Method for preparing oil-soluble modified collagen based on aqueous phase acylation method and application of oil-soluble modified collagen in cosmetics

Abstract

The invention discloses a method for preparing oil-soluble modified collagen based on a water phase acylation method and application of the oil-soluble modified collagen in cosmetics, and belongs to the technical field of protein modification and cosmetics. Hydrophilic protein is subjected to acylating chlorination in a water phase, the ratio of protein to acyl chloride and the adding rate of acyl chloride are improved, and a series of oil-soluble modified protein with improved oil solubility is prepared. The modified protein prepared by the invention not only can exert the original biological activity in cosmetics, but also can have other additional functions, meets the diversified requirements of consumers on cosmetics, and has extremely high practical value and economic value.

Description

Technical Field

[0001] This invention relates to a method for preparing oil-soluble modified collagen based on aqueous phase acylation and its application in cosmetics, belonging to the field of protein modification and cosmetics technology. Background Technology

[0002] In recent years, the application of macromolecular proteins in the cosmetics field has received increasing attention, mainly due to their natural origin, high safety, and rich bioactivity. These characteristics collectively endow macromolecular proteins with outstanding skincare potential. Common macromolecular protein ingredients in skincare formulations include collagen and elastin. These macromolecular proteins are renowned for their excellent moisturizing abilities, forming a dense protective film on the skin's surface, effectively reducing moisture evaporation, thereby significantly improving skin hydration and softness, and providing long-lasting moisture and protection.

[0003] Despite the numerous advantages that macromolecular proteins exhibit in cosmetics, their application still faces several challenges. Firstly, due to their hydrophilic nature, macromolecular proteins are primarily suited for water-based products such as serums and masks. In oil-based systems like water-in-oil (W / O) emulsions, facial oils, or sunscreens, macromolecular proteins are difficult to add stably, limiting the diversity of cosmetic formulations and hindering the full realization of the advantages of macromolecular proteins in oil-based products with specific functions. For example, in sunscreens, if macromolecular proteins cannot undergo oil-soluble modification, they will separate from the oil phase components, resulting in an uneven sunscreen texture, which in turn affects the user experience and sun protection effectiveness.

[0004] Secondly, when large protein molecules are physically mixed with oil-phase components, phase separation easily occurs, leading to protein aggregation and inactivation. This not only weakens the biological activity of large protein molecules but may also form particulate matter, affecting the appearance and texture of cosmetics and reducing consumer trust in the product.

[0005] Furthermore, the skin's surface lipid film is lipophilic, while hydrophilic macromolecular proteins have difficulty penetrating this barrier, thus affecting their anti-wrinkle and repair effects. Studies have shown that only active ingredients that can penetrate the lipid film can better reach the deeper layers of the skin and exert their biological effects. Therefore, improving the oil solubility of macromolecular proteins is crucial for enhancing their transdermal efficiency.

[0006] To address the hydrophilicity issue of large protein molecules, some existing technologies employ chemical phase modification methods, such as glutaraldehyde crosslinking and succinic anhydride acylation. However, these methods require the use of toxic organic solvents (such as DMF and THF), which not only pollute the environment but also pose potential health hazards, failing to meet cosmetic safety standards. Furthermore, the use of organic solvents increases production costs and operational complexity, limiting their large-scale application. Some existing technologies utilize liposomes or nanoemulsions to encapsulate large protein molecules, but these methods suffer from low encapsulation rates and susceptibility to demulsification under high temperatures, resulting in poor protein release and stability.

[0007] Therefore, developing a method to modify macromolecular proteins under mild conditions to improve their oil solubility, so that the modified macromolecular proteins can not only exert their original biological activity in cosmetics, but also have other additional functions, thus meeting the diverse needs of consumers for cosmetics, has extremely high practical and economic value. Summary of the Invention

[0008] To address the aforementioned problems, this invention involves acyl chloride treatment of hydrophilic proteins in an aqueous phase, improving the ratio of protein to acyl chloride and the rate of acyl chloride addition, thereby preparing a series of oil-soluble modified proteins with enhanced oil solubility.

[0009] The first objective of this invention is to provide a method for preparing oil-soluble modified proteins, the method comprising the steps of: (1) Disperse collagen powder in water and stir to obtain collagen solution; (2) Under pH 8~10 conditions, acyl chloride is added dropwise to collagen solution and reacted; after the reaction, ultrafiltration is performed, and 30% hydrochloric acid aqueous solution is added to adjust the pH to 3~3.5 before filtration to obtain filter cake. The filter cake is dried to obtain oil-soluble modified collagen. The ratio of collagen powder to acyl chloride is 50-100 mg: 50-130 μL. The molecular weight of the collagen is 5~100 kDa; Optionally, the molecular weight of collagen is 5 kDa, 50 kDa, or 100 kDa.

[0010] In one embodiment, the ratio of collagen to water in step (1) is 50~100 mg: 1~10 g: 10~120 mL.

[0011] In one embodiment, the stirring in step (1) is carried out at 10~25 °C for 20~35 min.

[0012] In one embodiment, the dropping rate of acyl chloride in step (2) is 0.28~2.17 μL / min.

[0013] Optionally, the dropping rate of acyl chloride is 0.8~1.5 μL / min.

[0014] In one embodiment, the reaction in step (2) is carried out at 5~25 °C for 240~480 min.

[0015] In one embodiment, the reaction in step (2) is carried out at 5~10 °C for 240~300 min.

[0016] In one embodiment, the acyl chloride is any one of palmitoyl chloride, lauroyl chloride, stearoyl chloride, cocoyl chloride, myristoyl chloride, and octanoyl chloride.

[0017] Preferably, the acyl chloride is palmitoyl chloride.

[0018] A second object of the present invention is to provide oil-soluble modified collagen prepared by any of the above methods.

[0019] A third object of the present invention is to provide the application of any of the methods described above or the oil-soluble modified collagen in the preparation of cosmetics.

[0020] The fourth objective of this invention is to provide a cosmetic product, characterized in that the cosmetic product contains the oil-soluble modified collagen as described in claim 7; the cosmetic product is an essential oil, sunscreen, foundation, or lipstick.

[0021] A fifth objective of this invention is to provide a method for improving the oil solubility of collagen, the method comprising the steps of: (1) Disperse collagen powder in water and stir until completely dissolved to obtain a collagen solution; (2) Under pH 8~10 conditions, acyl chloride is added dropwise to collagen solution and reacted; after the reaction, ultrafiltration is performed, and 30% hydrochloric acid aqueous solution is added to adjust the pH to 3~3.5 before filtration to obtain filter cake. The filter cake is dried to obtain oil-soluble modified collagen. The ratio of collagen powder to acyl chloride is 50-100 mg: 50-130 μL. The molecular weight of the collagen is 5~100 kDa; Optionally, the molecular weight of collagen is 5 kDa, 50 kDa, or 100 kDa; acyl chlorides include palmitoyl chloride, lauroyl chloride, cocoyl chloride, stearoyl chloride, myristoyl chloride, and capryloyl chloride; In step (2), the dropping rate of acyl chloride is 0.28~2.17 μL / min.

[0022] In one embodiment, the ratio of collagen to water in step (1) is 50~100 mg: 1~10 g: 10~120 mL.

[0023] In one embodiment, the stirring in step (1) is carried out at 10~25 °C for 20~35 min.

[0024] In one embodiment, the dropping rate of acyl chloride in step (2) is 0.28~2.17 μL / min.

[0025] Optionally, the dropping rate of acyl chloride is 0.8~1.5 μL / min.

[0026] In one embodiment, the reaction in step (2) is carried out at 5~25 °C for 240~480 min.

[0027] In one embodiment, the reaction in step (2) is carried out at 5~10 °C for 240~300 min.

[0028] Beneficial effects of the present invention This invention involves acyl chloride treatment of hydrophilic proteins in an aqueous phase, with improvements made to the ratio of protein to acyl chloride and the rate of acyl chloride addition, to prepare a series of oil-soluble modified proteins with enhanced oil solubility.

[0029] Specifically, (1) The oil-soluble modified collagen prepared by the present invention has excellent oil solubility and can be dissolved in common natural plant oils such as meadowfoam seed oil and camellia seed oil (solubility > 0.4% w / v). (2) The oil-soluble modified protein prepared by this invention has improved stability. After being dissolved in the oil phase and subjected to an accelerated test at 60°C for 30 days, the collagen structure retention rate is >85%. (3) The oil-soluble modified protein prepared in this invention enhances skin permeability. According to the Franz diffusion cell test, the skin permeability of the modified collagen increased by 8.4 times, and the cumulative permeability over 24 hours increased from 0.352 μg / cm³. 2 Increased to 2.96 μg / cm 2 ; (4) The oil-soluble modified protein prepared by the present invention has excellent formulation compatibility and can be stably present in high oil content systems such as W / O emulsions and sunscreens. Attached Figure Description

[0030] Figure 1 The results are shown in the infrared spectroscopy results. (a) shows the infrared spectra of collagen with different molecular weights; (b) shows the infrared spectra of oil-soluble modified collagen and unmodified collagen from Example 1.

[0031] Figure 2 The results show the penetration of oil-soluble modified collagen and unmodified collagen in Example 1.

[0032] Figure 3 The results show the repair efficacy test results of the oil-soluble modified collagen essence oil in Example 1.

[0033] Figure 4 The wrinkle-reducing effect of adding modified collagen essence oil in Example 1 is shown. Detailed Implementation

[0034] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0035] Raw materials used in the examples: Collagen (5 kDa, 50 kDa, 100 kDa) was purchased from Jiangsu Chuangjian Medical Technology Co., Ltd. Palmitoyl chloride was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Octanoyl chloride was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Lauroyl chloride was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Myristoyl chloride was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Cocoyl chloride was purchased from Hubei Xingdongcheng Chemical Co., Ltd. Stearoyl chloride was purchased from Hubei Chengfeng Chemical Co., Ltd.

[0036] Test method: 1. Detection method for promoting fibroblast proliferation The MTT (3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide) method is a classic cell proliferation / toxicity assay that indirectly reflects cell number and viability by detecting the ability of mitochondrial dehydrogenases in living cells to reduce MTT to formazan.

[0037] Take fibroblasts in the logarithmic growth phase and adjust the density (5×10⁻⁶). 3 ~1×10 4 Cells / well, 96-well plate); 100 μL of complete culture medium was added to each well, and the cells were cultured for 24 h to allow them to adhere; the experimental groups are as follows: Blank control group (culture medium only, no cells), negative control group (untreated cells), experimental group (different concentrations of test samples), positive control group (e.g., 10% FBS or PDGF stimulation); add 10 μL of MTT solution (5 mg / mL, prepared in PBS) to each well (final concentration 0.5 mg / mL); incubate at 37 ℃ for 4 h, then discard the supernatant; add 100~150 μL of DMSO to each well, and shake slowly for 10 min to dissolve the crystals; measure the absorbance at 490 nm using a microplate reader.

[0038] 2. Transdermal efficacy testing methods The Franz Diffusion Cell is a standard in vitro model for studying the transdermal absorption of drugs / active ingredients and is suitable for evaluating the transdermal permeability of oil-soluble modified proteins.

[0039] Take fresh hairless pig skin, remove subcutaneous fat, wash with physiological saline, check for integrity (no damage), and soak in receptor fluid for 30 minutes to achieve equilibrium. The skin is fixed between the donor pool and the recipient pool (with the stratum corneum facing the donor side). The recipient pool is filled with preheated recipient fluid (37±0.5℃), air bubbles are removed, and the fluid is magnetically stirred (500~600 rpm) to maintain its homogeneity. Add 200-500 μL of oil-soluble modified protein preparation to the donor pool (ensure even skin coverage), while the control group contains an unmodified protein aqueous solution. At time points of 0, 5, 10, 15, 20, 22, and 24 h, 200–500 μL of sample was taken from the receptor pool (with isothermal and equal-volume receptor solution added simultaneously).

[0040] Cumulative permeability Q n =(C n ×V0+Σ(i=1ton-1)(C i ×V i )) / A Among them, Q n C represents the cumulative permeability at the nth sampling time point; n Vn represents the concentration of the drug in the receiving cell at the nth time point; V0 represents the total volume of the receiving cell; Cn represents the total volume of the receiving cell. i For the i-th sampling point, the volume of the sample taken from the receiving cell; ∑(i=1 to n-1) is the summation symbol; C i × V i This represents the summation of all terms from the 1st to the (n-1th)th time point; A is the effective permeation area of ​​the diffusion pool.

[0041] 3. Stability testing methods Group 1 was divided into two fully packaged samples. One sample was placed at room temperature, and the other was placed in the freezer at a temperature of (-18±1)℃. After 24 hours, the sample in the freezer was removed and brought back to room temperature. The sample was then compared with the sample at room temperature to observe the differences in characteristics. Group 2 was divided into two fully packaged samples. One sample was placed at room temperature, and the other was placed in a constant temperature chamber at (40±1)℃. After 24 hours, the sample in the constant temperature chamber was removed and brought back to room temperature. The sample was then compared with the sample at room temperature to observe the differences. Samples from group 3 were placed in centrifuge tubes, and the centrifuge speed was set to 2000~4000 rpm. After 30 minutes of testing, the stratification and separation of the products were observed.

[0042] Example 1: Preparation of oil-soluble modified collagen The steps for modifying oil-soluble collagen using an aqueous phase acylation method are as follows: (1) Slowly disperse 100 mg of collagen (5 kDa) powder in 10 mL of pre-cooled deionized water and stir continuously on a magnetic stirrer at 8°C for 30 min until completely dissolved to form a clear and transparent collagen solution. (2) 120 μL palmitoyl chloride (C18) was added dropwise to the collagen solution at a flow rate of 1.0 μL / min using a micro-injection pump. The addition time was 120 min. After the addition was completed, the reaction was stirred continuously for 4 h. During this period, the temperature of the reaction system was maintained at 8℃±0.5℃ using an ice-water bath. (3) After the reaction was completed, the solution was purified by ultrafiltration centrifuge tube with a molecular weight cutoff of 5 kDa (3000 rpm, 4 cycles). The retentate was collected and 30% hydrochloric acid aqueous solution was added to adjust the pH to 3~3.5. The filter cake was then filtered and the filter cake was treated by freeze dryer (pre-cooled at -80℃, vacuum degree ≤5 Pa) for 24 hours to obtain a white fluffy powder product (i.e., oil-soluble modified collagen).

[0043] Example 2: Changing the molecular weight of collagen Based on Example 1, the molecular weight of collagen was changed to 50 kDa, and the remaining steps were the same as in Example 1 to prepare oil-soluble collagen.

[0044] Example 3: Changing the molecular weight of collagen Based on Example 1, the molecular weight of collagen was changed to 100 kDa, and the remaining steps were the same as in Example 1 to prepare oil-soluble collagen.

[0045] Comparative Example 1: Changing the ratio of collagen to palmitoyl chloride (1) Based on Example 1, the ratio of collagen to palmitoyl chloride was changed to 100 mg: 90 μL, and the remaining steps were the same as in Example 1.

[0046] (2) Based on Example 1, the ratio of collagen to palmitoyl chloride was changed to 100 mg: 115 μL, and the remaining steps were the same as in Example 1.

[0047] (3) Based on Example 1, the ratio of collagen to palmitoyl chloride was changed to 100 mg: 165 μL, and the remaining steps were the same as in Example 1.

[0048] Comparative Example 2: Using other acyl chlorides Based on Example 1, octanoyl chloride, lauroyl chloride, myristoyl chloride, cocoyl chloride, and stearoyl chloride were used respectively, and the remaining steps were the same as in Example 1.

[0049] Comparative Example 3: Using traditional acyl chloride modification methods The modification of collagen using the traditional water-organic solvent method involves the following steps: When modifying collagen using a water-acetone system, first dissolve the collagen in 0.5 M acetic acid solution (pH 3.0) or PBS buffer (pH 7.4) to prepare a 2.5 mg / mL solution, and stir overnight at 4°C to ensure complete dissolution. Then, slowly add pre-cooled acetone dropwise under ice bath conditions, gradually increasing the acetone ratio (water:acetone from 1:1 to 1:4) through a gradient dehydration method, controlling the temperature not to exceed 10°C to avoid protein denaturation. For further cross-linking, add 0.05% glutaraldehyde or EDC / NHS as a cross-linking agent to the system. After the reaction is complete, centrifuge to collect the precipitate, and wash with 70% cold acetone to remove residual reagents. Finally, choose reconstitution or freeze-drying according to application requirements.

[0050] Comparative Example 4: Changing the temperature Based on Example 1, the reaction temperature in step (2) was changed to 25°C, and the remaining steps were the same as in Example 1, to prepare oil-soluble modified collagen.

[0051] Comparative Example 5: Physical Embedding Method Following the literature "EDC / NHS Crosslinked Modified Regenerated Silkworm Silk Nanofibers; DOI: 10.16865 / j.cnki.1000-7555.2012.10.040", collagen was encapsulated using a physical encapsulation method, with the following steps: Liposomes were prepared by mixing collagen with lipid materials such as soybean lecithin and cholesterol using a high-pressure homogenization method. The particle size and encapsulation efficiency of the liposomes were controlled by optimizing parameters such as homogenization pressure, temperature, and time. The prepared liposomes were then added to the oil phase to form a water-in-oil emulsion.

[0052] First, collagen solution was mixed with lipid materials such as soybean lecithin and cholesterol in a certain proportion. High-pressure homogenization was then performed, cycling the mixture five times at 50-80 MPa while maintaining the temperature within 25 °C. By optimizing the homogenization parameters (pressure 60 MPa, temperature 15 °C, 8 cycles), liposomes with a particle size of 200 nm and an encapsulation efficiency of over 80% were obtained. Then, the prepared liposome dispersion was slowly added to an oil phase preheated to 45 °C, with the oil-to-water volume ratio controlled between 3:1 and 5:1. High-speed shear emulsification was carried out at 12000 rpm for 2-5 min, ultimately forming a stable water-in-oil emulsion. The emulsion particle size was determined by dynamic light scattering, and the encapsulation efficiency was quantitatively analyzed using ultrafiltration centrifugation combined with the BCA method. The entire preparation process required aseptic conditions, and the pH was controlled at 7.4 to maintain the stability of the collagen.

[0053] Example 4: Performance Testing of Oil-Soluble Modified Collagen The oil-soluble modified collagen prepared in Examples 1-3 and Comparative Examples 1-5 was tested for its properties.

[0054] 1. Infrared Spectroscopy The oil-soluble modified collagen prepared in Examples 1, 2, and 3 (with unmodified collagen as a control) was analyzed by Fourier transform infrared spectroscopy (FTIR). The results are as follows: Figure 1 As shown.

[0055] The results showed that at 1740 cm -1 A new ester bond characteristic peak was detected at 1650 cm⁻¹. -1 The decrease in the absorption peak intensity of amide I confirms that the acylation reaction successfully introduced long-chain fatty acid groups.

[0056] 2. Oil solubility test results The solubility of oil-modified collagen and unmodified collagen prepared in Examples 1 and 1-5 in different oils was tested, and the results are shown in Table 1.

[0057] Table 1. Solubility test results of different oils

[0058] The results showed that the solubility of oil-modified collagen in meadowfoam seed oil reached 0.45% (w / v, g / mL), which was significantly improved compared with unmodified collagen.

[0059] Among them, comparing Example 1 and Comparative Example 1, it can be seen that the solubility of the modified protein in Example 1 in meadowfoam seed oil and camellia seed oil is significantly better than the solubility of the products obtained in the three different proportions in Comparative Example 1. Comparing Example 1 and Comparative Example 2, it can be seen that octanoyl chloride (C8), lauroyl chloride (C12), myristoyl chloride (C14), cocoyl chloride (a mixture of C8-C18), and stearoyl chloride (C18) all have poor solubility in meadowfoam seed oil and camellia seed oil. Comparing Example 1 and Comparative Example 4, it can be seen that a higher reaction temperature will significantly affect the solubility of the prepared oil-soluble modified collagen. Comparing Example 1 with Comparative Examples 3 and 5, it can be seen that the collagen modification method of Example 1 is significantly better than the traditional acyl chloride modification method and physical modification method in terms of solubility.

[0060] 3. Bioactivity The effects of oil-soluble modified collagen and unmodified collagen prepared in Example 1 and Comparative Examples 1-5 on promoting fibroblast proliferation were tested, and the results are shown in Table 2.

[0061] Table 2. Proliferation-promoting effect on fibroblasts

[0062] The results showed that, comparing Example 1 and Comparative Example 1, the oil-soluble modified collagen prepared by collagen and palmitoyl chloride in different ratios had no effect on the proliferation of fibroblasts and was similar to that of unmodified collagen. Comparing Example 1 with Comparative Examples 2-4, it can be seen that the oil-soluble modified collagen prepared in Example 1 has a better proliferative effect on fibroblasts than the modified collagen prepared using other acyl chlorides and conventional methods.

[0063] 4. Stability testing Collagen was dissolved in meadowfoam seed oil at a concentration of 4.12 mg / mL; control group: refrigerated at 4℃ (untreated group); experimental group: placed in a constant temperature oven at 60℃ for 30 days; Temperature 60℃±1℃ (daily calibration temperature), time 30 days (it is recommended to record oven temperature fluctuations daily), sampling points 0 days (initial), 7 days, 15 days, 30 days (can be increased as needed).

[0064] Structural characterization of collagen samples was performed using solid-state circular dichroism spectroscopy (SS-CD). A certain amount of sample powder was compressed into tablets, ensuring uniform compression without cracks. A J-1500 circular dichroism chromatograph was used to scan within the wavelength range of 190–250 nm (parameter settings: bandwidth 1 nm, scan speed 50 nm / min, isothermal at 25℃). Baseline correction with a blank quartz slide was performed before testing, and the sample was placed perpendicular to the optical path to minimize scattering interference.

[0065] The results showed that unmodified collagen exhibited a typical negative peak at 222 nm (Δε = -12.3 deg·cm). 2 ·dmol -1 The retention rate of the triple helix structure was 82.1% according to CDPro software; however, the peak intensity at 222 nm of the sample treated at 60℃ for 30 days decreased to 85.2% of the initial value (Δε = -10.5 deg·cm). 2 ·dmol -1 This confirms that its structural stability meets the design requirement of >85%.

[0066] During the test, three repeated scans were performed to ensure data repeatability (RSD < 3%), and a solution CD test was performed simultaneously as a control. The results of the two tests showed the same trend, verifying the reliability of the solid CD data.

[0067] The results are shown in Table 3. The modified collagen can still effectively maintain the stability of its secondary structure under solid conditions.

[0068] Table 3 Stability Testing

[0069] 5. Transdermal Results The transdermal effect of the oil-soluble modified collagen prepared in Example 1 was tested, and the results are as follows: Figure 2 As shown, the results indicate that the cumulative penetration of oil-soluble modified collagen in pig skin increased by 8.4 times over 24 hours.

[0070] The transdermal effects of the oil-soluble modified collagen prepared by Comparative Example 3 and Comparative Example 5 were tested. The results showed that the transdermal effect of the oil-soluble modified collagen (Comparative Example 3) prepared by the traditional acyl chloride method was only 1.8 times that of natural collagen. At the same time, due to the use of organic solvents, trace amounts of DMF residue were present in the product, which did not meet the strict cosmetic safety standards. The transdermal efficiency of physically encapsulated modified collagen (Comparative Example 5) is only 1.5 times that of natural collagen. Furthermore, the physical encapsulation method is complex and has high production costs, making it unsuitable for large-scale production.

[0071] 6. Stability in water-in-oil emulsions The oil-soluble modified collagen prepared in Example 1 was used to prepare a water-in-oil emulsion, and the steps are as follows: 0.2% by mass of modified collagen and 3% by mass of Span 80 were dissolved in meadowfoam seed oil as the oil phase (70% oil phase), and homogenized with 30% of the aqueous phase containing 0.9% NaCl at 15000 rpm for 3 min to prepare an emulsion (70% oil phase and 30% water).

[0072] The results showed that the emulsion exhibited excellent stability: no stratification or oil separation occurred after storage at 40℃ for 30 days, and the water separation rate after centrifugation was only 3.2% ± 0.5%; microscopic observation confirmed that the droplets were uniformly distributed and did not aggregate. The modified collagen prepared in Example 1 effectively anchored the oil phase through hydrophobic chains, giving the emulsion good anti-agglomeration properties, which fully meets the application requirements of cosmetics.

[0073] The liposomes prepared in Comparative Example 5 had an average particle size of approximately 150 nm and an encapsulation rate of 28%. They were initially stable in water-in-oil emulsions, but demulsification occurred after 14 days of storage, leading to the release of collagen and the separation of the emulsion.

[0074] Example 3: Application of oil-soluble modified collagen in the preparation of cosmetics The oil-soluble modified collagen obtained in Examples 1-3 was used to prepare essential oils and sunscreens, and their efficacy was tested (Example 1 is used as an example below).

[0075] 1. Anti-aging essential oil (1) Preparation of anti-aging essential oil The anti-aging essence oil components include: oil-soluble modified collagen (0.15% by mass), meadowfoam seed oil (45%), squalane (30%), vitamin E (4.85%), and rosehip oil (20%), with an essence oil without added oil-soluble modified collagen serving as a control.

[0076] (2) Detection effect Subject selection criteria, basic conditions: Healthy adults aged 18-45 (with signed informed consent), without systemic diseases or immunodeficiency, and who have not taken anti-inflammatory / immunosuppressive drugs within the past month. Skin condition requirements are in accordance with GB / T 29665-2013. Smoothness: Score ≥3 points using palpation method (0-5 level scale, 3 points = texture can be perceived with slight touch but no roughness). Glossiness: Measured under standard lighting using a digital image analyzer (such as CK-MPA), the specular reflectance of the forehead area is ≥35%.

[0077] Exclusion criteria: The test site showed visible erythema, desquamation, or acne (confirmed by Wood's lamp test). Transepidermal water loss (TEWL) > 15 g·h -1 ·m -2 (Measured using MPA580 probe); A history of cosmetic allergy or positive dermatographia.

[0078] A double-blind, controlled trial was conducted on 30 female participants aged 35-55 for 4 weeks. The participants used the anti-aging serum oil twice a day, morning and evening, with each application consisting of two pumps.

[0079] The results show that Figure 4 As shown, anti-aging essential oil can significantly improve dynamic expression lines. The results of the VC20 wrinkle depth analyzer showed that the average depth of wrinkles at the corners of the eyes decreased by 25.6% (p<0.01), and the subjective scores of the subjects showed that more than 93% of the participants felt that their skin firmness was improved.

[0080] The results of a control group serum oil test without added oil-soluble sericin peptides showed that the serum oil had limited effect on improving dynamic expression lines. VC20 wrinkle depth analyzer results showed no significant change in the average depth of crow's feet wrinkles, decreasing by only 4.7% (p>0.05). Meanwhile, subjective ratings from participants indicated that only about 36% reported an improvement in skin firmness.

[0081] (3) Accelerated stability test Further validation of its commercial potential: After storage at 40℃ / 75% RH for 3 months, the formula remained homogeneous and transparent, with no oil separation or precipitation, indicating that the long-chain acylation modification effectively enhanced the dispersion stability of collagen in complex lipid systems. This formula provides an innovative solution for developing gentle and effective naturally derived anti-wrinkle products, especially suitable for the anti-aging needs of sensitive and oily skin types.

[0082] 2. Sunscreen (1) Preparation of sunscreen The sunscreen ingredients include: modified collagen (0.2%), titanium dioxide (5%), ethylhexyl methoxycinnamate (7%), and silicone oil matrix (87.8%), with an essential oil without added oil-soluble modified collagen as a control.

[0083] (2) Detection effect Subject selection criteria, basic conditions: Healthy volunteers aged 18-50 (gender balanced), Fitzpatrick skin type III-IV (common in Asians), signed informed consent forms and passed ethical review (refer to the Declaration of Helsinki); skin condition requirements refer to GB / T39665-2020; test site: inner forearm (not exposed to strong light for ≥7 days); Exclusion criteria: The test area shows pigmentation, scarring, or inflammation (confirmed by dermoscopy); Have you recently used whitening or spot-removing products? (Questionnaire screening) History of ultraviolet allergy or photosensitivity.

[0084] The methods for testing sun protection effectiveness are as follows: In vitro testing (preliminary experiment), SPF value determination: Instrument: UV-2000S Sun Protection Factor Tester Method: According to ISO 24444:2019, the sample was coated on a PMMA plate (2 mg / cm²) and the transmittance at wavelengths of 290-400 nm was measured.

[0085] Calculation: SPF = MED (after protection) / MED (without protection).

[0086] Human testing Grouping: Experimental group vs. control group, with at least 30 people in each group (double-blind randomization); Single dose: 2 mg / cm² (applied to a 5×5 cm test area); Exposure time: 30 minutes after application, irradiate with ultraviolet light (simulating sunlight, UVA+UVB). Evaluation indicators: MED determination: minimum erythema dose (mainly UVB); PPD assay: Persistent pigmentation (primarily UVA, assessed after 24 hours); Skin tolerance: Immediate reaction (1 hour after application): erythema, burning sensation (0-3 scale); Long-term observation (continuous use for 28 days): drying, peeling.

[0087] The results are as follows Figure 3 As shown, the results indicate that, while maintaining the original SPF value of the sunscreen system (base value of approximately 30), the formula increases the final SPF value by 15% (measured value of 34.5±1.2) through the nanoscale synergistic effect of collagen and titanium dioxide. The mechanism may stem from the optimized regulation of the ultraviolet scattering path by the collagen molecular chain.

[0088] In the water resistance test, after being treated in a 40 ℃ water bath for 30 min, the retention rate of modified collagen in the formulation was still as high as 92.3%, which is significantly better than that of traditional water-soluble collagen (retention rate <60% under the same conditions). This is due to the long-chain alkyl modification enhancing the intermolecular interaction between collagen and silicone oil matrix, forming a stable hydrophobic protective layer.

[0089] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing oil-soluble modified collagen, characterized in that, The method includes the following steps: (1) Disperse collagen powder in water and stir to obtain collagen solution; (2) Under pH 8~10 conditions, acyl chloride is added dropwise to collagen solution and reacted; after the reaction, ultrafiltration is performed, and 30% hydrochloric acid aqueous solution is added to adjust the pH to 3~3.5 before filtration to obtain filter cake. The filter cake is dried to obtain oil-soluble modified collagen. The ratio of collagen powder to acyl chloride is 50-100 mg: 50-130 μL. The collagen has a molecular weight of 5-100 kDa; the acyl chloride includes any one of palmitoyl chloride, lauroyl chloride, cocoyl chloride, myristoyl chloride, and capryloyl chloride.

2. The method according to claim 1, characterized in that, In step (1), the ratio of collagen to water is 1~10g:10~120mL.

3. The method according to claim 1, characterized in that, In step (1), the stirring is carried out at 10~25 ℃ for 20~35 min.

4. The method according to claim 1, characterized in that, In step (2), the dropping rate of acyl chloride is 0.28~2.17 μL / min.

5. The method according to claim 1, characterized in that, In step (2), the reaction is carried out at 5~25℃ for 240~480 min.

6. The method according to claim 1, characterized in that, The acyl chloride is palmitoyl chloride.

7. Oil-soluble modified collagen prepared by the method according to any one of claims 1 to 6.

8. The application of the method according to any one of claims 1 to 6 or the oil-soluble modified collagen according to claim 7 in the preparation of cosmetics.

9. A cosmetic product, characterized in that, The cosmetic contains the oil-soluble modified collagen as described in claim 7; the cosmetic is an essential oil, sunscreen, foundation, or lipstick.

10. A method for improving the oil solubility of collagen, characterized in that, The method includes the following steps: (1) Disperse collagen powder in water and stir until completely dissolved to obtain a collagen solution; (2) Under pH 8~10 conditions, acyl chloride is added dropwise to collagen solution and reacted; after the reaction, ultrafiltration is performed, 30% hydrochloric acid aqueous solution is added to adjust the pH to 3~3.5, and then the filter cake is obtained by filtration and drying the filter cake to obtain oil-soluble modified protein; The ratio of collagen powder to acyl chloride is 50-100 mg: 50-130 μL. The collagen has a molecular weight of 5-100 kDa; the acyl chlorides include palmitoyl chloride, lauroyl chloride, stearoyl chloride, cocoyl chloride, myristoyl chloride, and capryloyl chloride; In step (2), the dropping rate of acyl chloride is 0.28~2.17 μL / min.

Images