Stabilized protease for skin care and method of making and use thereof

Abstract

The present application belongs to the technical field of stabilized protease, and specifically discloses a stabilized protease for skin care, a preparation method and application thereof. The preparation method comprises a primary cross-linking reaction and a secondary cross-linking reaction. The primary cross-linking reaction is a cross-linking reaction between the amino group of the protease and the carboxyl group of carbomer, or a cross-linking reaction between the sulfydryl group of the protease and allyl-modified carbomer, or a cross-linking reaction between the hydroxyl group of the protease and the carboxyl group of carbomer; and the secondary cross-linking reaction is a secondary cross-linking reaction of the product obtained in the primary cross-linking reaction under an amine-reactive cross-linking reagent. The stabilized protease obtained by the technology has low skin permeability, which is mainly due to its immobilization characteristics; at the same time, the enzyme activity is retained through cross-linking and, in some embodiments, through the stabilization treatment of a physical additive. The present application is particularly related to an immobilized papain product suitable for local skin care.

Description

Technical Field

[0001] This invention belongs to the field of stabilized protease technology, specifically a stabilized protease for skin care, its preparation method, and its application. Background Technology

[0002] Protease activity is crucial for epidermal homeostasis. Therefore, proteases offer various potential benefits when applied to the skin, but are limited by certain constraints. Papain is a potent protease derived from papaya and some other plants. However, it rapidly loses its activity in solution. This is because, similar to all proteases, papain undergoes self-digestion and denaturation. Furthermore, other problems associated with conventional papain products can be encountered when using them as topical skin care agents related to skin penetration and irritation. Therefore, it is essential to develop protease products that do not have these limitations for use in skin care.

[0003] To overcome the aforementioned difficulties in conventional technologies, the present invention provides a stable cross-linked protease product. Summary of the Invention

[0004] In view of the deficiencies in the existing technology, the purpose of this invention is to provide a stabilized protease for skin care, its preparation method and application.

[0005] The first objective of this invention is to provide a method for preparing a stabilized protease for skin care, characterized by comprising the following steps: S1: Primary cross-linking reaction The amino group of the protease is cross-linked with the carboxyl group of carbomer using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysulfosuccinimide as cross-linking agents in a mass ratio of 3:1.75. The amount of protease added is 0.1%-2%. Alternatively, the cross-linking reaction between the thiol groups of the protease and the allyl-modified carbomer requires ammonium persulfate as an initiator, with the amount used being 0.5 mol% of the protease's thiol groups, and the amount of protease added being 0.1%-2%. Alternatively, the hydroxyl groups of the protease are cross-linked with the carboxyl groups of carbomer, using DCC (N,N'-dicyclohexylcarbodiimide) and DMAP (4-dimethylaminopyridine) as cross-linking agents in a mass ratio of 20:1, and the amount of protease added is 0.1%-2%. S2: Secondary crosslinking reaction The product obtained from the primary crosslinking reaction undergoes a secondary crosslinking reaction under an amine reactive crosslinking reagent. The reaction is promoted by adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysulfosuccinimide in a mass ratio of 3:1.75.

[0006] Preferably, the amount of crosslinking agent added in step S2 is 1%-5%, and the crosslinking agent in step S2 is dimethyl hexamethylenediimide (DMA), bis(sulfosuccinimide) octanoate (BS3), dimethyl octanoate (DMS), dimethyl heptamethine (DMP), or disuccinimide octanoate (DSS).

[0007] Preferably, it further includes S3: adding a physical stabilizer after the secondary crosslinking reaction.

[0008] Preferably, the amount of the physical stabilizer added is 0.1%-5% of the mass of the secondary crosslinking reaction product.

[0009] Preferably, the physical stabilizer is a sugar or a sugar polymer.

[0010] Preferably, the physical stabilizer is sodium alginate, trehalose, mannitol, glycerol, xanthan gum, sucrose, or sorbitol.

[0011] Preferably, the protease is papain, fig protease, bromelain, or actinidin.

[0012] A second objective of the present invention is to provide a stabilized protease for skin care prepared by the above method.

[0013] A third objective of the present invention is to provide the application of the above-mentioned stabilized protease for skin care in skin care products for relieving dry, aging or damaged skin, wherein the amount of the stabilized protease added is 0.0001%.

[0014] A fourth objective of this invention is to provide the application of the above-mentioned stabilized protease for skin care in wound or burn debridement ointment, wherein the amount of stabilized protease added is 0.0001%.

[0015] The outermost layer of the epidermis, the stratum corneum, is composed of dead cells that have migrated upwards from below within a few days. These dead cells are typically shed from the skin surface through a process called epidermal exfoliation, which stimulates the growth of new cells at deeper levels. Younger skin is more efficient at this process than older or damaged skin. Consequently, older skin appears dull, thick, and less radiant. This can be exacerbated by environmental factors such as sun exposure; hormonal influences such as androgens, estrogens, and epidermal growth factor; and vitamin deficiencies such as vitamins A and D. Protease activity is a key factor in the exfoliation process. Therefore, applying proteases to the skin for cosmetic effects such as skin smoothing and anti-aging is desirable. However, the use of proteases in cosmetics and other applications is known to have three main limitations: instability, easy loss of activity, and skin penetration.

[0016] The substances involved in this invention are explained as follows: (1) Protease Proteases are enzymes that catalyze the breakdown of proteins. As proteins themselves, proteases have a tendency to self-degrade and are inherently unstable. This proteinaceous nature also makes them allergenic. Furthermore, because of their ability to degrade proteins, they can penetrate deeper into the epidermis and cause damage to the underlying layers. Suitable proteases for use in this invention include papain. Papain is a cysteine ​​protease obtained from the latex of papaya and mountain papaya, most often from the green, unripe fruit. These proteases are present at concentrations between approximately 0.1% and 5% by weight.

[0017] (2) Carbomer The protease is first immobilized on the carbomer. The carbomer is a homopolymer of acrylic acid with a high molecular weight, crosslinked with any of several polyol allyl ethers (e.g., allyl ether pentaerythritol, allyl ether of sucrose, or allyl ether of propylene). Suitable examples of carbomers are carbomer 910, carbomer 934, carbomer 934p, carbomer 940, and carbomer 941, where the numerical suffix indicates the average molecular weight of the polymer chain.

[0018] As used herein, "immobilized" protease refers to a protease that has already undergone a primary cross-linking reaction with a carbomer. A suitable cross-linking agent for this primary cross-linking reaction is a reagent capable of cross-linking a carboxyl group to a primary amine. An example of a suitable cross-linking agent is the water-soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC, a zero-length cross-linking agent used to couple a carboxyl group to a primary amine. This group forms a cross-link bond upon reaction with a free amine group. In this embodiment, EDC is the primary cross-linking agent that transforms into a disubstituted urea during the reaction. NHS is an ester and catalyst that increases the cross-linking rate and remains unchanged during the cross-linking reaction.

[0019] Alternatively, a suitable crosslinking agent for this primary crosslinking reaction is one capable of crosslinking and coupling the double-bonded functional group to the thiol group of the protease. An example of a suitable crosslinking agent is allyl alcohol. Modifying carbomer with allyl alcohol introduces an allyl functional group (-CH=CH2) by reacting the carbomer's carboxyl group with the alcohol's hydroxyl group. Acryloyl chloride introduces an allyl functional group to carbomer and acts as a "bridging precursor" for the crosslinking reaction, forming an ester bond (-CO-O-CH=CH2) with the carbomer carboxyl group during the reaction. In the primary crosslinking reaction, the click reaction is used directly, through the reaction of the protease's thiol group with the modified carbomer.

[0020] Alternatively, a suitable crosslinking agent for this primary crosslinking reaction is a reagent capable of crosslinking and coupling the carboxyl group to the hydroxyl group of the protease. An example of a suitable crosslinking agent is the use of DCC (N,N'-dicyclohexylcarbodiimide) and DMAP (4-dimethylaminopyridine) as efficient condensing agents to catalyze the esterification reaction between the hydroxyl groups (such as serine and threonine residues on the side chains) in the protease molecule and the carboxyl group of carbomer, forming an ester bond (-CO-O-) and achieving covalent crosslinking.

[0021] (3) Crosslinking agent Once immobilized on a carbomer, the immobilized protease reacts with an amine-reactive crosslinking agent in a secondary crosslinking reaction to form a protease-carbomer copolymer, also referred to herein as a "stabilized protease". The crosslinking agent is preferably a low molecular weight crosslinking agent, such that it will react completely or nearly completely in the secondary crosslinking reaction. Examples of suitable crosslinking agents that can be used include iminoester crosslinking agents such as dimethyl hexamethyleneimine (DMA), dimethyl heptaethyleneimine (DMP), dimethyl octylimine (DMS), etc. The amine-reactive crosslinking agent can be provided at concentrations ranging from 0.05% to about 5%, preferably between about 1% and 5%, by weight. In some embodiments, DMP is used.

[0022] (4) Physical stabilizers Following chemical cross-linking, the immobilized and cross-linked protease is optionally further stabilized with a physical stabilizer such as a sugar or sugar polymer. Suitable examples of sugars or sugar polymers are sodium alginate, trehalose, mannitol, glycerol, and xanthan gum. In one embodiment, sodium alginate is used. The concentration of this physical stabilizer can be between about 0.1% and 5% by weight.

[0023] (5) Application / Formulation The stabilized protease of the present invention retains protease activity in solution or dry form, exhibits minimal skin irritation, and is relatively easy to provide in formulations. Therefore, exemplary embodiments of the present invention provide much more stable and safer protease products compared to protease products of conventional techniques, particularly embodiments of stabilized papain products.

[0024] Stabilizing proteases are suitable for use in cosmetics and personal care formulations, such as exfoliating products, anti-wrinkle and / or anti-aging products, bath additives, hair care products, liquid soaps and commercial soaps, cleansing solutions, moisturizing cleansing cloths, oils or powders, and anti-acne products. Stabilizing proteases are particularly suitable for treating dry, aged, or damaged skin by applying one or more of the stabilizing proteases of the present invention to dry, aged, or damaged skin requiring relief.

[0025] Cosmetic and / or personal care formulations may be in the form of, for example, water-in-oil or oil-in-water emulsions, alcoholic or alcohol-containing formulations, vesicular dispersions or gels of ionic or nonionic amphoteric lipids. Exemplary cosmetic and / or personal care formulations contain between 0.5% and 5% by weight, preferably between 1% and 3%, of stabilized protease.

[0026] The stabilized proteases of the present invention are also suitable for pharmaceutical applications, such as debridement applications. Debridement is the removal of dead or damaged tissue from a wound (e.g., an ulcerated wound or a burn) to aid healing. In one embodiment of the invention, one or more stabilized proteases of the present invention are applied to a skin wound or burn requiring debridement. Exemplary formulations and products comprising stabilized proteases for treating wounds or burns include bandages / dressings, patches, washing solutions, ointments or gels, or synthetic tissues. In some embodiments, the debridement composition, such as a bandage, dressing, or patch, may optionally include an antimicrobial agent. For debridement applications, the amount of stabilized protease included in the pharmaceutical composition will be an amount that effectively removes necrotic tissue and liquefies pus in the wound and achieves substantially complete removal of such substances within a reasonable time (e.g., within seven days).

[0027] In addition to skin care applications, the stabilized protease of the present invention can also be adapted to other applications known in the art where a stable form of the protease is desired. An exemplary application is an oral care composition.

[0028] Topical compositions containing stabilized proteases may further include a variety of other ingredients conventionally used in cosmetic, personal care, or pharmaceutical formulations, provided they do not unacceptably alter the benefits of the invention. Non-limiting examples of optional conventional ingredient categories include fragrances, pigments, colorants, essential oils, astringents, anti-aging agents, anti-acne agents, anti-caking agents, defoamers, antimicrobial agents, antioxidants, binders, pH adjusters, skin bleaching and brightening agents, skin conditioning agents, sunscreens, anti-inflammatory agents, humectants, thickeners, and vitamins.

[0029] The beneficial effects of this invention are: (1) The stabilized protease for skin care prepared in this invention has minimal skin penetration and minimal skin irritation.

[0030] (2) The stabilized protease for skin care prepared in this invention retains protease activity in solution or in dry form. Attached Figure Description

[0031] Figure 1 This is the stabilized protease solution of the present invention.

[0032] Figure 2 This is a comparison chart of the activity of the stabilized protease and the free protease of the present invention on day 0.

[0033] Figure 3 The present invention aims to stabilize the enzyme activity stability of papain under different storage conditions.

[0034] Figure 4 This invention aims to improve the activity of the stabilized papain diluent.

[0035] Figure 5 This is the FITC standard curve in the experimental examples of this invention.

[0036] Figure 6 This is a before-and-after comparison of the acne-removing effect of the product in Experiment Example 2.

[0037] Figure 7 This is a before-and-after comparison of the whitening effect of the product in Experiment Example 2. Detailed Implementation

[0038] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0039] The technical solution of the present invention is as follows: A stabilized protease for skin care, its preparation method, and its application.

[0040] The first objective of this invention is to provide a method for preparing a stabilized protease for skin care, characterized by comprising the following steps: S1: Primary cross-linking reaction The amino group of the protease is cross-linked with the carboxyl group of carbomer using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysulfosuccinimide as cross-linking agents in a mass ratio of 3:1.75. The amount of protease added is 0.1%-2%. Alternatively, the cross-linking reaction between the thiol groups of the protease and the allyl-modified carbomer requires ammonium persulfate as an initiator, with the amount used being 0.5 mol% of the protease's thiol groups, and the amount of protease added being 0.1%-2%. Alternatively, the hydroxyl groups of the protease are cross-linked with the carboxyl groups of carbomer, using DCC (N,N'-dicyclohexylcarbodiimide) and DMAP (4-dimethylaminopyridine) as cross-linking agents in a mass ratio of 20:1, and the amount of protease added is 0.1%-2%. S2: Secondary crosslinking reaction The product obtained from the primary crosslinking reaction undergoes a secondary crosslinking reaction under an amine reactive crosslinking reagent. The reaction is promoted by adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysulfosuccinimide in a mass ratio of 3:1.75.

[0041] In some embodiments, the amount of crosslinking agent added in step S2 is 1%-5%, and the crosslinking agent in step S2 is dimethyl hexamethyleneimine, bis(sulfosuccinimide) octanoate, dimethyl octanoate, dimethyl heptamethine, or disuccinimide octanoate.

[0042] In some embodiments, S3 is further included: adding a physical stabilizer after the secondary crosslinking reaction.

[0043] In some embodiments, the amount of the physical stabilizer added is 0.1%-5% of the mass of the secondary crosslinking reaction product.

[0044] In some embodiments, the physical stabilizer is a sugar or a sugar polymer.

[0045] In some embodiments, the physical stabilizer is sodium alginate, trehalose, mannitol, glycerol, xanthan gum, sucrose, or sorbitol.

[0046] In some embodiments, the protease is papain, fig protease, bromelain, or actinidin.

[0047] The stabilized protease for skin care was prepared by the above method.

[0048] The above-mentioned stabilized protease for skin care is used in skin care products to relieve dry, aging or damaged skin, wherein the amount of stabilized protease added is 0.0001%.

[0049] A fourth objective of this invention is to provide the application of the above-mentioned stabilized protease for skin care in wound or burn debridement ointment, wherein the amount of stabilized protease added is 0.0001%.

[0050] Example 1 A stable papain product, retaining its protease activity and safer than that containing free papain, was produced through a primary cross-linking reaction. In the primary cross-linking reaction, 1% papain was cross-linked with carbomer using carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) along with N-hydroxysulfosuccinimide (NHS). Next, a secondary cross-linking reaction was carried out, in which the immobilized papain was reacted with dimethyl heptamethrin (DMP) to further cross-link the cross-linked papain, thereby forming a stable cross-linked papain product. DMP and papain were provided at a concentration of 0.25% by weight. The final stabilized protease solution was dialyzed to remove free small molecules, etc.

[0051] The enzyme activity / stability of the stabilized papain products were tested after storage.

[0052] Example 2 A primary cross-linking reaction yielded a stable papain product that retains its protease activity and is safer than that containing free papain. In the primary cross-linking reaction, carbomer was first conjugated with 0.25% allyl alcohol via esterification; subsequently, it was mixed with 0.25% papain in an aqueous solution, and a thiol-ene click reaction was performed to form a stable carbon-sulfur bond between the allyl group of carbomer and the thiol group of papain, achieving primary cross-linking. Next, a secondary cross-linking reaction was carried out, in which the immobilized papain reacted with dimethyl heptanediimide (DMP) to further cross-link the cross-linked papain, thereby forming a stable cross-linked papain product. By weight, DMP was provided at a concentration of 1%, papain at a concentration of 0.25%, and allyl alcohol-modified carbomer at a concentration of 1%.

[0053] Example 3 A primary cross-linking reaction yielded a stable papain product that retains its protease activity and is safer than that containing free papain. In this primary cross-linking reaction, DCC (N,N'-dicyclohexylcarbodiimide) was used in combination with 0.2% 4-dimethylaminopyridine (DMAP) as a catalytic synergist, causing esterification of 0.25% papain in a 1% carbomer 980 aqueous system. The hydroxyl groups of the papain formed stable ester bonds with the carboxyl groups of the carbomer, achieving covalent cross-linking fixation. Next, a secondary cross-linking reaction was carried out, in which the immobilized papain reacted with dimethyl heptamethrin (DMP) to further cross-link the cross-linked papain, thereby forming a stable cross-linked papain product. By weight, DMP was provided at a concentration of 0.25%, papain at a concentration of 0.25%, and carbomer at a concentration of 1%.

[0054] Figure 1 This is the stabilized protease solution of the present invention. Figure 2 This demonstrates that the stabilized protease group prepared by this invention has higher activity compared to free papain. Figure 3 The stability of stabilized papain under different storage conditions was tested over time. The graph shows that sealed storage resulted in better stability. Figure 4 This illustrates the changes in enzyme activity after different dilutions of a concentrated solution of stabilized papain. After an 8-fold dilution, the papain activity showed a linear relationship with the dilution factor. However, at dilutions less than 8 times, the activity increased with increasing dilution, indicating that the stabilized papain exhibits a protective effect on enzyme activity. Figure 5 This is the standard curve of the test method used in the process of testing enzyme activity. The curve equation is: y = -0.006x + 0.6517. All papain activities are converted from this standard curve.

[0055] Experimental Example 1 The 0.25% stabilized papain and 0.1% sodium alginate prepared in the above three examples were combined. The activity percentages of the experimental sample of stabilized papain stored with 0.1% sodium alginate and the control sample of stabilized papain product not stored with sodium alginate are detailed in Table 1.

[0056] Table 1

[0057] Sodium alginate increases the stability of proteases. As shown in Table 1, the addition of sodium alginate did not significantly reduce protease activity.

[0058] The following methods were used to test the activity and stability of papain: Papain activity was tested using a protease activity assay kit (fluorescence method).

[0059] (1) Setting up the FITC (fluorescein isothiocyanate) standard curve. Take FITC (1 mM), add 495 μl of AssayBuffer, mix well, and prepare 500 μl of 10 μM FITC standard solution. Take 0, 1.56, 3.12, 6.25, 12.5, 25, 50, and 100 μL of 10 μM FITC standard solution and add them to the standard wells of a 96-well plate, and make up to 100 μL with AssayBuffer (or prepare the standard solution by serial dilution). At this time, the concentration and amount of FITC in each well of the standard curve are 0, 0.16, 0.31, 0.62, 1.25, 2.5, 5, 10 M or 0, 0.016, 0.031, 0.062, 0.125, 0.25, 0.5, 1 nmol, respectively.

[0060] (2) Take an appropriate amount of proteinase K (2.5 mg / ml) and dilute it 50-fold with Assay Buffer. For example, take 2 μl of Proteinase K (2.5 mg / ml), add 98 μl of Assay Buffer, and mix by inversion to obtain 50 μg / ml Proteinase K. Then dilute it 50-fold with Assay Buffer. For example, take 2 μl of Proteinase K (50 μg / ml), add 98 ml of Assay Buffer, and mix by inversion to obtain 1 μg / ml Proteinase K. When used as a positive control, use 50 μl per well, i.e., 50 ng / well. The dilution factor for the positive control can be adjusted according to the activity of the proteinase in the sample.

[0061] (3) Take 1-50 μl of sample or diluted sample into the wells of a 96-well plate, and add Assay Buffer to bring the total volume to 50 ml. Simultaneously, set up wells containing only Assay Buffer as blank controls. (4) Add 50 μl of Protease Substrate working solution to each well except the FITC standard curve and mix well.

[0062] (5) Immediately use an appropriate microplate reader, set the excitation wavelength to 485 nm and the emission wavelength to 530 nm for fluorescence detection. Record the initial reading as F1. React at 37℃ for 10-30 min, and record the reaction time as T. Usually, measure the fluorescence value every 2 min or 5 min, and record the nth fluorescence reading as Fn. The enhancement of fluorescence depends on the amount of FITC generated after the protease hydrolyzes the substrate, ΔF = Fn - F1; finally, take the data within the time range where the reaction is linear after the start of the reaction for analysis or calculation. Establish a FITC standard curve, substitute ΔF into the standard curve, and the amount of FITC generated by the protease catalysis in the sample during the reaction time can be calculated (referred to as Sa). The formula for calculating protease activity is as follows: Protease Activity (mU / ml) = Sa * n / (T * Vsample (ml)). Note: Sa is the amount of FITC generated (nmol) determined according to the standard curve; the standard curve is as follows. Figure 6 As shown.

[0063] Experimental Example 2 The stabilized protease obtained in Example 1 was used to make a skin care product, with an addition amount of 0.0001%. It was applied twice daily, 2 ml each time, for 30 consecutive days. Skin condition was observed before and after application. See details below. Figure 6 and Figure 7 As can be seen from the picture, after 30 days of application, the acne on the forehead was significantly reduced, and the skin became whiter, indicating that the product has a good effect on acne removal and whitening.

[0064] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0065] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing a stabilized protease for skin care, characterized in that, Includes the following steps: S1: Primary cross-linking reaction The amino group of the protease is cross-linked with the carboxyl group of carbomer using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysulfosuccinimide as cross-linking agents in a mass ratio of 3:1.

75. The amount of protease added is 0.1%-2%. Alternatively, the cross-linking reaction between the thiol groups of the protease and the allyl-modified carbomer requires ammonium persulfate as an initiator, with the amount used being 0.5 mol% of the protease's thiol groups, and the amount of protease added being 0.1%-2%. Alternatively, the hydroxyl groups of the protease are cross-linked with the carboxyl groups of carbomer, using DCC (N,N'-dicyclohexylcarbodiimide) and DMAP (4-dimethylaminopyridine) as cross-linking agents in a mass ratio of 20:1, and the amount of protease added is 0.1%-2%. S2: Secondary crosslinking reaction The product obtained from the primary crosslinking reaction undergoes a secondary crosslinking reaction under an amine reactive crosslinking reagent. The reaction is promoted by adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysulfosuccinimide in a mass ratio of 3:1.

75.

2. The method for preparing a stabilized protease for skin care as described in claim 1, characterized in that, The amount of crosslinking agent added in step S2 is 1%-5%, and the crosslinking agent in step S2 is dimethyl hexamethyleneimine, bis(sulfosuccinimide) octanoate, dimethyl octanoate, dimethyl heptamethine, or disuccinimide octanoate.

3. The method for preparing a stabilized protease for skin care as described in claim 2, characterized in that, It also includes S3: adding a physical stabilizer after the secondary crosslinking reaction.

4. The method for preparing a stabilized protease for skin care as described in claim 3, characterized in that, The amount of the physical stabilizer added is 0.1%-5% of the mass of the secondary crosslinking reaction product.

5. The method for preparing a stabilized protease for skin care as described in claim 4, characterized in that, The physical stabilizer is a sugar or a sugar polymer.

6. The method for preparing a stabilized protease for skin care as described in claim 5, characterized in that, The physical stabilizer is sodium alginate, trehalose, mannitol, glycerin, xanthan gum, sucrose, or sorbitol.

7. The method for preparing a stabilized protease for skin care as described in claim 6, characterized in that, The protease is papain, fig protease, bromelain, or kiwi protease.

8. A stabilized protease for skin care prepared by the method of any one of claims 1-7.

9. The use of a stabilized protease for skin care as described in claim 8 in a skin care product for relieving dry, aging, or damaged skin, characterized in that, The amount of the stabilized protease added is 0.0001%.

10. The application of the stabilized protease for skin care as described in claim 8 in a wound or burn debridement ointment, characterized in that, The amount of the stabilized protease added is 0.0001%.

Images