Method for preparing and application of pH-responsive core-shell composite enzyme composition

Abstract

The application relates to the technical field of skin care products, in particular to a preparation method and application of a pH-responsive core-shell composite enzyme composition; carboxymethyl chitosan is dissolved in deionized water, papain and keratinase are added, and stirring is carried out until the mixture is uniform to obtain an enzyme-containing solution; under stirring, the obtained solution is added dropwise into a sodium tripolyphosphate aqueous solution to generate an ionic gelation reaction and form an inner core microsphere loaded with the proteinase; superoxide dismutase is added into the inner core microsphere suspension to obtain a composite enzyme composition suspension; a stabilizer is added into the suspension, and freeze-drying is carried out to obtain a composite enzyme composition powder; the papain and the keratinase in the inner core synergistically decompose aged keratin; the SOD in the outer layer synchronously removes free radicals and relieves inflammatory reactions in the keratin metabolism process; the composition has precise pH response characteristics, can rapidly release active ingredients in the weak alkaline region of acne skin, slowly release the active ingredients in the healthy weak acidic skin region, and realizes targeted repair.

Description

Technical Field

[0001] This invention relates to the field of skincare technology, and in particular to the preparation method and application of pH-responsive core-shell complex enzyme compositions. Background Technology

[0002] With the increasing sophistication of skincare needs, enzyme preparations, with their advantages of gentleness, high efficiency, and good biocompatibility, have gradually become core active ingredients in the skincare industry, especially in improving abnormal keratin metabolism, enlarged pores, and treating acne and inflammation. However, many common enzyme-based skincare products on the market suffer from the following technical shortcomings: First, single enzyme preparations have limited efficacy, only achieving single exfoliation or single antioxidant effects, failing to address the multiple needs of acne-prone skin, including pore unclogging and antioxidant repair. Second, free-state enzyme preparations have poor stability, easily deactivated by temperature, pH, and external environmental factors, making it difficult to meet the requirements for long-term storage and room-temperature use. Third, they lack targeted intelligent release mechanisms, resulting in indiscriminate action of the enzyme active ingredients on the entire face, potentially causing excessive irritation to healthy skin.

[0003] While some existing encapsulation techniques can improve enzyme stability, most lack environmental responsiveness, have uncontrollable release rates, and cannot adapt to the pH differences in different areas of the skin (healthy skin has a slightly acidic pH, while acne-prone skin has a neutral to slightly alkaline pH). Therefore, developing a complex enzyme composition with strong stability, pH-targeted release, and multiple functions including gentle regulation of abnormal keratin metabolism and anti-inflammatory repair has become an urgent technical problem to be solved in this field. Summary of the Invention

[0004] In view of the shortcomings of the prior art, one objective of the present invention is to provide a method for preparing a pH-responsive core-shell complex enzyme composition, another objective is to provide a pH-responsive core-shell complex enzyme composition obtained by the preparation method, and a third objective is to apply the composition to cosmetics.

[0005] One of the objectives of this invention is achieved through the following technical solution: A method for preparing a pH-responsive core-shell complex enzyme composition includes the following steps: Step S1 (Core Preparation): Dissolve carboxymethyl chitosan in deionized water, add papain and keratinase, stir and mix evenly to obtain an enzyme-containing solution; Step S2 (ionic crosslinking): Under stirring conditions, the solution obtained in step S1 is added dropwise to an aqueous solution of sodium tripolyphosphate, and an ionic gelation reaction occurs to form microspheres with a core loaded with protease. Step S3 (Surface Loading): Add superoxide dismutase (SOD) to the core microsphere suspension obtained in step S2. Utilize electrostatic adsorption to bind SOD to the surface of the microspheres or the outer network to obtain a composite enzyme composition suspension. Step S4 (Post-processing): Add a stabilizer to the suspension obtained in step S3, freeze-dry to obtain a composite enzyme composition powder, which is a pH-responsive core-shell composite enzyme composition.

[0006] Preferably, in step S1, the mass concentration of carboxymethyl chitosan is 0.5%-2.0%; the mass ratio of papain and keratinase to carboxymethyl chitosan is 1:10 to 1:30.

[0007] Preferably, the carboxymethyl chitosan is one or more of O-carboxymethyl chitosan, N-carboxymethyl chitosan, or N,O-carboxymethyl chitosan.

[0008] Preferably, in step S2, the concentration of the sodium tripolyphosphate aqueous solution is 0.5%-2.0%; the reaction temperature is 20℃-35℃; the reaction time is 30-60 minutes; and the pH value is adjusted to 5.0-6.0.

[0009] Preferably, in step S4, the stabilizer is one or more of trehalose, sucrose, glycerol or lycine glycosyl polypeptide, and the amount added is 2%-5% of the mass of the suspension.

[0010] Preferably, the mass ratio of papain, keratinase and superoxide dismutase is (0.1-1):(1-2):(0.5-2).

[0011] The second objective of this invention is achieved through the following technical solution: A pH-responsive core-shell complex enzyme composition was obtained by the above preparation method.

[0012] Preferably, the pH-responsive core-shell complex enzyme composition uses carboxymethyl chitosan as the wall matrix and forms a three-dimensional network structure through sodium tripolyphosphate ion crosslinking; the core of the pH-responsive core-shell complex enzyme composition is loaded with a protease composition, which includes papain and keratinase; the outer network of the composition is bound to superoxide dismutase (SOD) by electrostatic adsorption; the composition slowly releases its internal enzyme activity in an environment of pH 5.5-6.5, and rapidly swells and releases its internal enzyme activity in an environment of pH 6.5-7.5.

[0013] The third objective of this invention is achieved through the following technical solution: A cosmetic product comprising the above-described pH-responsive core-shell complex enzyme composition, and a cosmetically acceptable carrier.

[0014] Preferably, the cosmetic is a toner, exfoliating gel, face mask, or serum.

[0015] The beneficial effects of this invention are as follows: 1. Significant synergistic effects of multiple enzymes: Papain and keratinase in the core work together to decompose aged keratin, while SOD in the outer layer simultaneously removes free radicals and alleviates inflammatory reactions during keratin metabolism. Exfoliation has both antioxidant and soothing effects, avoiding the irritation problems of traditional exfoliating products; 2. Targeted release of pH: The composition has precise pH response characteristics. Active ingredients are quickly released in the weakly alkaline areas of acne-prone skin and slowly released in the weakly acidic areas of healthy skin, achieving targeted repair, reducing ineffective release and skin irritation, and improving efficacy utilization. Detailed Implementation

[0016] The following is a further explanation with reference to specific implementation methods: Example 1

[0017] The preparation method of the complex enzyme composition in this embodiment is as follows: S1: Dissolve N,O-carboxymethyl chitosan in deionized water to prepare a 1% (w / w) solution. Add papain and keratinase in a 1:2 (w / w) ratio. The total amount added is 1:20 (w / w) of carboxymethyl chitosan. Stir until homogeneous to obtain an enzyme-containing solution.

[0018] S2: Add the above solution dropwise to a 1% sodium tripolyphosphate aqueous solution, adjust the pH to 5.5, and react at 25°C for 45 minutes to obtain a core microsphere suspension.

[0019] S3: Add SOD to the suspension. The mass ratio of papain, keratinase and SOD is 1:2:2. Stir for 30 minutes to allow SOD to be electrostatically adsorbed onto the outer layer of the microspheres.

[0020] S4: Add 2% trehalose by mass as a stabilizer to the suspension, and freeze-dry to obtain the composition powder.

[0021] Example 2

[0022] The difference between this embodiment and Embodiment 1 is that the mass ratio of papain, keratinase and SOD is 0.1:1:0.5, while the other steps are the same.

[0023] Example 3

[0024] The difference between this embodiment and Embodiment 1 is that the mass ratio of papain, keratinase and SOD is 0.5:2:1, while the other steps are the same.

[0025] Example 4

[0026] The difference between this embodiment and Embodiment 1 is that in step S4, 2% by mass of Lycium barbarum glycosyl peptides are added to the suspension as a stabilizer (that is, Lycium barbarum glycosyl peptides replace trehalose). The Lycium barbarum glycosyl peptides were purchased from Fufeng Sinote Biotechnology Co., Ltd., product name: Lycium barbarum polysaccharide peptides, with an effective content ≥98%.

[0027] Comparative Example 1 The difference from Example 1 is that it does not contain papain, and the original amount of papain is replaced with an equivalent amount of keratinase.

[0028] Comparative Example 2 The difference from Example 1 is that it does not contain keratinase, and the original amount of keratinase is replaced with an equivalent amount of papain.

[0029] Comparative Example 3 The difference from Example 1 is that it does not contain superoxide dismutase. Comparative Example 4 The difference from Example 1 is that it does not contain a stabilizer. Comparative Example 5 The difference from Example 1 is that it is a free enzyme composition, without composition encapsulation, and the enzyme ratio is the same as in Example 1.

[0030] Test 1: Mildness Test - Human Patch Test Human skin patch test, the trial method is based on: "Cosmetic Safety Technical Specifications" (2015 edition).

[0031] General testing method: Select a suitable patch tester and use a closed patch test method. Select at least 30 qualified volunteers to participate in the test. Add the test substance and control sample to the square chamber of the patch tester. Apply the patch tester containing the test substance and control sample to the inner forearm of the subject, and gently press it with the palm of your hand to make it evenly applied to the skin. After 24 hours, remove the patch tester and observe the skin reaction according to the standards in Table 1 at 30 minutes (after the indentation disappears), 24 hours, and 48 hours, and record the observation results.

[0032] Following the general method described above, 34 qualified volunteers were selected to participate in the test. The test samples prepared from the complex enzyme compositions of each embodiment and comparative example were used as test substances, and sterile deionized water was used as the control sample. The patch test device containing the test substance and the control sample was applied to the inner forearm of the subject and removed after 24 hours. The skin reaction was observed 30 minutes, 24 hours and 48 hours after the patch test device was removed, according to the grading standard of skin reaction for closed patch test in Table 1, and the observation results were recorded as shown in Table 2.

[0033] Test sample: 2g of compound enzyme composition, 98g of deionized water, mixed evenly to obtain the test sample.

[0034] Table 1 Rating levels Skin reaction 0 negative reaction 1 Suspicious reaction, only slight erythema 2 Weak positive reaction (erythema reaction): erythema, infiltration, edema, and papules may be present. 3 Strong positive reaction (herpes reaction): erythema, infiltration, edema, papules; the reaction may extend beyond the test area. 4 Extremely strong positive reaction (confluent herpes simplex reaction): obvious erythema, severe infiltration, edema, confluent herpes simplex; reaction extends beyond the test area. Table 2

[0035] As can be seen from Table 2, the encapsulation compositions of Examples 1-4 and Comparative Examples 1, 3, and 4 did not cause skin irritation, while Comparative Example 5, which was not encapsulated, showed obvious irritation. This proves that the core-shell structure can effectively reduce the direct effect of enzymes on the skin. Comparative Example 2 showed slight irritation due to the absence of keratinase (containing only papain), indicating that the synergy between papain and keratinase can balance the exfoliating efficacy and gentleness.

[0036] Test 2: Release effect of active ingredients at different pH levels Using the amount of protease activity released from the composite enzyme composition as the evaluation index, the release capacity of the pH-responsive core-shell composite enzyme under different pH conditions was investigated: First, fluorescence spectrophotometry was used, with fluorescein isothiocyanate (FITC)-labeled casein as the specific protease substrate. A standard curve was established based on the change in fluorescence signal after protease digestion of the substrate. The protease content in the unencapsulated comparative example 5 was determined as the total activity content. 1g of the test substance from Example 1 was weighed and immersed in 20mL of [a solution / concentration solution]. In the corresponding buffer systems with pH=6.0 and pH=7.5, the samples were allowed to stand for 1 h, 2 h, 3 h, and 4 h to reach the preset soaking time. After that, each system was centrifuged at 2000 rpm for 10 min, and the supernatant of each group was collected for testing. An equal amount of FITC-labeled casein substrate was added to the supernatant of each group, and the mixture was thoroughly mixed and incubated at 37℃. After the enzymatic reaction was complete, the fluorescence intensity of the system was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The content of active protease in the supernatant was calculated according to the standard curve. Combined with the total protease activity in the test substance, the cumulative release rate of protease under different pH conditions was calculated to determine its pH-responsive release performance.

[0037] The test results are shown in Table 3 below:

[0038] Normal skin has a slightly acidic surface, while the pH value of oily and acne-prone skin can rise to neutral or even slightly alkaline.

[0039] As shown in Table 3, the pH-responsive core-shell complex enzyme composition exhibits significant pH-responsive release characteristics: in the weakly alkaline environment of oily and acne-prone skin at pH 7.5, the release rate and cumulative release amount of the protease are much higher than in the normal skin environment at pH 6.0, with a release rate of 91.67% after 4 hours, which is nearly three times that under pH 6.0 conditions (31.76%). This differentiated release mechanism achieves "release on demand"—slow release on the surface of normal skin to avoid irritation, and rapid swelling and release of active ingredients in oily and acne-prone skin areas, thereby precisely enhancing the keratin-clearing effect and solving the problem of indiscriminate release of traditional enzyme skincare products.

[0040] Test 3: Evaluation of Human Efficacy Sixty volunteers aged 25-40 (all with enlarged pores, dull skin, and closed comedones) were randomly divided into 6 groups (1 example group + 4 comparative groups + 1 blank control group). They used essence water containing the composition of Example 1 and Comparative Examples 1-4 (added at 4%) and blank essence water without the composition, respectively, once in the morning and once in the evening for 28 consecutive days. The efficacy of each example and comparative example was evaluated.

[0041] Test sample: 3g glycerol, 0.02g disodium EDTA, 0.13g sodium polyacrylamide dimethyl taurate, 4g 1,3-butanediol, 0.5g fentanyl ketone, 0.5g hexanediol, 0.03g sodium hyaluronate, 4g of the complex enzyme composition obtained in Example 1 or Comparative Examples 1-4, and 87.43g deionized water, mixed evenly.

[0042] Blank control: 3g glycerol, 0.02g disodium EDTA, 0.13g sodium polyacrylamide dimethyl taurate, 4g 1,3-butanediol, 0.5g succinate, 0.5g hexanediol, 0.03g sodium hyaluronate, and 92.43g deionized water were mixed thoroughly.

[0043] The improvement in skin condition is represented by the improvement rate of each value, calculated as follows: The improvement rate relative to the blank value is calculated as follows: (X before use - X after use) / X before use × 100%.

[0044] The rate of change in fat content = (average fat content of 10 people in each group after using the test sample - average fat content of 10 people in each group before using the test sample, i.e., at T0) / average fat content of 10 people in each group before using the test sample, i.e., at T0.

[0045] TWEL value change rate = [(Average transdermal water loss of 10 people in each group after using the test sample - Average transdermal water loss of 10 people in each group before using the test sample (T0)) / Average transdermal water loss of 10 people in each group before using the test sample (T0)] × 100% Skin smoothness change rate = [(Average skin smoothness SEsm of 10 people in each group after using the test sample - Average skin smoothness SEsm of 10 people in each group before using the test sample (T0)) / Average skin smoothness SEsm of 10 people in each group before using the test sample (T0)] × 100% Pore ​​area change rate = [(average skin pore area of ​​10 people in each group after using the test sample - average skin pore area of ​​10 people in each group before using the test sample, i.e., T0) / average skin pore area of ​​10 people in each group before using the test sample, i.e., T0] × 100%.

[0046] Change rate of skin redness = [(Average skin redness of 10 people in each group after using the test sample - Average skin redness of 10 people in each group before using the test sample (T0)) / Average skin redness of 10 people in each group before using the test sample (T0)] × 100%.

[0047] Table 4 Changes in skin smoothness (%) Change rate of oil content (%) Change in pore area (%) TEWL value change rate (%) Changes in skin redness (%) Example 1 -14.31 -32.28 -54.55 -25.20 -28.77 Comparative Example 1 -11.34 -25.23 -37.25 -23.45 -26.45 Comparative Example 2 -9.75 -16.22 -34.54 -23.98 -23.45 Comparative Example 3 -10.34 -10.13 -23.21 -14.25 -13.71 Comparative Example 4 -5.13 -14.63 -12.35 -11.21 -10.35 Blank example -0.25 -0.37 -0.08 -0.51 -0.66 As shown in Table 4, the pH-responsive core-shell complex enzyme composition of Example 1 exhibited the best overall efficacy in human trials: compared to the blank sample and each comparative example, it improved skin smoothness by 14.31%, reduced pore area by 54.55%, decreased oil content by 32.28%, improved skin redness by 28.77%, and reduced TEWL value by 25.20%. This indicates that the composition effectively unclogs pores through the synergistic effect of papain and keratinase, combined with the anti-inflammatory and oil-controlling effects of SOD, and the gentle release characteristics of the core-shell structure, successfully achieving multiple effects of "gentle exfoliation-oil control-anti-inflammation-barrier repair." Meanwhile, the efficacy indicators of each comparative example (lacking papain, keratinase, SOD, or stabilizer, respectively) were significantly reduced, further confirming the necessity of multi-enzyme synergy, pH-responsive core-shell structure, and stabilizer for product efficacy, effectively addressing the pain points of traditional enzyme skincare products being single-function and highly irritating.

[0048] Test 4: SOD enzyme activity of pH-responsive core-shell complex enzyme composition after freeze-drying Since freeze-drying of daily chemical products is usually carried out at extreme low temperatures of -50°C, SOD is prone to permanent inactivation due to ice crystals and other factors at such extreme temperatures. Therefore, it is necessary to determine the SOD enzyme activity of the pH-responsive core-shell composite enzyme composition obtained in this invention.

[0049] The pH-responsive core-shell complex enzyme compositions obtained in Examples 1, 4, and 4 were used to determine the enzyme activity using the pyrogallol method. The results were repeated three times and the average value was taken.

[0050] The results are shown in Table 5.

[0051] Table 5 sample Example 1 Example 4 Comparative Example 4 Enzyme activity (U / g) 1626 2009 1372 As shown in Table 5, the enzyme activity of Comparative Example 4 (without stabilizer) was significantly lower than that of Example 1 or Example 4, indicating that the stabilizer played a certain protective role for SOD during the freeze-drying process. Furthermore, the enzyme activity of Comparative Example 1 was significantly lower than that of Example 4. Since the only difference between Examples 1 and 4 was the type of stabilizer, it can be inferred that the addition of Lycium barbarum glycosyl peptides provided better protection for SOD at extreme temperatures than conventional stabilizers such as trehalose.

[0052] The embodiments and descriptions above are merely illustrative of the principles and preferred embodiments of the present invention. Various changes and modifications may be made to the present invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. A method for preparing a pH-responsive core-shell complex enzyme composition, characterized in that, Includes the following steps: Step S1: Dissolve carboxymethyl chitosan in deionized water, add papain and keratinase, stir and mix evenly to obtain an enzyme-containing solution; Step S2: Under stirring conditions, the solution obtained in step S1 is added dropwise to an aqueous solution of sodium tripolyphosphate, which causes an ion gelation reaction to form core microspheres loaded with protease. Step S3: Add superoxide dismutase to the core microsphere suspension obtained in step S2. Utilize electrostatic adsorption to bind SOD to the surface of the microspheres or the outer network to obtain a composite enzyme composition suspension. Step S4: Add a stabilizer to the suspension obtained in step S3, freeze dry, and obtain a composite enzyme composition powder, which is a pH-responsive core-shell composite enzyme composition.

2. The method for preparing the pH-responsive core-shell complex enzyme composition according to claim 1, characterized in that: In step S1, the mass concentration of carboxymethyl chitosan is 0.5%-2.0%; the mass ratio of papain and keratinase to carboxymethyl chitosan is 1:10 to 1:

30.

3. The method for preparing the pH-responsive core-shell complex enzyme composition according to claim 1, characterized in that: The carboxymethyl chitosan is one or more of O-carboxymethyl chitosan, N-carboxymethyl chitosan, or N,O-carboxymethyl chitosan.

4. The method for preparing the pH-responsive core-shell complex enzyme composition according to claim 1, characterized in that: In step S2, the concentration of sodium tripolyphosphate aqueous solution is 0.5%-2.0%; the reaction temperature is 20℃-35℃; the reaction time is 30-60 minutes; and the pH value is adjusted to 5.0-6.

0.

5. The method for preparing the pH-responsive core-shell complex enzyme composition according to claim 1, characterized in that: In step S4, the stabilizer is one or more of trehalose, sucrose, glycerol or lycine glycosyl polypeptide, and the amount added is 2%-5% of the mass of the suspension.

6. The method for preparing the pH-responsive core-shell complex enzyme composition according to claim 1, characterized in that: The mass ratio of papain, keratinase and superoxide dismutase is (0.1-1):(1-2):(0.5-2).

7. A pH-responsive core-shell complex enzyme composition obtained by the preparation method according to any one of claims 1-6.

8. The pH-responsive core-shell complex enzyme composition according to claim 7, characterized in that, The pH-responsive core-shell complex enzyme composition uses carboxymethyl chitosan as the wall matrix and forms a three-dimensional network structure through sodium tripolyphosphate ion crosslinking. The core of the pH-responsive core-shell complex enzyme composition is loaded with a protease composition, which includes papain and keratinase. The outer network of the composition is bound to superoxide dismutase by electrostatic adsorption. The composition slowly releases its internal enzyme activity in an environment of pH 5.5-6.5 and rapidly swells and releases its internal enzyme activity in an environment of pH 6.5-7.

5.

9. A cosmetic product, characterized in that, This includes pH-responsive core-shell complex enzyme compositions prepared by any one of claims 1-6, and cosmetically acceptable carriers.

10. The cosmetic product according to claim 9, characterized in that, The cosmetics mentioned are toners, exfoliating gels, face masks, or serums.

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