Keratinase-containing composition and application thereof in preparation of painless depilatory paste

Abstract

The invention provides a composition containing keratinase. The composition is prepared from the following components: 5000 to 20000 U / g of keratinase, 2 to 10 wt% of calcium thioglycolate, 0.5 to 5 wt% of folium cortex eucommiae extract, 0.3 to 2 wt% of eucalyptus multibracteata essential oil, more than or equal to 70% of cineole and 0.05 to 1 wt% of hyaluronic acid binding peptide HABP. The composition is used for preparing the painless depilatory paste. The keratinase is innovatively compounded with the calcium thioglycolate and other components, so that the skin irritation is remarkably reduced while efficient hair removal is ensured, and the keratinase hair removal cream has the multiple advantages of long-acting hair inhibition, immediate repair, use experience improvement and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of daily necessities, and particularly relates to a composition containing keratinase and application of the composition in preparing a painless depilatory cream. Background Art

[0002] Depilatory creams are personal care products that remove hair by chemically dissolving hair keratin. They are widely used on the limbs, underarms, lips, and other areas. Their core function is to disrupt the disulfide bonds in hair, making it fragile and easily breakable, allowing it to be removed by physical wiping or rinsing.

[0003] In actual application, the limitations of traditional depilatory creams are particularly prominent. First, although thioglycolic acid ingredients can effectively destroy disulfide bonds, their highly alkaline environment can easily damage the skin barrier, leading to dryness, redness, swelling, and even burns after use. The risk is especially high for users with sensitive skin. Secondly, depilatory creams are less effective on coarse and hard hair, often requiring extended dwell time, further exacerbating skin irritation. In addition, existing products mostly rely on a single chemical ingredient and lack a sustained inhibitory effect on hair follicles, resulting in rapid hair regeneration and requiring frequent use.

[0004] To address these problems, existing technologies attempt to relieve irritation by adding soothing ingredients such as aloe vera extract or vitamin E, or by adjusting the pH value to a "safe range" of 10.5-12.5 to balance hair removal effect and mildness. However, these improvements still cannot fundamentally solve the potential damage to the skin caused by chemical hair removal.

[0005] In terms of hair removal efficiency, traditional depilatory creams have limited penetration into hair follicles, acting only on the surface and failing to inhibit hair growth at its source. Some studies have attempted to introduce proteases, such as papain, to aid in the breakdown of keratin, but their activity is unstable and easily affected by other ingredients in the formula, resulting in limited effectiveness. The viscous texture of existing depilatory creams can lead to uneven application, affecting even hair removal. If left unwashed, any residual cream can clog pores and cause folliculitis.

[0006] Another technical difficulty lies in post-hair removal skin repair. Some high-end products incorporate moisturizers such as glycerin or hyaluronic acid, but these moisturizers are short-lived and fail to synergize with the hair removal ingredients to enhance their effectiveness. In recent years, a small number of studies have explored the use of bio-enzymes such as keratinase, which can specifically degrade keratin under milder conditions. However, existing keratinase preparations suffer from low activity and poor stability, and are particularly susceptible to inactivation in complex formulations, limiting their commercial application.

[0007] The odor problem of existing depilatory creams has yet to be effectively addressed. The sulfide produced by the decomposition of thioglycolic acid has a strong odor that is difficult to completely mask even with the addition of fragrance, impacting the user experience. Some products attempt to mask the odor with natural essential oils, but these ingredients can further irritate the skin or react with the depilatory ingredients, reducing the product's effectiveness.

[0008] Therefore, it is necessary to design a composition containing keratinase and use thereof in preparing a painless depilatory cream. Summary of the Invention

[0009] In order to overcome the defects in the prior art, a composition containing keratinase and its application in preparing a painless depilatory cream are provided.

[0010] In order to achieve the above object, the present invention provides the following technical solutions: A keratinase-containing composition comprises the following components: keratinase: 5000-20000 U / g, calcium thioglycolate: 2-10 wt%, eucommia ulmoides leaf extract: 0.5-5 wt%, eucalyptus multibracte essential oil: 0.3-2 wt%, cineole content ≥70%, and hyaluronic acid binding peptide HABP: 0.05-1 wt%.

[0011] The keratinase is prepared by the following steps: (1) Inoculate the recombinant Bacillus subtilis SCK6 into a fermentation medium containing 40-60 g / L oatmeal and 70-90 g / L soy peptone and culture at 37°C for 48-72 hours; (2) Collect the fermentation broth by centrifugation, add ammonium sulfate to 30% saturation to precipitate impurities, and then adjust to 50% saturation to precipitate the target enzyme; (3) Dissolve the precipitate in 50 mM HAc-NaAc buffer, purify it through an ion exchange column, and collect the 0.25 M NaCl elution peak to obtain keratinase.

[0012] The fermentation medium in step (1) further comprises: 0.1-0.3 mM calcium chloride and 0.5-0.7 g / L magnesium sulfate heptahydrate, with a pH value of 6.8-7.2.

[0013] The preparation method of the Eucommia ulmoides leaf extract comprises the following steps: (1) Crush the dried Eucommia ulmoides leaves and add 60-80% ethanol at a material-liquid ratio of 1:8-1:12; (2) Reflux extraction twice, each time for 1-3 hours, the first extraction temperature is 78-82℃, and the second extraction temperature is 68-72℃; (3) The extracts were combined, concentrated under reduced pressure, and then freeze-dried to obtain the Eucommia ulmoides leaf extract.

[0014] The composition further comprises the following auxiliary components: 2-6 wt % of cetearyl alcohol; 0.3-1.2 wt % of carbomer 940, which is pre-swelled with glycerol at a mass ratio of 1:2-4; and 0.5-2 wt % of sodium hydroxide.

[0015] The hyaluronic acid binding peptide HABP is compounded with keratinase by the following steps: (1) Mix hyaluronic acid binding peptide HABP and keratinase at a molar ratio of 0.8-1.2:1; (2) Incubate at 35-40°C and pH 7.5-8.5 for 45-90 minutes.

[0016] During the incubation process, ultrasonic treatment was applied at 10-20 kHz and the power density was controlled at 50-100 W / L.

[0017] The composition further comprises 0.3-2 wt % of nano-sized trehalose.

[0018] The nano-sized trehalose is prepared by supercritical CO2-assisted spray drying under the following conditions: inlet temperature of 120-150°C, CO2 pressure of 8-12 MPa, and the particle size of the nano-sized trehalose is less than 200 nm.

[0019] The invention discloses an application of a composition containing keratinase, wherein the composition is used for preparing a painless depilatory cream.

[0020] Compared with the prior art, the advantages and beneficial effects of the present invention are: 1. The composition of the present application, through the synergistic action of keratinase and calcium thioglycolate, retains the high efficiency of chemical depilation while significantly reducing the irritation of traditional depilatory products. Keratinase can accurately identify and decompose the unique structure of hair keratin, allowing the amount of calcium thioglycolate used to be significantly reduced, thereby controlling the pH value of the formula system to a range closer to the physiological environment of the skin, avoiding the damage of the skin barrier caused by strong alkalinity. This dual mechanism of action is not only suitable for ordinary hair, but is particularly effective for treating coarse and hard hair, greatly shortening the product's residence time on the skin.

[0021] 2. The composition of the present application creatively integrates hair removal efficacy and repair functions. The complex of hyaluronic acid-bound peptide and keratinase can form a protective network during the hair removal process, continuously release moisturizing factors, and effectively relieve the dryness and discomfort caused by chemical hair removal. The active ingredients rich in Eucommia ulmoides leaf extract can not only soothe the skin, but also regulate the metabolic activity of hair follicle cells, delaying the rate of hair regeneration from the root. The high-purity eucalyptol component of polybracteate essential oil plays a dual role, acting as a natural antibacterial agent to prevent infection and promoting the penetration of other active ingredients, making the hair removal effect more uniform and thorough.

[0022] 3. Keratinase, derived from an optimized fermentation process, exhibits excellent storage stability under specific ionic conditions, overcoming the vulnerability of traditional enzyme preparations to inactivation in complex formulations. Ultrasound-assisted compounding ensures uniform bonding of the functional peptide and enzyme molecules, maintaining the stability of the active ingredient during storage and use. The application of nanomaterials not only improves the bioavailability of protective agents such as trehalose but also enhances the physical stability of the entire formulation, effectively preventing stratification of the paste or sedimentation of the active ingredient.

[0023] 4. The introduction of keratinase reduces reliance on chemical depilatories, ultimately reducing exposure to irritants. The meticulously purified enzyme removes impurities that could cause allergic reactions. Combined with the anti-allergic properties of Eucommia ulmoides leaf extract, the product is suitable for a wider range of patients. Nano-scale protective ingredients rapidly repair damaged skin microstructures during the depilatory process, reducing the likelihood of inflammatory reactions.

[0024] 5. The addition of natural essential oils cleverly neutralizes the chemical odors often found in traditional hair removal products, making the entire experience more pleasant. The specially designed gel matrix has unique rheological properties, allowing for easy and even application and rapid dissolution during rinsing, eliminating the worry of residual cream residue. This texture design also ensures even distribution of the active ingredients across the skin's surface, resulting in more consistent hair removal results.

[0025] 6. This invention innovatively combines keratinase with ingredients such as calcium thioglycolate, which significantly reduces skin irritation while ensuring efficient hair removal. It also has multiple advantages such as long-lasting hair suppression, instant repair and improved user experience. DETAILED DESCRIPTION

[0026] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0027] In this application, the sources of various raw materials are briefly described as follows: Calcium thioglycolate: Wuhan Kemik Biopharmaceutical Technology Co., Ltd., purity ≥99%; Eucalyptus polyphylla essential oil: Shandong Longchang Animal Health Products Co., Ltd., cineole content ≥ 70%; Hyaluronic acid binding peptide HABP: Nanjing Senbeijia Biotechnology Co., Ltd.; Cetearyl alcohol: Jinan Zhonghe Chemical Co., Ltd., Lanette 16; Carbomer 940: Wuhan Desheng Biotechnology Co., Ltd., viscosity 40000-60000 mPa·s; Sodium hydroxide: Cangzhou Liushi Chemical Co., Ltd., food grade flake caustic soda.

[0028] A keratinase-containing composition comprises the following components: keratinase: 5000-20000 U / g, calcium thioglycolate: 2-10 wt%, eucommia ulmoides leaf extract: 0.5-5 wt%, eucalyptus multibracte essential oil: 0.3-2 wt%, cineole content ≥70%, and hyaluronic acid binding peptide HABP: 0.05-1 wt%.

[0029] The keratinase is prepared by the following steps: (1) Inoculate the recombinant Bacillus subtilis SCK6 into a fermentation medium containing 40-60 g / L oatmeal and 70-90 g / L soy peptone and culture at 37°C for 48-72 hours; (2) Collect the fermentation broth by centrifugation, add ammonium sulfate to 30% saturation to precipitate impurities, and then adjust to 50% saturation to precipitate the target enzyme; (3) Dissolve the precipitate in 50 mM HAc-NaAc buffer, purify it through an ion exchange column, and collect the 0.25 M NaCl elution peak to obtain keratinase.

[0030] The fermentation medium in step (1) further comprises: 0.1-0.3 mM calcium chloride and 0.5-0.7 g / L magnesium sulfate heptahydrate, with a pH value of 6.8-7.2.

[0031] The preparation method of the Eucommia ulmoides leaf extract comprises the following steps: (1) Crush the dried Eucommia ulmoides leaves and add 60-80% ethanol at a material-liquid ratio of 1:8-1:12; (2) Reflux extraction twice, each time for 1-3 hours, the first extraction temperature is 78-82℃, and the second extraction temperature is 68-72℃; (3) The extracts were combined, concentrated under reduced pressure, and then freeze-dried to obtain the Eucommia ulmoides leaf extract.

[0032] The composition further comprises the following auxiliary components: 2-6 wt % of cetearyl alcohol; 0.3-1.2 wt % of carbomer 940, which is pre-swelled with glycerol at a mass ratio of 1:2-4; and 0.5-2 wt % of sodium hydroxide.

[0033] The hyaluronic acid binding peptide HABP is compounded with keratinase by the following steps: (1) Mix hyaluronic acid binding peptide HABP and keratinase at a molar ratio of 0.8-1.2:1; (2) Incubate at 35-40°C and pH 7.5-8.5 for 45-90 minutes.

[0034] During the incubation process, ultrasonic treatment was applied at 10-20 kHz and the power density was controlled at 50-100 W / L.

[0035] The composition further comprises 0.3-2 wt % of nano-sized trehalose.

[0036] The nano-sized trehalose is prepared by supercritical CO2-assisted spray drying under the following conditions: inlet temperature of 120-150°C, CO2 pressure of 8-12 MPa, and the particle size of the nano-sized trehalose is less than 200 nm.

[0037] The invention discloses an application of a composition containing keratinase, wherein the composition is used for preparing a painless depilatory cream.

[0038] The present invention combines keratinase with calcium thioglycolate to reduce alkaline irritation while ensuring hair removal efficiency, and introduces natural ingredients such as eucommia leaf extract and eucalyptus multiflora essential oil to synergistically soothe and repair the skin. The efficient preparation process of keratinase enables it to maintain stable activity in the formula, and the introduction of hyaluronic acid-bound peptides further enhances the targeting of skin moisturizing and hair removal ingredients. In addition, the addition of nano-trehalose improves the stability and permeability of the formula, and ultrasonic treatment technology optimizes the composite efficiency of enzymes and peptides. These improvements jointly solve the technical bottlenecks of traditional depilatory creams in terms of irritation, residual black spots, odor and long-term hair suppression, providing a safer and more efficient solution for painless hair removal.

[0039] The technical solution of the present invention is further illustrated by the following examples and comparative examples, but the protection scope of the present invention is not limited thereto.

[0040] Example 1 The keratinase composition of this embodiment comprises the following components: keratinase 20,000 U / g, calcium thioglycolate 6wt%, eucommia leaf extract 5wt%, eucalyptus multiflora essential oil 0.3wt%, and hyaluronic acid binding peptide HABP 0.525wt%. The preparation of keratinase uses recombinant Bacillus subtilis SCK6 to culture in a fermentation medium containing 60 g / L oatmeal and 80 g / L soy peptone for 72 hours. After ammonium sulfate fractionation precipitation, the fermentation broth is dissolved in 50 mM HAc-NaAc buffer and purified by ion exchange column to collect the 0.25 M NaCl elution peak. The fermentation medium is supplemented with 0.1 mM calcium chloride and 0.7 g / L magnesium sulfate heptahydrate, pH 7.0. The eucommia leaf extract is extracted with 70% ethanol reflux at a 1:12 material-liquid ratio, the first extraction is at 82°C for 1 hour, and the second extraction is at 68°C for 3 hours. The auxiliary components include 6 wt% cetearyl alcohol, 0.75 wt% carbomer 940, and 1.25 wt% sodium hydroxide. A hyaluronic acid-binding peptide and keratinase are mixed in a 1:1 molar ratio and incubated at 37.5°C, pH 8.0, for 67.5 minutes while being ultrasonically treated at 15 kHz and a power density of 75 W / L. The composition also contains 1.15 wt% nano-sized trehalose and was prepared using supercritical CO2-assisted spray drying at an inlet temperature of 135°C and a CO2 pressure of 10 MPa.

[0041] Example 2 The composition of this example contains 5000 U / g of keratinase, 10 wt% calcium thioglycolate, 0.5 wt% of Eucommia ulmoides leaf extract, 2 wt% of Eucalyptus polybracte essential oil, and 0.05 wt% of hyaluronic acid-binding peptide (HABP). The keratinase fermentation medium contains 40 g / L oatmeal and 90 g / L soy peptone. The culture is incubated for 48 hours and supplemented with 0.3 mM calcium chloride and 0.5 g / L magnesium sulfate heptahydrate, pH 6.8. Eucommia ulmoides leaf extraction uses 80% ethanol at a 1:8 ratio of solid to liquid, with the first extraction at 78°C for 3 hours and the second extraction at 72°C for 1 hour. Auxiliary components include 2 wt% cetearyl alcohol, 1.2 wt% carbomer 940, and 0.5 wt% sodium hydroxide. The peptide and enzyme are combined in a 1.2:1 molar ratio, incubated at 40°C, pH 7.5 for 45 minutes, and ultrasonicated at 20 kHz with a power density of 50 W / L. Nano-sized trehalose 0.3wt% was prepared under the following conditions: inlet temperature 120℃, CO2 pressure 12 MPa.

[0042] Example 3 The composition of this example contains 12,500 IU / g keratinase, 2 wt% calcium thioglycolate, 2.75 wt% eucommia leaf extract, 1.15 wt% eucalyptus essential oil, and 1 wt% hyaluronic acid-binding peptide (HABP). The fermentation medium contains 50 g / L oatmeal and 70 g / L soy peptone. The culture is incubated for 60 hours and supplemented with 0.2 mM calcium chloride and 0.6 g / L magnesium sulfate heptahydrate, pH 7.2. The eucommia leaf extract uses 60% ethanol at a 1:10 ratio of solid to liquid, with the first extraction at 80°C for 2 hours and the second extraction at 70°C for 2 hours. Auxiliary components include 4 wt% cetearyl alcohol, 0.3 wt% carbomer 940, and 2 wt% sodium hydroxide. The peptide and enzyme are combined in a 0.8:1 molar ratio, incubated at 35°C, pH 8.5 for 90 minutes, and ultrasonicated at 10 kHz with a power density of 100 W / L. Nano-trehalose 2 wt%, preparation conditions are inlet temperature 150℃, CO2 pressure 8 MPa.

[0043] Comparative Example 1

[0044] Compared with Example 1, this comparative example does not contain keratinase, and only uses 15 wt % of calcium thioglycolate as the depilatory active ingredient. Other components are the same as those in Example 1.

[0045] Comparative Example 2

[0046] Compared with Example 2, this comparative example used ordinary papain instead of keratinase with an enzyme activity of 5000 U / g, and did not adopt an ultrasonic-assisted compounding process.

[0047] Comparative Example 3

[0048] Compared with Example 3, this comparative example did not add nano-trehalose, and the Eucommia ulmoides leaf extract was replaced by an equal amount of aloe vera extract.

[0049] Comparative Example 4

[0050] Compared with Example 1, the cineole content of the Eucalyptus polybracteraceus essential oil in this comparative example is only 30%, and the hyaluronic acid binding peptide HABP is not used.

[0051] Comparative Example 5

[0052] Compared with Example 2, the keratinase in this comparative example was not purified by ion exchange, and the crude enzyme preparation after ammonium sulfate precipitation was directly used.

[0053] Performance test results and analysis

[0054] The keratinase-containing compositions obtained in the Examples and Comparative Examples were subjected to performance testing using the following methods to evaluate product performance: skin irritation was tested using a patch test, hair removal efficiency was assessed by hair breakage time, hair reduction efficacy was evaluated by observing the hair regrowth cycle, and stability was tested by examining enzyme activity retention after six months of room temperature storage. The test results are shown in Table 1.

[0055] Table 1 Performance test results

[0056] As can be seen from Table 1, Examples 1-3 showed significant advantages in all tests. The skin irritation index was much lower than that of Comparative Examples 1-5, confirming that the synergistic effect of keratinase and calcium thioglycolate can effectively reduce irritation. The hair breakage time of Example 1 was the shortest at only 5 minutes, which was 58% shorter than that of Comparative Example 1, indicating that high enzyme activity combined with an optimized formula can significantly improve hair removal efficiency. The hair suppression period of the three examples all reached more than 8 days, which was 60% longer than that of Comparative Example 1, reflecting the synergistic hair suppression effect of Eucommia ulmoides leaf extract and Eucalyptus multi-bracte essential oil. In terms of enzyme activity retention rate, Example 1 reached 95%, which was 30 percentage points higher than that of Comparative Example 2, proving the protective effect of ion exchange purification and nano-trehalose on enzyme stability. The odor score showed that the combination of high-purity eucalyptol and hyaluronic acid-binding peptide can effectively neutralize sulfide odor.

[0057] Comparative Example 1 demonstrates the high irritation of single chemical depilatories. While effective, they suffer from poor skin tolerance. Comparative Example 2 demonstrates the vulnerability of conventional proteases to inactivation in complex systems, and the lack of ultrasound-assisted compounding reduces their effectiveness. Comparative Example 3 demonstrates the important role of nano-sized trehalose in skin protection and the unique advantages of Eucommia ulmoides leaf extract in hair removal. Comparative Example 4 confirms that low-purity essential oils and the lack of HABP can compromise overall product performance. Comparative Example 5 demonstrates that crude enzyme preparations containing impure proteins can increase the risk of allergies.

[0058] These results fully demonstrate the beneficial effects of this invention: the scientifically formulated combination of keratinase and chemical depilatory agents achieves efficient and gentle hair removal, the innovative composite process ensures the stability of the active ingredients, and the synergistic effect of natural components addresses odor and hair loss issues. The parameters in this example are rationally set, and the optimized combination of component contents and process conditions achieves an optimal balance of product performance.

[0059] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A composition containing keratinase, characterized in that The composition comprises the following components: keratinase: 5000-20000 U / g, calcium thioglycolate: 2-10 wt%, eucommia leaf extract: 0.5-5 wt%, eucalyptus multibracte essential oil: 0.3-2 wt%, cineole content ≥70%, and hyaluronic acid binding peptide HABP: 0.05-1 wt%.

2. A keratinase-containing composition according to claim 1, characterized in that The keratinase is prepared by the following steps: (1) Inoculate the recombinant Bacillus subtilis SCK6 into a fermentation medium containing 40-60 g / L oatmeal and 70-90 g / L soy peptone and culture at 37°C for 48-72 hours; (2) Collect the fermentation broth by centrifugation, add ammonium sulfate to 30% saturation to precipitate impurities, and then adjust to 50% saturation to precipitate the target enzyme; (3) Dissolve the precipitate in 50 mM HAc-NaAc buffer, purify it through an ion exchange column, and collect the 0.25 M NaCl elution peak to obtain keratinase.

3. A keratinase-containing composition according to claim 2, characterized in that The fermentation medium in step (1) further comprises: 0.1-0.3 mM calcium chloride and 0.5-0.7 g / L magnesium sulfate heptahydrate, with a pH value of 6.8-7.

2.

4. A keratinase-containing composition according to claim 1, characterized in that The preparation method of the Eucommia ulmoides leaf extract comprises the following steps: (1) Crush the dried Eucommia ulmoides leaves and add 60-80% ethanol at a material-liquid ratio of 1:8-1:12; (2) Reflux extraction twice, each time for 1-3 hours, the first extraction temperature is 78-82℃, and the second extraction temperature is 68-72℃; (3) The extracts were combined, concentrated under reduced pressure, and then freeze-dried to obtain the Eucommia ulmoides leaf extract.

5. A keratinase-containing composition according to claim 1, characterized in that The composition further comprises the following auxiliary components: cetearyl alcohol: 2-6 wt%; carbomer 940: 0.3-1.2 wt%, which is pre-swollen with glycerin at a mass ratio of 1:2-4; Sodium hydroxide: 0.5-2 wt%.

6. A keratinase-containing composition according to claim 1, characterized in that The hyaluronic acid binding peptide HABP is compounded with keratinase by the following steps: (1) Mix hyaluronic acid binding peptide HABP and keratinase at a molar ratio of 0.8-1.2:1; (2) Incubate at 35-40°C and pH 7.5-8.5 for 45-90 minutes.

7. A keratinase-containing composition according to claim 6, characterized in that: During the incubation process, ultrasonic treatment was applied at 10-20 kHz and the power density was controlled at 50-100 W / L.

8. The keratinase-containing composition according to claim 1, characterized in that: The composition further comprises 0.3-2 wt % of nano-sized trehalose.

9. A keratinase-containing composition according to claim 8, characterized in that: The nano-sized trehalose is prepared by supercritical CO2-assisted spray drying under the following conditions: inlet temperature of 120-150°C, CO2 pressure of 8-12 MPa, and the particle size of the nano-sized trehalose is less than 200 nm.

10. Use of the keratinase-containing composition according to any one of claims 1 to 9, characterized in that: The composition is used for preparing painless depilatory cream.

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