage site for exfoliating the stratum corneum is single, and the effect is poor; although a few patents use complex proteases, such as CN120458978A, CN117946986A and CN115581632A, the stability of the enzymes is not solved because the optimal conditions for various proteases are not consistent, and different proteases will degrade each other, and even peptides; in addition, the penetration of enzymes as biological macromolecules is also a big difficulty.

[0004] Therefore, it undoubtedly has great potential to prepare a complex protease exfoliation product that can stably exist, does not degrade each other, and can penetrate into the skin. SUMMARY

[0005] The purpose of the present application is to provide a complex protease composition, in which the proteases can stably exist and do not degrade each other, and can penetrate into the skin to play a role.

[0006] To solve the above technical problems, the present application adopts the following technical solutions: A complex protease composition comprises trypsin, bromelain and papain, and different protease liposomes are prepared according to the characteristics of each protease, the liposomes are wrapped to form a physical barrier to proteases to play a protective role, and a suitable microenvironment for wrapping enzymes is formed.

[0007] The present application prepares different liposomes according to the characteristics of each protease, provides a buffer environment, forms an optimal microenvironment (local pH, micro-pH environment formed by the wrapped molecules) for wrapping the enzyme, thereby playing a role of physical isolation and protection of the protease; in order to further improve the stability, hydroxypropyl silanized dextran is added in the process of preparing the liposome in the present application, which can be adsorbed on the surface of the protease, stabilize the spatial structure of the enzyme and further provide a microenvironment, in addition, the substance can help the enzyme to be adsorbed on the skin and prolong the residence time.

[0008] In the present application, the liposome molecules are selected as follows: 1) dioleoyl phosphatidylglycerol (DOPG), the liposome formed by the molecule, due to the negative charge of the hydrophilic head, will adsorb hydrogen ions, in order to maintain the electrical neutrality, the water environment inside the liposome will be rich in OH - , thereby creating an alkaline microenvironment; 2) cholesterol, adding cholesterol molecules, due to the amphiphilic property and rigid structure of cholesterol, it is helpful to stabilize the bilayer structure of the liposome; 3) (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP), the liposome formed by the molecule, due to the positive charge of the hydrophilic head, will adsorb hydroxyl ions, in order to maintain the electrical neutrality, the water environment inside the liposome will be rich in hydrogen ions, thereby creating an acidic microenvironment; 4) hydrogenated lecithin, commonly used as raw material for preparing liposomes, which is used to wrap the protease to form a relatively stable buffer environment.

[0009] The preparation method of pancreatin liposome comprises the following steps: 1) two schemes are adopted, hydrogenated lecithin or a mixture of hydrogenated lecithin and DOPG in a molar ratio of 4:6-6:4 is selected as the phospholipid, then cholesterol is added, the molar ratio of cholesterol to phospholipid is 0.3-0.5:1, an organic solvent (such as diethyl ether, not limited to diethyl ether) is added, and a phospholipid solution is obtained by dissolving, the total lipid concentration in the phospholipid solution is 10-15 mg / ml; 2) prepare an aqueous solution of pancreatin by using 100 mM carbonate buffer with pH 8.0, prepare a 2-3% hydroxypropyl silanized dextran aqueous solution under the condition of 50℃ and 500 rpm stirring, then reduce the temperature to room temperature, and then add the aqueous solution of pancreatin under the condition of 30℃ and 300 rpm stirring to dissolve the pancreatin, the concentration of pancreatin is 20-30 mg / ml, after the preparation is completed, filter the solution through a 0.22 μm membrane to remove insoluble substances, and obtain a pancreatin solution; 3) The pancreatin liposome is prepared by mixing the pancreatin solution and the phospholipid solution in a mass ratio of 1:1, and the reverse evaporation method is selected to complete the preparation. The method is suitable for preparing the liposome of a biological macromolecule. An ice water bath is used, and the low-power ultrasonic emulsification method is used to emulsify for 5-10 min under the condition of 40-60 W to form a single phase, a milky white and viscous W / O type primary emulsion. A rotary evaporator is used, the temperature is set to 25-30 DEG C, and the organic solvent is removed under vacuum for 15-20 min to form the liposome (the pancreatin is wrapped in the liposome). Then, the liposome suspension is loaded into a dialysis bag. The molecular weight of the pancreatin is between 10-100 kda, and the diameter is less than 10 nm. The liposome preparation prepared by the present application is about 150 nm, and therefore, a 200 kda dialysis bag is selected to separate the liposome.

[0010] 4) The pancreatin is dialyzed in a large amount of buffer (4 DEG C) for 12-24 hours, and the buffer is replaced for 3-4 times to remove the free pancreatin in the water phase. Then, the dialysis bag is placed in 20 W high molecular weight PEG to remove water, so that the mass is controlled to be the same as that of the pancreatin solution added in step 3).

[0011] The preparation process of the bromelain liposome is basically the same as that of the pancreatin liposome, and the difference is that different liposome molecules are selected in step 1). The two schemes are the hydrogenated lecithin and the mixed phospholipid of hydrogenated lecithin:DOTAP in a molar ratio of 4:6-6:4. In step 2), the protease is bromelain, and the buffer is 100 mM phosphate buffer with a pH of 6.0. The rest is the same.

[0012] The preparation process of the papain liposome is basically the same as that of the bromelain liposome, and the difference is that the protease is papain in step 2). The rest is the same.

[0013] Application of the complex protease composition Based on the protection of the protease liposome on the protease, the complex protease composition can be applied in the keratolytic product. The preparation method of the keratolytic product includes the following steps. The prepared protease liposomes are mixed in equal volumes, and slightly stirred to mix; The outer phase material solution is dissolved, and the outer phase material includes the following components: disodium azelate, lysozyme, ethylhexylglycerin, phenoxyethanol and nicotinamide. After the protease liposome solution is mixed, the outer phase material is added into the mixed liposome solution, and stirred at 200 rpm at room temperature until a transparent and uniform liquid is formed. The mass percentage of each outer phase material is as follows: 2-5% disodium azelate, 0.5-1% lysozyme, 0.1% ethylhexylglycerin, 0.5-1% phenoxyethanol and 2-5% nicotinamide.

[0014] The technical advantages of the present application are: 1) The synergistic effect of complex proteases broadens the range of proteins involved in exfoliation and improves exfoliation efficiency; 2) The enzyme is isolated and protected by liposome formulation technology, providing the enzyme with the optimal microenvironment and significantly improving the enzyme's stability; 3) The stability and retention of the enzyme were further improved by introducing the protective agent hydroxypropyl silanized dextran; 4) The complex protease composition prepared in this invention can enter the skin epidermis and exert its effect under the mediation of liposomes; 5) The preparation method is relatively simple, the quality is controllable, the repeatability is good, and it is easy to standardize production. Detailed Implementation

[0015] To enable those skilled in the art to understand the present invention more clearly and intuitively, the present invention will be further described below.

[0016] This invention proposes a complex protease composition comprising trypsin, bromelain, and papain, and different protease liposomes are prepared according to the characteristics of each protease. The liposomes encapsulate and form a physical barrier to protect the protease and create the optimal microenvironment for encapsulating the enzyme.

[0017] Based on the characteristics of each protease, this invention prepares different liposomes to provide a buffer environment and form an optimal microenvironment (local pH, formed by the encapsulated molecules) for encapsulating the enzyme, thereby achieving the effect of physical isolation and protection of the protease. To further improve stability, this invention also adds hydroxypropyl silanized dextran, which can adsorb onto the protein surface, stabilize the enzyme's spatial structure, and further provide a microenvironment. In addition, this substance can help the enzyme adsorb onto the skin and prolong its retention time.

[0018] The preparation method of pancreatic enzyme liposomes includes the following steps: 1) Two options are adopted: hydrogenated lecithin or a mixture of hydrogenated lecithin and DOPG in a molar ratio of 4:6-6:4, then cholesterol is added, with a cholesterol:phospholipid molar ratio of 0.3-0.5:1, and ether is added to dissolve and obtain a phospholipid solution. The total lipid concentration in the phospholipid solution is 10-15 mg / ml. 2) Prepare an aqueous solution of trypsin using a 100 mM carbonate buffer solution at pH 8.0. Prepare a 2-3% aqueous solution of hydroxypropyl silanized dextran by stirring at 50°C and 500 rpm. Cool to room temperature, then add the aqueous solution of trypsin at 30°C and 300 rpm to dissolve the trypsin. The concentration of trypsin should be 20-30 mg / ml. After preparation, filter through a 0.22 μm membrane to remove insoluble substances and obtain the trypsin solution. 3) Pancreatic enzyme liposomes are prepared by mixing pancreatic enzyme solution and phospholipid solution at a mass ratio of 1:1. This process is carried out using the reverse evaporation method, which is suitable for preparing biomolecular liposomes: Emulsification is performed using an ice-water bath and low-power ultrasonic emulsification at 40-60W for 5-10 minutes to form a single-phase, milky white, viscous W / O type proemulsion; then, using a rotary evaporator at a temperature of 25-30℃ under vacuum, continuous rotary evaporation is performed for 15-20 minutes to remove diethyl ether, thus forming liposomes (with pancreatic enzyme encapsulated within). The liposome suspension is then placed into dialysis bags. Since the molecular weight of pancreatic enzyme is between 10-100 kDa and its diameter is less than 10 nm, and the liposome formulation prepared in this invention is approximately 150 nm, a 200 kDa dialysis bag is selected for liposome separation.

[0019] 4) Dialyze in a large volume buffer (4°C) for 12-24 hours, changing the buffer 3-4 times in between, to remove free trypsin in the aqueous phase. Then, place the dialysis bag in 20W high molecular weight PEG to remove moisture, so that the quality control is the same as the trypsin solution added in step 3).

[0020] The preparation process of bromelain liposomes is basically the same as that of pancreatic enzyme liposomes. The difference is that step 1) is divided into two schemes, namely hydrogenated lecithin and hydrogenated lecithin:DOTAP mixed phospholipids with a molar ratio of 4:6-6:4. In step 2), the protease is bromelain and the buffer is phosphate buffer at pH 6.0. The rest are the same.

[0021] The preparation process of papain liposomes is basically the same as that of bromelain liposomes. The only difference is that the protease in step 2) is papain, while the rest are the same.

[0022] Application of complex protease compositions Based on the protection of proteases by liposomes, complex protease compositions can be used in exfoliating products. Methods for preparing exfoliating products include: Mix the protease liposomes prepared above in equal volumes and stir gently until homogeneous. The external phase substances are dissolved and include the following components: disodium azelate, lysozyme, ethylhexylglycerol, phenoxyethanol, and nicotinamide. After the liposome solutions of each protease are mixed, the external phase substances are added to the mixed liposome solution and stirred at 200 rpm at room temperature until a transparent and homogeneous liquid is formed. The mass percentage of each external phase substance is as follows: 2-5% disodium azelate, 0.5-1% lysozyme, 0.1% ethylhexylglycerol, 0.5-1% phenoxyethanol, and 2-5% nicotinamide.

[0023] The present invention further describes the preparation of exfoliating products in Examples 1-7, the main components of which are shown in Table 1.

[0024] Table 1

[0025] The preparation methods of pancreatic enzyme liposomes in Examples 4 and 5 include the following steps: 1) Mix hydrogenated lecithin and DOPG in a molar ratio of 1:1, add cholesterol (cholesterol:phospholipid molar ratio of 0.5:1), add organic solvent (ether), and dissolve to obtain a phospholipid solution with a total lipid concentration of 15 mg / ml. 2) Prepare an aqueous solution of trypsin using 100 mM carbonate buffer at pH 8.0. Prepare a 2% aqueous solution of hydroxypropyl silanized dextran at 50°C and 500 rpm. Cool to room temperature and then dissolve the trypsin at 30°C and 300 rpm. The concentration of trypsin is 30 mg / ml. After preparation, filter through a 0.22 μm membrane to remove insoluble substances to obtain trypsin. 3) Pancreatic enzyme liposomes are prepared by mixing pancreatic enzyme solution and phospholipid solution at a mass ratio of 1:1. This process is carried out by reverse evaporation, which is suitable for preparing biomacromolecule liposomes: using an ice-water bath and low-power ultrasonic emulsification at 40-60W for 5-10 minutes, a single-phase, milky white, viscous W / O type proemulsion is formed; using a rotary evaporator, the temperature is set to 25-30℃, and under vacuum, the organic phase is removed by continuous rotary evaporation for 15-20 minutes to form liposomes. The liposome suspension is then placed into a dialysis bag. The molecular weight of pancreatic enzyme is between 10-100 kDa and the diameter is less than 10 nm. The liposome formulation prepared in this invention is about 150 nm in diameter. Therefore, a 200 kDa dialysis bag is selected to separate the liposomes.

[0026] 4) Dialyze in a large volume buffer (4°C) for 12-24 hours, changing the buffer 3-4 times in between, to remove free trypsin in the aqueous phase. Then, place the dialysis bag into 20W high molecular weight PEG to remove moisture, so that the quality control is the same as the trypsin solution added in step 3).

[0027] The preparation process of bromelain liposomes in Examples 4 and 7 is basically the same as that of pancreatic enzyme liposomes. The difference is that different liposome molecules and ratios are used in step 1): hydrogenated lecithin and DOTAP are mixed at a molar ratio of 1:1. In step 2), the protease is bromelain and the buffer is phosphate buffer at pH 6.0. The rest are the same.

[0028] The preparation process of papain liposomes in Examples 4 and 6 is basically the same as that of bromelain liposomes. The difference is that in step 2), the protease is papain and the buffer is phosphate buffer at pH 6.0. The rest are the same.

[0029] In Example 2, the preparation method for encapsulating each protease liposome did not involve the addition of hydroxypropyl silanized dextran, and the other steps were the same as those in Example 4.

[0030] In Example 3, none of the proteases were encapsulated with liposomes. Therefore, when preparing the protease solutions, water was used as the solvent and the buffer system was 100 mM pH 7.0 phosphate. 2% hydroxypropyl silanized dextran was dissolved at 50°C and 500 rpm. After the solution was cooled to room temperature, each protease was added to water at a concentration of 10 mg / ml and dissolved by stirring at 500 rpm. After complete dissolution, different protease solutions were obtained.

[0031] The preparation methods of each protease liposome in Example 1 are basically the same as those in Example 4. The difference is that the phospholipid molecules used in step 1) are all hydrogenated lecithin, and the remaining steps are the same as in Example 4.

[0032] The preparation method of the exfoliating products in Examples 1-7 involves mixing equal volumes of the protease liposomes (Examples 1, 2, 4-7) or protease solutions (Example 3) prepared above, and stirring gently to mix them evenly. Dissolve the external phase substances by adding disodium azelate, lysozyme, ethylhexylglycerol, phenoxyethanol, and nicotinamide to the mixed liposome solution and stirring at 200 rpm at room temperature until a transparent and homogeneous liquid is formed. The mass percentage of each external phase substance in the solution is as follows: 2-5% disodium azelate, 0.5-1% lysozyme, 0.1% ethylhexylglycerol, 0.5-1% phenoxyethanol, and 2-5% nicotinamide.

[0033] In addition, comparative examples 1-3 (Table 2) are set up based on Chinese patent applications with publication numbers CN120458978A, CN117946986A, and CN115581632A.

[0034] Table 2

[0035] The preparation methods and formulations of Comparative Examples 1-3 are based on the published texts of relevant patents.

[0036] Test Example 1: Protein Content Detection BCA kit for detecting protein content Solution preparation: Prepare a 2 mg / ml bovine serum albumin (BSA) solution. Calculate the total volume of BCA working solution required based on the quantity of standards and test samples. Typically, 2 mL of working solution is needed for each reaction. Mix BCA solution A and solution B at a volume ratio of 50:1 and mix thoroughly.

[0037] Preparation of standard curve: The 2 mg / ml BSA standard was diluted a series of times with phosphate buffer to prepare at least 6 standard points of different concentrations, as shown in the table.

[0038] Table 3

[0039] Take 100 µl of each concentration standard and add it to a clean test tube or microplate. Use an ELISA reader to detect the absorbance at 562 nm.

[0040] Detection: Add 2 mL of BCA working solution to all test tubes / wells containing standards and samples, mix thoroughly (vortex or pipette), cover with a cap or sealing film, and incubate at 60°C for 30 minutes. After incubation, remove and cool to room temperature, measure absorbance, calculate the standard curve based on the absorbance value, and calculate the sample protein concentration based on the standard curve and sample absorbance value. Dilute the sample to be tested with phosphate to ensure that the absorbance value is between 0.2 and 0.8.

[0041] Test Example 2: Encapsulation efficiency of protease Each protease liposome sample was placed in a 100 kDa dialysis bag and dialyzed in a large volume buffer. Free small proteins diffused out of the bag, while the liposomes, due to their large size, remained inside. 10% Triton X-100 solution was added to the liposome suspension to achieve a final concentration of 0.5%–1%. The solution was vortexed; the solution changed from opalescent to clear, indicating that the liposomes were completely destroyed. Finally, the protein content was determined using the BCA method from Test Example 1, and the results are shown in Table 4.

[0042] Table 4

[0043] Test Example 3: Liposome Stability The exfoliating product in Example 1 was tested using dynamic light scattering (DLS) to determine its Z-average diameter at 25°C and 48°C for 0, 14, and 28 days. The results are shown in Table 5.

[0044] Table 5

[0045] Results: After 28 days, the particle size did not change significantly, but increased slightly, and was less than 16%.

[0046] Test Example 4: Enzyme Activity and Enzyme Activity Retention Rate The exfoliating products from Examples 1-7 were placed for 0 hours and 28 days. BSA solutions were prepared using 100 mM pH 8.0 carbonate buffer and 100 mM pH 6.0 phosphate buffer, respectively, and keratin solutions were prepared using pH 7.0 phosphate buffer. The concentrations of BSA and keratin were 30 mg / L. Then, the exfoliating products from each example and comparative example were mixed with keratin and BSA solutions at a volume ratio of 1:2. 10% Triton X-100 solution was added to the composition to achieve a final concentration of 0.5%-1%, which disrupted liposomes and released proteases. After reacting at 37°C for 10 min, TCA was added to precipitate unreacted proteins. The precipitate was removed by filtration through a 0.22 μm aqueous filter membrane. Small molecule peptides were quantitatively analyzed by BCA method. Based on the absorbance value, relative enzyme activity was calculated using the 0-hour sample as a baseline, and the enzyme activity retention rate was calculated. The absorbance value A1 in Example 1 is used as the baseline, and Ai is the absorbance value in Example 1. Relative enzyme activity = Ai / A1 × 100%; enzyme activity retention rate = relative enzyme activity. 28d / relative enzyme activity 0h ×100%, the calculation results are shown in Tables 6 and 7.

[0047] Table 6 Relative enzyme activity results

[0048] Table 7 Retention Rate Test Results

[0049] In Table 7, " / " indicates that the relative enzyme activity is less than 20%, indicating no obvious activity, so no retention rate test was performed.

[0050] Test Example 5: Exfoliation Effect The test sample consisted of 60 participants aged 20-40 years, 15 men and 15 women, with an additional 30 participants serving as controls. Examples 1-7 and Comparative Examples 1-3 were used to treat the face. The treatment method involved applying each composition to the face for 10 minutes, followed by washing it off. After 0, 2, 4, and 6 days, high-resolution imaging and quantification of skin texture, pores, and pigmentation on the skin surface were performed using VISIA® Complexion Analysis image analysis technology. The results are shown in Table 8.

[0051] Table 8 Exfoliation Effects

[0052] Test Example 6: Permeability The Franz diffusion cell assay, used to analyze transdermal efficiency, consists of a donor and a receiver chamber separated by a pigskin skin sample. The protease liposome suspension (donor) is added to the donor chamber. The receiver chamber is filled with receiver solution: 50 mM pH 7.0 PBS + 1% phenoxyethanol + 0.1% ethylhexylglycerin, maintained at a constant temperature of 37°C. Receiver solution samples were taken at time points 2, 4, 8, 12, and 24 h, and isothermal fresh receiver solution was added. The protein content in the receiver solution was determined using the BCA method: this represents the amount absorbed transdermally, i.e., the amount of drug that actually penetrates the skin and enters the systemic circulation. Protein content in the skin was also determined: after the experiment, the skin sample was homogenized, perforated, and the protein content was measured. This represents the amount of drug retained in each layer of the skin, which is crucial for localized effects such as exfoliation. The protein detection method is the same as in Test Example 1. The test results are shown in Table 9.

[0053] Table 9 Permeability Test Results

[0054] Results Analysis: Comparing the "relative enzyme activity at 0 hours" of Examples 1 (hydrogenated lecithin liposome encapsulation), Example 4 (liposome encapsulation), and Example 3 (unencapsulated) in Table 6, it can be found that although the direct activity of Example 3 is slightly higher, combined with the enzyme activity retention rate data in Table 7, the enzyme activity retention rates of Examples 1 and 4 for the three substrates after 28 days (>89%) are much higher than that of Example 3 (<32%). Furthermore, the difference between Examples 1 and 4 is not significant. This demonstrates that liposomes effectively protect the protease, preventing its inactivation during storage and providing product shelf-life stability.

[0055] The advantages of multi-enzyme synergy are obvious: broad-spectrum exfoliation ability. The results of Examples 5, 6 and 7 show that the complex protease broadens the protein range of exfoliation and enhances the exfoliation effect.

[0056] Excellent stability: The results in Table 5 show that the liposomes of Example 1, after being stored at 25°C and 48°C for 28 days, only increased in particle size by less than 16%, indicating that the liposome structure is very stable and does not easily aggregate or break down. This is the physical basis for its ability to protect enzyme activity for a long time.

[0057] Excellent skin penetration and retention: Example 1: Only 34% of the protein remained in the supply chamber, while a high percentage (41.2%) remained in the skin layer, and 24.8% entered the receiving chamber. This demonstrates that liposomes, as carriers, can significantly promote the penetration of proteases into the stratum corneum and accumulate in large quantities within the skin, thereby exerting a continuous and efficient exfoliating effect. In Example 3 (unencapsulated) and Comparative Examples 1-3, the vast majority of proteins (>79%) remained in the supply chamber and could not effectively penetrate, with extremely low retention in the skin layer (<16%). This indicates that without the assistance of liposomes, free enzymes cannot effectively penetrate the skin. The VISIA clinical test data in Table 8 directly confirm that all the above-mentioned technical advantages are translated into excellent practical efficacy.

[0058] Examples 1 and 2 showed significantly higher improvement rates (up to 37.8%) in three dimensions: skin texture, pore permeability, and pigmentation, compared to all other groups. The regimens in Examples 1 and 2 demonstrated remarkable efficacy in significantly improving skin texture, minimizing pores, and lightening pigmentation in clinical trials (Table 8), far exceeding the effects of the unencapsulated complex enzyme formulation (Example 3) and existing technologies (Comparative Examples 1-3).

[0059] Based on the above conclusions, this invention provides a superior, efficient, and stable enzymatic exfoliation product solution that is better than existing technologies and has broad application prospects in the fields of cosmetics and personal care.

[0060] The above description of the embodiments is provided to enable those skilled in the art to understand and apply the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the embodiments described herein, and any improvements and modifications made to the present invention by those skilled in the art based on the disclosure thereof should be within the scope of protection of the present invention.

Claims

1. A complex protease composition, characterized in that, The pancreatin, bromelain and papain are included, and each of the proteases is encapsulated by liposome to obtain pancreatin liposome, bromelain liposome and papain liposome respectively; wherein, The liposome encapsulating pancreatin in the pancreatin liposome comprises monophospholipid component of hydrogenated lecithin or dipospholipid component of hydrogenated lecithin, dioleoylphosphatidylglycerol and cholesterol; The liposome encapsulating bromelain in the bromelain liposome comprises monophospholipid component of hydrogenated lecithin or dipospholipid component of hydrogenated lecithin and (2,3-dioleoyl-propyl)-trimethylammonium chloride and cholesterol; The liposome encapsulating papain in the papain liposome comprises monophospholipid component of hydrogenated lecithin or dipospholipid component of hydrogenated lecithin and (2,3-dioleoyl-propyl)-trimethylammonium chloride.

2. The complex protease composition of claim 1, wherein, The preparation method of the pancreatin liposome, the bromelain liposome and the papain liposome comprises the following steps respectively: 1) selecting monophospholipid component or dipospholipid component corresponding to the composition of the protease liposome, then adding cholesterol, the molar ratio of cholesterol to phospholipid being 0.3-0.5:1, then adding an organic solvent to obtain a phospholipid solution, the total lipid concentration in the phospholipid solution being 10-15 mg / ml; the molar ratio of hydrogenated lecithin to another phospholipid component in the dipospholipid component being 4:6-6:4; 2) first preparing an aqueous solution of the corresponding protease with a buffer, then preparing a 2-3% hydroxypropyl silanized dextran aqueous solution, and reducing the temperature to room temperature; under stirring, the aqueous solution of the corresponding protease is added to the hydroxypropyl silanized dextran aqueous solution to dissolve the protease, so that the concentration of the corresponding protease is 20-30 mg / ml, after preparation, the insoluble substances are removed by filtration with a 0.22 μm water-based membrane to obtain the corresponding protease solution; 3) mixing the corresponding protease solution and the phospholipid solution in a mass ratio of 1:1 to prepare the liposome encapsulating the corresponding protease, and the process is completed by reverse evaporation; 4) loading the suspension of the liposome into a dialysis bag to separate free protease and concentrate.

3. The complex protease composition of claim 2, wherein, In step 3), in the reverse evaporation method, first, an ice water bath is used, low-power ultrasonic emulsification is used, and emulsification is performed for 5-10 min under the condition of 40-60 W to form a single phase, milky white and viscous W / O type primary emulsion; then a rotary evaporator is used, the temperature is set to 25-30°C, and the organic solvent is removed under vacuum for 15-20 min to form the liposome.

4. The complex protease composition of claim 2, wherein, In step 4), the suspension of the liposome is first dialyzed in a buffer for 12-24 hours, and the buffer is replaced 3-4 times to remove free protease in the water phase, and then the dialysis bag is placed in 20W high molecular weight PEG to remove water, so that the mass is controlled to be the same as that of the corresponding protease solution added in step 3).

5. The complex protease composition of claim 2, wherein, In step 2), the aqueous solution of pancreatin is prepared by using 100 mM carbonate buffer with pH 8.0; The aqueous solution of bromelain is prepared by using 100 mM phosphate buffer with pH 6.0; The aqueous solution of papain is prepared by using 100 mM phosphate buffer with pH 6.

0.

6. Use of the complex protease composition according to any one of claims 1-5 in a keratolytic product and / or a product for inhibiting hyperkeratosis.

7. A keratolytic product characterized in that, The complex protease composition according to any one of claims 1-5.

8. The keratolytic product of claim 7, wherein, The pancreatin liposome, bromelain liposome and papain liposome are mixed in equal volume.

9. The keratolytic product of claim 8, wherein, The outer phase material further comprises disodium azelate, lysozyme, ethylhexylglycerin, phenoxyethanol, nicotinamide; The mass percentage of each component in the outer phase material in the keratolytic product is as follows: 2-5% disodium azelate, 0.5-1% lysozyme, 0.1% ethylhexylglycerin, 0.5-1% phenoxyethanol, 2-5% nicotinamide.

10. The method of preparing an exfoliating product according to claim 9, wherein the exfoliating product is a cosmetic product. The method comprises the following steps: 1) The pancreatin liposome, bromelain liposome and papain liposome are mixed in equal volume and stirred slightly; 2) The outer phase material is dissolved, and disodium azelate, lysozyme, ethylhexylglycerin, phenoxyethanol and nicotinamide are added into the mixed liposome solution respectively, and stirred at 200 rpm at room temperature until a transparent and uniform liquid is formed; the mass percentage of each component in the outer phase material is as follows: 2-5% disodium azelate, 0.5-1% lysozyme, 0.1% ethylhexylglycerin, 0.5-1% phenoxyethanol, 2-5% nicotinamide.

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