Oxidation-resistant polypeptide from sheep placenta, enzymatic preparation method thereof and application of the polypeptide in anti-aging cosmetics

Abstract

The present application relates to the technical field of biological peptide preparation, and particularly relates to a sheep placenta antioxidant polypeptide, an enzymatic preparation method thereof and application of the sheep placenta antioxidant polypeptide in anti-aging cosmetics; fresh sheep placenta subjected to pretreatment is low-temperature pulverized, and then is transferred to a buffer solution to which a protease inhibitor is added; after repeated freeze-thawing, the sheep placenta is transferred to a high-speed tissue homogenizer, ultrasonic crushing is performed to release proteins, low-temperature centrifugation is performed to obtain supernatant, and the supernatant is transferred to a PEG / ammonium sulfate aqueous two-phase system, selective screening is performed on target proteins to obtain a target precursor protein; the precursor protein is enzymatically hydrolyzed into an antioxidant polypeptide; temperature is controlled, and the precursor protein is subjected to enzymatic hydrolysis under the assistance of ultrasonic waves by using a composite enzyme; after the enzymatic hydrolysis is completed, a target polypeptide component is collected; the polypeptide component is wrapped in a nano carrier to obtain a nano wrapped body, and after low-temperature vacuum freeze drying, a stable antioxidant polypeptide freeze-dried powder is obtained; by using a gradient cooling method to perform repeated freeze-thawing, by accurately controlling a cooling rate and stepwise freezing, the protection on protein activity is realized.

Description

Technical Field

[0001] This invention relates to the field of biopeptide preparation technology, and in particular to a sheep placenta antioxidant polypeptide, its enzymatic preparation method, and its application in anti-aging cosmetics. Background Technology

[0002] Sheep placental antioxidant peptides are primarily used as a highly active natural bio-functional ingredient in anti-aging cosmetics. Their core value lies in delaying skin aging through two pathways: scavenging free radicals and repairing cell damage. Common product forms include lotions, night creams, eye creams, serums, and masks.

[0003] Currently, the mainstream method for preparing sheep placental antioxidant peptides is enzymatic hydrolysis, especially ultrasound-assisted enzymatic hydrolysis. Although enzymatic hydrolysis is widely used, it still has several drawbacks in the preparation of sheep placental antioxidant peptides: (1) Due to the complex structure of sheep placenta, the existing enzymatic hydrolysis process has a low extraction rate of protein components, resulting in a large amount of active protein not being utilized. (2) The production process usually requires a large amount of protease, which increases the cost of industrial production; (3) The freeze-thaw process of raw materials and processing, as well as the heating and extreme pH conditions during enzymatic hydrolysis, may damage the heat-sensitive active ingredients and cause the loss of active substances. Summary of the Invention

[0004] The purpose of this invention is to provide a sheep placenta antioxidant polypeptide, its enzymatic preparation method, and its application in anti-aging cosmetics, thereby solving at least one of the above-mentioned technical problems.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: An enzymatic hydrolysis method for preparing antioxidant peptides from sheep placenta, the method comprising the following steps: S1 involves pulverizing pretreated fresh sheep placenta at low temperature, then transferring it to a buffer solution containing protease inhibitors. After repeated freeze-thaw cycles, the mixture is transferred to a high-speed tissue homogenizer, where it is sonicated to release proteins. The supernatant is then collected by low-temperature centrifugation and transferred to a PEG / ammonium sulfate aqueous two-phase system for selective screening of target proteins to obtain target precursor proteins. S2 hydrolyzes the precursor protein into antioxidant peptides: the temperature is controlled at 24~25℃, and the precursor protein is hydrolyzed by a complex enzyme under the assistance of ultrasound. After the hydrolysis is completed, the target peptide components are collected. S3 encapsulates peptide components in a nanocarrier to obtain nano-encapsulations, which are then freeze-dried at low temperature under vacuum to obtain stable antioxidant peptide freeze-dried powder.

[0006] Furthermore, the repeated freeze-thaw cycles in step S1 employ a gradient cooling method, first cooling to -20℃ at a rate of 1-5℃ / min, holding for 2-4 hours, then cooling to -40℃ at a rate of 1-5℃ / min, holding for 2-4 hours, then cooling to -80℃ and holding for another 2-4 hours at a rate of 1-5℃ / min.

[0007] Furthermore, the mass composition of the protease inhibitor in step S1 is as follows: 4-6 parts of benzoamidine hydrochloride 12-15 parts of phenanthroline 5-8 parts of aprotinin Leucopeptidase 6-10 parts, 2-5 parts of gastric enzyme inhibitors Trehalose 75-85 parts, Hydroxypropyl methylcellulose 12-18 parts.

[0008] Furthermore, the complex enzyme in step S2 is a complex enzyme composed of alkaline protease, chymotrypsin and flavor protease in a mass ratio of 1:1:1.16.

[0009] Furthermore, in step S2, the end of enzymatic hydrolysis is indicated by the degree of hydrolysis and peptide yield, with a degree of hydrolysis of 45.32% and a peptide yield of 25.23%.

[0010] Furthermore, after enzymatic hydrolysis in step S2, insoluble particles are removed by centrifugation, and the polypeptide components are collected by fractionation according to molecular weight using an ultrafiltration membrane.

[0011] Furthermore, in step S1, the PEG / ammonium sulfate aqueous two-phase system uses PEG 4000 with a concentration of 16%, the ammonium sulfate concentration is 18%, the pH is 7.0, the extraction time is 90-120 min, and the extraction conditions are 35℃.

[0012] Furthermore, step S3 specifically includes: the polypeptide component is first mixed with hyaluronic acid and ceramide, and then PEG-modified liposomes are used as carrier materials to prepare nano-encapsulations with a particle size of 50~150nm by high-pressure nano-sizing technology.

[0013] Sheep placental antioxidant peptides, wherein the antioxidant peptides are prepared by any of the methods described above.

[0014] Application of sheep placental antioxidant peptides in anti-aging cosmetics.

[0015] The beneficial effects of adopting the technical solution of the present invention are: The aqueous two-phase system in this invention has a water content of over 80%, and its interfacial tension is much lower than that of the water / organic solvent two-phase system, making proteins less prone to denaturation and inactivation. This invention employs a gradient cooling method with repeated freeze-thaw cycles, precisely controlling the cooling rate and using stepwise freezing to protect protein activity.

[0016] Liposome nanoparticles have a structure similar to human cell membranes, exhibiting excellent biocompatibility. This allows peptides to easily penetrate the gaps in the stratum corneum of the skin, increasing transdermal absorption several times over. Compared to the difficulty of free peptides penetrating the skin barrier, nano-encapsulation enables highly efficient penetration and deposition of active ingredients deep within the skin.

[0017] Liposome nanoparticles encapsulate easily inactivated peptides, isolating them from harmful external factors such as oxygen, moisture, and light. Combined with low-temperature vacuum freeze-drying technology, the bioactivity of the peptides can be maximized, achieving an activity retention rate of ≥90% at room temperature for 12 months. Functional ingredients such as hyaluronic acid and ceramides can be simultaneously added during nano-encapsulation, achieving synergistic effects between peptides and auxiliary ingredients, thus achieving moisturizing and skin barrier repair effects and enhancing the overall skincare efficacy of the product. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to specific examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example

[0019] An enzymatic hydrolysis method for preparing antioxidant peptides from sheep placenta includes the following steps: S1 involves pulverizing pretreated fresh sheep placenta at low temperature, then transferring it to a buffer solution containing protease inhibitors. After repeated freeze-thaw cycles, the mixture is transferred to a high-speed tissue homogenizer, where it is sonicated to release proteins. The supernatant is then collected by low-temperature centrifugation and transferred to a PEG / ammonium sulfate aqueous two-phase system for selective screening of target proteins to obtain target precursor proteins. The repeated freeze-thaw cycles in step S1 employ a gradient cooling method. First, the temperature is lowered to -20℃ at a rate of 1-2℃ / min, held for 2 hours, then lowered to -40℃ at a rate of 1-2℃ / min, held for 2 hours, and then lowered to -80℃ and held for another 2 hours at a rate of 2-3℃ / min.

[0020] The mass fraction composition of the protease inhibitor in step S1 is as follows: 4 parts of benzoamidine hydrochloride 12 parts of phenanthroline, 5 parts of aprotinin Six portions of leucopeptidase. Two portions of gastric inhibitory enzymes, 75 parts trehalose 12 parts of hydroxypropyl methylcellulose.

[0021] In step S1, the PEG / ammonium sulfate aqueous two-phase system used PEG 4000 with a concentration of 16%, ammonium sulfate with a concentration of 18%, pH 7.0, extraction time of 90 min, and extraction conditions of 35℃.

[0022] S2 hydrolyzes the precursor protein into antioxidant peptides: the temperature is controlled at 24℃, and the precursor protein is hydrolyzed by a complex enzyme under the assistance of ultrasound. After the hydrolysis is completed, the target peptide component is collected. The peptide sequence is Glu-Pro-Val-Ser-His-Phe. The complex enzyme in step S2 is a complex enzyme composed of alkaline protease, chymotrypsin and flavor protease in a mass ratio of 1:1:1.16.

[0023] In step S2, the end of enzymatic hydrolysis was measured by the degree of hydrolysis and peptide yield. The degree of hydrolysis was 45.32%, and the peptide yield was 25.23%.

[0024] After enzymatic hydrolysis in step S2, insoluble particles are removed by centrifugation, and the polypeptide components are collected by fractionation according to molecular weight using an ultrafiltration membrane.

[0025] S3 encapsulates peptide components in nanocarriers to obtain nano-encapsulated particles, and then freeze-dries them at low temperature and vacuum to obtain stable antioxidant peptide lyophilized powder. Specifically, the peptide components are first mixed with hyaluronic acid and ceramide, and then PEG-modified liposomes are used as carrier materials to prepare nano-encapsulated particles with a particle size of 50 nm through high-pressure nano-sizing technology. Example

[0026] An enzymatic hydrolysis method for preparing antioxidant peptides from sheep placenta, the method comprising the following steps: S1 involves pulverizing pretreated fresh sheep placenta at low temperature, then transferring it to a buffer solution containing protease inhibitors. After repeated freeze-thaw cycles, the mixture is transferred to a high-speed tissue homogenizer, where it is sonicated to release proteins. The supernatant is then collected by low-temperature centrifugation and transferred to a PEG / ammonium sulfate aqueous two-phase system for selective screening of target proteins to obtain target precursor proteins. The repeated freeze-thaw cycles in step S1 employ a gradient cooling method. First, the temperature is lowered to -20℃ at a rate of 2-3℃ / min, held for 3 hours, then lowered to -40℃ at a rate of 2-3℃ / min, held for 3 hours, and then lowered to -80℃ and held for another 3 hours at a rate of 2-35℃ / min.

[0027] The mass fraction composition of the protease inhibitor in step S1 is as follows: 5 parts of benzalkonium hydrochloride 14 parts of phenanthroline, 7 portions of aprotinin. Leucopeptidase 8 parts, 3 portions of gastric inhibitory enzymes, 80 parts trehalose 15 parts of hydroxypropyl methylcellulose.

[0028] In step S1, the PEG / ammonium sulfate aqueous two-phase system uses PEG 4000 with a concentration of 16%, ammonium sulfate with a concentration of 18%, pH 7.0, extraction time of 100 min, and extraction conditions of 35℃.

[0029] S2 hydrolyzes the precursor protein into antioxidant peptides: the temperature is controlled at 24~25℃, and the precursor protein is hydrolyzed by a complex enzyme under the assistance of ultrasound. After the hydrolysis is completed, the target peptide component is collected. The peptide sequence is Glu-Pro-Val-Ser-His-Phe. The complex enzyme in step S2 is a complex enzyme composed of alkaline protease, chymotrypsin and flavor protease in a mass ratio of 1:1:1.16.

[0030] In step S2, the end of enzymatic hydrolysis was measured by the degree of hydrolysis and peptide yield. The degree of hydrolysis was 45.32%, and the peptide yield was 25.23%.

[0031] After enzymatic hydrolysis in step S2, insoluble particles are removed by centrifugation, and the polypeptide components are collected by fractionation according to molecular weight using an ultrafiltration membrane.

[0032] S3 encapsulates peptide components in nanocarriers to obtain nano-encapsulated particles, and then freeze-dries them at low temperature and vacuum to obtain stable antioxidant peptide lyophilized powder. Specifically, the peptide components are first mixed with hyaluronic acid and ceramide, and then PEG-modified liposomes are used as carrier materials to prepare nano-encapsulated particles with a particle size of 100 nm through high-pressure nano-sizing technology. Example

[0033] An enzymatic hydrolysis method for preparing antioxidant peptides from sheep placenta, the method comprising the following steps: S1 involves pulverizing pretreated fresh sheep placenta at low temperature, then transferring it to a buffer solution containing protease inhibitors. After repeated freeze-thaw cycles, the mixture is transferred to a high-speed tissue homogenizer, where it is sonicated to release proteins. The supernatant is then collected by low-temperature centrifugation and transferred to a PEG / ammonium sulfate aqueous two-phase system for selective screening of target proteins to obtain target precursor proteins. The repeated freeze-thaw cycles in step S1 employ a gradient cooling method. First, the temperature is lowered to -20℃ at a rate of 4-5℃ / min, held for 4 hours, then lowered to -40℃ at a rate of 4-5℃ / min, held for 4 hours, and then lowered to -80℃ and held for another 4 hours at a rate of 4-5℃ / min.

[0034] The mass fraction composition of the protease inhibitor in step S1 is as follows: 6 parts of benzoamidine hydrochloride 15 parts of phenanthroline, 8 portions of aprotinin. Leucopeptidase 10 parts, 5 parts of gastric inhibitory enzymes, 85 parts trehalose 18 parts of hydroxypropyl methylcellulose.

[0035] In step S1, the PEG / ammonium sulfate aqueous two-phase system used PEG 4000 with a concentration of 16%, ammonium sulfate with a concentration of 18%, pH 7.0, extraction time of 120 min, and extraction conditions of 35℃.

[0036] S2 hydrolyzes the precursor protein into antioxidant peptides: the temperature is controlled at 24~25℃, and the precursor protein is hydrolyzed by a complex enzyme under the assistance of ultrasound. After the hydrolysis is completed, the target peptide component is collected. The peptide sequence is Glu-Pro-Val-Ser-His-Phe. The complex enzyme in step S2 is a complex enzyme composed of alkaline protease, chymotrypsin and flavor protease in a mass ratio of 1:1:1.16.

[0037] In step S2, the end of enzymatic hydrolysis was measured by the degree of hydrolysis and peptide yield. The degree of hydrolysis was 45.32%, and the peptide yield was 25.23%.

[0038] After enzymatic hydrolysis in step S2, insoluble particles are removed by centrifugation, and the polypeptide components are collected by fractionation according to molecular weight using an ultrafiltration membrane.

[0039] S3 encapsulates peptide components in nanocarriers to obtain nano-encapsulated particles, and then freeze-dries them at low temperature and vacuum to obtain stable antioxidant peptide lyophilized powder. Specifically, the peptide components are first mixed with hyaluronic acid and ceramide, and then PEG-modified liposomes are used as carrier materials to prepare nano-encapsulated particles with a particle size of 150 nm through high-pressure nano-sizing technology.

[0040] The enzymatic hydrolysis temperature in step S2 was increased from 24~25℃ to 50℃ (the optimal temperature range for conventional trypsin / alkaline protease), and the rest of the operation was exactly the same as in Example 2.

[0041] The complex enzyme (alkaline protease: chymotrypsin: flavor protease = 1:1:1.16) in step S2 was replaced with a single alkaline protease (although its enzymatic hydrolysis product has certain antioxidant activity). The total amount of enzyme added was the same as in Example 2, and the rest of the operation was exactly the same as in Example 2.

[0042] Process differences: The PEG-modified liposomes in step S3 were replaced with ordinary liposomes without PEG modification. The remaining operations (including liposome composition, particle size of 100 nm, high-pressure nano-sizing technology, etc.) were exactly the same as in Example 2.

[0043] Experimental methods: Degree of hydrolysis (DH): The degree of hydrolysis during enzymatic hydrolysis was determined using the pH-Stat method or the ninhydrin colorimetric method. Calculation formula: DH (%) = (Number of peptide bonds broken by hydrolysis / Total number of peptide bonds) × 100%.

[0044] Peptide yield determination: The TCA method (trichloroacetic acid method) was used. After enzymatic hydrolysis, an equal volume of 15% TCA solution was added to precipitate unhydrolyzed large molecular weight proteins. After centrifugation, the supernatant was collected, and the peptide content in the supernatant was determined by the Kjeldahl method or the biuret method. Peptide yield (%) = (mass of peptides in supernatant / mass of substrate protein) × 100%.

[0045]

[0046] 2.1 Determination of DPPH free radical scavenging rate Experimental Methods: Following GB / T39100-2020, "Determination of Antioxidant Activity of Peptides: DPPH and ABTS Methods". Prepare 0.1 mM DPPH methanol solution (stored protected from light). Prepare a series of concentration gradients (0.05, 0.10, 0.20, 0.40, 0.80 mg / mL) for each sample peptide. Mix the sample with the DPPH solution at a 1:4 volume ratio. After reacting in the dark for 30 min, measure the absorbance at 517 nm. Vitamin C was used as a positive control.

[0047]

[0048] 2.2 Determination of hydroxyl radical (·OH) scavenging rate Experimental method: The Fenton reaction system was used. Different concentrations of sample peptides were added to the reaction system, and H2O2 reacted with Fe²⁺ to generate ·OH. The ·OH oxidized salicylic acid to generate a colored product, and the absorbance was measured at 510 nm.

[0049]

[0050] 2.3 Determination of superoxide anion radical scavenging rate Experimental method: The auto-oxidation method of pyrogallol was used. Pyrogallol was added to Tris-HCl buffer (pH 8.2). Under alkaline conditions, pyrogallol was auto-oxidized to generate superoxide anion radicals, and the absorbance change was measured at 325 nm.

[0051]

[0052] 2.4 Determination of Total Antioxidant Capacity (T-AOC) Experimental methods: Total antioxidant capacity (T-AOC) was determined using a kit (Nanjing Jiancheng Bioengineering Institute). FeSO4 was used as the standard, and the unit was mmol / g.

[0053] Experimental methods: Skin model: Ex vivo porcine or mouse skin was used, and after removing subcutaneous fat, it was fixed in the Franz diffusion cell.

[0054] Experimental procedure: Add equal amounts (containing the same peptide concentration) of the nano-encapsulated samples from the examples and ordinary liposomes (Comparative Example 3) to the supply pool, add PBS buffer (pH 7.4) to the receiving pool, and magnetically stir at 32±0.5℃ (simulating skin surface temperature).

[0055] Sampling and testing: The receiving solution was collected at 0, 2, 4, 6, 8, 12 and 24 h, and the cumulative transdermal transdermal amount of peptides in the receiving solution was determined by HPLC.

[0056]

[0057] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent claim. 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 anti-oxidative polypeptides from sheep placenta via enzymatic hydrolysis, characterized in that: The preparation method includes the following steps: S1 involves pulverizing pretreated fresh sheep placenta at low temperature, then transferring it to a buffer solution containing protease inhibitors. After repeated freeze-thaw cycles, the mixture is transferred to a high-speed tissue homogenizer, where it is sonicated to release proteins. The supernatant is then collected by low-temperature centrifugation and transferred to a PEG / ammonium sulfate aqueous two-phase system for selective screening of target proteins to obtain target precursor proteins. S2 hydrolyzes the precursor protein into antioxidant peptides: the temperature is controlled at 24~25℃, and the precursor protein is hydrolyzed by a complex enzyme under the assistance of ultrasound. After the hydrolysis is completed, the target peptide components are collected. S3 encapsulates peptide components in a nanocarrier to obtain nano-encapsulations, which are then freeze-dried at low temperature under vacuum to obtain stable antioxidant peptide freeze-dried powder.

2. The enzymatic preparation method of sheep placental antioxidant polypeptide according to claim 1, characterized in that: The repeated freeze-thaw cycles in step S1 employ a gradient cooling method. First, the temperature is lowered to -20°C at a rate of 1-5°C / min, held for 2-4 hours, then lowered to -40°C at a rate of 1-5°C / min, held for 2-4 hours, and then lowered to -80°C and held for another 2-4 hours at a rate of 1-5°C / min.

3. The enzymatic preparation method for sheep placental antioxidant polypeptides according to claim 1, characterized in that: The mass fraction composition of the protease inhibitor in step S1 is as follows: 4-6 parts of benzoamidine hydrochloride 12-15 parts of phenanthroline 5-8 parts of aprotinin Leucopeptidase 6-10 parts, 2-5 parts of gastric enzyme inhibitors Trehalose 75-85 parts, Hydroxypropyl methylcellulose 12-18 parts.

4. The enzymatic preparation method for sheep placental antioxidant polypeptides according to claim 3, characterized in that: The complex enzyme in step S2 is a complex enzyme composed of alkaline protease, chymotrypsin and flavor protease in a mass ratio of 1:1:1.

16.

5. The enzymatic preparation method for sheep placental antioxidant polypeptides according to claim 1, characterized in that: In step S2, the end of enzymatic hydrolysis was indicated by the degree of hydrolysis and peptide yield. The degree of hydrolysis was 45.32%, and the peptide yield was 25.23%.

6. The enzymatic preparation method of sheep placental antioxidant polypeptide according to claim 1, characterized in that: After enzymatic hydrolysis in step S2, insoluble particles are removed by centrifugation, and the polypeptide components are collected by fractionation according to molecular weight using an ultrafiltration membrane.

7. The enzymatic preparation method of sheep placental antioxidant polypeptide according to claim 1, characterized in that: In step S1, the PEG / ammonium sulfate aqueous two-phase system uses PEG 4000 with a concentration of 16%, ammonium sulfate with a concentration of 18%, pH 7.0, extraction time of 90-120 min, and extraction conditions of 35℃.

8. The enzymatic preparation method of sheep placental antioxidant polypeptide according to claim 1, characterized in that: Step S3 specifically includes: the polypeptide component is first mixed with hyaluronic acid and ceramide, and then PEG-modified liposomes are used as carrier materials to prepare nano-encapsulations with a particle size of 50~150nm by high-pressure nano-sizing technology.

9. Sheep placental antioxidant polypeptide, characterized in that: The antioxidant polypeptide is prepared by any one of the methods described in claims 1-8.

10. The use of the sheep placental antioxidant polypeptide obtained according to any one of claims 1-8 in anti-aging cosmetics.

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