A method for preparing pearl polypeptide by using active oxygen for efficient depolymerization

Abstract

The present application relates to the technical fields of bioactive substance extraction and preparation, and discloses a method for preparing pearl polypeptide by using active oxygen for efficient depolymerization, which comprises the following steps: (1) raw material pretreatment: washing and drying freshwater pearls, crushing, and sieving to obtain pearl powder; (2) preparation of suspension liquid: mixing and dispersing the pearl powder with water to obtain a pearl suspension liquid; (3) efficient depolymerization: adding hydrogen peroxide aqueous solution to the pearl suspension liquid, treating at 15-35 DEG C for 5-30 min, and releasing polypeptide active ingredients by the action of active oxygen to make the organic matrix and inorganic minerals in the pearl dissociate; the mass fraction of the hydrogen peroxide aqueous solution is 0.1%-3.0%, and the addition amount is 1%-10% of the volume of the pearl suspension liquid. The present application uses an aqueous system as a reaction medium, uses active oxygen formed by hydrogen peroxide to promote the dissociation of the organic-inorganic composite structure in the pearl, and can realize the release of active ingredients such as amino acids and polypeptides in the pearl under relatively mild conditions, and reduce the adverse effects of strong acid and strong base conditions on the active ingredients.

Description

Technical Field

[0001] This invention relates to the field of bioactive substance extraction and preparation technology, specifically a method for preparing pearl polypeptides using efficient depolymerization of reactive oxygen species. Background Technology

[0002] Pearls are natural organic-inorganic composite materials formed by mollusks during biomineralization. They are mainly composed of calcium carbonate inorganic mineral phase (90%–95%) and a small amount of organic matrix (5%–10%). The organic matrix contains active ingredients such as proteins, peptides, and amino acids, which have various biological activities such as anti-oxidation and repair promotion, and have broad application prospects in the cosmetics and food industries. However, the dense and stable internal structure of pearls, with the inorganic mineral phase tightly bound to the organic matrix, makes it difficult to directly release the active components such as proteins, peptides, and amino acids. Existing pearl active ingredient extraction technologies mainly include acid hydrolysis, alkaline hydrolysis, and enzymatic hydrolysis. Acid hydrolysis or alkaline hydrolysis usually uses strong acids (such as hydrochloric acid and sulfuric acid) or strong bases (such as sodium hydroxide) and reacts at high temperatures for a long time. Although it can decompose the calcium carbonate mineral phase and release some organic components, it has problems such as intense reaction conditions, easy destruction of active ingredients, complex subsequent neutralization and desalination processes, and large wastewater discharge. Although enzymatic hydrolysis has relatively mild reaction conditions, the enzyme preparations are expensive, the reaction time is long (usually 4 to 24 hours), and the enzymes have poor permeability to the dense structure of pearls, making industrial control complex.

[0003] In recent years, some studies have attempted to combine physical aids (such as ultrasound and microwave) with chemical / enzymatic methods, but these methods generally suffer from problems such as long process flows, significant introduction of exogenous substances, heavy post-processing burdens, and insufficient product composition stability. Therefore, existing processes for extracting active pearl components and preparing pearl peptides still struggle to simultaneously achieve gentle depolymerization, retention of active components, high raw material utilization, and industrial applicability. It is necessary to provide a method for preparing pearl peptides that can promote the dissociation of the organic matrix and inorganic mineral phase within the pearl under gentler conditions, improve the release efficiency of amino acids and peptides, and reduce the introduction of exogenous chemical reagents. Summary of the Invention

[0004] To address the problems mentioned in the background section, this invention provides a method for preparing pearl polypeptides using efficient depolymerization of reactive oxygen species, comprising the following steps:

[0005] (1) Raw material pretreatment: Freshwater pearls are washed, dried, crushed, and sieved to obtain pearl powder;

[0006] (2) Preparation of suspension: The pearl powder is mixed with water and dispersed to obtain a pearl suspension;

[0007] (3) High-efficiency depolymerization: Add hydrogen peroxide aqueous solution to the pearl suspension and treat at 15-35℃ for 5-30 min. The organic matrix and inorganic mineral phase in the pearl are dissociated by active oxygen, releasing polypeptide active ingredients. The mass fraction of the hydrogen peroxide aqueous solution is 0.1%-3.0%, and the amount added is 1%-10% of the volume of the pearl suspension.

[0008] (4) Solid-liquid separation: The depolymerized system is subjected to solid-liquid separation, and the filtrate is collected;

[0009] (5) Post-processing: The filtrate is concentrated, desalted and dried in sequence to obtain pearl polypeptide extract.

[0010] Preferably, in step (3), the mass fraction of the hydrogen peroxide aqueous solution is 0.3% to 1.5%, and the amount added is 3% to 8% of the volume of the pearl suspension; the treatment temperature is 20 to 30°C, the treatment time is 15 to 25 min, and the stirring speed is 400 to 600 r / min.

[0011] Preferably, in step (3), the mass fraction of the hydrogen peroxide aqueous solution is 0.5%, the amount added is 5% of the volume of the pearl suspension, the treatment temperature is 25°C, the treatment time is 20 min, and the stirring speed is 500 r / min.

[0012] Preferably, in step (1), the particle size of the pearl powder is 50-150 μm; in step (2), the concentration of the pearl suspension is 5-20 g / L.

[0013] Preferably, the solid-liquid separation adopts a method of centrifugation followed by filtration; the centrifugation speed is 4000-6000 r / min, and the centrifugation time is 8-15 min; the filtration adopts a microporous filter membrane with a pore size of 0.22-0.45 μm.

[0014] Preferably, the concentration is performed under reduced pressure at a temperature of 45–55°C, concentrating to 1 / 4–1 / 8 of the original volume; the desalting is performed by dialysis with a dialysis bag having a molecular weight cutoff of 500 Da for 24–36 hours; and the drying is performed by freeze drying.

[0015] A pearl polypeptide extract prepared by the method described above has the following characteristics:

[0016] (1) The peptide content is ≥8%, and the peak area of ​​the component with a molecular weight of 500-3000 Da accounts for ≥80% when determined by gel permeation chromatography;

[0017] (2) Contains glycine, alanine, glutamic acid and aspartic acid, of which glycine accounts for ≥15% of the total amino acids;

[0018] (3) At a concentration of 1 mg / mL, the DPPH free radical scavenging rate is ≥60%;

[0019] (4) Residual hydrogen peroxide ≤ 0.005%.

[0020] Preferably, the total content of the four amino acids glycine, alanine, glutamic acid and aspartic acid in the extract accounts for more than 40% of the total amino acid content.

[0021] Preferably, the amount of pearl polypeptide extract added to the cosmetic is 0.1% to 5%.

[0022] Preferably, the amount of pearl polypeptide extract added to the food is 1% to 20%.

[0023] Compared with existing technologies, this invention provides a method for preparing pearl polypeptides using efficient depolymerization of reactive oxygen species, which has the following beneficial effects:

[0024] 1. The method provided by this invention has mild reaction conditions. Under normal temperature conditions of 15-35°C, hydrogen peroxide is used as the active oxygen source, and the treatment time is only 5-30 minutes. This avoids the damage of active ingredients such as polypeptides and heat-sensitive amino acids in pearls caused by high temperature, strong acid, and strong alkali, and preserves the bioactivity of the product to the greatest extent.

[0025] 2. This invention does not require the introduction of strong acids, strong bases or exogenous enzyme preparations. It can effectively depolymerize the organic-inorganic composite structure of pearls by using only a small amount of hydrogen peroxide (mass fraction 0.1% to 3.0%), which significantly reduces the introduction of exogenous chemical reagents and reduces the burden of subsequent post-processing such as neutralization, desalination and removal. The process is more green and environmentally friendly.

[0026] 3. The extraction efficiency of the method of the present invention is high, the extraction rate of pearl polypeptide can reach 8% to 15%, and the content of active ingredients in the obtained extract can reach 55% to 75%. Compared with the traditional acid hydrolysis method and enzymatic hydrolysis method, the extraction rate is increased by about 100% and 60% respectively, the reaction time is shortened by 99% and 72%, and the reagent cost is reduced by 62% and 80%, resulting in significant comprehensive economic benefits.

[0027] 4. The pearl polypeptide extract obtained by this invention has clear quality characteristics: polypeptide content ≥8%, peak area ratio of components with molecular weight of 500-3000 Da ≥80%, rich in characteristic amino acids such as glycine, alanine, glutamic acid and aspartic acid, DPPH free radical scavenging rate ≥60% (at a concentration of 1 mg / mL), residual hydrogen peroxide ≤0.005%, and the product has good stability and high safety.

[0028] 5. The method of the present invention is simple and has a short process (only requiring pretreatment, suspension preparation, efficient depolymerization, solid-liquid separation, post-treatment, etc.), low equipment requirements, and the raw materials can be reused 1 to 2 times. It is easy to realize industrial-scale production and has good industrial application prospects.

[0029] 6. The pearl polypeptide extract obtained by this invention has a good effect on promoting fibroblast migration and promoting type III collagen expression. Adding 0.1% to 5% can be used to prepare cosmetics such as serums, masks, and creams, with significant moisturizing, anti-wrinkle, elasticity and luster-enhancing effects. At the same time, it has good antioxidant activity. Adding 1% to 20% can be used to prepare nutritional supplements or functional foods, with a wide range of applications. Attached Figure Description

[0030] Figure 1 This is a process flow diagram of the present invention for preparing pearl polypeptide extract by efficient depolymerization of reactive oxygen species;

[0031] Figure 2 This is a schematic diagram illustrating the effect of pearl suspension concentration on the release of active ingredients in this invention.

[0032] Figure 3 This is a schematic diagram illustrating the effect of contact time on the release of active ingredients in this invention;

[0033] Figure 4 This is a schematic diagram illustrating the effect of the number of times raw materials are reused on the release efficiency of active ingredients in this invention.

[0034] Figure 5 This is a bar chart comparing the extraction effects of the present invention with those of acid hydrolysis / enzymatic hydrolysis.

[0035] Figure 6 This is the HPLC amino acid analysis chromatogram of the pearl polypeptide obtained in this invention;

[0036] Figure 7 This is a molecular weight distribution (GPC) map of the pearl polypeptide obtained in this invention.

[0037] Figure 8 A schematic diagram showing the effect of different hydrogen peroxide concentrations and amounts on the extraction efficiency;

[0038] Figure 9 A diagram illustrating the comparison of the effects of different extraction methods;

[0039] Figure 10 This is a schematic diagram showing the comparison results between the hydrogen peroxide method and the ozone method. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Please see Figure 1-10 :

[0042] Unless otherwise stated, the raw materials, reagents, and equipment used in the following examples are all commercially available products in the art. Experimental methods not specified in the examples are generally performed under standard conditions or as recommended by the manufacturer.

[0043] Example 1: Preparation of Pearl Polypeptide Extract

[0044] This embodiment illustrates the method for preparing pearl polypeptides using efficient depolymerization of reactive oxygen species according to the present invention, specifically including the following steps:

[0045] (1) Raw material pretreatment: Natural freshwater pearl raw materials were selected and repeatedly rinsed with pure water to remove dust and impurities attached to the surface. The cleaned pearls were placed in a forced-air drying oven and dried at 50°C for 6 hours until there was no obvious residual moisture on the surface. The pearls were then pulverized using a pulverizer and passed through a 200-mesh sieve to obtain pearl powder with a particle size of approximately 75 μm.

[0046] (2) Preparation of suspension: Weigh the above pearl powder and add it to ultrapure water with a resistivity of 18.25 MΩ·cm (25℃) to prepare a pearl suspension with a mass concentration of 10 g / L. Stir at 500 r / min for 20 min using a magnetic stirrer to ensure uniform dispersion of the pearl powder.

[0047] (3) Efficient depolymerization: The above pearl suspension was placed in a reaction vessel, and at 25°C and a stirring speed of 500 r / min, a 0.5% (w / w) hydrogen peroxide aqueous solution was added, with the addition amount being 5% of the suspension volume. The mixture was stirred for another 20 min. During the treatment, hydrogen peroxide decomposed to produce reactive oxygen species such as hydroxyl radicals (·OH) and superoxide anion radicals (·O2⁻), which attacked the protein / peptide chains in the pearl organic matrix, promoting the dissociation of the interface between the organic matrix and the inorganic calcium carbonate mineral phase, and releasing active ingredients such as amino acids and peptides into the liquid phase.

[0048] (4) Solid-liquid separation: After the depolymerization treatment, the reaction system was transferred to a centrifuge tube and centrifuged at 5000 r / min for 10 min using a benchtop centrifuge. The supernatant was collected. The supernatant was filtered through a 0.22 μm microporous membrane to obtain a clear filtrate.

[0049] (5) Post-processing: The above filtrate was concentrated to 1 / 5 of its original volume using a rotary evaporator under reduced pressure in a 50°C water bath. The concentrate was transferred to a dialysis bag with a molecular weight cutoff of 500 Da and dialyzed in pure water for 24 h to remove salts and residual hydrogen peroxide. After dialysis, the solution in the bag was collected and freeze-dried to obtain a pale yellow powdery pearl polypeptide extract.

[0050] The obtained pearl polypeptide extract was analyzed:

[0051] Polypeptide content (BCA method): 12.5%.

[0052] Molecular weight distribution (GPC method): The peak area of ​​components with molecular weights of 500–3000 Da accounted for 86%.

[0053] Amino acid composition (PITC-derived RP-HPLC method): Contains 15 amino acids including glycine, alanine, glutamic acid, aspartic acid, leucine, and arginine, of which glycine accounts for 18.5% of the total amino acids.

[0054] DPPH free radical scavenging rate (1 mg / mL concentration): 68%.

[0055] Residual hydrogen peroxide (titanium salt colorimetric method): <0.001%.

[0056] The results show that the method of this embodiment can efficiently obtain high-purity and high-activity pearl polypeptide extracts under mild conditions.

[0057] Example 2: Effect of different pearl suspension concentrations on extraction efficiency

[0058] This embodiment sets up multiple concentration points for comparison to verify the effect of pearl suspension concentration on extraction efficiency.

[0059] Pearl powder was mixed with ultrapure water to prepare pearl suspensions with mass concentrations of 0, 1, 2, 3, 5, 10, 20, 30, and 50 g / L, respectively. The remaining steps were the same as in Example 1.

[0060] Test results as follows Figure 2 As shown, when the concentration of the pearl suspension was increased from 0 g / L to 1, 2, 3, and 5 g / L, the total amino acid content in the extract increased rapidly; when the concentration was increased to 10 g / L, the total amino acid content reached approximately 25 mg / L; thereafter, under conditions of 20 g / L, 30 g / L, and 50 g / L, the total amino acid content fluctuated only slightly within the range of 24–26 mg / L, without a significant further increase. Considering both the extraction effect and the suitability of the process, controlling the pearl suspension concentration within the range of 5–20 g / L can better achieve the objectives of this invention.

[0061] Example 3: Effects of different hydrogen peroxide concentrations and dosages on extraction efficiency

[0062] This embodiment sets up comparisons with different hydrogen peroxide concentrations and amounts to verify the optimal range of hydrogen peroxide usage conditions.

[0063] A pearl suspension with a mass concentration of 10 g / L was prepared according to the method in Example 1. The suspension was treated at 25°C for 20 min. The mass fractions of added hydrogen peroxide aqueous solution were set to 0.1%, 0.5%, 1.0%, and 3.0%, respectively, and the amounts added based on the suspension volume were set to 1%, 3%, 5%, and 10% for comparison. All other conditions remained the same.

[0064] Test results as follows Figure 8 As shown.

[0065] Depend on Figure 8 It was found that the extraction effect was optimal when the hydrogen peroxide concentration was 0.5% and the addition amount was 5%, achieving high levels of peptide content and DPPH scavenging rate, with extremely low residual hydrogen peroxide. When the hydrogen peroxide concentration was too low (0.1%), the reactive oxygen species activity was insufficient, and the depolymerization effect was not significant; when the hydrogen peroxide concentration was too high (3.0%), the improvement in extraction effect was limited, and the residual amount increased, potentially adversely affecting product safety.

[0066] Example 4: Effect of different processing times on extraction efficiency

[0067] This embodiment sets up a comparison of different efficient depolymerization processing times to verify the optimal range of processing time.

[0068] A pearl suspension with a mass concentration of 10 g / L was prepared according to the method in Example 1. At 25°C, a 0.5% (w / w) aqueous solution of hydrogen peroxide was added at 5% of the suspension volume, and the suspension was treated for 0, 1, 3, 5, 8, 10, 15, 20, 30, 60, 90, 120, and 180 min, respectively, with the remaining steps being the same.

[0069] Test results as follows Figure 3 As shown, when the treatment time was extended from 0 min to 20 min, the total amino acid content in the extract increased rapidly; after 20 min, the total amino acid content tended to stabilize, and further extending the treatment time had no significant effect on increasing the release of active ingredients. The total amino acid content obtained under the conditions of 20, 30, 60, 90, 120, and 180 min showed little difference and was at a relatively high level. Considering extraction efficiency, energy consumption, and process suitability, a treatment time controlled within the range of 15–25 min can better achieve the purpose of this invention, with 20 min achieving a good release effect.

[0070] Example 5: Investigation on the number of times raw materials can be reused

[0071] This embodiment is used to verify the change in the release efficiency of active ingredients when pearl raw materials are reused under the same conditions.

[0072] A pearl suspension with a mass concentration of 10 g / L was prepared according to the method in Example 1. A first extraction was performed under the conditions of Example 1, and the extract was collected. The solid sediment after centrifugation and filtration was collected and added back into ultrapure water at the same material-to-liquid ratio. The extraction steps were repeated to obtain the second, third, and fourth extracts. The total amino acid content of each extract was determined.

[0073] The results are as follows Figure 4 As shown, the release of active ingredients was highest after the first treatment of the raw material; after the second reuse, the total amino acid content in the extract decreased significantly (approximately 40% of the first time); after the third and fourth reuses, the release of active ingredients further decreased and tended to stabilize (approximately 10%–15% of the first time). The results indicate that pearl raw materials can still release a certain amount of active ingredients after 1–2 reuses, but the release efficiency gradually decreases with increasing reuse frequency. Considering both extraction efficiency and process economy, the optimal number of reuses for the raw material is 1–2 times.

[0074] Example 6: Comparison of the method of the present invention with existing technologies (acid hydrolysis, enzymatic hydrolysis)

[0075] This embodiment is used to compare the extraction effect of the method of the present invention with that of existing acid hydrolysis and enzymatic hydrolysis methods.

[0076] Acid hydrolysis method control: Take 10 g of pearl powder, add 1 L of 2 mol / L HCl, react at 80℃ for 4 h, then neutralize to pH 7.0, centrifuge and filter, concentrate under reduced pressure, dialyze to desalt, and freeze dry.

[0077] Enzymatic hydrolysis control: Take 10 g of pearl powder, add 1 L of pure water, adjust the pH to 7.5, add protease (100 U / g pearl powder), react at 50℃ for 12 h, then centrifuge and filter, concentrate under reduced pressure, dialyze to desalt, and freeze dry.

[0078] The method of this invention is carried out according to the method of Example 1.

[0079] See the comparison of test results. Figure 9 and Figure 5

[0080] Compared with acid hydrolysis, the method of the present invention increases the extraction rate by approximately 101%, the content of active ingredients in the extract by approximately 51%, shortens the reaction time by approximately 99%, and reduces reagent costs by approximately 62%. Compared with enzymatic hydrolysis, the method of the present invention increases the extraction rate by approximately 60%, increases the content of active ingredients in the extract by approximately 17%, shortens the reaction time by approximately 72%, and reduces reagent costs by approximately 80%. These results demonstrate that the method of the present invention has significant advantages in terms of extraction efficiency, reaction time, and cost.

[0081] Example 7: Comparison of Hydrogen Peroxide Method and Ozone Method

[0082] This embodiment compares the effects of hydrogen peroxide and ozone methods on the extraction and activity retention of pearl polypeptides.

[0083] Hydrogen peroxide method: Performed according to the method in Example 1.

[0084] Ozone method control: A pearl suspension with a mass concentration of 10 g / L was prepared according to the method in Example 1. Ozone was introduced into the pearl suspension through an ozone generator at 25°C to control the ozone concentration in the system to 0.3 mg / L or 1.0 mg / L, and the treatment was carried out for 20 min. After treatment, solid-liquid separation and post-treatment were performed according to the method in Example 1.

[0085] Test results are shown Figure 10 .

[0086] Depend on Figure 10 It is evident that while the ozone method can promote the release of some active ingredients, the peptide content and DPPH free radical scavenging rate of the resulting product are significantly lower than those obtained by the hydrogen peroxide method; the decrease in activity is even more pronounced when the ozone concentration increases. This indicates that excessive ozone oxidation can easily lead to further oxidative degradation of pearl peptides, affecting the retention of activity. In contrast, this invention uses an appropriate amount of hydrogen peroxide treatment, which can better maintain peptide activity while promoting the release of pearl active ingredients.

[0087] Example 8: Application of pearl polypeptide extract in cosmetics (serum)

[0088] This embodiment illustrates the application of the pearl polypeptide extract of the present invention in the preparation of cosmetics that promote fibroblast migration and / or promote type III collagen expression.

[0089] Pearl polypeptide essence formula (mass fraction):

[0090] Pearl polypeptide extract (prepared in Example 1): 2.0%

[0091] Hyaluronic acid: 0.5%

[0092] Glycerin: 5.0%

[0093] 1,3-Butanediol: 3.0%

[0094] Deionized water: Balance

[0095] Preparation method: Pearl polypeptide extract, hyaluronic acid, glycerin and 1,3-butanediol are added to deionized water in sequence, stirred and dissolved evenly, the pH is adjusted to 5.5-6.5, filtered and sterilized to obtain the final product.

[0096] In vitro efficacy evaluation:

[0097] Promotes human fibroblast migration rate (24 h, scratch assay): 45%.

[0098] Type III collagen mRNA expression was increased (48 h, qPCR): 2.8-fold.

[0099] DPPH free radical scavenging rate (1 mg / mL): 72%.

[0100] ABTS free radical scavenging rate (1 mg / mL): 65%.

[0101] Human trial evaluation (28 days, 30 healthy volunteers, twice daily):

[0102] Skin hydration (Corneometer): Increased by 28%.

[0103] Wrinkle depth (Visia): Reduced by 15%.

[0104] Skin elasticity (Cutometer): Increased by 12%.

[0105] Skin radiance: Increased by 18%.

[0106] The results show that the pearl polypeptide extract of the present invention has good antioxidant and collagen synthesis-promoting activities, and is suitable for the cosmetic field.

[0107] Example 9: Application of pearl polypeptide extract in food (nutritional supplement)

[0108] This embodiment illustrates the application of the pearl polypeptide extract of the present invention in the preparation of food with antioxidant properties.

[0109] Pearl polypeptide nutritional supplement formula (mass fraction):

[0110] Pearl polypeptide extract (prepared in Example 1): 10%

[0111] Maltodextrin: 80%

[0112] Vitamin C: 5%

[0113] Magnesium stearate: 5%

[0114] Preparation method: Mix all raw materials evenly, pass through a 60-mesh sieve, granulate by dry method, compress into tablets or fill into capsules, each tablet (capsule) contains 100 mg of pearl polypeptide extract.

[0115] Antioxidant efficacy evaluation (in vitro):

[0116] DPPH free radical scavenging rate (1 mg / mL): 65%.

[0117] ABTS free radical scavenging rate (1 mg / mL): 58%.

[0118] Total antioxidant capacity (FRAP method): 0.45 mmol FeSO4 / g.

[0119] Stability test (40℃, 75% relative humidity, accelerated for 6 months):

[0120] The decrease rate of peptide content is <5%.

[0121] Moisture increase: <3%.

[0122] Microbial limits: Compliant with national standards.

[0123] The results show that the pearl polypeptide extract of the present invention can be stably applied in nutritional supplements or functional foods and has a clear antioxidant effect.

[0124] Example 10: Application of Pearl Peptide Extract in Facial Masks

[0125] This embodiment illustrates another application of the pearl polypeptide extract of the present invention in cosmetics.

[0126] Pearl polypeptide facial mask liquid formula (mass fraction):

[0127] Pearl polypeptide extract (prepared in Example 1): 3.0%

[0128] Glycerin: 8.0%

[0129] Propylene glycol: 5.0%

[0130] Carbomer: 0.2%

[0131] Triethanolamine: 0.2%

[0132] Phenoxyethanol: 0.5%

[0133] Deionized water: Balance

[0134] Preparation method: Carbomer is dispersed in deionized water and swollen. Glycerin and propylene glycol are added and stirred evenly. Triethanolamine is added to neutralize to pH 6.0-7.0. Pearl polypeptide extract and phenoxyethanol are then added and stirred evenly. The mixture is then filled into mask bags, each containing 25 mL of mask liquid and one non-woven mask sheet.

[0135] Human trial evaluation (28 days, 30 healthy women, 3 times a week):

[0136] Skin hydration improvement rate: 32%.

[0137] Skin brightness (L* value) improved by 8.5%.

[0138] Skin roughness parameter (SEr): reduced by 22%.

[0139] Subject satisfaction: 93% (students felt their skin became smoother and more radiant).

[0140] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing pearl polypeptides using efficient depolymerization of reactive oxygen species, characterized in that, Includes the following steps: (1) Raw material pretreatment: Freshwater pearls are washed, dried, crushed, and sieved to obtain pearl powder; (2) Preparation of suspension: The pearl powder is mixed with water and dispersed to obtain a pearl suspension; (3) High-efficiency depolymerization: Add hydrogen peroxide aqueous solution to the pearl suspension and treat at 15-35℃ for 5-30 min. The organic matrix and inorganic mineral phase in the pearl are dissociated by active oxygen, releasing polypeptide active ingredients. The mass fraction of the hydrogen peroxide aqueous solution is 0.1%-3.0%, and the amount added is 1%-10% of the volume of the pearl suspension. (4) Solid-liquid separation: The depolymerized system is subjected to solid-liquid separation, and the filtrate is collected; (5) Post-processing: The filtrate is concentrated, desalted and dried in sequence to obtain pearl polypeptide extract.

2. The method according to claim 1, characterized in that, In step (3), the mass fraction of the hydrogen peroxide aqueous solution is 0.3% to 1.5%, and the amount added is 3% to 8% of the volume of the pearl suspension; the treatment temperature is 20 to 30°C, the treatment time is 15 to 25 min, and the stirring speed is 400 to 600 r / min.

3. The method according to claim 2, characterized in that, In step (3), the mass fraction of the hydrogen peroxide aqueous solution is 0.5%, the amount added is 5% of the volume of the pearl suspension, the treatment temperature is 25℃, the treatment time is 20 min, and the stirring speed is 500 r / min.

4. The method according to claim 1, characterized in that, In step (1), the particle size of the pearl powder is 50-150 μm; in step (2), the concentration of the pearl suspension is 5-20 g / L.

5. The method according to claim 1, characterized in that, In step (4), the solid-liquid separation is carried out by centrifugation followed by filtration; the centrifugation speed is 4000-6000 r / min and the centrifugation time is 8-15 min; the filtration is carried out using a microporous membrane with a pore size of 0.22-0.45 μm.

6. The method according to claim 1, characterized in that, In step (5), the concentration is vacuum concentration at a temperature of 45-55°C, concentrated to 1 / 4-1 / 8 of the original volume; the desalting is performed by dialysis with a dialysis bag with a molecular weight cutoff of 500 Da for 24-36 h; and the drying is freeze drying.

7. A pearl polypeptide extract prepared by the method according to any one of claims 1-6, characterized in that, It meets the following characteristics: (1) The peptide content is ≥8%, and the peak area of ​​the component with a molecular weight of 500-3000 Da accounts for ≥80% when determined by gel permeation chromatography; (2) Contains glycine, alanine, glutamic acid and aspartic acid, of which glycine accounts for ≥15% of the total amino acids; (3) At a concentration of 1 mg / mL, the DPPH free radical scavenging rate is ≥60%; (4) Residual hydrogen peroxide ≤ 0.005%.

8. The pearl polypeptide extract according to claim 7, characterized in that, The extract contains more than 40% of the total content of four amino acids: glycine, alanine, glutamic acid, and aspartic acid.

9. The use of the pearl polypeptide extract according to claim 7 or 8 in the preparation of cosmetics having the effect of promoting fibroblast migration and / or promoting type III collagen expression, characterized in that, The amount of pearl polypeptide extract added to the cosmetic is 0.1% to 5%.

10. The use of the pearl polypeptide extract according to claim 7 or 8 in the preparation of food with antioxidant properties, characterized in that, The amount of pearl polypeptide extract added to the food is 1% to 20%.

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