Preparation method and application of andrias davidianus active peptide with cell repair and anti-aging functions

Abstract

The present application belongs to the technical field of bioactive peptide preparation and biological function application, and particularly relates to a preparation method and application of giant salamander active peptide with cell repair and anti-aging functions. The present application uses legal second-generation giant salamander muscle and cartilage mixed tissues as raw materials, removes grease and fishy smell impurities through low-temperature pretreatment, adopts two-stage gradient composite enzyme directional enzymolysis to precisely enrich active peptide segments, then carries out multi-level membrane fractionation purification through microfiltration, ultrafiltration and nanofiltration to intercept 1000-3000 Da high-activity components, and finally carries out low-temperature vacuum freeze drying to lock the bioactivity, thereby preparing giant salamander active peptide powder. The present application is stable and controllable, and the product has high active peptide purity and high bioactivity retention rate, is rich in various anti-aging active amino acid groups, can effectively remove free radicals, repair skin cell oxidative damage and inhibit cell aging and apoptosis, is suitable for the fields of anti-aging health-care food, skin care cosmetics and cell repair biological pharmaceutical preparations, and has good industrial application prospects.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide preparation and biofunctional application technology, specifically referring to the preparation method and application of giant salamander active peptides with cell repair and anti-aging functions. Background Technology

[0002] The Chinese giant salamander is a rare and unique amphibious aquatic organism in my country, rich in high-quality collagen, functional proteins, and various amino acids in its muscle and cartilage tissues. These make it a superior natural raw material for preparing bioactive peptides. Chinese giant salamander bioactive peptides possess advantages such as low molecular weight, easy absorption, good biocompatibility, and low toxicity, demonstrating potential application value in cell regulation, anti-oxidation, and tissue repair. This is currently a hot research area for bioactive raw materials.

[0003] Existing publicly available technologies for preparing active peptides from giant salamanders mostly employ traditional processes involving single-protease hydrolysis, crude purification at room temperature, and high-temperature drying. These methods suffer from several technical shortcomings: First, the hydrolysis method is singular, only randomly cleaving protein peptide bonds and failing to selectively enrich cell repair and anti-aging specific active peptides. The resulting products have a disordered composition of active components, a high proportion of ineffective impurities, weak cell repair targeting, and only a single antioxidant effect, failing to achieve multi-dimensional anti-aging effects such as cell damage repair, delayed cell aging, and inhibition of telomere loss. Second, the raw material pretreatment process is rudimentary, lacking precise degreasing and deodorization. First, residual oils and fishy odors in the product greatly limit its high-end applications in the food, cosmetics, and biopharmaceutical fields. Second, the purification process is crude and lacks precise molecular weight classification, leaving behind large-molecule impurities and small-molecule ineffective fragments, resulting in low product purity and unstable biological activity. Third, the high-temperature drying process easily damages the active structure of peptides, causing a large loss of active components and a significant decrease in product efficacy. At the same time, the existing technology has not formed an integrated process for raw material pretreatment, targeted enzymatic hydrolysis, precise purification, and activity lock-in, resulting in low product conversion rate, poor batch stability, and difficulty in achieving large-scale industrial production.

[0004] A search of existing patents and literature revealed no complete technical solution for preparing high-purity, high-activity giant salamander active peptides with both targeted cell repair and multi-dimensional anti-aging functions through a two-stage gradient complex enzyme directed enzymatic hydrolysis combined with precise membrane fractionation purification and low-temperature activity lock-in process. Existing technologies have significant process gaps and efficacy shortcomings, failing to meet the application needs of high-end bioactive products. Summary of the Invention

[0005] To address the problems of low activity, poor purity, limited efficacy, insufficient stability, and limited application in existing giant salamander active peptide preparation processes, this invention provides a method and application for preparing giant salamander active peptides with cell repair and anti-aging functions. Through process innovation, highly active peptides are precisely enriched to achieve dual effects of targeted cell repair and multi-dimensional anti-aging, while improving product purity, stability, and industrial applicability, and expanding its high-end application scenarios.

[0006] To achieve the above functions, the technical solution adopted by the present invention is as follows: a method for preparing and applying giant salamander active peptides with cell repair and anti-aging functions, comprising the following steps: Low-temperature pretreatment of raw materials: The raw materials are a mixture of muscle and cartilage from second-generation artificially bred giant salamanders. The fascia, blood clots and impurities are removed, and the mixture is rinsed with sterile water at low temperature. It is then chopped into a paste at a low temperature of 0–4℃. Sterile ultrapure water of 3–5 times the weight of the raw materials is added and mixed evenly to make a raw material homogenate. Subsequently, a combination of low-temperature centrifugal degreasing and activated carbon adsorption is used to remove grease and fishy impurities, and a pure pretreated raw material liquid is obtained. Gradient-based complex enzyme-directed enzymatic hydrolysis: The pretreated raw material solution is adjusted to pH and temperature, and then enzymatically hydrolyzed using a two-stage gradient complex enzyme process. The first stage uses a complex enzyme of neutral protease and papain to gently hydrolyze large molecular proteins at low temperature. The second stage uses a complex enzyme of flavor protease and trypsin to directionally cleave specific peptide bonds and enrich cell repair and anti-aging active peptides. Temperature and pH are controlled throughout the enzymatic hydrolysis process. After the enzymatic hydrolysis is completed, the enzyme is inactivated by high temperature and cooled to room temperature to obtain crude peptide solution. Multi-stage membrane fractionation purification: The crude peptide solution is sequentially passed through a multi-stage membrane separation system of microfiltration, ultrafiltration and nanofiltration to accurately retain active peptide components with a molecular weight of 1000-3000 Da, and remove large molecular weight impurities, small molecular weight ineffective fragments and inorganic salt impurities to obtain a high-purity active peptide purified solution. Low-temperature activity lock-in drying: The purified liquid is dried using low-temperature vacuum freeze-drying technology, and the drying temperature and vacuum degree are controlled to retain the bioactivity of peptides to the greatest extent, and finally obtain powdered giant salamander active peptide products.

[0007] Furthermore, the low-temperature centrifugation parameters in step (1) are: temperature 2–4℃, rotation speed 8000–10000 r / min, and centrifugation time 15–20 min; the activated carbon adsorption deodorization conditions are: activated carbon addition amount is 0.8%–1.2% of the raw material liquid mass, low-temperature adsorption in the dark for 30–40 min, and activated carbon is removed by filtration, leaving no fishy residue and without damaging the active protein components.

[0008] Furthermore, the two-stage gradient complex enzymatic hydrolysis process in step (2) specifically includes: First stage of enzymatic hydrolysis: Adjust the pH of the raw material solution to 6.8–7.2 and the temperature to 45–50℃, add 0.2%–0.4% of a neutral protease and papain complex enzyme in a mass ratio of 2:1, and hydrolyze at a constant temperature for 90–120 min. Second stage of enzymatic hydrolysis: Adjust the pH of the system to 7.5–8.0 and the temperature to 50–55℃, and add 0.15%–0.25% of the total enzyme amount of flavor protease and trypsin complex enzyme at a mass ratio of 1:1.5. Perform enzymatic hydrolysis at a constant temperature for 60–80 min. The enzyme inactivation conditions are: heating in a water bath at 90–95℃ for 10–15 minutes to completely inactivate the enzyme activity and terminate the enzymatic hydrolysis reaction.

[0009] Furthermore, the multi-stage membrane fractionation purification process in step (3) is as follows: First, a 0.22μm microfiltration membrane is used to filter and remove suspended impurities and undigested solid particles; then, a 3000Da ultrafiltration membrane is used to retain large molecular weight proteins, and a 1000Da ultrafiltration membrane is used to remove small molecular weight ineffective peptides and ionic impurities; finally, the solution is concentrated by nanofiltration to obtain a high-purity active peptide purified solution with a solid content ≥15%, of which the 1000–3000Da active peptide component accounts for ≥92%.

[0010] Furthermore, the low-temperature vacuum freeze-drying parameters in step (4) are: vacuum degree 0.08–0.10 MPa, pre-freezing temperature -35 to -30℃, drying temperature -20 to -15℃, drying time 24–30 h, and the resulting active peptide powder has a moisture content ≤3% and a bioactivity retention rate ≥95%.

[0011] This invention also includes the giant salamander active peptide obtained by the above preparation method. The molecular weight of the active peptide is precisely concentrated in the optimal activity range of 1000–3000 Da, and the amino acid sequence of the peptide is rich in a high proportion of active functional groups such as proline, glycine, and lysine. Based on the specific molecular weight distribution and active amino acid composition, this active peptide possesses excellent biological activities in targeting and repairing cellular oxidative damage, scavenging free radicals in vivo and in vitro, and inhibiting cell senescence and apoptosis. It has strong targeted efficacy and stable effects.

[0012] The highly active giant salamander peptides prepared by this invention can be widely used in the preparation of cell repair and anti-aging products, specifically covering three core areas: anti-aging health foods, skin care and anti-aging cosmetics, and biopharmaceutical preparations for repairing oxidative damage cells. They are suitable for multiple industrialization scenarios such as food, beauty, and biopharmaceuticals.

[0013] Compared with the prior art, the present invention achieves the following beneficial effects by adopting the above solution: 1. This invention adopts a combined pretreatment process of low-temperature chopping, low-temperature centrifugal degreasing, and low-temperature light-proof deodorization with activated carbon. The entire process is carried out under low-temperature and sterile conditions, which not only thoroughly removes oil, fishy smell, impurities and waste tissues from the raw materials, solving the problem of strong fishy smell and many impurities in traditional process products, but also completely preserves the complete structure of active proteins in the raw materials, laying the foundation for the subsequent preparation of highly active peptides. 2. It adopts a two-stage gradient complex enzymatic hydrolysis process. The first stage is low-temperature and gentle cleavage of large molecular proteins, and the second stage is directional cleavage of specific peptide bonds. Compared with the traditional single enzymatic hydrolysis process, it can accurately enrich the core active peptides of 1000-3000 Da. The content of anti-aging and cell repair active groups such as proline, glycine, and lysine in the peptides is greatly increased, and the product's efficacy in targeting and repairing oxidatively damaged cells, scavenging free radicals, and inhibiting cell aging is significantly enhanced. 3. Through multi-stage precise separation via microfiltration, ultrafiltration, and nanofiltration, solid impurities, large molecular weight proteins, small molecular weight fragments, and inorganic salts are removed in stages. The peptide components with the optimal active molecular weight range are precisely targeted, with the core active peptides accounting for ≥92%, completely solving the defects of traditional products such as mixed components, many ineffective impurities, and low activity. 4. The product adopts a low-temperature vacuum freeze-drying process, which avoids the problems of peptide denaturation and activity loss caused by high-temperature drying. The product has a bioactivity retention rate of ≥95%, a moisture content of ≤3%, strong storage stability, and can be stored for a long time without diminishing efficacy. 5. The giant salamander active peptides prepared by this invention have precise efficacy, high purity, and good safety. They can be applied to multiple fields such as anti-aging health foods, skin care cosmetics, and cell repair biopharmaceutical preparations. They have strong industrial adaptability and extremely high market application value. Attached Figure Description

[0014] Figure 1 This is a process flow diagram of the method for preparing giant salamander active peptides according to the present invention; Figure 2 This is a comparison chart of the active component content of the giant salamander active peptide product of this invention and traditional products; Figure 3 This is a curve comparing the cell repair and antioxidant effects of the giant salamander active peptide product of this invention. Detailed Implementation

[0015] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1:

[0016] The present invention discloses a method for preparing giant salamander active peptides with cell repair and anti-aging functions, comprising the following steps: Low-temperature pretreatment of raw materials: 100g of mixed muscle and cartilage from legally farmed second-generation giant salamanders was selected, fascia impurities were removed, and the mixture was rinsed clean with sterile water at 4℃. The mixture was then minced into a fine paste at 4℃ and mixed with 400g of sterile ultrapure water to form a homogenate. The homogenate was centrifuged at 3℃ and 9000r / min for 18min to separate and remove the upper layer of oil. 1.0% activated carbon was added to the lower layer of liquid and adsorbed at 4℃ in the dark for 35min. The activated carbon was then removed by filtration to obtain a pure pretreated raw material liquid. Gradient-directed enzymatic hydrolysis using a complex enzyme: The pretreated raw material solution was adjusted to pH 7.0 and temperature 48℃, and 0.3% of a neutral protease and papain complex enzyme (mass ratio 2:1) was added. The solution was hydrolyzed at a constant temperature for 100 min. Subsequently, the pH of the system was adjusted to 7.8 and temperature 52℃, and 0.2% of a flavor protease and trypsin complex enzyme (mass ratio 1:1.5) was added. The solution was hydrolyzed at a constant temperature for 70 min. After hydrolysis, the solution was heated in a 92℃ water bath for 12 min to inactivate the enzymes, and then allowed to cool naturally to room temperature to obtain the crude peptide solution. Multi-stage membrane fractionation purification: The crude peptide solution is first filtered through a 0.22μm microfiltration membrane to remove solid impurities; then it is sequentially filtered through 3000Da and 1000Da ultrafiltration membranes to retain peptide components of 1000–3000Da; finally, it is concentrated through a nanofiltration membrane to obtain a high-purity active peptide purified solution with a solid content of 16%. Low-temperature active lock-in drying: The purified liquid was placed in a vacuum freeze dryer, with a pre-freezing temperature of -32℃, a vacuum degree of 0.09MPa, and a drying temperature of -18℃ for 28 hours to obtain a white powdery giant salamander active peptide product with a water content of 2.5%, of which the 1000–3000Da active peptide component accounted for 93.2%. Example 2:

[0017] The present invention discloses a method for preparing giant salamander active peptides with cell repair and anti-aging functions, comprising the following steps: Low-temperature pretreatment of raw materials: 200g of mixed muscle and cartilage of legally artificially bred second-generation giant salamanders was selected, washed and chopped at 0℃ under sterile conditions, and then homogenized with 5 times the amount of sterile ultrapure water; degreased by low-temperature centrifugation at 2℃ and 10000r / min for 15min, and then deodorized by adding 0.8% activated carbon at 4℃ for 40min adsorption, and filtered to obtain pure raw material liquid; Gradient-directed enzymatic hydrolysis with complex enzymes: First stage: pH 6.8, 45℃, add 0.2% complex enzyme (neutral protease: papain = 2:1) and hydrolyze for 120 min; Second stage: pH 7.5, 50℃, add 0.15% complex enzyme (flavor protease: trypsin = 1:1.5) and hydrolyze for 80 min; Inactivate enzyme at 90℃ for 15 min, cool to obtain crude peptide solution; Multi-stage membrane fractionation purification: 0.22μm microfiltration for impurity removal, 3000Da and 1000Da ultrafiltration fractionation, and nanofiltration concentration to obtain a 15.5% solids purified solution; Low-temperature drying: Pre-freeze at -30℃, vacuum degree 0.08MPa, dry at -15℃ for 30h to obtain an active peptide product with a water content of 2.8% and an effective component ratio of 92.5%.

[0018] Comparative Example Giant salamander active peptides were prepared using a traditional single trypsin hydrolysis, room temperature precipitation and defatting, and high-temperature spray drying process. This method lacked gradient enzymatic hydrolysis, precise membrane fractionation, and low-temperature lock-in processes, while maintaining consistent amounts of other raw materials. The resulting product contained only 65.3% effective peptide components, had a noticeable fishy odor, and exhibited only 62% activity retention. Its cell repair and antioxidant effects were significantly lower than those of the products in Examples 1 and 2 of this invention.

[0019] Efficacy verification test Cell repair activity assay Using an H2O2-induced oxidative damage model of human skin fibroblasts, the cell repair rates of the products from Examples 1 and 2 of this invention, as well as the comparative products, were tested. The results showed that the cell damage repair rate of the product of this invention reached 89.2%–91.5%, while that of the comparative product was only 52.3%, indicating a significant improvement in the cell repair performance of this invention.

[0020] 2. Antioxidant and anti-aging test The DPPH and ABTS free radical scavenging rates and telomere protection effects of the product of this invention were tested. The DPPH free radical scavenging rate was ≥92% and the ABTS free radical scavenging rate was ≥90%, which can significantly inhibit telomere loss and delay the cell aging cycle. The comparative product had a free radical scavenging rate of only 55%-60%, no obvious telomere protection effect, and its anti-aging effect was singular and weak.

[0021] 3. Product stability test After being stored at room temperature in a sealed container for 6 months, the product of this invention retains ≥90% of its activity, with no clumping, no odor, and stable components; while the product of traditional processes loses more than 40% of its activity, exhibits slight clumping and a fishy smell, and has extremely poor stability. Application Examples

[0022] 1. Cosmetic Application: Adding the active peptides of the giant salamander of this invention at a mass ratio of 0.5%–2% to anti-aging serums, creams, and masks can significantly enhance the skin cell repair ability, improve fine lines, dullness, and skin damage, and is gentle and non-irritating, suitable for all skin types.

[0023] 2. Application in health food: Active peptide powder can be made into tablets, powders, and oral liquids. Long-term use can eliminate free radicals in the body, repair oxidative damage to cells, delay aging, and improve the body's metabolic capacity.

[0024] 3. Biomedical applications: It can be used to prepare oxidative damage cell repair agents to help repair skin trauma, mucous membrane damage and metabolic cell damage in the body. It has good biocompatibility and no toxic side effects.

[0025] The present invention and its embodiments have been described above. This description is not restrictive, and the embodiments shown are only one of the embodiments of the present invention, and are not actually limited thereto. In short, if those skilled in the art are inspired by this description and design similar embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for preparing giant salamander active peptides with cell repair and anti-aging functions, characterized in that, Includes the following steps: (1) Low-temperature pretreatment of raw materials: The muscle and cartilage mixture of artificially bred second-generation giant salamanders were selected as raw materials. The fascia, blood clots and impurities were removed. The raw materials were rinsed with sterile water at low temperature and chopped into meat paste at 0-4℃. Sterile ultrapure water of 3-5 times the weight of the raw materials was added and mixed evenly to make a raw material homogenate. Then, low-temperature centrifugation degreasing and activated carbon adsorption combined deodorization treatment were used to remove oil and fishy impurities to obtain pure pretreated raw material liquid. (2) Gradient complex enzyme targeted enzymatic hydrolysis: The pH and temperature of the pretreated raw material solution are adjusted, and a two-stage gradient complex enzymatic hydrolysis process is adopted for enzymatic hydrolysis. The first stage uses a complex enzyme of neutral protease and papain to hydrolyze macromolecular proteins at low temperature and gently. The second stage uses a complex enzyme of flavor protease and trypsin to cleave specific peptide bonds and enrich cell repair and anti-aging active peptides. The temperature and pH are controlled throughout the enzymatic hydrolysis process. After the enzymatic hydrolysis is completed, the enzyme is inactivated by high temperature and cooled to room temperature to obtain crude peptide solution. (3) Multi-stage membrane fractionation purification: The crude peptide solution is sequentially passed through a multi-stage membrane separation system of microfiltration, ultrafiltration and nanofiltration to accurately retain active peptide components with a molecular weight of 1000–3000 Da, remove large molecular weight impurities, small molecular weight ineffective fragments and inorganic salt impurities, and obtain a high-purity active peptide purified solution. (4) Low-temperature activity lock-in drying: The purified liquid is dried using low-temperature vacuum freeze-drying technology, and the drying temperature and vacuum degree are controlled to retain the bioactivity of peptides to the greatest extent, and finally obtain powdered giant salamander active peptide products.

2. The method for preparing giant salamander active peptides according to claim 1, characterized in that, The low-temperature centrifugation parameters in step (1) are: temperature 2–4℃, rotation speed 8000–10000 r / min, and centrifugation time 15–20 min; the activated carbon adsorption deodorization conditions are: activated carbon addition amount is 0.8%–1.2% of the raw material liquid mass, low-temperature adsorption in the dark for 30–40 min, and activated carbon is removed by filtration, leaving no fishy odor residue and without damaging the active protein components.

3. The method for preparing giant salamander active peptides according to claim 1, characterized in that, The two-stage gradient complex enzymatic hydrolysis process in step (2) is specifically as follows: First stage of enzymatic hydrolysis: Adjust the pH of the raw material solution to 6.8–7.2 and the temperature to 45–50℃, add 0.2%–0.4% of a neutral protease and papain complex enzyme in a mass ratio of 2:1, and hydrolyze at a constant temperature for 90–120 min. Second stage of enzymatic hydrolysis: Adjust the pH of the system to 7.5–8.0 and the temperature to 50–55℃, and add 0.15%–0.25% of the total enzyme amount of flavor protease and trypsin complex enzyme at a mass ratio of 1:1.

5. Perform enzymatic hydrolysis at a constant temperature for 60–80 min. The enzyme inactivation conditions are: heating in a water bath at 90–95℃ for 10–15 minutes to completely inactivate the enzyme activity and terminate the enzymatic hydrolysis reaction.

4. The method for preparing giant salamander active peptides according to claim 1, characterized in that, The multi-stage membrane fractionation purification process in step (3) is as follows: First, a 0.22μm microfiltration membrane is used to filter and remove suspended impurities and undigested solid particles; then, a 3000Da ultrafiltration membrane is used to retain large molecular weight proteins, and a 1000Da ultrafiltration membrane is used to remove small molecular weight ineffective peptides and ionic impurities; finally, the solution is concentrated by nanofiltration to obtain a high-purity active peptide purified solution with a solid content ≥15%, of which the 1000–3000Da active peptide component accounts for ≥92%.

5. The method for preparing giant salamander active peptides according to claim 1, characterized in that, The low-temperature vacuum freeze-drying parameters in step (4) are: vacuum degree 0.08–0.10 MPa, pre-freezing temperature -35 to -30℃, drying temperature -20 to -15℃, drying time 24–30 h, and the resulting active peptide powder has a moisture content ≤3% and a bioactivity retention rate ≥95%.

6. The giant salamander active peptide prepared by the preparation method according to any one of claims 1 to 5, characterized in that, The active peptides have molecular weights concentrated in the range of 1000–3000 Da, and the amino acids in the peptide segments contain a high proportion of proline, glycine, and lysine active groups, possessing biological activities such as targeted repair of cellular oxidative damage, free radical scavenging, and inhibition of cell senescence and apoptosis.

7. The application of the giant salamander active peptide according to claim 6 in the preparation of cell repair and anti-aging products, characterized in that, The products include anti-aging health foods, skin care and anti-aging cosmetics, and biopharmaceutical preparations for repairing oxidatively damaged cells.

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