Compact anti-wrinkle elastin peptide freeze-dried powder and preparation method thereof

Abstract

The invention discloses compact anti-wrinkle elastin peptide freeze-dried powder and a preparation method thereof, and belongs to the technical field of bioactive peptides. The freeze-dried powder is prepared by taking core tubes and / or throat tubes of bred animals as raw materials through cleaning and impurity removal, crushing and homogenizing, degreasing and impurity removal, composite enzymolysis, enzymolysis inactivation, centrifugal separation, gradient filtration, concentration, sterilization and vacuum freeze drying, and has remarkable elastinase inhibitory activity and tightening and anti-wrinkle effects. The livestock and poultry slaughtering byproducts which are wide in source and low in cost are selected as raw materials, high-value utilization of the livestock and poultry slaughtering byproducts is achieved, the optimized preparation technology solves the problems that in the prior art, enzymolysis efficiency is low, product purity is low, activity is insufficient, and stability is poor, the technology is simple, large-scale production is easy, and the method can be widely applied to the fields of cosmetics, food and health care products.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, and in particular to a firming and anti-wrinkle elastin peptide freeze-dried powder and its preparation method. Background Technology

[0002] Skin firmness and elasticity are primarily maintained by elastin and collagen in the dermis. Elastin accounts for only 2% to 4% of total skin protein but is responsible for over 90% of the skin's elastic function. Its reduced content and structural degradation are among the core reasons for wrinkles, sagging, and drooping skin. With age and the influence of external environmental factors (UV radiation, pollution, stress, etc.), the body's own ability to synthesize elastin declines, while the activity of matrix metalloproteinases (MMPs) increases, accelerating elastin degradation and leading to loss of skin elasticity and wrinkle formation. Therefore, supplementing with exogenous elastin peptides is an effective way to improve skin condition and achieve firmness and anti-wrinkle effects.

[0003] Currently, the raw materials for preparing elastin peptides mainly come from bovine cervical ligaments, porcine aortas, and skipjack tuna heart vessels. These raw materials have limited sources, high costs, and complex extraction processes, resulting in low protein recovery rates and insufficient active peptide content. Porcine, bovine, and ovine heart tubes and larynxes, as major byproducts of livestock slaughtering and processing, are widely available, inexpensive, and rich in elastin, with elastin content reaching 18%–25% of the total protein in the heart tubes and larynxes. They are high-quality raw materials for preparing elastin peptides. However, there is currently no mature process for specifically preparing firming and anti-wrinkle elastin peptide freeze-dried powder using porcine, bovine, and ovine heart tubes and larynxes as core raw materials.

[0004] Existing elastin peptide preparation processes mostly employ simple enzymatic hydrolysis and filtration purification, which have the following drawbacks: First, the enzymatic hydrolysis efficiency is low, elastin degradation is incomplete, the resulting peptides have uneven molecular weight distribution, large peptides are difficult to be absorbed by the skin, and the content of small active peptides (especially characteristic peptides with elastase inhibitory activity) is low; second, the purification process is rudimentary and cannot effectively remove impurities such as proteins, fats, and nucleic acids, resulting in low product purity and affecting activity and safety; third, the drying process is unreasonable, which can easily lead to denaturation and inactivation of elastin peptides, destroying their biological activity; fourth, the product has poor stability and is prone to moisture absorption and degradation during storage, affecting its performance.

[0005] In addition, although chemical synthesis of elastin-like peptides can achieve precise structural control, it has problems such as high synthesis cost, difficulty in large-scale production, and potential physiological toxicity, making it difficult to apply to large-scale production in the fields of cosmetics and health products.

[0006] Therefore, developing a freeze-dried elastin peptide powder that uses widely available and inexpensive pig, cattle, and sheep heart tubes and trachea as raw materials, and optimizes the production process through technological innovation to produce a high-purity, high-activity, high-absorption, and stable product with clear firming and anti-wrinkle effects, can solve the problems of low raw material utilization, outdated processes, and poor product performance in existing technologies. This has significant industrial application value and market prospects. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a freeze-dried elastin peptide powder for firming and anti-wrinkle purposes and its preparation method. Using pig, bovine, and ovine heart tubes and tracheae as raw materials, and through optimized cleaning, compound enzymatic hydrolysis, gradient purification, and vacuum freeze-drying processes, the invention solves the problems of low raw material utilization, poor enzymatic hydrolysis efficiency, low product purity, insufficient activity, and poor stability in existing technologies. The resulting freeze-dried elastin peptide powder has a concentrated molecular weight, is easily absorbed, and has significant firming and anti-wrinkle effects. At the same time, it realizes the high-value utilization of livestock and poultry by-products and reduces production costs.

[0008] To achieve the above objectives, the present invention provides a firming and anti-wrinkle elastin peptide freeze-dried powder, which is made from the heart tube and / or larynx of farmed animals as raw materials, and is produced by washing and removing impurities, crushing and homogenizing, defatting and removing impurities, compound enzymatic hydrolysis, enzymatic inactivation, centrifugation, gradient filtration, concentration, sterilization, and vacuum freeze drying; the elastin peptide freeze-dried powder has an elastin peptide content ≥92%, a peptide segment with a molecular weight distribution of 100~1000Da accounting for ≥88%, a desmokinin content ≥0.8%, a moisture content ≤3.0%, and a total bacterial count ≤100CFU / g.

[0009] On the other hand, the present invention also provides a method for preparing the above-mentioned firming and anti-wrinkle elastin peptide lyophilized powder, comprising the following steps: S1. Chop the cleaned and impurity-removed raw materials, add 2 to 3 times the weight of the raw materials of deionized water, and crush into a homogenous paste to obtain a heart tube and throat tube homogenate. S2. Add 0.3-0.5% (by mass) of compound degreasing agent to the homogenate in the centrifugal tube throat, adjust the pH to 6.5-7.0, stir at 40-45℃ for 30-40 min, centrifuge at 8000-10000 r / min and 4℃ to remove the upper fat and lower precipitate, and collect the intermediate clear liquid. S3. Adjust the pH of the supernatant collected in S2 to 7.5-8.5, add 1.2-1.8% of the supernatant mass of compound enzyme, and enzymatically hydrolyze at 50-55℃ and 180-220r / min for 4-6 hours, adjusting the pH every hour during this period to maintain it at 7.5-8.5. S4. Inactivate the enzyme hydrolysate and rapidly cool it to below 40°C. Centrifuge the inactivated enzyme hydrolysate at 10000~12000r / min and 4°C for 20~30min to remove the precipitate and collect the supernatant. S5. The supernatant in S4 is passed through a 0.45μm filter membrane and a 0.22μm filter membrane for microfiltration, and then through an ultrafiltration membrane with a molecular weight cutoff of 1000Da for ultrafiltration. The permeate is collected. S6. Concentrate the permeate in S5 under conditions of vacuum degree of 0.08~0.10MPa and 50~55℃ until the solid content is 25~30% to obtain elastin peptide concentrate. S7. The sterilized elastin peptide concentrate is first pre-frozen at -40~-35℃ for 2~3h, then sublimated and dried at a vacuum of 0.01~0.02MPa and a sublimation temperature of -20~-15℃ for 8~10h, and finally desorbed and dried at a desorption temperature of 30~35℃ for 2~3h to obtain lyophilized elastin peptide powder.

[0010] Preferably, in S1, the raw material cleaning and impurity removal includes: removing attached fat and connective tissue from fresh heart tubes and / or trachea, rinsing with deionized water, soaking in 0.8~1.2% physiological saline for 20~30 minutes, gently stirring 2~3 times during soaking, rinsing with deionized water until the water is clear, and draining the water; adding 0.05~0.1% vitamin C to the physiological saline.

[0011] Preferably, in S2, the composite degreasing agent is a mixture of sodium citrate and sodium dihydrogen phosphate in a mass ratio of 2:1.

[0012] Preferably, in S3, the complex enzyme is composed of elastase, trypsin, and flavor protease mixed in a mass ratio of 3:2:1; the elastase activity is ≥2000U / g, the trypsin activity is ≥1500U / g, and the flavor protease activity is ≥1000U / g.

[0013] Preferably, in step S4, the inactivation of the enzymatic hydrolysate includes: heating the enzymatic hydrolysate to 90-95°C and holding it at that temperature for 15-20 minutes.

[0014] Preferably, in S5, during the ultrafiltration process, the flow rate of the permeate is controlled to be 10~15 mL / min.

[0015] Preferably, in S7, the elastin peptide concentrate is sterilized by microwave with a microwave power of 300~400W, a sterilization time of 8~12s, and a sterilization temperature controlled at 60~65℃.

[0016] Preferably, in S7, the pre-freezing rate is 5~8℃ / h.

[0017] Therefore, the lyophilized firming and anti-wrinkle elastin peptide powder and its preparation method of the present invention have the following beneficial effects: (1) This invention uses the heart tubes and trachea of ​​pigs, cattle, and sheep as raw materials, which are widely available, inexpensive, and have high elastin content. This solves the problem of limited sources and high costs of existing elastin peptide raw materials. At the same time, it realizes the high-value utilization of livestock and poultry by-products, reduces resource waste, and conforms to the green and environmentally friendly industrial development trend.

[0018] (2) Taking into account the tough texture, slightly high fat content, and easy residue of impurities on the inner wall of the heart tube and throat tube, the cutting size, homogenization particle size, and soaking method were adjusted to suit the characteristics of the raw materials; physiological saline + vitamin C was used for soaking and cleaning to prevent oxidative denaturation of elastin. A compound degreasing agent is used to effectively remove fat and impurities from the raw materials; a compound enzymatic hydrolysis of elastase, trypsin, and flavor protease is used to synergistically improve the hydrolysis efficiency and increase the content of small molecule active peptides (100~1000Da), especially the content of characteristic peptides with elastase inhibitory activity such as VPGXG repeat sequences and GLPY. Gradient filtration and ultrafiltration purification are used to precisely control the molecular weight distribution of peptides and improve product purity; microwave low-temperature sterilization and vacuum freeze-drying are used to maximize the preservation of the bioactivity of elastin peptides, solving the problems of low enzymatic hydrolysis efficiency, low product purity, insufficient activity and poor stability of existing processes.

[0019] (3) The lyophilized elastin peptide powder prepared by the present invention has an elastin peptide content of ≥92%, a peptide segment with a molecular weight of 100~1000Da accounting for ≥88%, and a desmokinin content of ≥0.8%. It is easily absorbed by the skin and can effectively supplement the elastin peptides required by the skin, inhibit the activity of matrix metalloproteinase-1 (MMP-1), reduce the degradation of skin elastin, promote the proliferation of skin fibroblasts and the synthesis of collagen and elastin, significantly improve skin laxity, reduce wrinkles, and has a firming and anti-wrinkle effect that is superior to existing similar products. At the same time, the product has low moisture content, meets the standard for total bacterial count, has good stability, is not easy to absorb moisture or degrade during storage, and has high safety in use.

[0020] (4) The preparation process of the present invention is clear and easy to operate. The required equipment is conventional equipment in the food and biological fields. No special expensive equipment is required. The production cost is low, the enzymatic hydrolysis efficiency is high, and the product recovery rate is ≥85%. It is suitable for large-scale industrial production and has broad market application prospects.

[0021] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Elastase inhibition rate; Figure 2 This refers to the content of hydroxyproline. Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0025] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0026] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0027] Example 1 A method for preparing lyophilized elastin peptide powder for firming and anti-wrinkle purposes includes the following steps: S1. Cleaning and removing impurities: Take fresh pig heart tube and pig trachea (without lesions or odors), remove attached fat and connective tissue, rinse 4 times with deionized water, soak in 1.0% physiological saline (with 0.08% vitamin C added) for 25 minutes, rinse with deionized water until the water is clear, and drain.

[0028] S2. Homogenization: Cut the pig heart tube and pig trachea mixed in a 1:1 mass ratio into 1.2cm pieces, put them into a high-speed tissue homogenizer, add deionized water equivalent to 2.5 times the mass of the raw materials, and homogenize until a homogenate is formed (particle size ≤ 80μm) to obtain the raw material homogenate.

[0029] S3. Degreasing and impurity removal: Add 0.4% of the mass of the compound degreasing agent (sodium citrate: sodium dihydrogen phosphate mass ratio = 2:1) to the raw material homogenate, adjust the pH to 6.8, stir at 42℃ for 35 min, and then centrifuge at 9000 r / min and 4℃ for 18 min to remove the upper fat and lower precipitate, and collect the intermediate clear liquid.

[0030] S4. Compound enzymatic hydrolysis: Adjust the pH of the clear liquid to 8.0, add 1.5% of the mass of the clear liquid with a compound enzyme (elastase:trypsin:flavor protease mass = 3:2:1, elastase activity 2000U / g, trypsin activity 1500U / g, flavor protease activity 1000U / g), and enzymatically hydrolyze at 52℃ and 200r / min for 5h, adjusting the pH every 1h to maintain it at 8.0.

[0031] S5. Enzymatic inactivation: Heat the enzyme hydrolysate to 92°C, keep it at that temperature for 18 minutes, and then quickly cool it down to below 40°C.

[0032] S6. Centrifugation: Centrifuge the inactivated enzyme hydrolysate at 11000 r / min and 4℃ for 25 min to remove the precipitate and collect the supernatant.

[0033] S7. Gradient filtration: The supernatant is passed through 0.45μm and 0.22μm filter membranes for microfiltration, and then through an ultrafiltration membrane (molecular weight cutoff of 1000Da) for ultrafiltration. The permeate flow rate is controlled at 12mL / min, and the permeate is collected.

[0034] S8. Concentration: The permeate is placed in a vacuum concentration tank and concentrated to a solid content of 28% under vacuum conditions of 0.09 MPa and 52°C to obtain a concentrated solution.

[0035] S9. Sterilization: Sterilize using 350W microwave for 10 seconds at a temperature of 62℃.

[0036] S10. Vacuum freeze drying: The concentrate is placed in a vacuum freeze dryer and cooled to -38°C at a rate of 6°C / h for 2.5h. Then, it is sublimated and dried at a vacuum of 0.015MPa and a sublimation temperature of -18°C for 9h. Finally, it is desorbed and dried at a desorption temperature of 32°C for 2.5h to obtain elastin peptide freeze-dried powder.

[0037] Example 2 A method for preparing lyophilized elastin peptide powder for firming and anti-wrinkle purposes includes the following steps: S1. Cleaning and removing impurities: Take fresh bovine heart tube and trachea, remove attached fat, connective tissue and diseased parts, rinse 3 times with deionized water, soak in 0.8% physiological saline (with 0.05% vitamin C added) for 20 minutes, gently stir twice during soaking, rinse with deionized water until the water is clear, and drain.

[0038] S2. Crushing and homogenizing: Cut the bovine heart tube and bovine throat tube, which are mixed in a 1:1 mass ratio, into small pieces of 0.8cm, add deionized water equivalent to twice the mass of the raw materials, and crush into a homogenous slurry (particle size ≤80μm) to obtain a heart tube and throat tube homogenate.

[0039] S3. Degreasing and impurity removal: Add 0.3% of the mass of the compound degreasing agent (sodium citrate: sodium dihydrogen phosphate mass ratio = 2:1) to the homogenate of the centrifuge tube and throat tube, adjust the pH to 6.5, stir at 40℃ for 30 min, and then centrifuge at 8000 r / min and 4℃ for 15 min to remove the upper fat and lower precipitate, and collect the intermediate clear liquid.

[0040] S4. Compound enzymatic hydrolysis: Adjust the pH of the clear liquid to 7.5, add 1.2% of the weight of the clear liquid with a compound enzyme (elastase:trypsin:flavor protease weight = 3:2:1, elastase activity 2200U / g, trypsin activity 1600U / g, flavor protease activity 1100U / g), and enzymatically hydrolyze at 50℃ and 180r / min for 4 hours, adjusting the pH every 1 hour to maintain it at 7.5.

[0041] S5. Enzymatic inactivation: Heat the enzyme hydrolysate to 90°C, keep it warm for 15 minutes, and then quickly cool it down to below 40°C.

[0042] S6. Centrifugation: Centrifuge the inactivated enzyme hydrolysate at 10000r / min and 4℃ for 20min to remove the precipitate and collect the supernatant.

[0043] S7. Gradient filtration: The supernatant is passed through 0.45μm and 0.22μm filter membranes for microfiltration, and then through an ultrafiltration membrane (molecular weight cutoff of 1000Da) for ultrafiltration. The permeate flow rate is controlled at 10mL / min, and the permeate is collected.

[0044] S8. Concentration: The permeate is placed in a vacuum concentration tank and concentrated to a solid content of 25% under vacuum conditions of 0.08 MPa and 50°C to obtain a concentrated solution.

[0045] S9. Sterilization: Sterilize using a 300W microwave for 8 seconds at a temperature of 60℃.

[0046] S10. Vacuum freeze drying: The concentrate is placed in a vacuum freeze dryer and cooled to -40°C at a rate of 5°C / h for 2 hours. Then, it is sublimated and dried at a vacuum of 0.01MPa and a sublimation temperature of -20°C for 8 hours. Finally, it is desorbed and dried at a desorption temperature of 30°C for 2 hours to obtain elastin peptide freeze-dried powder.

[0047] Example 3 A method for preparing lyophilized elastin peptide powder for firming and anti-wrinkle purposes includes the following steps: S1. Cleaning and removing impurities: Take fresh sheep heart tube and sheep trachea, remove attached fat, connective tissue and diseased parts, rinse 5 times with deionized water, soak in 1.2% physiological saline (with 0.1% vitamin C added) for 30 minutes, gently stir 3 times during soaking, rinse with deionized water until the water is clear, and drain the water.

[0048] S2. Crushing and homogenizing: Cut the sheep heart tube and sheep larynx tube, which are mixed in a mass ratio of 1:1, into small pieces of 1.5cm, add deionized water equivalent to 3 times the mass of the raw materials, and crush into a homogenous slurry (particle size ≤80μm) to obtain heart tube and larynx tube homogenate.

[0049] S3. Degreasing and impurity removal: Add 0.5% of the mass of the compound degreasing agent (sodium citrate: sodium dihydrogen phosphate mass ratio = 2:1) to the homogenate of the centrifuge tube and throat tube, adjust the pH to 7.0, stir at 45℃ for 40 min, and then centrifuge at 10000 r / min and 4℃ for 20 min to remove the upper fat and lower precipitate, and collect the intermediate clear liquid.

[0050] S4. Compound enzymatic hydrolysis: Adjust the pH of the clear liquid to 8.5, add 1.8% of the clear liquid mass of compound enzyme (elastase:trypsin:flavor protease mass ratio = 3:2:1, elastase activity 2500U / g, trypsin activity 1800U / g, flavor protease activity 1200U / g), and enzymatically hydrolyze for 6 hours at 55℃ and 220r / min, adjusting the pH every 1 hour to maintain it at 8.5.

[0051] S5. Enzymatic inactivation: Heat the enzyme hydrolysate to 95°C, keep it warm for 20 minutes, and then quickly cool it down to below 40°C.

[0052] S6. Centrifugation: Centrifuge the inactivated enzyme hydrolysate at 12000 r / min and 4℃ for 30 min to remove the precipitate and collect the supernatant.

[0053] S7. Gradient filtration: The supernatant is passed through 0.45μm and 0.22μm filter membranes for microfiltration, and then through an ultrafiltration membrane (molecular weight cutoff of 1000Da) for ultrafiltration. The permeate flow rate is controlled at 15mL / min, and the permeate is collected.

[0054] S8. Concentration: The permeate is placed in a vacuum concentration tank and concentrated to a solid content of 30% under a vacuum of 0.10 MPa and a temperature of 55°C to obtain a concentrated solution.

[0055] S9. Sterilization: Sterilize using a 400W microwave for 12 seconds at a temperature of 65℃.

[0056] S10. Vacuum freeze drying: The concentrate is placed in a vacuum freeze dryer and cooled to -35°C at a rate of 8°C / h for 3 hours. Then, it is sublimated and dried for 10 hours under a vacuum of 0.02MPa and a sublimation temperature of -15°C. Finally, it is desorbed and dried for 3 hours at a desorption temperature of 35°C to obtain elastin peptide freeze-dried powder.

[0057] Example 4 A method for preparing lyophilized elastin peptide powder for firming and anti-wrinkle is basically the same as that in Example 1, except that the raw materials are pig heart tube, cow heart tube, sheep heart tube, pig trachea, cow trachea and sheep trachea mixed in a mass ratio of 1:1:1:1:1:1, and the remaining steps and parameters are the same as in Example 1.

[0058] Comparative Example 1 A method for preparing lyophilized elastin peptide powder includes the following steps: S1. Cleaning and removing impurities: Same as S1 in Example 1.

[0059] S2, crushing and homogenization: Same as S2 in Example 1.

[0060] S3. Enzymatic hydrolysis: Adjust the pH of the cardiac tube and larynx homogenate to 8.0, add 1.5% elastase (activity 2000U / g) by weight of the supernatant, and hydrolyze for 5 hours at 52℃ and 200r / min, adjusting the pH every 1 hour during the process.

[0061] S4, Enzymatic inactivation: Same as S5 in Example 1.

[0062] S5. Centrifugal separation: Same as S6 in Example 1.

[0063] S6. Filtration: Filter the supernatant through a 0.22μm filter membrane without ultrafiltration.

[0064] S7. Concentration, sterilization, and freeze drying: Same as S8-S10 in Example 1.

[0065] Comparative Example 2 A method for preparing lyophilized elastin peptide powder is basically the same as that in Example 1, except that: in S7, the filter is only passed through a 0.22μm filter membrane and no ultrafiltration is performed; in S9, the product is sterilized by high-pressure steam at 121℃ and 0.1MPa for 20 minutes. The remaining steps and parameters are the same as in Example 1.

[0066] Comparative Example 3 A method for preparing lyophilized elastin peptide powder is basically the same as that in Example 1, except that: S1 is soaked in pure physiological saline without adding vitamin C; S10 is dried with hot air (60°C, 8h) instead of vacuum freeze drying, and the remaining steps and parameters are the same as in Example 1.

[0067] Comparative Example 4 A method for preparing lyophilized elastin peptide powder is basically the same as that in Example 1, except that: the block size in S2 is 2cm, the particle size of the homogenate is ≤100μm, and no stirring is performed during soaking; the enzymatic hydrolysis time in S4 is 3h, and the remaining steps and parameters are the same as in Example 1.

[0068] Test Example 1 Test indicators: elastin peptide content, molecular weight distribution, desmokinin content, moisture content, and total bacterial count; The testing method is as follows: ① Elastin peptide content: determined by the biuret method (refer to GB 5009.5-2025); ② Molecular weight distribution: Determined by high performance liquid chromatography (HPLC) using a TSKgel G2000SWxl column (7.8 mm × 300 mm), a mobile phase of acetonitrile:water:trifluoroacetic acid = 30:70:0.1 (v / v / v), a flow rate of 1.0 mL / min, and a detection wavelength of 220 nm. ③ Desmocin content: determined by high performance liquid chromatography (refer to GB / T 22729-2008). ④ Moisture content: determined by vacuum drying method (refer to GB / T 5009.3-2016). ⑤ Total bacterial count: determined by plate count method (refer to GB 17399-2016).

[0069] The test results are shown in Table 1: Table 1 Measurement Indicators of Elastin Peptides

[0070] As shown in Table 1, the lyophilized elastin peptide powders prepared in Examples 1-4 have an elastin peptide content of ≥92%, a 100~1000Da peptide fraction of ≥88%, a desmokinin content of ≥0.8%, a moisture content of ≤2.5%, and a total bacterial count of ≤75CFU / g. All of these indicators are superior to those of Comparative Examples 1-4.

[0071] Comparative Example 1, due to its single enzymatic hydrolysis without defatting and impurity removal steps, suffered from low hydrolysis efficiency and numerous impurities, resulting in a significant decrease in elastin peptide content and the proportion of small molecule peptides, and a high total bacterial count. Comparative Example 2, lacking ultrafiltration purification, failed to remove large molecule peptides, and traditional high-temperature sterilization destroyed some active ingredients, leading to a decrease in desmokinin content and the proportion of small molecule peptides. Comparative Example 3, lacking vitamin C soaking, experienced partial oxidation of elastin, and hot air drying caused peptide denaturation, resulting in excessive moisture content; all indicators were inferior to the examples. Comparative Example 4, not adapted to the characteristics of the cardiac and tracheal tubes, had excessively large pieces, insufficient homogenate particle size, and insufficient enzymatic hydrolysis time, resulting in incomplete elastin degradation and poor product performance.

[0072] Test Example 2 Elastase can degrade elastin, producing a detectable yellow product. The anti-wrinkle potential of a product can be evaluated by measuring the inhibition rate of elastase activity in the sample.

[0073] The specific test method is as follows: Accurately weigh 10 mg of sample, dissolve and dilute to 10 mL with phosphate buffer (pH=7.5, 0.05 mol / L) to prepare a 1.0 mg / mL sample solution for later use. Take 0.2 mL of elastase solution (concentration 0.1 mg / mL, prepared with the above phosphate buffer), add 0.2 mL of sample solution, and incubate at 37°C for 15 min. Then add 0.4 mL of substrate solution (0.5 mg / mL succinyl-alanine-alanine-proline-leucine-p-nitroaniline, prepared with the above phosphate buffer), and continue incubation at 37°C for 30 min. Add 0.2 mL of glacial acetic acid to terminate the reaction, and measure the absorbance at 410 nm (denoted as A). 样品 ).

[0074] A blank control group was set up (using 0.2 mL of phosphate buffer instead of the sample solution, with all other operations the same, and the absorbance recorded as A). 空白 The enzyme control group (using 0.2 mL of phosphate buffer instead of the sample solution, without adding substrate solution, with all other operations the same, absorbance recorded as A) and the enzyme control group (using 0.2 mL of phosphate buffer instead of the sample solution, without adding substrate solution, with all other operations the same, absorbance recorded as A) 酶 The formula for calculating the inhibition rate is: Inhibition rate (%) = [1 - (A)] 样品 -A 酶 ) / (A 空白 -A 酶 )]×100%.

[0075] Each sample was tested in triplicate, and the average value was taken. The test concentration was 1.0 mg / mL.

[0076] Test results are as follows Figure 1 As shown, the lyophilized elastin peptide powders prepared in Examples 1-4 all exhibited elastase inhibition rates ≥76.5%, significantly higher than those in Comparative Examples 1-4. This indicates that the product of the present invention can effectively inhibit elastase activity, reduce the degradation of skin elastin, and possesses good anti-wrinkle potential. Comparative Example 1, due to single enzymatic hydrolysis, had a low content of active peptides and the lowest inhibition rate; Comparative Example 2, due to high-temperature sterilization destroying the structure of active peptides, showed a decreased inhibition rate; Comparative Example 3, due to elastin oxidation and peptide denaturation, showed reduced inhibitory activity; and Comparative Example 4, due to inadequate enzymatic hydrolysis and insufficient content of active peptides, had a poor inhibitory effect because it was not adapted to the characteristics of the cardiac and larynx.

[0077] Test Example 3 Test materials: Human skin fibroblasts (HSF), DMEM culture medium, fetal bovine serum, MTT reagent, hydroxyproline assay kit; Test method is as follows: Fibroblast proliferation assay: HSF cells were seeded in 96-well plates and cultured for 24 h. Then, sample solutions of different concentrations (0.25, 0.5, 1.0 mg / mL) were added and cultured for another 48 h. The absorbance (OD490) was measured by the MTT assay and the cell proliferation rate was calculated.

[0078] Collagen synthesis test: HSF cells were seeded in 6-well plates and cultured for 24 h. Then, 1.0 mg / mL of sample solution was added and cultured for another 72 h. The cell culture medium was collected and the hydroxyproline content was determined using a hydroxyproline detection kit.

[0079] The test results are shown in Table 2 (cell proliferation rate) and Figure 2 (Hydroxyproline content): Table 2 Cell proliferation rate

[0080] From Table 2 and Figure 2 It can be seen that the lyophilized elastin peptide powder prepared in Examples 1-4 can significantly promote the proliferation of human skin fibroblasts, and the proliferation rate increases with increasing sample concentration, reaching ≥159.3% at a concentration of 1.0 mg / mL. Simultaneously, it can significantly promote collagen synthesis by fibroblasts, with the hydroxyproline content increasing by ≥16.7 μg / mL compared to the blank control group. This indicates that the product of this invention can further enhance skin firmness and reduce wrinkles by promoting fibroblast proliferation, increasing collagen synthesis, and repairing the dermal structure of the skin. This is consistent with the results of the elastase inhibitory activity test mentioned above, fully demonstrating its excellent firming and anti-wrinkle effects. The products of Comparative Examples 1-4 have significantly weaker promoting effects on fibroblast proliferation and collagen synthesis than the examples. Among them, Comparative Example 1, due to single enzymatic hydrolysis without defatting and impurity removal, has a low content of active peptides and thus the worst promoting effect on cell proliferation and collagen synthesis. Comparative Example 2, due to high-temperature sterilization destroying the active peptide structure and lack of ultrafiltration purification resulting in a low proportion of effective ingredients, has a significantly reduced promoting effect. Comparative Example 3 suffered from impaired activity due to elastin oxidation and peptide denaturation, thus failing to effectively stimulate cell activity. Comparative Example 4, due to its incompatibility with the characteristics of the cardiac and tracheal tubes, resulted in insufficient enzymatic hydrolysis and a lack of effective active peptides, making it difficult to exert the desired cell regulatory effect.

[0081] Test Example 4 The lyophilized powder samples prepared in Examples 1 and 3 and Comparative Examples 3 and 4 were sealed and stored in an environment with a temperature of 25±2℃ and a relative humidity of 60±5% for 6 months. Samples were taken at 0 months, 3 months and 6 months to detect the content of elastin peptides, desmokine, moisture content and total bacterial count. The appearance of the samples was observed (whether they absorbed moisture, clumped, or changed color) to evaluate the product stability. The results are shown in Table 3. Table 3 Stability Test Results

[0082] As shown in Table 3, the elastin peptide freeze-dried powders prepared in Examples 1 and 3 showed minimal changes in various indicators during a 6-month storage period. The elastin peptide content and deskeletonin content decreased only slightly, while the moisture content and total bacterial count consistently met the standards. The powders maintained a white, loose powder appearance without moisture absorption, clumping, or discoloration, indicating excellent stability and a long shelf life, meeting the needs of industrial production and market applications. In contrast, Comparative Example 3, which used hot air drying instead of vacuum freeze drying and lacked vitamin C soaking, resulted in easy oxidation of the elastin, leading to significant clumping and moisture absorption after 3 months of storage. After 6 months, the indicators severely exceeded standards and an off-odor appeared. Comparative Example 4, due to its incompatibility with the characteristics of the heart and throat, had insufficient product purity and activity, was prone to degradation and clumping during storage, exhibiting poor stability. This further demonstrates the rationality and necessity of the process optimization in this invention.

[0083] Test Example 5 Test subjects: Thirty female volunteers aged 25-55 were randomly divided into 5 groups of 6 each. Each group used the products from Example 1, Example 3, Comparative Example 1, Comparative Example 3, and the blank control group (a placebo without elastin peptides), respectively. The products were used twice daily, morning and evening, for 28 consecutive days. Before use (day 0), after 14 days, and after 28 days, the facial skin elasticity value (R² value, higher values ​​indicate better skin elasticity) was measured using a skin elasticity meter. The number and depth of fine lines around the eyes were measured using a skin wrinkle meter. The test results were recorded and analyzed, and adverse skin reactions were observed. The test results are shown in Table 4. Table 4. Results of Skin Firming and Anti-wrinkle Efficacy Test

[0084] As shown in Table 4, after 28 days of continuous use of the products of Examples 1 and 3 of this invention, the volunteers' facial skin elasticity R2 value significantly increased (increased by ≥40%), the number of fine lines around the eyes decreased by ≥45%, and the depth of wrinkles decreased by ≥33%, with noticeable improvement observed as early as 14 days of use. In contrast, the products of Comparative Examples 1 and 3, as well as the blank control group, showed little or no improvement in skin elasticity and wrinkles. Furthermore, no adverse reactions such as skin redness or itching occurred in any of the volunteers during the use of the products of the examples, indicating that the products of this invention not only have significant firming and anti-wrinkle effects but also have high safety, making them widely applicable in cosmetics, food, health products, and other fields.

[0085] In summary, through raw material innovation and process optimization, the freeze-dried elastin peptide powder prepared by this invention is significantly superior to existing technologies in terms of basic properties, elastase inhibitory activity, cell proliferation and collagen synthesis, and firming and anti-wrinkle effects on the human body. It effectively solves the technical problems in the existing elastin peptide preparation process, such as low raw material utilization, poor enzymatic hydrolysis efficiency, low product purity, insufficient activity, poor stability, and unsatisfactory firming and anti-wrinkle effects. It realizes the high-value utilization of livestock and poultry by-products, and the process is simple and easy to scale up, with significant technical advantages and broad market application prospects.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A compacted anti-wrinkle elastin peptide lyophilized powder, characterized in that: The elastic protein peptide freeze-dried powder is prepared from the heart tube and / or throat tube of a farmed animal by cleaning and removing impurities, crushing and homogenizing, defatting and removing impurities, composite enzymolysis, enzyme inactivation, centrifugal separation, gradient filtration, concentration, sterilization, and vacuum freeze-drying; in the elastic protein peptide freeze-dried powder, the content of elastic protein peptide is greater than or equal to 92%, the proportion of peptide segments with a molecular weight distribution of 100-1000 Da is greater than or equal to 88%, the content of tropocollagen is greater than or equal to 0.8%, the water content is less than or equal to 3.0%, and the total number of colonies is less than or equal to 100 CFU / g.

2. A process for the preparation of a compacted anti-wrinkle elastin peptide lyophilized powder according to claim 1, characterized in that, The method comprises the following steps: S1, chopping the raw material after cleaning and removing impurities, adding deionized water equivalent to 2-3 times the mass of the raw material, and pounding to a homogenate to obtain a heart tube and throat tube homogenate; S2, adding 0.3-0.5% of a composite defatting agent to the heart tube and throat tube homogenate, adjusting the pH to 6.5-7.0, stirring at 40-45°C for 30-40 min, and centrifuging at 8000-10000 r / min and 4°C to remove the upper layer of fat and the lower layer of sediment, and collecting the middle clear liquid; S3, adjusting the pH of the clear liquid collected in S2 to 7.5-8.5, adding 1.2-1.8% of a composite enzyme based on the mass of the clear liquid, and performing enzymolysis at 50-55°C and 180-220 r / min for 4-6 h, during which the pH is adjusted every 1 h and maintained at 7.5-8.5; S4, inactivating the enzyme solution and rapidly cooling it to below 40°C; centrifuging the inactivated enzyme solution at 10000-12000 r / min and 4°C for 20-30 min to remove the sediment and collect the supernatant; S5, sequentially microfiltering the supernatant in S4 through 0.45μm and 0.22μm filter membranes, and ultrafiltering it through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da to collect the permeate; S6, concentrating the permeate in S5 to a solid content of 25-30% at a vacuum of 0.08-0.10 MPa and a temperature of 50-55°C to obtain an elastic protein peptide concentrate; S7, pre-freezing the sterilized elastic protein peptide concentrate at -40--35°C for 2-3 h, sublimation drying it at a vacuum of 0.01-0.02 MPa and a sublimation temperature of -20--15°C for 8-10 h, and finally desorption drying it at a desorption temperature of 30-35°C for 2-3 h to obtain the elastic protein peptide freeze-dried powder.

3. The method of claim 2, wherein the method is characterized by, In S1, the cleaning and removal of impurities of the raw material comprises: rinsing the fresh heart tube and / or throat tube after removing the attached fat and connective tissue with deionized water, soaking it in 0.8-1.2% physiological saline for 20-30 min, stirring it gently 2-3 times during the soaking, rinsing it with deionized water until the water is clear, and draining the water; 0.05-0.1% vitamin C is added to the physiological saline.

4. The method of claim 2, wherein the method is characterized by: In S2, the composite defatting agent is a mixture of sodium citrate and sodium dihydrogen phosphate in a mass ratio of 2:

1.

5. The method for preparing a firming and anti-wrinkle elastin peptide freeze-dried powder according to claim 2, characterized in that: In S3, the composite enzyme is a mixture of elastase, trypsin, and flavor protease in a mass ratio of 3:2:1; the activity of the elastase is greater than or equal to 2000 U / g, the activity of the trypsin is greater than or equal to 1500 U / g, and the activity of the flavor protease is greater than or equal to 1000 U / g.

6. The method of claim 2, wherein the method is characterized by the steps of: In S4, the inactivation of the enzyme solution comprises: heating the enzyme solution to 90-95°C and maintaining it for 15-20 min.

7. The method for preparing a firming and anti-wrinkle elastin peptide freeze-dried powder according to claim 2, characterized in that: In S5, during the ultrafiltration process, the flow rate of the permeate is controlled at 10~15 mL / min.

8. The method for preparing a firming and anti-wrinkle elastin peptide freeze-dried powder according to claim 2, characterized in that: In S7, the elastin peptide concentrate is sterilized by microwave with a microwave power of 300~400W, a sterilization time of 8~12s, and a sterilization temperature controlled at 60~65℃.

9. The method for preparing a firming and anti-wrinkle elastin peptide freeze-dried powder according to claim 2, characterized in that: In S7, the pre-freezing rate is 5~8℃ / h.

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