Preparation method and application of a beauty and skin care cubilose composite peptide

Abstract

The application provides a preparation method and application of a beauty and skin care bird's nest composite peptide, which comprises the following raw materials in parts by weight: 0.15-0.25 parts of bird's nest peptide, 2.0-3.0 parts of elastin peptide, 90.5-91.5 parts of cod collagen peptide and 6.0-6.5 parts of eggshell membrane; the components are scientifically compounded, and a specific processing technology is used to obtain a finished product. The application can fully retain effective active substances in various raw materials, improve the human body absorption and utilization rate of nutritional ingredients, effectively promote the generation of skin collagen, relieve the skin condition, repair the skin barrier and has good anti-aging and skin care effects. The components of the product are uniformly mixed, the overall quality is stable and reliable, meanwhile, the raw materials are effectively removed from the bad flavors such as fishy and astringent flavors, the taste is refreshing and pure, the preparation process of the application is simple and standard, the production operability is strong, and the application is convenient for large-scale processing and production.

Description

Technical Field

[0001] This invention relates to the fields of bioengineering and health food technology, and in particular to a method for preparing a beauty-enhancing bird's nest complex peptide and its application. Background Technology

[0002] With rising living standards and increased health awareness, the oral beauty product market is experiencing rapid growth. Among them, collagen peptide products have become mainstream products in the beauty and anti-aging field due to their high safety, good absorption, and definite efficacy. Currently, most oral beauty peptide products on the market use single collagen peptides as their main ingredient. Although they can replenish skin collagen to some extent, they suffer from problems such as limited efficacy and poor synergistic effects, making it difficult to meet consumers' needs for comprehensive beauty and anti-aging.

[0003] To enhance product efficacy, some companies have begun to experiment with compounding collagen peptides with other active ingredients, such as adding elastin peptides, bird's nest peptides, and eggshell membrane extracts. However, existing technologies still have several drawbacks: traditional enzymatic hydrolysis processes retain less than 50% of the sialic acid from bird's nest and less than 1% of the extraction rate of the characteristic elastin components, desmokinin and isodesmokinin, resulting in low product bioactivity; ordinary eggshell membrane powder is simply pulverized, leaving the highly cross-linked keratin structure intact, resulting in extremely low water solubility and bioavailability, making it difficult to exert its skin barrier repair effects; traditional dry powder mixing processes cannot solve the problem of uniform distribution of trace active ingredients added at concentrations below 0.5%, causing large batch-to-batch quality variations and unstable efficacy; at the same time, the products generally have a strong bitter or fishy taste, requiring the addition of large amounts of flavorings and sweeteners to mask it, which not only affects the naturalness of the product but may also burden the health of some consumers. Summary of the Invention

[0004] In view of this, the present invention proposes a method for preparing a beauty-enhancing bird's nest complex peptide and its application, thereby solving the above problems.

[0005] The technical solution of the present invention is as follows: a beauty and skin care bird's nest compound peptide, comprising the following raw materials in parts by weight: 0.15-0.25 parts bird's nest peptide, 2.0-3.0 parts elastin peptide, 90.5-91.5 parts cod collagen peptide, and 6.0-6.5 parts eggshell membrane.

[0006] Furthermore, the compound peptide comprises the following raw materials in parts by weight: 0.2 parts bird's nest peptide, 2.5 parts elastin peptide, 91.05 parts cod collagen peptide, and 6.25 parts eggshell membrane.

[0007] Furthermore, the bird's nest peptide is a small molecule peptide prepared by a stepwise enzymatic hydrolysis process. Specifically, after cleaning and removing impurities from the bird's nest, soaking it in purified water at 35-45℃ to soften it, adding purified water at a material-to-liquid ratio of 1:10-12 (g / mL) to form a slurry, first adding 0.15-0.2% of the dry weight of the bird's nest with trypsin, and enzymatically hydrolyzing for 1.2-1.5 hours at 42-45℃ and pH 7.0-7.5; then adding 0.1-0.15% of the dry weight of the bird's nest with a compound enzyme, wherein the compound enzyme is a mixture of fig protease and aminopeptidase at a mass ratio of (2-4):1, and continuing enzymatic hydrolysis at the same temperature for 0.8-1.2 hours; the enzymatic hydrolysate is then subjected to instantaneous enzyme inactivation at 90-100℃ for 4-6 minutes, centrifuged to collect the supernatant, ultrafiltered to retain components with a molecular weight ≤1000Da, and spray-dried to obtain the final product.

[0008] Furthermore, the elastin peptide is a specific elastin peptide derived from the bulbus arteriosus of the skipjack tuna heart, and the specific extraction method is as follows: (a) The defatted bob of the skipjack tuna heart was placed in a buffer solution of pH 3.5-4.5 and subjected to pulsed electric field-assisted swelling. The process parameters for pulsed electric field assisted swelling are: electric field strength 10-20kV / cm, pulse width 10-50μs, frequency 1-10Hz, processing time 1-5 minutes, and material temperature controlled below 20℃.

[0009] (b) Add elastase to the swollen material at an enzyme-to-substrate mass ratio of 1:(30-80), and hydrolyze at pH 7.5-8.5 and 40-50℃ for 0.5-1.5 hours. Then add trypsin and continue hydrolysis for 1-3 hours at an enzyme-to-substrate mass ratio of 1:(80-150). Inactivate the enzyme, centrifuge, and collect the supernatant. (c) Pass the supernatant through ultrafiltration membranes with molecular weight cutoffs of 3000 Da and 500 Da in sequence, and collect the retentate of 500-3000 Da; (d) Freeze-dry the retentate to obtain elastin peptide powder.

[0010] Furthermore, the cod collagen peptides are type I collagen peptides derived from deep-sea cod skin, and the specific extraction method is as follows: (a) After removing the scales and flesh from the deep-sea cod skin, chop it up and soak it in a 0.05-0.1 mol / L NaOH solution pre-cooled at 4°C for 4-8 hours to remove non-collagenous proteins. Wash it with water until neutral. (b) Place the treated fish skin in a 0.3-0.5 mol / L acetic acid solution and allow it to swell for 2-4 hours at a material-to-solution mass ratio of 1:8-12 and a temperature of 15-25℃. Then add pepsin at an enzyme-to-solution mass ratio of 1:(40-60) and enzymatically hydrolyze it for 3-6 hours at a pH of 2.0-3.5 and a temperature of 25-35℃. (c) Adjust the pH of the enzyme hydrolysate to 7.5-8.5, add trypsin at an enzyme-to-substrate mass ratio of 1:(80-120), continue enzymatic hydrolysis at 40-50℃ for 2-4 hours, inactivate the enzyme, centrifuge, and collect the supernatant. (d) The supernatant is decolorized and impurities are removed by passing it through a ceramic membrane with a pore size of 0.1-0.2 μm, and the permeate is collected; (e) Pass the permeate through an ultrafiltration membrane with a molecular weight cutoff of 800 Da, collect the permeate with a molecular weight cutoff of less than 800 Da, and concentrate it under reduced pressure to 1 / 5 to 1 / 10 of the original volume. (f) Slowly add ethanol to the concentrate until the ethanol volume fraction is 65%-75%, stir and let stand for 2-4 hours, collect the precipitate, wash with anhydrous ethanol and freeze dry to obtain cod collagen peptide powder with an average molecular weight ≤800Da.

[0011] Furthermore, the eggshell membrane powder is a soluble eggshell membrane extract obtained through low-temperature complex enzymatic hydrolysis, specifically through the following steps: (a) Eggshell membranes were subjected to airflow ultrafine grinding at a low temperature of 0-8℃ to obtain micro powder with a particle size D90≤20μm; (b) Disperse the micro powder in Tris-HCl buffer at a mass ratio of 1:(10-20) to pH 7.0-8.0, add glutathione or cysteine ​​to a final concentration of 5-15 mmol / L, and gently stir at 30-40℃ for 1-3 hours to open the keratin disulfide bond cross-linking structure. (c) Add keratinase to the mixture at an enzyme-to-base mass ratio of 1:(50-100) and hydrolyze at pH 7.5-8.5 and 40-50℃ for 2-4 hours; then add papain at an enzyme-to-base mass ratio of 1:(100-200) and continue hydrolyzing for 2-3 hours. Inactivate the enzyme, centrifuge, and collect the supernatant. (d) The supernatant was passed through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da. The permeate was collected, concentrated under reduced pressure, and then freeze-dried to obtain a soluble eggshell membrane extract with good water solubility and high bioavailability.

[0012] A method for preparing a beauty-enhancing bird's nest complex peptide includes the following steps: S1. Premixing: Weigh out cod collagen peptides and eggshell membrane extract according to the ratio, and premix for 6-10 minutes under vacuum of -0.08MPa, 20-25℃ and 15-20rpm to form a uniform matrix. S2, Gradient Mixing: (1) Prepare an aqueous solution of elastin peptide with a mass concentration of 8-12%, and spray it evenly onto the matrix surface through an atomizing nozzle at a flow rate of 0.2-0.4 L / min. Maintain a rotation speed of 20 rpm during the spraying process, and continue mixing for 4-6 minutes after the spraying is completed. (2) Prepare an aqueous solution of bird's nest peptide with a mass concentration of 4-6%, and spray it evenly onto the matrix surface at a flow rate of 0.1-0.2 L / min. Maintain a rotation speed of 20 rpm during the spraying process. After spraying, continue mixing for 10 minutes and vacuum dry until the moisture content is ≤3%. S3. Post-processing: Continue mixing the mixed materials at the same temperature for 15-20 minutes, pass through an 80-mesh sieve, and aseptically dispense under nitrogen protection throughout the process to obtain the finished compound peptide product.

[0013] The above-mentioned beauty and skin-nourishing bird's nest complex peptide is used in the preparation of products with beauty and skin-nourishing functions, wherein the products are solid beverages, tablets, capsules, oral liquids or cosmetics.

[0014] Compared with the prior art, the beneficial effects of the present invention are: This invention utilizes a scientific blend of cod collagen peptides, elastin peptides, bird's nest peptides, and eggshell membrane extracts to construct a system that integrates exogenous supplementation, structural repair, and endogenous activation, achieving synergistic effects across multiple targets. A low-temperature stepwise enzymatic hydrolysis and flavor modification process thoroughly removes odors while maximally preserving core active ingredients such as sialic acid, desmodium / isodesmodium, and hydroxyproline, ensuring high bioactivity. Vacuum gradient spray mixing technology forms uniform particles, solving the problem of distributing trace active ingredients and ensuring consistent quality across batches. All peptides are optimized to a small molecular size range, allowing for direct absorption without digestion, significantly improving bioavailability. A comprehensive oxygen and moisture-proof stabilization process ensures the product maintains excellent physicochemical stability and sensory experience even after long-term storage at room temperature. Detailed Implementation

[0015] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0016] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0017] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0018] Example 1 A beauty and skin-nourishing bird's nest elastin complex peptide is composed of the following raw materials in parts by weight: 0.2 parts bird's nest peptide, 2.5 parts elastin peptide, 91.05 parts cod collagen peptide, and 6.25 parts eggshell membrane.

[0019] Preparation method: 1. Preparation of bird's nest peptides: After cleaning and removing impurities, bird's nest is soaked in purified water at 40℃ to soften it. Then, purified water is added at a material-to-liquid ratio of 1:11 (g / mL) to form a slurry. First, trypsin (enzyme activity 2500U / mg) at 0.18% of the dry weight of bird's nest is added, and enzymatic hydrolysis is carried out at 43℃ and pH 7.2 for 1.3 hours. Then, a compound enzyme (figase: aminopeptidase = 3:1, figase activity 800,000 U / g, aminopeptidase activity 20,000 U / g) at 0.12% of the dry weight of bird's nest is added, and enzymatic hydrolysis is continued at the same temperature for 1 hour. The enzymatic hydrolysate is then subjected to instantaneous enzyme inactivation at 95℃ for 5 minutes, centrifuged at 4000r / min for 15 minutes, and the supernatant is collected. The fraction with a molecular weight ≤1000Da is retained by ultrafiltration and spray-dried to obtain the final product.

[0020] Tests showed that 93.5% of the peptides had a molecular weight of ≤1000 Da, and the sialic acid retention rate was 88.2%.

[0021] 2. Preparation of elastin peptides: (a) The defatted bob heart arterial bulb was placed in an acetate-sodium acetate buffer solution at pH 4.0 and subjected to pulsed electric field-assisted swelling. The process parameters were: electric field strength 15kV / cm, pulse width 30μs, frequency 5Hz, processing time 3 minutes, and material temperature controlled below 18℃. (b) Add elastase (enzyme activity 30 U / mg) to the swollen material at an enzyme-to-substrate mass ratio of 1:50. After enzymatic hydrolysis at pH 8.0 and 45°C for 1 hour, add trypsin (enzyme activity 2500 U / mg) and continue enzymatic hydrolysis for 2 hours at an enzyme-to-substrate mass ratio of 1:100. Inactivate the enzyme at 90°C for 3 minutes and centrifuge at 4000 r / min for 15 minutes to collect the supernatant. (c) Pass the supernatant through ultrafiltration membranes with molecular weight cutoffs of 3000 Da and 500 Da in sequence, and collect the retentate of 500-3000 Da; (d) The retentate was freeze-dried to obtain elastin peptide powder. The total content of desmosin and isodesmosin was determined to be 16.8%.

[0022] 3. Preparation of cod collagen peptides: (a) After removing the scales and flesh from the deep-sea cod skin, chop it up and soak it in a 0.08 mol / L NaOH solution pre-cooled at 4°C for 6 hours to remove non-collagenous proteins. Wash it with water until neutral. (b) The treated fish skin was placed in a 0.4 mol / L acetic acid solution and swollen for 3 hours at a material-to-solution mass ratio of 1:10 and a temperature of 20°C. Then, pepsin (enzyme activity 3000 U / mg) was added at an enzyme-to-solution mass ratio of 1:50 and enzymatically hydrolyzed for 4.5 hours at a pH of 2.5 and a temperature of 30°C. (c) Adjust the pH of the enzyme hydrolysate to 8.0, add trypsin at an enzyme-to-bottom mass ratio of 1:100, continue enzymatic hydrolysis at 45°C for 3 hours, inactivate the enzyme at 95°C for 5 minutes, and centrifuge at 4000 r / min for 15 minutes to collect the supernatant. (d) The supernatant was decolorized and impurities removed by passing it through a ceramic membrane with a pore size of 0.15 μm, and the permeate was collected; (e) Pass the permeate through an ultrafiltration membrane with a molecular weight cutoff of 800 Da, collect the permeate with a molecular weight cutoff of less than 800 Da, and concentrate it under reduced pressure to 1 / 8 of the original volume. (f) Slowly add ethanol to the concentrate until the ethanol volume fraction reaches 70%, stir, let stand for 3 hours, collect the precipitate, wash twice with anhydrous ethanol, and freeze-dry to obtain cod collagen peptide powder. Analysis showed that the average molecular weight was 650 Da, the proportion of peptides with molecular weights of 300-800 Da was 94.2%, and the hydroxyproline content was 10.8%.

[0023] 4. Preparation of soluble eggshell membrane extract: (a) Eggshell membranes were subjected to airflow ultrafine grinding at a low temperature of 4℃ to obtain micro powder with a particle size D90=15μm; (b) The micro powder was dispersed in Tris-HCl buffer at a mass ratio of 1:15 to 1:15, and glutathione was added to a final concentration of 10 mmol / L. The mixture was then gently stirred at 35°C for 2 hours to open the keratin disulfide bond cross-linking structure. (c) Add keratinase (enzyme activity 100,000 U / g) to the mixture, with an enzyme-to-substrate mass ratio of 1:75, and hydrolyze at pH 8.0 and 45℃ for 3 hours; then add papain (enzyme activity 800,000 U / g), with an enzyme-to-substrate mass ratio of 1:150, and continue hydrolysis for 2.5 hours. Inactivate the enzyme at 95℃ for 5 minutes, centrifuge at 4000 r / min for 15 minutes, and collect the supernatant. (d) The supernatant was passed through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da. The permeate was collected, concentrated under reduced pressure, and then freeze-dried to obtain a soluble eggshell membrane extract.

[0024] 5. Preparation of complex peptides: S1. Premixing: Weigh out cod collagen peptides and eggshell membrane extract according to the ratio, add them to a vacuum double helix conical mixer, and premix for 8 minutes at a vacuum of -0.08MPa, 22℃ and 18rpm to form a uniform matrix. S2, Gradient Mixing: (1) Prepare an aqueous solution of 10% by mass of elastin peptides and spray it evenly onto the matrix surface through an atomizing nozzle at a flow rate of 0.3 L / min. Maintain a rotation speed of 20 rpm during the spraying process and continue mixing for 5 minutes after the spraying is completed. (2) Prepare a 5% aqueous solution of bird's nest peptides and spray it evenly onto the matrix surface at a flow rate of 0.15 L / min. Maintain a rotation speed of 20 rpm during the spraying process. After spraying, continue mixing for 10 minutes and vacuum dry to a moisture content of 2.5%. S3. Post-processing: The mixed materials are mixed for another 18 minutes at the same temperature, passed through an 80-mesh sieve, and aseptically packaged under nitrogen protection throughout the process to obtain the finished compound peptide product. Example 2 A beauty and skin-nourishing bird's nest complex peptide is composed of the following raw materials in parts by weight: 0.15 parts bird's nest peptide, 2.0 parts elastin peptide, 90.5 parts cod collagen peptide, and 6.0 parts eggshell membrane.

[0025] The preparation method is the same as in Example 1.

[0026] Example 3 A beauty and skin-nourishing bird's nest complex peptide is composed of the following raw materials in parts by weight: 0.25 parts bird's nest peptide, 3.0 parts elastin peptide, 91.5 parts cod collagen peptide, and 6.5 parts eggshell membrane.

[0027] The preparation method is the same as in Example 1.

[0028] Comparative Example 1 The difference between this comparative example and Example 1 is that it was prepared using a traditional direct mixing method. All four raw materials prepared in Example 1 were directly mixed at 22°C and 20 rpm for 30 minutes.

[0029] Comparative Example 2 The difference between this comparative example and Example 1 is that the bird's nest peptide was prepared using a traditional single enzymatic hydrolysis process: after cleaning and removing impurities from the bird's nest, soaking and softening it, pure water was added at a material-to-liquid ratio of 1:11 (g / mL) to form a slurry, and 0.3% papain (from papaya latex, food grade, enzyme activity 800,000 U / g) was added. Enzymatic hydrolysis was carried out at 55℃ and pH 6.5 for 3 hours, followed by enzyme inactivation at 95℃ for 10 minutes. The final product was obtained by centrifugation, ultrafiltration, and spray drying. The preparation methods for the remaining raw materials and the complex peptide were the same as in Example 1.

[0030] Comparative Example 3 The difference between this comparative example and Example 1 is that the soluble eggshell membrane extract is replaced with ordinary eggshell membrane powder, which is only pulverized at 60 mesh and not subjected to enzymatic hydrolysis modification. The rest of the formulation and preparation method are exactly the same as in Example 1.

[0031] Comparative Example 4 The difference between this comparative example and Example 1 is that the pulsed electric field-assisted swelling step was removed during the preparation of the elastin peptides. Instead, the defatted bonito heart arterial bulb was directly immersed in a pH 4.0 buffer solution at room temperature for 3 hours. The remaining steps and parameters were exactly the same as in Example 1.

[0032] Comparative Example 5 The difference between this comparative example and Example 1 is that the glutathione step and the keratinase hydrolysis step were removed during the preparation of the eggshell membrane extract. Only papain was used for hydrolysis under the same conditions for 5 hours. The remaining steps were exactly the same as in Example 1.

[0033] Comparative Example 6 The difference between this comparative example and Example 1 is that the cod collagen peptide preparation does not involve the second enzymatic hydrolysis step with trypsin, but only uses pepsin for 7.5 hours under the same conditions. The remaining steps are exactly the same as in Example 1.

[0034] I. Physicochemical Properties and Active Ingredient Testing 1. Mixing uniformity test Ten batches each of the composite peptide products prepared in Examples 1-3 and Comparative Examples 1-6 were taken. Using sialic acid as an indicator (representing the distribution of bird's nest peptides), the sialic acid content at each sampling point was determined by high-performance liquid chromatography. 1.1 Chromatographic conditions Chromatographic column: C18 column (4.6mm × 250mm, 5μm) Mobile phase: 0.05 mol / L potassium dihydrogen phosphate solution (pH 3.0) - methanol (95:5, v / v) Detection wavelength: 220nm Flow rate: 1.0 mL / min Column temperature: 30℃ Injection volume: 20 μL 1.2 Operating Procedures Preparation of standard solution: Accurately weigh 10 mg of bird's nest peptide standard (N-acetylneuraminic acid, purity ≥99%), place it in a 10 mL volumetric flask, add ultrapure water to dissolve and dilute to the mark, shake well to prepare a 1 mg / mL standard stock solution.

[0035] Preparation of test solution: Accurately weigh 0.5g of sample from 10 different positions (top, middle, bottom, left, right, front, back, center, corner 1, and corner 2) of the sample to be tested, place them in 50mL volumetric flasks, add 40mL of ultrapure water, sonicate for 10 minutes, dilute to the mark, shake well, and filter through a 0.22μm microporous membrane to obtain the test solution.

[0036] 1.3 Determination: Accurately pipette 20 μL of the standard solution and each test solution into the liquid chromatograph, record the chromatogram, and calculate the average content based on the peak area of ​​the characteristic peak of bird's nest peptide (retention time of about 12.5 min).

[0037] Calculate the relative standard deviation (RSD%). The smaller the RSD value, the higher the uniformity.

[0038] 2. Detection of core active ingredient content 2.1 Sialic acid content: Separated and determined by high performance liquid chromatography (HPLC), and quantified by external standard method.

[0039] (1) Chromatographic conditions: Chromatographic column: C18 column (4.6 mm × 250 mm, 5 μm) Mobile phase: 0.02 mol / L potassium dihydrogen phosphate buffer (pH 2.5) - acetonitrile (95:5) Detection wavelength: 205nm Flow rate: 1.0 mL / min Column temperature: 30℃ Injection volume: 20 μL.

[0040] (2) Procedure: Prepare a series of standard solutions of 0.05-0.5 mg / mL using N-acetylneuraminic acid (purity ≥98%) as the standard. Weigh 0.2 g of sample, add 5 mL of 0.1 mol / L sulfuric acid, hydrolyze in a water bath at 80 °C for 1 h, cool, neutralize to neutral with 0.1 mol / L sodium hydroxide, make up to 10 mL, and filter through a 0.22 μm filter membrane.

[0041] (3) Calculation: The sialic acid content in the sample was calculated using the external standard method. Calculation of sialic acid retention rate: Sialic acid retention rate (%) = Sialic acid content of sample / Sialic acid content of bird's nest raw material × 100%.

[0042] 2.2 Detection of elastin peptide desmokinin and isodesmokinin content Desmolysin and isodesmolysin are cross-linked amino acids unique to elastin. After acid hydrolysis, the samples were separated and determined by high performance liquid chromatography, and quantified by external standard method.

[0043] (1) Chromatographic conditions Chromatographic column: C18 column (4.6mm × 250mm, 5μm) Mobile phase A: 0.1% pentafluoropropionic acid aqueous solution; Mobile phase B: acetonitrile Gradient elution program: 0-15 min, B5% to 25%; 15-25 min, B25% to 5%; 25-30 min, B5% Flow rate: 1.0 mL / min Detection wavelength: 275nm (ultraviolet) Column temperature: 35℃ Injection volume: 20 μL (2) Operating steps Preparation of standard solutions: Accurately weigh 5.0 mg each of desmodium and isodesmodium standards, dissolve them in 0.01 mol / L hydrochloric acid, and dilute to 5 mL to obtain 1.0 mg / mL single standard stock solutions. Take appropriate amounts of each stock solution, mix them, and dilute with 0.01 mol / L hydrochloric acid to prepare a series of mixed standard working solutions containing 5, 10, 20, 50, and 100 μg / mL of desmodium and isodesmodium.

[0044] Sample solution preparation: Accurately weigh approximately 0.2 g of elastin peptide sample and place it in a vacuum hydrolysis tube. Add 10 mL of 6 mol / L hydrochloric acid, evacuate, seal, and place in a 110℃ constant temperature drying oven for 24 hours. Remove and cool, transfer the hydrolysate to an evaporating dish, and evaporate to dryness in an 80℃ water bath. Dissolve the residue in 0.01 mol / L hydrochloric acid and transfer to a 10 mL volumetric flask, dilute to volume, and filter through a 0.45 μm microporous membrane for analysis.

[0045] (3) Measurement and Calculation Inject 20 μL each of the mixed standard working solution and the sample solution, and record the peak areas of desmolide and isodesmolide. Plot standard curves with standard solution concentration on the x-axis and peak area on the y-axis.

[0046] Calculation formula: Desmodium content (%) = C1 × V × 10 -6 / m×100% Isodesin content (%) = C² × V × 10 -6 / m×100% Total content of desmokine + isodesmokine (%) = Desmokine content + isodesmokine content In the formula: C1 and C2 are the concentrations (μg / mL) of desmodium and isodesmodium in the sample solution, respectively; V is the final volume (10mL); and m is the sample weight (g).

[0047] 2.3 Detection of hydroxyproline content in cod collagen peptides The spectrophotometric method of chloramine T-p-dimethylaminobenzaldehyde was used for detection. The operation steps are as follows: (1) Preparation of standard curve: Accurately weigh 10 mg of L-hydroxyproline standard, place it in a 100 mL volumetric flask, add 0.01 mol / L hydrochloric acid to dissolve it and dilute to the mark to prepare a 100 μg / mL standard solution.

[0048] Accurately pipette 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the standard solution into 10 mL stoppered test tubes, and add ultrapure water to each to a final volume of 1.0 mL.

[0049] Add 1.0 mL of chloramine T solution, shake well, and let stand at room temperature for 20 minutes.

[0050] Add 1.0 mL of 3.5 mol / L perchloric acid solution, shake well, and let stand at room temperature for 5 minutes.

[0051] Add 2.0 mL of p-dimethylaminobenzaldehyde solution, shake well, heat in a 60°C water bath for 20 minutes, and immediately cool in ice water for 5 minutes.

[0052] The absorbance was measured at a wavelength of 560 nm, and a standard curve was plotted.

[0053] (2) Sample hydrolysis: Accurately weigh 50 mg of the sample to be tested, place it in a hydrolysis tube, add 5 mL of 6 mol / L hydrochloric acid, seal with nitrogen, and hydrolyze at 110 °C for 16 hours. After cooling, transfer to a 50 mL volumetric flask, dilute to the mark with ultrapure water, and shake well.

[0054] (3) Determination: Accurately pipette 1.0 mL of hydrolysate into a 10 mL stoppered test tube, perform the colorimetric reaction according to the standard curve preparation steps, measure the absorbance at a wavelength of 560 nm, and calculate the hydroxyproline content according to the standard curve.

[0055] 3. Water-soluble transmittance test Operating steps: Accurately weigh 0.5 g of the sample to be tested and place it in a 100 mL beaker. Add 50 mL of ultrapure water at 25℃±1℃. Turn on the magnetic stirrer and stir at 300 rpm for 30 seconds. Immediately stop stirring and let stand for 5 minutes. Take the supernatant and place it in a 1 cm cuvette. Using ultrapure water as a blank control, measure the transmittance at a wavelength of 600 nm.

[0056] Each sample was measured in triplicate, and the average value was taken.

[0057] 4. Test Results

[0058] Meanwhile, visual inspection of the solution clarity showed that the Example group was clear and transparent with no precipitation, Comparative Example 3 had a white precipitate, and Comparative Example 5 was slightly turbid with a small amount of suspended matter. Results analysis: The mixing uniformity RSD of the example group was ≤1.0%, which proves that the vacuum gradient spray mixing process of the present invention can ensure excellent uniformity throughout the entire formulation range.

[0059] Compared with Comparative Example 1, Example 1 demonstrates that the vacuum gradient spray mixing process of this invention solves the industry problem of uneven mixing of trace amounts of bird's nest peptides, resulting in product quality uniformity far superior to traditional processes. Compared with Comparative Example 2, this invention's stepwise enzymatic hydrolysis process significantly reduces sialic acid degradation. Compared with Comparative Examples 3 and 5, the soluble eggshell membrane extract preparation process of this invention more fully degrades eggshell membrane keratin than single enzymatic hydrolysis, significantly improving water solubility and solving the problems of poor solubility and easy precipitation of ordinary eggshell membrane powder. Compared with Comparative Example 4, this invention demonstrates that pulsed electric field-assisted swelling can effectively disrupt the dense structure of the skipjack tuna's cardiac arterial bulb, significantly increasing the extraction rate of elastin characteristic components. Compared with Comparative Example 6, this invention's stepwise enzymatic hydrolysis process more fully hydrolyzes collagen, increasing the content of the characteristic amino acid hydroxyproline and enhancing product bioactivity.

[0060] II. In vitro bioactivity testing (I) Test Items and Methods Using a human skin fibroblast (HSF) in vitro culture model, samples were prepared into a 100 μg / mL complete culture medium and cultured for 48 hours before measurement. 1. Cell proliferation rate: MTT assay 1.1 Operating Steps: (1) Cell seeding: HSF cells in the logarithmic growth phase were digested with 0.25% trypsin to prepare a single-cell suspension, and the cell concentration was adjusted to 5×10⁻⁶. 4 The cells were seeded at a rate of 100 μL per well in a 96-well plate and incubated for 24 hours to allow them to adhere to the plate.

[0061] (2) Sample processing: Discard the old culture medium, add 100 μL of culture medium containing 100 μg / mL sample to each well of the experimental group, and add 100 μL of culture medium without sample to the blank control group, and continue to culture for 48 hours.

[0062] (3) MTT incubation: Add 20 μL of 5 mg / mL MTT solution to each well and continue incubation for 4 hours.

[0063] (4) Dissolving MTT: Carefully aspirate the supernatant from the wells, add 150 μL of DMSO to each well, and place on a shaker to shake at low speed for 10 minutes to fully dissolve the purple crystals.

[0064] (5) Measurement: The absorbance (OD) value of each well was measured at a wavelength of 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0065] 1.2 Calculation Cell proliferation rate (%) = (OD value of experimental group / OD value of blank control group) × 100% 2. Type I collagen synthesis: ELISA kit method 2.1 Operating Procedures (1) Sample collection: After culturing the cells for 48 hours according to the cell proliferation experiment, collect the supernatant from each well, centrifuge at 4℃ and 3000r / min for 10 minutes, and take the supernatant for testing.

[0066] (2) Preparation of standard: Dilute the kit standard to concentrations of 0, 5, 10, 20, 40 and 80 ng / mL.

[0067] (3) Add 100 μL of standard or test sample to each well, seal the plate, and incubate at 37°C for 90 min.

[0068] (4) Washing the plate: Discard the liquid in the well, wash the plate with washing solution 3 times, soaking for 1 minute each time, and pat dry.

[0069] (5) Add primary antibody: Add 100 μL of biotinylated anti-human type I collagen antibody to each well, incubate at 37°C for 60 min, and wash the plate repeatedly.

[0070] (6) Add enzyme conjugate: Add 100 μL of HRP-labeled streptavidin to each well, incubate at 37°C for 30 min, and wash the plate repeatedly.

[0071] (7) Color development termination: Add 90 μL LMB color development solution to each well and incubate at 37°C in the dark for 15 min; add 50 μL of termination solution and mix well.

[0072] (8) Measurement: The OD value of each well was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0073] 2.2. Calculation Plot a standard curve with the concentration of the standard on the x-axis and the OD value on the y-axis. Calculate the concentration (μg / mL) of type I collagen in the sample based on the standard curve.

[0074] 3. Inhibition rate of matrix metalloproteinase-1 (MMP-1): ELISA kit method 3.1 Operating Procedures (1) Sample collection: Same as the test of type I collagen synthesis.

[0075] (2) Standard dilution: Prepare a series of standard solutions of 0, 0.5, 1, 2, 4 and 8 ng / mL using the standards in the kit.

[0076] (3) Sample addition and incubation: The operation steps are the same as those for type I collagen testing, using the corresponding MMP-1 antibody and enzyme conjugate.

[0077] (4) Measurement: The OD value of each well was measured at a wavelength of 450 nm.

[0078] 3.2 Calculation The concentration of MMP-1 in the sample was calculated based on the standard curve. MMP-1 inhibition rate (%) = (MMP-1 concentration in blank control group - MMP-1 concentration in experimental group) / MMP-1 concentration in blank control group × 100% (II) Test Results and Analysis

[0079] Results analysis: The compound peptide of the present invention can increase the proliferation rate of fibroblasts and the amount of collagen synthesis, and the MMP-1 inhibition rate reaches 46.9%, which shows significant anti-aging activity.

[0080] Compared with Comparative Example 1, Example 1 shows that the gradient spray mixing process of the present invention ensures the uniform distribution of trace active ingredients, avoiding the decrease in efficacy caused by insufficient local concentration. Compared with Comparative Example 2, the stepwise enzymatic hydrolysis process of the present invention improves the sialic acid retention rate, significantly enhancing the product's ability to promote cell proliferation and collagen synthesis. Compared with Comparative Examples 3 and 5, the enzymatic hydrolysis process of the present invention can more thoroughly degrade eggshell membrane keratin than single enzymatic hydrolysis, releasing more bioactive small molecule peptides. The prepared soluble eggshell membrane extract can fully release natural repair factors and synergistically enhance fibroblast activity. Compared with Comparative Example 4, pulsed electric field-assisted swelling can improve the extraction rate of elastin characteristic components desmolide and isodesmolide. These components can not only replenish skin elasticity but also synergistically inhibit the activity of collagen-degrading enzymes. Compared with Comparative Example 6, the stepwise enzymatic hydrolysis process of the present invention increases the hydroxyproline content in cod collagen peptides.

[0081] III. Skin Barrier Repair Function Test The test was conducted using an EpiSkin3D reconstructed human skin model. 1. Operating Steps (1) Barrier damage modeling: The skin model was placed in a culture plate, 200 μL of 1% SDS solution was added to each well, and the plate was treated at 37°C for 2 hours. The plate was then gently rinsed 3 times with PBS to establish a skin barrier damage model.

[0082] (2) Sample administration: 200 μL of 5% sample solution was added to each well of the injury model group, and the normal control group and the injury control group were added with the same amount of PBS. The samples were then placed in an incubator and cultured for 24 hours.

[0083] (3) TEWL value determination: Take out the skin model, dry the surface liquid with filter paper, and use a skin water flow meter to measure the transepidermal water loss (TEWL) value. Each model is measured 3 times and the average value is taken.

[0084] 2. Calculation Barrier repair rate (%) = (TEWL value of damaged control group - TEWL value of experimental group) / (TEWL value of damaged control group - TEWL value of normal control group) × 100% 3. Test Results

[0085] Results analysis: The barrier repair rates of Examples 1, 2 and 3 were all ≥77.5%, with minimal differences between groups, indicating that the soluble eggshell membrane extract has a good skin barrier repair function.

[0086] Compared with Comparative Examples 3 and 5, Example 1 demonstrates that the soluble eggshell membrane extract prepared by the present invention can fully release hyaluronic acid, chondroitin sulfate and keratin small molecule peptides, significantly promoting the repair of damaged skin barriers; the compound enzymatic hydrolysis process can completely decompose keratin into bioactive small molecule peptides, greatly improving the repair effect.

[0087] IV. Sensory Evaluation This test strictly followed GB / T 16291.1-2012 "General Guidelines for the Selection, Training and Management of Sensory Analysis Evaluators". Twenty healthy volunteers aged 20-40 with no smoking history and no taste or smell disorders were recruited. After three days of professional training, 16 evaluators with high sensory sensitivity and good scoring consistency were selected to form a group. A three-point test combined with descriptive analysis was used for blind evaluation. During the test, all samples were randomly numbered. 5g of each sample was weighed and dissolved in 100mL of 40℃ purified water. The sample temperature was controlled at 35℃±1℃ and placed in identical white paper cups. A 10-point scale was used to evaluate the bitterness, fishiness, and overall acceptability. Evaluators fasted for one hour before the test and tasted the samples in random order. After tasting each sample, they rinsed their mouths with purified water and waited two minutes. They independently filled out the scoring sheet and calculated the average score for each indicator. Rating criteria: A 10-point scale is used for rating. Bitterness and fishiness: 10 points means no odor at all, 7-9 points means very slight and almost imperceptible, 4-6 points means slightly acceptable, and 1-3 points means obviously unacceptable. Overall acceptance is based on taste, flavor, color, etc. 10 points means very satisfied and willing to buy often, 7-9 points means relatively satisfied, 4-6 points means average, and 1-3 points means unsatisfied and will not buy.

[0088] The results showed that the bitterness score of the products in Examples 1-3 of this invention was 9.2-9.4, the fishy smell score was 9.5-9.6, and the overall acceptability score was 9.3-9.4. This is mainly because the stepwise enzymatic hydrolysis process of this invention hydrolyzes bitter peptides with aminopeptidase, while completely removing unsaturated fatty acids from fish raw materials, thus fundamentally eliminating bitterness and fishy smell. The products have a light and natural taste and can be consumed directly without the addition of flavorings or sweeteners, resulting in extremely high consumer acceptance.

[0089] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A beauty-enhancing bird's nest complex peptide, characterized in that, It includes the following ingredients by weight: 0.15-0.25 parts bird's nest peptide, 2.0-3.0 parts elastin peptide, 90.5-91.5 parts cod collagen peptide, and 6.0-6.5 parts eggshell membrane.

2. The complex peptide as described in claim 1, characterized in that, The ingredients include the following parts by weight: 0.2 parts bird's nest peptide, 2.5 parts elastin peptide, 91.05 parts cod collagen peptide, and 6.25 parts eggshell membrane.

3. The complex peptide as described in claim 1, characterized in that, The bird's nest peptides are small molecule peptides prepared using a stepwise enzymatic hydrolysis process. Specifically, after cleaning and removing impurities from the bird's nest, and soaking it in purified water at 35-45℃ to soften it, purified water is added at a material-to-liquid ratio of 1:10-12 (g / mL) to form a slurry. First, 0.15-0.2% of the dry weight of the bird's nest is added to the slurry, and enzymatic hydrolysis is carried out at 42-45℃ and pH 7.0-7.5 for 1.2-1.5 hours. Then, 0.1-0.15% of the dry weight of the bird's nest is added to the slurry to form a complex enzyme, which is a mixture of fig protease and aminopeptidase at a mass ratio of (2-4):

1. Enzymatic hydrolysis is continued at the same temperature for 0.8-1.2 hours. The hydrolysate is then subjected to instantaneous enzyme inactivation at 90-100℃ for 4-6 minutes, centrifuged to collect the supernatant, ultrafiltered to retain the fraction with a molecular weight ≤1000Da, and spray-dried to obtain the final product.

4. The complex peptide as described in claim 1, characterized in that, The elastin peptides are specific elastin peptides derived from the bulbus arteriosus of the skipjack tuna heart, and the specific extraction method is as follows: (a) The defatted bob of the skipjack tuna heart was placed in a buffer solution of pH 3.5-4.5 and subjected to pulsed electric field-assisted swelling. (b) Add elastase to the swollen material at an enzyme-to-substrate mass ratio of 1:(30-80), and hydrolyze at pH 7.5-8.5 and 40-50℃ for 0.5-1.5 hours. Then add trypsin and continue hydrolysis for 1-3 hours at an enzyme-to-substrate mass ratio of 1:(80-150). Inactivate the enzyme, centrifuge, and collect the supernatant. (c) Pass the supernatant through ultrafiltration membranes with molecular weight cutoffs of 3000 Da and 500 Da in sequence, and collect the retentate of 500-3000 Da; (d) Freeze-dry the retentate to obtain elastin peptide powder.

5. The complex peptide as described in claim 4, characterized in that, The process parameters for pulsed electric field assisted swelling are: electric field strength 10-20kV / cm, pulse width 10-50μs, frequency 1-10Hz, processing time 1-5 minutes, and material temperature controlled below 20℃.

6. The complex peptide as described in claim 1, characterized in that, The cod collagen peptides are type I collagen peptides derived from deep-sea cod skin, and the specific extraction method is as follows: (a) After removing the scales and flesh from the deep-sea cod skin, chop it up and soak it in a 0.05-0.1 mol / L NaOH solution pre-cooled at 4°C for 4-8 hours to remove non-collagenous proteins. Wash it with water until neutral. (b) Place the treated fish skin in a 0.3-0.5 mol / L acetic acid solution and allow it to swell for 2-4 hours at a material-to-solution mass ratio of 1:8-12 and a temperature of 15-25℃. Then add pepsin at an enzyme-to-solution mass ratio of 1:(40-60) and enzymatically hydrolyze it for 3-6 hours at a pH of 2.0-3.5 and a temperature of 25-35℃. (c) Adjust the pH of the enzyme hydrolysate to 7.5-8.5, add trypsin at an enzyme-to-substrate mass ratio of 1:(80-120), continue enzymatic hydrolysis at 40-50℃ for 2-4 hours, inactivate the enzyme, centrifuge, and collect the supernatant. (d) The supernatant is decolorized and impurities are removed by passing it through a ceramic membrane with a pore size of 0.1-0.2 μm, and the permeate is collected; (e) Pass the permeate through an ultrafiltration membrane with a molecular weight cutoff of 800 Da, collect the permeate with a molecular weight cutoff of less than 800 Da, and concentrate it under reduced pressure to 1 / 5 to 1 / 10 of the original volume. (f) Slowly add ethanol to the concentrate until the ethanol volume fraction is 65%-75%, stir and let stand for 2-4 hours, collect the precipitate, wash with anhydrous ethanol and freeze dry to obtain cod collagen peptide powder with an average molecular weight ≤800Da.

7. The complex peptide as described in claim 1, characterized in that, The eggshell membrane powder is a soluble eggshell membrane extract obtained through low-temperature compound enzymatic hydrolysis. The specific steps are as follows: (a) Eggshell membranes were subjected to airflow ultrafine grinding at a low temperature of 0-8℃ to obtain micro powder with a particle size D90≤20μm; (b) Disperse the micro powder in Tris-HCl buffer solution at a mass ratio of 1:(10-20) to pH 7.0-8.0, add glutathione or cysteine ​​to a final concentration of 5-15 mmol / L, and gently stir at 30-40℃ for 1-3 hours. (c) Add keratinase to the mixture at an enzyme-to-base mass ratio of 1:(50-100) and hydrolyze at pH 7.5-8.5 and 40-50℃ for 2-4 hours; then add papain at an enzyme-to-base mass ratio of 1:(100-200) and continue hydrolyzing for 2-3 hours. Inactivate the enzyme, centrifuge, and collect the supernatant. (d) The supernatant was passed through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da. The permeate was collected, concentrated under reduced pressure, and then freeze-dried to obtain a soluble eggshell membrane extract.

8. The preparation method of the beauty and skin-nourishing bird's nest complex peptide as described in claim 1, characterized in that, Includes the following steps: S1. Premixing: Weigh out cod collagen peptides and eggshell membrane extract according to the ratio, and premix for 6-10 minutes under vacuum of -0.08MPa, 20-25℃ and 15-20rpm to form a uniform matrix. S2, Gradient Mixing: (1) Prepare an aqueous solution of elastin peptide with a mass concentration of 8-12%, and spray it evenly onto the matrix surface through an atomizing nozzle at a flow rate of 0.2-0.4 L / min. Maintain a rotation speed of 20 rpm during the spraying process, and continue mixing for 4-6 minutes after the spraying is completed. (2) Prepare an aqueous solution of bird's nest peptide with a mass concentration of 4-6%, and spray it evenly onto the matrix surface at a flow rate of 0.1-0.2 L / min. Maintain a rotation speed of 20 rpm during the spraying process. After spraying, continue mixing for 10 minutes and vacuum dry until the moisture content is ≤3%. S3. Post-processing: Continue mixing the mixed materials at the same temperature for 15-20 minutes, pass them through an 80-mesh sieve, and aseptically dispense them under nitrogen protection throughout the process to obtain the finished compound peptide product.

9. The application of the beauty and skin-nourishing bird's nest complex peptide according to any one of claims 1-7 in the preparation of products with beauty and skin-nourishing functions.

10. The application according to claim 9, characterized in that, The product may be a solid beverage, tablet, capsule, oral liquid, or cosmetic.

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