Preparation and application of animal-derived collagen peptide containing e-xn-p short peptide sequence

Abstract

The application discloses preparation and application of animal-source collagen peptide containing E-Xn-P short peptide sequence and belongs to the technical field of preparation and application of bioactive peptides. The patent adopts the method of enzymolysis and chelation and dissociation, and separates and obtains animal-source collagen active peptide containing E-Xn-P short peptide sequence, wherein E represents glutamic acid, P represents proline, X is natural L-amino acid, n is 0-4, the animal-source collagen active peptide has strong divalent metal ion chelation capacity and metal ion absorption promotion capacity, immunoregulation activity and antioxidant and anti-inflammatory capacity. The animal-source collagen active peptide containing E-Xn-P short peptide sequence prepared in the application can be used for developing new health-care food, medicine and skin care products and has the characteristics of high bioactivity and rich nutritional value, contributes to product utilization rate of collagen and provides a train of thought and a way for development of functional health-care food, medicine and skin care products.

Description

Technical Field

[0001] This invention relates to the preparation and application of an animal-derived collagen peptide containing an E-Xn-P short peptide sequence, belonging to the field of bioactive peptide preparation technology and application. Background Technology

[0002] With increasing focus on health and beauty, collagen, as an important biomolecule, has received widespread attention. Collagen is a crucial structural protein, widely found in connective tissue, participating in maintaining the structure and function of multiple biological systems, including skin, bones, and joints. As we age, collagen synthesis gradually decreases, leading to sagging skin, wrinkles, and declining joint function. Therefore, collagen peptides, as low-molecular-weight products of collagen hydrolysis, are more easily absorbed by the body and are widely used in anti-aging, joint health, and wound healing. Collagen peptides, as a natural ingredient with significant bioactivity, have become a popular product in the market. The structural characteristics of collagen peptides determine their various physiological functions in the body, such as promoting skin elasticity, delaying aging, promoting bone health, and relieving joint pain. Therefore, the preparation process of collagen peptides and the research on their bioactive peptides have become important topics in the field of biopharmaceuticals.

[0003] Traditional animal-derived collagen peptides (such as fish scales, mammalian skin and bones) are widely used in food, cosmetics, and pharmaceutical products. However, existing collagen peptide products have certain limitations in terms of precise regulation of active ingredients and functional enhancement, such as limited functionality and insufficient structural optimization, which restricts the efficacy and market competitiveness of these products.

[0004] Collagen peptides are typically obtained by hydrolyzing collagen or gelatin, with the hydrolysis process usually using acids, alkalis, or enzymes as catalysts. Enzymatic hydrolysis, as a highly efficient and specific hydrolysis method, has been widely used in recent years. However, in traditional collagen hydrolysis, the enzymatically obtained peptide chains are diverse, and their biological activities vary considerably, making it difficult to effectively control the formation of specific bioactive peptides. This makes the screening and isolation of bioactive peptides with specific biological activities, such as anti-aging, antioxidant, and anti-inflammatory functions, from collagen peptides a current research challenge.

[0005] Currently, most techniques in this field only involve enzymatic hydrolysis to process collagen peptides, determining the molecular weight of the hydrolyzed peptides. This involves breaking down collagen into smaller peptides for easier digestion and absorption. However, the resulting peptides have relatively specific sequences and functions, and their molecular weights are mostly below 10,000 Da. Patent CN118879814 A provides a method for preparing anti-aging fish collagen peptides, which involves pre-treating collagen and then directly hydrolyzing it. The resulting peptides have relatively large molecular weights and limited functions. CN111635920 A discloses a method for preparing donkey bone collagen peptide powder using a two-stage biomimetic enzymatic hydrolysis technique. This patent provides a method for producing donkey bone collagen peptides that are more compatible with human absorption and biological action by simulating the physiological environment of the human stomach and intestines using a two-stage biological enzymatic hydrolysis process. While this method can yield relatively efficient collagen peptides, their functionality remains unclear. Summary of the Invention

[0006] This invention aims to address the shortcomings of existing technologies. Through experimental decomposition and optimization of collagen peptides from different sources, a novel short peptide sequence containing an E-Xn-P active structure was discovered. This peptide exhibits significant biological activity, and it is not included in the products directly obtained by conventional enzymatic hydrolysis in existing systems. Specifically, the E-Xn-P short peptide sequence consists of E (glutamate), Xn (natural L-amino acid sequence), and P (proline), where n ranges from 0 to 4. This peptide exhibits unique functionality in various physiological activity tests, such as improving immunity and enhancing the body's anti-inflammatory and antioxidant capabilities. The E-Xn-P short peptide is a novel collagen-derived active peptide. Its unique amino acid arrangement and activity characteristics provide a new direction for collagen peptide research. Furthermore, the special properties produced during collagen hydrolysis provide a new approach for preparing collagen peptides with higher activity. In traditional enzymatic hydrolysis, most common bioactive peptides are relatively simple amino acid chains. However, the unique structure of E-Xn-P short peptides gives them greater potential in the application of collagen peptides.

[0007] To address the existing problems, this patent employs an enzymatic hydrolysis and chelation followed by dissociation method to separate and obtain EX-containing compounds. n Animal-derived collagen bioactive peptides with a short P-P peptide sequence, where E represents glutamic acid, P represents proline, and X is a natural L-amino acid, with n ranging from 0 to 4. These peptides possess strong chelating ability for divalent metal ions and promote the absorption of divalent metal ions.

[0008] The purpose of this invention is to provide a product containing EX n Animal-derived collagen bioactive peptides with -P short peptide sequences, containing EX nThis paper describes the preparation method of animal-derived collagen bioactive peptides with -P short peptide sequences and their applications in improving immunity, enhancing anti-inflammatory and antioxidant capabilities. This provides theoretical support for the rational utilization of collagen resources and the development of novel dietary supplements and skincare products.

[0009] This invention provides a product containing EX n -P short peptide sequence active peptide, said active peptide is animal-derived collagen active peptide, wherein said animal-derived collagen peptide contains unique EX n The amino acid sequence is -P, where E represents glutamic acid, P represents proline, and X is a natural L-amino acid, with n ranging from 0 to 4. Acidic amino acids (aspartic acid and glutamic acid) account for 25-40% of the total amino acid content. Peptides with a relative molecular mass of 0-2000 Da account for 80-95% of the total donkey collagen peptide content, exhibiting strong chelating ability for divalent metal ions and promoting the absorption of divalent metal ions.

[0010] The active peptide was prepared according to the following method:

[0011] (1) Enzymatic hydrolysis: Add an enzyme preparation to a system containing animal-derived collagen peptides to hydrolyze the protein to obtain an animal-derived collagen peptide hydrolysate.

[0012] (2) Chelation: The animal-derived collagen peptide hydrolysate obtained in step (1) is chelated with metal ions to obtain animal-derived collagen peptide metal chelate.

[0013] (3) Dissociation: The animal-derived collagen peptide metal chelate obtained in step (2) is subjected to ethanol fractionation precipitation and acid dissociation to obtain animal-derived collagen active peptide.

[0014] In one embodiment of the present invention, the animal-derived collagen peptides include, but are not limited to, donkey-hide gelatin, fish scales, and sheepskin;

[0015] In one embodiment of the present invention, in step (1), when the enzyme is papain and alkaline protease, a stepwise enzymatic hydrolysis is adopted: the concentration of the hydrolysis substrate is 5-10%, 1500-2500 U / mL of papain is first added to the hydrolysis system, the pH is maintained at 7.0-9.0, the temperature is 40-50℃, and the hydrolysis is carried out for 1.5-3.0 h; after the one-step hydrolysis is completed, the pH is adjusted to maintain it at 11.0-12.0, and then 3000-5000 U / mL of alkaline protease is added to the hydrolysis system, the pH is adjusted to maintain it at 11.0-12.0, the temperature is 40-50℃, and the hydrolysis is carried out for 4.0-6.0 h;

[0016] In one embodiment of the present invention, in step (1), when the enzyme is a neutral protease and a hydrolytic protease, a stepwise enzymatic hydrolysis is adopted: the concentration of the enzymatic substrate is 5-10%, 1250-2000 U / mL of neutral protease is first added to the enzymatic hydrolysis system, the pH is maintained at 5.5-7.5, the temperature is 30-60℃, and the enzymatic hydrolysis is carried out for 1.5-3.0 h; after the one-step enzymatic hydrolysis is completed, the pH is adjusted to maintain it at 7.5-10.0, and then 2000-5000 U / mL of hydrolytic protease is added to the enzymatic hydrolysis system, the pH is adjusted to maintain it at 7.5-10.0 again, the temperature is 40-50℃, and the enzymatic hydrolysis is carried out for 4.0-6.0 h;

[0017] In one embodiment of the present invention, in step (1), when the enzyme is a flavor protease, a single enzymatic hydrolysis is performed, the concentration of the enzymatic hydrolysis substrate is 5-10%, 2000-3000 U / mL of flavor protease is added to the enzymatic hydrolysis system, the pH is maintained at 5.5-7.0, the temperature is 45-60℃, the enzymatic hydrolysis is performed for 3.0-6.0 h, and the pH is adjusted to maintain it at 5.5-7.0.

[0018] In one embodiment of the present invention, in step (2), the chelation reaction is as follows: the mass ratio of animal-derived collagen peptide hydrolysate to divalent metal is adjusted to 2:1 to 4:1, the pH is adjusted to 7.0 to 9.0, the temperature is 30 to 50°C, the shaking speed is 150 to 200 rpm, the chelation reaction time is 30 to 60 min, and the supernatant is filtered to obtain animal-derived collagen peptide metal chelate;

[0019] In one embodiment of the present invention, in step (2), the divalent metal ion is one or more of iron, calcium, and zinc.

[0020] In one embodiment of the present invention, in step (3), the ethanol fractionation precipitation is carried out by adding pre-cooled anhydrous ethanol to the animal-derived collagen peptide metal chelate, controlling the volume fraction of ethanol in the final system to be 35-85%, stirring and mixing, and then letting it stand at 4-25°C for 0.5-2 hours. The precipitate is collected by centrifugation, and the above operation is repeated until the metal ions attached to the surface of the mixed peptide that have not been chelated with the mixed peptide are completely washed away. The precipitate obtained from the last washing is placed in a water bath at 40-60°C to promote the ethanol to evaporate to dryness.

[0021] In one embodiment of the present invention, in step (3), the acidic dissociation is performed by re-dissolving the animal-derived collagen peptide metal chelate after ethanol fractionation precipitation, adjusting the pH to 3.0-5.0, and then subjecting it to microwave heating treatment at a temperature of 40-60°C, a power of 200-400W, and a heating time of 3-10 minutes. After the reaction, the supernatant is collected by centrifugation, the pH is adjusted to 7.0-9.0, and the solution is allowed to stand for 10-30 minutes before adjusting the pH to 3.0-5.0 again. The solution is then subjected to microwave heating treatment (at a temperature of 40-60°C, a power of 200-400W, and a heating time of 3-10 minutes). After the reaction is completed and cooled, the supernatant is collected by centrifugation. The above operation is repeated 3-5 times to obtain an active peptide solution.

[0022] In one embodiment of the present invention, the active peptide has a strong chelating ability for divalent metal ions such as iron, calcium, and zinc, with a chelating ability of 20-60 mg / mL, and can promote the absorption of divalent iron, calcium, and zinc ions.

[0023] This invention provides a method for preparing the above-mentioned active peptide containing the E-Xn-P short peptide sequence, the method comprising the following steps:

[0024] (1) Enzymatic hydrolysis: Select animal-derived collagen, add one or more enzyme preparations of hydrolytic protease, papain, alkaline protease, neutral protease and flavor protease to enzymatically hydrolyze the protein, inactivate the enzyme, centrifuge and take the supernatant to obtain animal-derived collagen peptide hydrolysate.

[0025] (2) Chelation and dissociation: Animal-derived collagen peptides are chelated with metal ions by shaking, precipitated by ethanol fractionation, dissociated by acid, and freeze-dried to obtain active peptides containing the E-Xn-P short peptide sequence.

[0026] In one embodiment of the present invention, the animal-derived collagen in step (1) is derived from the skin and bone collagen, connective tissue or collagen products of large-scale livestock, poultry and aquatic products, including but not limited to donkey-hide gelatin, fish scales and sheepskin.

[0027] In one embodiment of the present invention, when the enzyme is papain and alkaline protease, a stepwise enzymatic hydrolysis is adopted: the concentration of the hydrolysis substrate is 5-10%, firstly, 1500-2500 U / mL of papain is added to the hydrolysis system, the pH is maintained at 7.0-9.0, the temperature is 40-50°C, and the hydrolysis is carried out for 1.5-3.0 h. The pH is then adjusted to maintain it at 11.0-12.0. Next, 3000-5000 U / mL of alkaline protease is added to the hydrolysis system, the pH is adjusted to maintain it at 11.0-12.0 again, the temperature is 40-50°C, and the hydrolysis is carried out for 4.0-6.0 h.

[0028] In one embodiment of the present invention, when the enzyme is a neutral protease and a hydrolytic protease, a stepwise enzymatic hydrolysis is adopted: the concentration of the enzymatic substrate is 5-10%, firstly, 1250-2000 U / mL of neutral protease is added to the enzymatic hydrolysis system, the pH is maintained at 5.5-7.5, the temperature is 30-60°C, and the enzymatic hydrolysis is carried out for 1.5-3.0 h. The pH is then adjusted to maintain it at 7.5-10.0, and then 2000-5000 U / mL of hydrolytic protease is added to the enzymatic hydrolysis system. The pH is then adjusted to maintain it at 7.5-10.0, the temperature is 40-50°C, and the enzymatic hydrolysis is carried out for 4.0-6.0 h.

[0029] In one embodiment of the present invention, when the enzyme is a flavor protease, a single enzymatic hydrolysis is performed. The concentration of the hydrolysis substrate is 5-10%. 2000-3000 U / mL of flavor protease is first added to the hydrolysis system, the pH is maintained at 5.5-7.0, the temperature is 45-60°C, and the hydrolysis is carried out for 3.0-6.0 hours. The pH is then adjusted to maintain it at 5.5-7.0.

[0030] In one embodiment of the present invention, the enzyme inactivation temperature in step (1) is set to 95-100°C and the time is controlled to 15-30 min; after enzyme inactivation, the sample is placed at room temperature and then the centrifuge speed is set to 4000-6000 r / min and the centrifugation time is set to 15-30 min.

[0031] In one embodiment of the present invention, the divalent metal ion in step (2) is one or more of iron, calcium, and zinc, the mass ratio of collagen peptide to divalent metal is controlled to be 2:1 to 4:1, the pH is adjusted to 7.0 to 9.0, the temperature is 30 to 50°C, the shaking speed is 150 to 200 rpm, and the chelation reaction time is 30 to 60 min.

[0032] In one embodiment of the present invention, the anhydrous ethanol mentioned in step (2) should be pre-cooled at 4°C. The pre-cooled anhydrous ethanol is added to the supernatant, and the volume fraction of ethanol in the final system is controlled to be 35-85%. The mixture is stirred and mixed, and then allowed to stand at 4°C for 0.5-2 hours. The centrifuge speed is set to 4500-6000 r / min, and the centrifugation time is 15-30 min. After centrifugation, a precipitate is obtained. The precipitate is reconstituted with deionized water, and then anhydrous ethanol is added again to control the volume fraction of ethanol in the final system to be 35-85%. After standing for 20-40 minutes, the centrifuge speed is set to 3000-5000 r / min, and the centrifugation time is 15-30 min. The precipitate is collected by centrifugation. The above operation is repeated until the metal ions attached to the surface of the mixed peptide that have not been chelated with the mixed peptide are completely washed away. The precipitate obtained from the last washing is placed in a water bath at 40-60°C to allow the ethanol to evaporate to dryness.

[0033] In one embodiment, the acidic dissociation in step (2) involves reconstituted the animal-derived collagen peptide metal chelate after fractional precipitation with ethanol, adjusting the pH to 3.0–5.0 with 2 mol / L HCl solution, and then placing the collagen peptide metal chelate solution into a microwave reaction flask for heating treatment. The temperature is controlled at 40–60°C, the power is controlled at 200–400W, and the heating time is controlled at 3–10 min. After the reaction is complete and cooled, centrifuge at 4500–6000 r / min for 10–20 min. Collect the supernatant and adjust the pH to 7.0–9.0 with 0.5 mol / L NaOH solution. Let it stand for 10–30 min, then adjust the pH to 3.0–5.0. Microwave-assisted dissociation is performed (temperature controlled at 40–60℃, power controlled at 200–400 W, heating time controlled at 3–10 min). After the reaction is complete and cooled, centrifuge and collect the supernatant. Repeat the above operation 3–5 times to obtain an active peptide solution containing the E-Xn-P short peptide sequence.

[0034] This invention provides the application of the active peptide containing the E-Xn-P short peptide sequence and its products in the preparation of products that improve the body's immunity and enhance the body's antioxidant and anti-inflammatory capabilities.

[0035] In one embodiment of the present invention, the product includes, but is not limited to, pharmaceuticals, health products, enteral nutrition preparations, dietary supplements, food, and skin care products.

[0036] This invention provides the application of the active peptide containing the E-Xn-P short peptide sequence in the preparation of products that improve immunity, enhance antioxidant and anti-inflammatory effects, and promote the absorption of ferrous, calcium, and zinc ions.

[0037] In one embodiment of the present invention, the product includes, but is not limited to, pharmaceuticals, health products, enteral nutrition preparations, dietary supplements, food, and skin care products.

[0038] In one embodiment of the present invention, an active peptide containing the E-Xn-P short peptide sequence is used in product quality control and quality grading.

[0039] The present invention also provides the use of the above-mentioned active peptides in the preparation of products having any one or more of the following functions;

[0040] (a) Increases the number of white blood cells and lymphocytes in immunocompromised individuals, reduces the proportion of basophils in immunocompromised individuals, and stimulates the proliferation of splenic lymphocytes;

[0041] (b) Alleviate immune damage to individual spleen cell T lymphocytes and B lymphocytes; alleviate immune damage to individual spleen lymphocytes caused by cyclophosphamide;

[0042] (c) Enhance the body’s antioxidant capacity, including but not limited to increasing the DPPH free radical scavenging rate, reducing the level of malondialdehyde (MDA) in the serum of immunocompromised individuals, and increasing the level of superoxide dismutase (SOD) in the serum of immunocompromised individuals.

[0043] (d) Enhance the body's anti-inflammatory capabilities, including but not limited to increasing the expression of interleukin IL-6 and decreasing the expression of TNF-α;

[0044] (e) Enhances the body's immune function; alleviates the iron homeostasis imbalance caused by cyclophosphamide.

[0045] (f) As a biomarker for quality control.

[0046] In one embodiment of the present invention, the product contains an effective dose of active peptides, and the product includes, but is not limited to, one or more of the following: drugs, health foods, health products, foods for special medical purposes, food, functional foods, dietary supplements, feed, feed additives, or pet food.

[0047] In one embodiment of the present invention, the drug can alleviate bodily damage caused by immunodeficiency.

[0048] In one embodiment of the present invention, the dosage form of the drug is a liquid formulation or a solid formulation.

[0049] In one embodiment of the present invention, the dosage form of the drug includes, but is not limited to, granules, capsules, tablets, pills, or oral liquids.

[0050] In one embodiment of the invention, the drug further includes pharmaceutically acceptable excipients.

[0051] In one embodiment of the present invention, the pharmaceutical excipients include any one or more of the following: solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, pH adjusters, buffers, plasticizers, defoamers, thickeners, humectants, filter aids, and release inhibitors.

[0052] In one embodiment of the present invention, the food includes, but is not limited to, grain products, vegetable products, fruit products, fermented foods, meat products, egg products, dairy products, beverages, and snacks.

[0053] In one embodiment of the present invention, the food also includes special dietary foods.

[0054] In one embodiment of the present invention, the health product also contains acceptable excipients.

[0055] The present invention also provides an active peptide metal chelate containing the E-Xn-P short peptide sequence, wherein the active peptide metal chelate containing the E-Xn-P short peptide sequence is obtained by chelating the above-mentioned active peptide with metal ions.

[0056] In one embodiment of the present invention, the chelation reaction is as follows: the mass ratio of active peptide to divalent metal ion is adjusted to 2:1 to 4:1, the pH is adjusted to 7.0 to 9.0, the temperature is 30 to 50°C, the shaking speed is 150 to 200 rpm, the chelation reaction time is 30 to 60 min, the supernatant is filtered and then freeze-dried to obtain the active peptide metal chelate.

[0057] In one embodiment of the present invention, the divalent metal ion is one or more of iron, calcium, and zinc.

[0058] The present invention also provides the application of the above-mentioned active peptide, or the above-mentioned active peptide metal chelate containing the E-Xn-P short peptide sequence, or the product containing the active peptide or the active peptide metal chelate containing the E-Xn-P short peptide sequence in the preparation of products that improve the body's immunity and iron deficiency anemia; characterized in that the product contains an effective dose of active peptide, and the product includes, but is not limited to, one or more of the following: drugs, health foods, health products, foods for special medical purposes, foods, functional foods, dietary supplements, feed, feed additives, or pet foods.

[0059] In one embodiment of the present invention, the drug can alleviate bodily damage caused by immunodeficiency.

[0060] In one embodiment of the present invention, the dosage form of the drug is a liquid formulation or a solid formulation.

[0061] In one embodiment of the present invention, the dosage form of the drug includes, but is not limited to, granules, capsules, tablets, pills, or oral liquids.

[0062] In one embodiment of the invention, the drug further includes pharmaceutically acceptable excipients.

[0063] In one embodiment of the present invention, the pharmaceutical excipients include any one or more of the following: solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, pH adjusters, buffers, plasticizers, defoamers, thickeners, humectants, filter aids, and release inhibitors.

[0064] In one embodiment of the present invention, the food includes, but is not limited to, grain products, vegetable products, fruit products, fermented foods, meat products, egg products, dairy products, beverages, and snacks.

[0065] In one embodiment of the present invention, the food also includes special dietary foods.

[0066] In one embodiment of the present invention, the health product also contains acceptable excipients.

[0067] The present invention also provides an E-Xn-P short peptide sequence, wherein the E-Xn-P short peptide sequence is EGGGAEVPEGQR (SEQ ID NO.1), namely: Glu-Gly-Gly-Gly-Ala-Glu-Val-Pro-Glu-Gly-Gln-Arg.

[0068] Alternatively, the E-Xn-P short peptide can be obtained by mixing EEP, ERP, and EGTP in a mass ratio of 1:1:1.

[0069] Furthermore, the present invention also provides an application of the E-Xn-P short peptide sequence in the preparation of antioxidant or anti-inflammatory products, wherein the E-Xn-P short peptide sequence is EGGGAEVPEGQR (SEQ ID NO.1), or the E-Xn-P short peptide is obtained by mixing EEP, ERP and EGTP in a mass ratio of 1:1:1 to obtain a mixed short peptide.

[0070] In one embodiment of the present invention, the product contains an effective dose of E-Xn-P short peptide, and the product includes, but is not limited to, one or more of the following: drugs, health foods, health products, foods for special medical purposes, food, functional foods, dietary supplements, feed, feed additives, or pet food.

[0071] In one embodiment of the present invention, the drug can alleviate bodily damage caused by immunodeficiency.

[0072] In one embodiment of the present invention, the dosage form of the drug is a liquid formulation or a solid formulation.

[0073] In one embodiment of the present invention, the dosage form of the drug includes, but is not limited to, granules, capsules, tablets, pills, or oral liquids.

[0074] In one embodiment of the invention, the drug further includes pharmaceutically acceptable excipients.

[0075] In one embodiment of the present invention, the pharmaceutical excipients include any one or more of the following: solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, pH adjusters, buffers, plasticizers, defoamers, thickeners, humectants, filter aids, and release inhibitors.

[0076] In one embodiment of the present invention, the food includes, but is not limited to, grain products, vegetable products, fruit products, fermented foods, meat products, egg products, dairy products, beverages, and snacks.

[0077] In one embodiment of the present invention, the food also includes special dietary foods.

[0078] In one embodiment of the present invention, the health product also contains acceptable excipients.

[0079] Beneficial effects

[0080] (1) This invention provides a product containing EX n Animal-derived collagen bioactive peptides with a -P short peptide sequence, as determined by amino acid sequence analysis, contain a unique EX... n The amino acid sequence is -P, where E represents glutamic acid, P represents proline, and X is a natural L-amino acid, with n ranging from 0 to 4. Amino acid composition analysis shows that acidic amino acids aspartic acid and glutamic acid account for 25-40% of the total amino acid content. The molecular weight distribution is 0-2000 Da, exhibiting strong chelating ability for divalent metal ions and promoting iron absorption.

[0081] (2) The EX-containing invention provided by this invention n Animal-derived collagen bioactive peptides with a short P-P peptide sequence can activate immune cells such as lymphocytes and promote the secretion of cytokines such as IL-6, thereby enhancing the body's immune response. Compared with existing technologies, this bioactive peptide has shown stronger immunomodulatory capabilities in in vitro experiments, providing a possibility for the development of novel immunomodulators.

[0082] (3) The EX-containing invention provided by this invention nThe active peptide with the -P short peptide sequence exhibits significant antioxidant properties, enhancing superoxide dismutase (SOD) activity and reducing the content of lipid peroxidation products (MDA), thereby alleviating cell damage caused by oxidative stress and demonstrating its potential application in antioxidation. It also shows strong anti-inflammatory effects, effectively reducing inflammatory responses. This active peptide contains a specific amino acid sequence that can form stable complexes with various metal ions such as iron, copper, and zinc, thereby reducing the harmful accumulation of these metal ions in the body. This characteristic makes it potentially valuable in the treatment of metal ion poisoning and related neurodegenerative diseases.

[0083] (4) Compared with traditional immunomodulators, the active peptide of the present invention exhibits extremely low cytotoxicity while exerting immunomodulatory effects, and does not affect the proliferation of normal cells even at high concentrations. This characteristic makes the active peptide a safe and effective immunomodulator suitable for long-term use. Given the immunomodulatory, antioxidant, anti-inflammatory, and metal ion chelating functions of this active peptide, it has broad application prospects in the pharmaceutical field. It can be used as an adjuvant therapy to enhance the immunity of cancer patients and reduce the side effects caused by chemotherapy. Furthermore, it can be used as an ingredient in functional foods to improve the immunity of athletes and the elderly, and as an antioxidant and anti-inflammatory agent in skincare and beauty products.

[0084] (5) Since this bioactive peptide is derived from animal collagen, its production process does not involve the use of any harmful chemicals, and the byproducts are biodegradable, making it environmentally friendly. This aligns with current trends in sustainable development and green chemistry. The production process of this bioactive peptide is simple, inexpensive, and has a wide range of raw material sources, making it potential for large-scale production. Its application in the pharmaceutical and functional food fields is expected to bring significant economic benefits to enterprises. The widespread use of this bioactive peptide will help improve public health and reduce medical costs, particularly in immune regulation and anti-inflammatory treatment. Furthermore, its application in metal ion chelation helps reduce environmental pollution and improve food safety.

[0085] (6) Using collagen peptides containing the E-Xn-P short peptide sequence as biomarkers for quality control allows for more accurate assessment and monitoring of product quality, thereby ensuring that products meet predetermined quality standards. By detecting the presence and content of the E-Xn-P short peptide sequence in a product, the quality and origin of the product can be traced, providing consumers with more transparent product information. The method of this invention can classify products into different quality grades based on the characteristics of the E-Xn-P short peptide sequence in collagen peptides, thereby meeting market demands for products of different quality and contributing to the standardization and normalization of the entire industry. Attached Figure Description

[0086] Figure 1Prussian blue stained sections of mouse spleen showing iron levels. Detailed Implementation

[0087] The following embodiments better illustrate the content of the present invention, but the present invention is not limited to the following embodiments.

[0088] The donkey-hide gelatin powder, fish scale collagen powder, and sheepskin powder involved in the following examples were purchased from Suzhou Saimaier Biotechnology Co., Ltd.; the papain, alkaline protease, neutral protease, and flavor protease were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; and the hydrolytic protease was purchased from Novozymes.

[0089] The mouse model used in the following examples is the cyclophosphamide mouse model, a commonly used immunosuppressive model widely applied in studying the mechanisms of immunomodulators, antitumor drugs, and immune-related diseases. Cyclophosphamide (CTX) is an alkylating agent commonly used in anticancer therapy and the treatment of autoimmune diseases. The cyclophosphamide mouse model is a frequently used experimental model in researching immunotherapy for autoimmune diseases and cancer.

[0090] Cyclophosphamide induces immunodeficiency in mice by inhibiting DNA synthesis and interfering with cell division, leading to a decline in immune cell function. Studies have shown that continuous injection of 80 mg / kg for 3 days can cause thymic atrophy, splenomegaly, and a decrease in the proportion of immune cells.

[0091] Example 1: Containing EX n Preparation and application of -P short peptide sequence of donkey-hide gelatin active peptide I

[0092] 1. Contains EX n Preparation of -P short peptide sequence of donkey-hide gelatin active peptide I

[0093] (1) Enzymatic hydrolysis:

[0094] 1) First step enzymatic hydrolysis

[0095] Mix donkey-hide gelatin powder with ultrapure water until the substrate concentration is 5%, and place it at 37°C and shake to dissolve for 5 minutes to obtain a collagen solution.

[0096] Before enzymatic hydrolysis, 0.5% (by weight of substrate) of nicotinamide was dissolved in 2 mL of deionized water and then added to the collagen solution. After mixing, the pH of the solution was adjusted to 9.0 with 0.5 mol / L NaOH solution. Papain (2000 U / mL) was added and the mixture was placed in a benchtop constant temperature shaker at 40°C and 200 rpm for 2 hours. The pH was checked every 30 minutes during the reaction and kept at 9.0 throughout.

[0097] 2) Second step enzymatic hydrolysis

[0098] After the first enzymatic hydrolysis step, the system was removed, and the pH was adjusted to 12.0 again with 0.5 mol / L NaOH solution. Alkaline protease (3000 U / mL) was added, and the mixture was placed in a benchtop constant-temperature shaker at 40°C and 200 rpm for 5 hours. The pH was checked every 30 minutes during the reaction, and the pH was maintained at 12.0 throughout. After the reaction, the enzyme was inactivated at 95°C for 15 minutes, cooled to room temperature, and centrifuged at 4500 rpm for 20 minutes. The supernatant was collected, which is the donkey-hide gelatin polypeptide enzymatic hydrolysate.

[0099] (2) Oscillatory chelation:

[0100] Take the enzymatic hydrolysate of donkey-hide gelatin polypeptide obtained in step (1) and mix it with 0.2 mol / L FeSO4 solution. Control the mass ratio of donkey-hide gelatin polypeptide enzymatic hydrolysate to FeSO4 solution to 4:1. Adjust the pH to 7.5. React in a benchtop constant temperature shaker at 37℃ and 200 rpm for 30 min. Filter and take the supernatant to obtain the donkey-hide gelatin peptide iron chelate solution.

[0101] (3) Low-temperature ethanol fractionation and precipitation:

[0102] Take the donkey-hide gelatin peptide iron chelate solution obtained in step (2), add the pre-cooled anhydrous ethanol solution to obtain a reaction system with an ethanol concentration of 50%, stir and mix well, place it at 4℃ and let it stand for 120 min, then set the centrifuge speed to 4500 r / min and the centrifugation time to 20 min, and collect the precipitate by centrifugation.

[0103] The collected precipitate was redissolved: anhydrous ethanol was added again to make the volume fraction of anhydrous ethanol in the system 50%. The remaining steps were the same as above. After the fourth wash (each time the volume fraction of anhydrous ethanol was 50%), sodium sulfide solution was added to the supernatant after centrifugation. The solution was observed to see if it turned black. If it did not turn black, the washing was stopped. The precipitate after centrifugation was placed in a 40℃ water bath to allow the ethanol to evaporate to dryness, thus obtaining the donkey-hide gelatin iron chelate. If the solution turned black, the washing was continued until the solution reacted with the sodium sulfide solution and did not change color, which indicated that the washing was complete.

[0104] (4) Microwave-assisted acid dissociation:

[0105] The dried donkey-hide gelatin iron chelate was redissolved in deionized water to make the concentration of the chelate 0.2 g / mL. The pH was adjusted to 3.0 with 2 mol / L HCl solution to obtain a collagen peptide metal chelate solution.

[0106] The obtained collagen peptide metal chelate solution was placed in a microwave reaction flask and heated at 55°C, with a power of 400W and a heating time of 3 minutes.

[0107] After the reaction was completed and cooled, the centrifuge speed was set to 4500 r / min and the centrifugation time was 10 min. After centrifugation, the supernatant was collected, and the pH of the supernatant was adjusted to 8.0 with 0.5 mol / L NaOH solution. After standing for 10 min, the pH of the solution was adjusted to 3.0, and microwave-assisted dissociation was performed for 3 min (conditions: temperature controlled at 55℃, power controlled at 400W). After the reaction was completed and cooled, the supernatant was collected by centrifugation. The above operation was repeated 3 times to obtain the product containing EX. n -P short peptide sequence of donkey-hide gelatin active peptide I solution.

[0108] 2. For the obtained EX n Amino acid sequence analysis of the -P short peptide sequence in the solution of donkey-hide gelatin active peptide I.

[0109] The amino acid sequence of peptides also affects their biological activity. The obtained peptides containing EX... n After freeze-drying the solution of the -P short peptide sequence of donkey-hide gelatin active peptide I, the amino acid sequences of the peptide chains in the polypeptide mixture were sequenced and identified using liquid chromatography-mass spectrometry and proteomics. The solution of donkey-hide gelatin active peptide I was dissolved in methanol to prepare a 10 mg / mL collagen peptide solution.

[0110] Specific chromatographic conditions: Acclaim PepMap C18 column (75 μm × 25 cm), mobile phase A: aqueous solution containing 0.1% formic acid, mobile phase B: acetonitrile solution containing 0.1% formic acid; sample loading volume 5.0 μL, elution flow rate 300.0 nL / min. Mass spectrometry ionization mode was ESI+, positive charge spray voltage was 2.0 kV.

[0111] A database of relevant fish proteins was established from the Uniprot database, and then database alignment was performed using PEAKS Studio software. De novo sequencing analysis was conducted to perform amino acid sequence analysis, and the results are shown in Table 1.

[0112] Table 1 contains EX. n The -P short peptide sequence showed a high matching degree with the active peptide I of donkey-hide gelatin, representing 12 peptide sequences with higher concentrations among the top 20 peptide sequences. These peptide sequences collectively contain EX... n -P short sequence, where E represents glutamic acid, P represents proline, and X is a natural L-amino acid, where n is 0 to 2. This EX... n The -P sequence structure is a retained feature in every peptide chain, and short sequences may play a role in protein or peptide binding capacity, antioxidant activity, and cell signaling.

[0113] Table 1: Table containing EX n The -P short peptide sequence has a high degree of matching with the active peptide I of donkey-hide gelatin.

[0114]

[0115] Example 2: Containing EX n Preparation and application of fish scale bioactive peptide II with -P short peptide sequence

[0116] 1. Contains EX n Preparation of fish scale bioactive peptide II with -P short peptide sequence

[0117] (1) Enzymatic hydrolysis:

[0118] 1) First step enzymatic hydrolysis

[0119] Fish scale collagen powder was mixed with ultrapure water until the substrate concentration was 7%, and then placed at 30°C and shaken to dissolve for 8 minutes to obtain a collagen solution.

[0120] Before enzymatic hydrolysis, dissolve 0.8% (by weight of substrate) of nicotinamide in 2 mL of deionized water, then add it to the collagen solution above, ensuring uniform distribution. Adjust the pH of the solution to 7.0 with 0.5 mol / L NaOH solution, add neutral protease (1500 U / mL), and react in a benchtop constant-temperature shaker at 40°C and 200 rpm for 2 hours. Check the pH every 30 minutes during the reaction, always maintaining the pH at 7.0.

[0121] 2) Second step enzymatic hydrolysis

[0122] After the first enzymatic hydrolysis step, the system was removed, and the pH was adjusted to 9.0 again with 0.5 mol / L NaOH solution. Hydrolytic protease (3000 U / mL) was added, and the mixture was incubated in a benchtop shaker at 50°C and 200 rpm for 6 hours. The pH was checked every 30 minutes during the reaction, and maintained at 9.0 throughout. After the reaction, the enzyme was inactivated at 95°C for 20 minutes, cooled to room temperature, and centrifuged at 6000 rpm for 15 minutes. The supernatant was then collected as the fish scale polypeptide enzymatic hydrolysate.

[0123] (2) Oscillatory chelation:

[0124] Take the fish scale polypeptide enzymatic hydrolysate obtained in step (1) and mix it with 0.25 mol / L FeSO4 solution. Control the mass ratio of fish scale polypeptide enzymatic hydrolysate to FeSO4 solution to 3:1. Adjust the pH to 8.0. React in a benchtop constant temperature shaker at 45℃ and 180 rpm for 45 min. Filter and take the supernatant to obtain fish scale peptide iron chelate solution.

[0125] (3) Low-temperature ethanol fractionation and precipitation:

[0126] Take the fish scale peptide iron chelate solution obtained in step (2) and add 1.5 times the volume of the solution to a pre-cooled anhydrous ethanol solution to obtain a reaction system with an ethanol concentration of 60%. Stir and mix well, place at 4℃ and let stand for 60 min. Then set the centrifuge speed to 5000 r / min and the centrifugation time to 15 min. Centrifuge and collect the precipitate.

[0127] The collected precipitate was redissolved: anhydrous ethanol was added again to bring the volume fraction of anhydrous ethanol in the system to 60%. The remaining steps were the same as above. After the fourth wash (each time with an anhydrous ethanol volume fraction of 60%), sodium sulfide solution was added to the supernatant after centrifugation. The solution was observed to see if it turned black. If it did not turn black, the washing was stopped. The centrifuged precipitate was placed in a 50°C water bath to allow the ethanol to evaporate to dryness, yielding fish scale peptide iron chelate. If the solution turned black, the washing continued until the solution reacted with the sodium sulfide solution without changing color, indicating that the washing was complete.

[0128] (4) Microwave-assisted acid dissociation:

[0129] The dried fish scale peptide iron chelate was redissolved in deionized water to make the concentration of the chelate 0.5 g / mL, and the pH was adjusted to 3.5 with 2 mol / L HCl solution to obtain the collagen peptide metal chelate solution.

[0130] The obtained fish scale peptide metal chelate solution was placed in a microwave reaction flask and heated at 45°C, with a power of 300W and a heating time of 8 minutes.

[0131] After the reaction is complete and cooled, centrifuge at 5000 rpm for 15 minutes. Collect the supernatant and adjust its pH to 7.5 with 0.5 mol / L NaOH solution. Let it stand for 15 minutes, then adjust the pH to 3.5. Microwave-assisted dissociation is performed for 8 minutes (temperature controlled at 45℃, power controlled at 300W). After the reaction is complete and cooled, centrifuge again and collect the supernatant. Repeat this process 5 times. The supernatant collected after the final centrifugation contains EX. n Fish scale active peptide II solution with -P short peptide sequence.

[0132] 2. For the obtained EX n Amino acid sequence analysis of fish scale active peptide II solution containing the -P short peptide sequence.

[0133] The amino acid sequence of peptides also affects their biological activity. The obtained peptides containing EX... n After freeze-drying the fish scale active peptide II solution containing the -P short peptide sequence, the amino acid sequences of the peptide chains in the peptide mixture were sequenced and identified using liquid chromatography-mass spectrometry and proteomics. The fish scale active peptide II solution was dissolved in methanol to prepare a 10 mg / mL collagen peptide solution.

[0134] Specific chromatographic conditions: Acclaim PepMap C18 column (75 μm × 25 cm), mobile phase A: aqueous solution containing 0.1% formic acid, mobile phase B: acetonitrile solution containing 0.1% formic acid; sample loading volume 5.0 μL, elution flow rate 300.0 nL / min. Mass spectrometry ionization mode was ESI+, positive charge spray voltage was 2.0 kV.

[0135] A database of relevant fish proteins was established from the Uniprot database, and then database alignment was performed using PEAKS Studio software. De novo sequencing analysis was conducted to perform amino acid sequence analysis, and the results are shown in Table 2.

[0136] Table 2 contains EX. n The -P short peptide sequence showed a high degree of matching with the fish scale active peptide II, representing the 12 peptide sequences with the highest concentrations among the first 18 peptide sequences. These peptide sequences collectively contain EX... n -P short sequence, where E represents glutamic acid, P represents proline, and X is a natural L-amino acid, where n is 0–3. This EX... n The -P sequence structure is a retained feature in every peptide chain, and short sequences may play a role in protein or peptide binding capacity, antioxidant activity, and cell signaling.

[0137] Table 2: Table containing EX n The -P short peptide sequence has a high degree of matching with the fish scale active peptide II peptide sequence.

[0138]

[0139] Example 3: Containing EX n Preparation and application of -P short peptide sequence of sheepskin active peptide III

[0140] 1. Contains EX n Preparation of α-P short peptide sequence of sheepskin active peptide III

[0141] (1) Enzymatic hydrolysis:

[0142] The processed sheepskin powder was mixed with ultrapure water until the substrate concentration was 10%, and then placed at 25°C and shaken to dissolve for 10 minutes to obtain a collagen solution.

[0143] Before enzymatic hydrolysis, dissolve 1.0% (by weight of substrate) of nicotinamide in 2 mL of deionized water, then add it to the collagen solution above, ensuring uniform distribution. Adjust the pH of the solution to 6.0 using 1.0 mol / L NaOH solution, add flavor protease (2500 U / mL), and incubate in a benchtop constant-temperature shaker at 45°C and 200 rpm for 5 hours. Check the pH every 30 minutes during the reaction, always maintaining the pH at 6.0.

[0144] After the process, the enzyme was inactivated at 95℃ for 15 minutes, cooled to room temperature, and centrifuged at 4500 r / min for 20 minutes. The supernatant was then collected as the sheepskin polypeptide enzymatic hydrolysate.

[0145] (2) Oscillatory chelation:

[0146] Take the sheepskin polypeptide enzymatic hydrolysate obtained in step (1) and mix it with 0.3 mol / L FeSO4 solution. Control the mass ratio of sheepskin polypeptide enzymatic hydrolysate to FeSO4 solution to 2:1. Adjust the pH to 9.0. React in a benchtop constant temperature shaker at 37°C and 150 rpm for 60 min. Filter and take the supernatant to obtain sheepskin peptide iron chelate solution.

[0147] (3) Low-temperature ethanol fractionation and precipitation:

[0148] Take the sheep skin peptide iron chelate solution obtained in step (2), add a certain volume of pre-cooled anhydrous ethanol solution to obtain a reaction system with an ethanol concentration of 80%, stir and mix well, place it at 4℃ and let it stand for 100 min, then set the centrifuge speed to 6000 r / min and the centrifugation time to 15 min, and collect the precipitate by centrifugation.

[0149] The collected precipitate was redissolved: anhydrous ethanol was added again to bring the volume fraction of anhydrous ethanol in the system to 80%. The remaining steps were the same as above. After the fourth wash (each time the volume fraction of anhydrous ethanol was 80%), sodium sulfide solution was added to the supernatant after centrifugation. The solution was observed to see if it turned black. If it did not turn black, the washing was stopped. The precipitate after centrifugation was placed in a 60°C water bath to allow the ethanol to evaporate to dryness, yielding the sheepskin peptide iron chelate. If the solution turned black, the washing continued until the solution reacted with the sodium sulfide solution without changing color, indicating that the washing was complete.

[0150] (4) Microwave-assisted acid dissociation:

[0151] The dried sheepskin peptide iron chelate was reconstituted in deionized water at a concentration of 0.8 g / mL, and the pH was adjusted to 3.5 with 1.0 mol / L HCl solution to obtain a collagen peptide metal chelate solution.

[0152] The obtained collagen peptide metal chelate solution was placed in a microwave reaction flask and heated at 40°C, with a power of 200W and a heating time of 10 minutes.

[0153] After the reaction is complete and cooled, centrifuge at 5000 rpm for 15 minutes. Collect the supernatant and adjust its pH to 8.5 with 0.5 mol / L NaOH solution. Let it stand for 15 minutes, then adjust the pH to 3.5 and microwave-assisted dissociation for 10 minutes (conditions: temperature controlled at 40℃, power controlled at 200W). Centrifuge and collect the supernatant, repeating the above operation four times. Collect the supernatant from the final centrifugation; this is the supernatant containing EX. n -P short peptide sequence of sheepskin collagen active peptide III solution.

[0154] 2. For the obtained EX n Amino acid sequence analysis of the -P short peptide sequence in a solution of sheepskin active peptide III.

[0155] The amino acid sequence of peptides also affects their biological activity. The obtained peptides containing EX... n After freeze-drying, the amino acid sequences of the peptide chains in the polypeptide mixture were identified by liquid chromatography-mass spectrometry (LC-MS) and proteomics. The sheepskin active peptide III solution was dissolved in methanol to prepare a 10 mg / mL collagen peptide solution.

[0156] Specific chromatographic conditions: Acclaim PepMap C18 column (75 μm × 25 cm), mobile phase A: aqueous solution containing 0.1% formic acid, mobile phase B: acetonitrile solution containing 0.1% formic acid; sample loading volume 5.0 μL, elution flow rate 300.0 nL / min. Mass spectrometry ionization mode was ESI+, positive charge spray voltage was 2.0 kV.

[0157] A database of relevant fish proteins was established from the Uniprot database, and then database alignment was performed using PEAKS Studio software. De novo sequencing analysis was conducted to perform amino acid sequence analysis, and the results are shown in Table 3.

[0158] Table 3 contains EX. n The -P short peptide sequence showed a high matching degree with the sheepskin active peptide III, and the 12 peptide sequences with the highest concentrations among the top 20 peptide sequences. These peptide sequences collectively contain EX... n -P short sequence, where E represents glutamic acid, P represents proline, and X is a natural L-amino acid, where n is 0–4. This EX... nThe -P sequence structure is a retained feature in every peptide chain, and short sequences may play a role in protein or peptide binding capacity, antioxidant activity, and cell signaling.

[0159] Table 3: Table containing EX n The -P short peptide sequence has a high degree of matching with the sheepskin active peptide III peptide sequence.

[0160]

[0161] Example 4: EX n Analysis of animal-derived collagen bioactive peptides with -P short peptide sequences

[0162] EX obtained from Examples 1-3 n The properties of animal-derived collagen bioactive peptides with -P short peptide sequences (donkey-hide gelatin bioactive peptide I, fish scale bioactive peptide II, and sheepskin bioactive peptide III, named bioactive peptide I, bioactive peptide II, and bioactive peptide III, respectively) were analyzed as follows:

[0163] 1. Contains EX n Relative molecular weight distribution of animal-derived collagen bioactive peptides with -P short peptide sequences

[0164] The molecular weight distribution was determined using high-performance liquid chromatography (HPLC). The specific steps were as follows: the prepared active peptides I, II, and III were freeze-dried, dissolved in ultrapure water, and prepared into clear solutions with a concentration of 1.0 mg / mL. Their relative molecular weight distribution was then determined using HPLC. The chromatographic column was a TSK gel G2000 SWXL (7.8 mm × 300 mm), and the mobile phase was acetonitrile / water / trifluoroacetic acid = 40:60:0.1 (v / v / v). The sample was filtered through a 0.45 μm filter before injection at a volume of 10 μL and a flow rate of 0.5 mL / min. The UV detection wavelength was 220 nm. The results are shown in Table 4.

[0165] Table 4 contains EX n Relative molecular weight distribution of animal-derived collagen bioactive peptides with -P short peptide sequences

[0166]

[0167] As shown in Table 4, the content containing EX n The relative molecular weight distribution of animal-derived collagen bioactive peptides with -P short peptide sequences is mainly concentrated in the range of 0–2000 Da, accounting for 80–95% of the total peptide content. After delipidation enzymatic hydrolysis, ethanol fractionation precipitation, and acidic dissociation, more low molecular weight peptides are retained. These retained low molecular weight peptides can form strong chelation reactions with metal ions, making them more stable and with higher biological nutritional value.

[0168] 2. Contains EXn Amino acid composition analysis of animal-derived collagen bioactive peptides with -P short peptide sequences

[0169] The amino acid composition was analyzed using a dedicated high-performance liquid chromatography (HPLC) instrument for amino acid hydrolysis analysis. The specific steps were as follows: Active peptides I, II, and III were freeze-dried separately. 120.0 mg of each active peptide sample was accurately weighed into a hydrolysis tube, and 8 mL of 6 mol / L HCl solution was added. After purging with nitrogen for 3 min, the mixture was hydrolyzed at 120℃ for 22 h. After cooling, the mixture was transferred to a 25 mL volumetric flask, neutralized with 4.80 mL of 10 mol / L NaOH solution, and then diluted to 25 mL with distilled water. After mixing and filtering, 1 mL of the filtrate was collected and centrifuged at 10000 r / min for 10 min. 400 μL of the supernatant was collected in a liquid chromatography vial and analyzed using the dedicated HPLC instrument for amino acid hydrolysis analysis. The results are shown in Table 5.

[0170] Table 5: Table containing EX n -P short peptide sequence, animal-derived collagen bioactive peptide amino acid composition

[0171]

[0172] Table 5 shows that aspartic acid and glutamic acid together account for a relatively high proportion of the overall amino acid composition, totaling 25-40%. This indicates that acidic amino acids are present in the EX... n -P short peptide sequences constitute a significant proportion of bioactive peptides derived from animal collagen. The high content of acidic amino acids helps enhance the hydrophilicity and charge properties of collagen, contributing to its elasticity and stability. Particularly in bioactive peptides derived from animal collagen, the abundance of acidic amino acids may enhance their activity in vivo. This suggests that these collagen peptides may possess strong structural stability and good biological activity, playing a crucial role, especially in enhancing collagen functionality.

[0173] 3. Contains EX n Detection of the anti-inflammatory and antioxidant capacity of animal-derived collagen bioactive peptides with -P short peptide sequences in cell experiments.

[0174] Caco-2 cells were placed in high-glucose DMEM medium containing 20% ​​FBS and 1% penicillin antibiotics and cultured at 37°C in a 5% CO2 incubator for 24 h. LPS was prepared at a concentration of 1.0 μg / mL, and Caco-2 cells were divided into five groups: normal group, model group, active peptide I group, active peptide II group, and active peptide III group.

[0175] Normal group: Add 2 mL of cell suspension (concentration of 1×10⁻⁶) to each well of a 6-well plate. 5Cells were added to each well with 0.25 mL of LPS solution (1.0 μg / mL) for 4 h, and then cultured at 37 °C and 5% CO2 for 24 h. The cell supernatant was then collected.

[0176] Model group: Add 2 mL of cell suspension (concentration of 1×10⁻⁶) to each well of a 6-well plate. 5 Cells were added to each well with 0.25 mL of LPS solution (1.0 μg / mL) for 4 h, and then cultured at 37 °C and 5% CO2 for 24 h. The cell supernatant was then collected.

[0177] Active peptide group I: Add 2 mL of cell suspension (concentration of 1×10⁻⁶) to each well of a 6-well plate. 5 Cells were treated with 0.25 mL of LPS solution (1.0 μg / mL) per well for 4 h, followed by the addition of 200 μL of 600 mg / L active peptide I solution. After culturing at 37 °C and 5% CO2 for 24 h, the cell supernatant was collected.

[0178] Active peptide II group: Add 2 mL of cell suspension (concentration of 1×10⁻⁶) to each well of a 6-well plate. 5 Cells were added to each well with 0.25 mL of LPS solution (1.0 μg / mL) for 4 h, followed by the addition of 200 μL of 600 mg / L active peptide II solution. After culturing at 37 °C and 5% CO2 for 24 h, the cell supernatant was collected.

[0179] Active peptide group III: Add 2 mL of cell suspension (concentration of 1×10⁻⁶) to each well of a 6-well plate. 5 Cells were treated with 0.25 mL of LPS solution (1.0 μg / mL) per well for 4 h, followed by the addition of 200 μL of 600 mg / L active peptide III solution. After incubation at 37 °C and 5% CO2 for 24 h, the cell supernatant was collected.

[0180] The cell supernatants were collected and used to measure relevant indicators of inflammatory cytokines. The results are shown in Table 6.

[0181] Table 6: Table containing EX n Effects of -P short peptide sequences on the anti-inflammatory activity of lipopolysaccharide-induced Caco-2 cells

[0182]

[0183] Table 6 shows that LPS stimulation significantly increased the expression of inflammatory cytokines IL-6 and TNF-α, while intervention with active peptides I-III significantly reduced the expression of LPS-induced inflammatory cytokines. These results indicate that the enzymes containing EX... nAnimal-derived collagen bioactive peptides with -P short peptide sequences possess certain anti-inflammatory capabilities.

[0184] 5. Contains EX n Evaluation of the chelation of divalent iron ions by -P short peptide sequences of collagen-active peptides

[0185] The in vitro ferrous ion chelating ability of active peptides I, II, and III was detected using the o-phenanthroline colorimetric method. The specific method is as follows:

[0186] (1) Plotting the standard curve

[0187] 10 mg / mL Fe 2+ As a standard solution, gradient concentrations were set at 0, 0.40, 0.80, 1.20, 1.60, and 2.00 μg / mL. [The solution was then applied to Fe...] 2+ Add 1 mL of 0.2 mol / L acetate-sodium acetate buffer (pH 5.0), 200 μL of 20 mg / mL ascorbic acid to the solution sequentially, mix thoroughly, and then add 400 μL of 5 mg / mL o-phenanthroline solution to prepare the working colorimetric solution. Let the reaction stand for 15 min. Use ultrapure water as a blank control. Measure the absorbance of the reaction solution at 510 nm using a 96-well plate and a microplate reader. Based on Fe... 2+ Plot a standard curve using concentration and absorbance values.

[0188] (2) The active peptide I, II, and III solutions prepared in Examples 1-3 were freeze-dried and then dissolved in ultrapure water to prepare 4% solutions by mass. The pH was adjusted to 5.5. The peptides and Fe... 2+ The mass ratio is 4:1. To prevent oxidation, Fe... 2 + Add 1% ascorbic acid solution to the solution, mix thoroughly, and chelate at 37℃ for 30 min with shaking. After chelation, centrifuge at 6000 r / min for 15 min, take 1 mL of supernatant, add working colorimetric solution according to the standard solution procedure, and measure the absorbance at 510 nm. Calculate the dissolved iron content in the supernatant by substituting the absorbance into the standard curve.

[0189] The preparation method of the control group (donkey-hide gelatin peptide) is as follows: the specific steps are the same as step (1) in step 1 of Example 1. The donkey-hide gelatin peptide enzymatic hydrolysate is obtained. The enzyme hydrolysate is inactivated at 95℃ for 15 min, cooled to room temperature, centrifuged at 4500 r / min for 20 min, the supernatant is collected, and freeze-dried to obtain donkey-hide gelatin peptide.

[0190] The results are shown in Table 7.

[0191] Table 7: Table containing EX n Iron chelating ability of collagen-active peptides with -P short peptide sequences

[0192]

[0193] Compared with donkey-hide gelatin peptides, the prepared active peptides I, II, and III all exhibited stronger ferrous ion chelating abilities, with active peptide I reaching as high as 52 μg / mL. The metal chelating ability of peptides not only affects their antioxidant capacity but also their other biological activities.

[0194] In Chinese invention patent publication CN112812175B, the iron chelating activity of component B (high glutamic acid donkey collagen peptide III) in Example 3 is 32.8 μg / mg. After conversion according to the method of this application, its ferrous ion chelating capacity of high glutamic acid donkey collagen peptide III is 26.58 μg / mL.

[0195] 6. Contains EX n Evaluation of the iron absorption-promoting capacity of fish scale active peptide II with -P short peptide sequence

[0196] The iron absorption-promoting capacity of the enzymatic hydrolysis products and the purified products after enzymatic hydrolysis of fish scale active peptides was evaluated using a using chamber system. The specific method is as follows:

[0197] (1) Preparation method of peptides by enzymatic hydrolysis only

[0198] Following the method in step 1(1) of Example 2, a fish scale polypeptide hydrolysate was prepared and named only hydrolyzed peptide.

[0199] (2) Prepare solutions of only enzymatically hydrolyzed peptide and fish scale active peptide II at a concentration of 0.5 mg / mL for experimental use.

[0200] Healthy SD rats were selected and anesthetized with ether after a 12-hour fast. The duodenum was quickly separated and cut into 2.0 cm segments. The serosa and part of the muscle layer were removed with forceps. The prepared intestinal mucosa was then placed in the perfusion chamber of the using chamber, with an effective permeability area of ​​0.5 cm². 2 Add 5 mL of preheated buffer solution (37°C) to both sides of the chamber, and continuously purge with a mixture of 95% O2 and 5% CO2. Maintain the system temperature at 37°C and equilibrate for 30 minutes to allow the intestinal mucosa to reach a stable state.

[0201] 5.0 mL of enzymatically hydrolyzed peptide (final concentration: 10 mg / mL), 5.0 mL of fish scale active peptide II (final concentration: 10 mg / mL), and 5.0 mL of FeSO4·7H2O (final concentration: 100.0 μg / mL) solution were added to the mucosal side, and an equal volume of buffer solution was added to the serous side. After the absorption experiment began, 1.0 mL of serous membrane sample was collected at 0, 15, 30, 60, 90, and 120 minutes to determine the iron permeability. Simultaneously, an equal volume of buffer solution was added to the serous side at each sampling point. The solution from the mucosal side was collected, and the cumulative iron absorption rate was determined according to the above method. The cumulative permeability and apparent permeability coefficient of iron to the slurry membrane side were calculated using methods such as [method name missing]. The iron absorption-promoting capacity of the fish scale active peptide enzymatic hydrolysis product and the purified product after enzymatic hydrolysis was evaluated by measuring the cumulative iron permeability. The results are shown in Table 8.

[0202] Table 8. Iron absorption-promoting capacity of fish scale active peptide II

[0203]

[0204] As shown in Table 8, compared with FeSO4·7H2O, the iron absorption rate of peptides hydrolyzed by enzymes alone is poor. After separation, the iron absorption capacity of fish scale active peptide II is significantly improved, reaching 26.04%, indicating that the iron absorption capacity of the prepared peptide is strong, which is 2.15 times that of the peptide after enzymatic hydrolysis.

[0205] 7. Contains EX n Evaluation of the antioxidant capacity of animal-derived collagen bioactive peptides with -P short peptide sequences

[0206] By detecting EX n The in vitro antioxidant capacity was analyzed by measuring the DPPH free radical scavenging rate of animal-derived collagen active peptides I, II, and III with -P short peptide sequences.

[0207] The specific method is as follows: Animal-derived collagen active peptides I, II, and III solutions were freeze-dried to obtain active peptide I, II, and III powders. DPPH was dissolved in anhydrous ethanol to prepare a 0.2 mmol / L solution. Active peptide I, II, and III powders were dissolved in deionized water to prepare sample solutions of 2.0 mg / mL. 2 mL of DPPH solution was placed in a test tube, and 2 mL of active peptide I, II, and III solutions were added respectively. The mixture was shaken and incubated at room temperature in the dark for 30 min. The absorbance of each sample was then measured at a wavelength of 517 nm. The free radical scavenging rate was calculated using the formula. A0 represents the absorbance value of the blank control group, and A1 represents the absorbance value of the sample solution. The results are shown in Table 9.

[0208] Table 9. In vitro antioxidant capacity of animal-derived collagen bioactive peptides I, II, and III.

[0209]

[0210]

[0211] As shown in Table 9, the content containing EX n The DPPH free radical scavenging rate of animal-derived collagen bioactive peptides with -P short peptide sequences all reached over 73%, and those containing EX... n Animal-derived collagen active peptide I with the -P short peptide sequence has a free radical scavenging rate of nearly 80%, indicating that it has strong in vitro antioxidant capacity.

[0212] Experimental Example 5: Containing EX n -P short peptide sequence animal-derived collagen bioactive peptides improve immune status in mice

[0213] 1. Animal experiments

[0214] The EX-containing samples prepared in Examples 1, 2, and 3 n Animal-derived collagen bioactive peptides I, II, and III (named as bioactive peptide I from donkey-hide gelatin, bioactive peptide II from fish scales, and bioactive peptide III from sheepskin, respectively) with short P-peptide sequences were applied to cyclophosphamide (CTX)-induced immunosuppressed mice to investigate their immunomodulatory effects. The experimental methods and results are as follows:

[0215] Experimental Methods: Mice were pre-fed for 7 days to allow them to acclimatize to the environment. After one week of pre-feding, the mice were randomly divided into 5 groups: normal control group (CON group), cyclophosphamide immunodeficiency group (CTX group), active peptide I group (Group I), active peptide II group (Group II), and active peptide III group (Group III), with 10 mice in each group. The specific treatment methods for each group after the pre-feding period are as follows:

[0216] Normal control group (CON group): From day 1 to day 12, CON group mice were orally administered 200 μL of physiological saline daily; from day 10 to day 12 of oral administration, CON group mice were intraperitoneally injected with 200 μL of physiological saline daily.

[0217] Cyclophosphamide immunodeficiency group (CTX group): From day 1 to day 12, mice in the CTX group were orally administered 200 μL of physiological saline daily; during day 10 to day 12 of oral administration, mice in the CTX group were intraperitoneally injected with 200 μL of cyclophosphamide (80 mg / kg·bw) daily.

[0218] Group I: From day 1 to day 12, mice in Group I were orally administered the donkey-hide gelatin active peptide I solution prepared in step 1 of Example 1 via gavage. The prepared solution was freeze-dried and then dissolved in physiological saline. The dosage was 1.2 g / kg·bw, and 200 μL was administered via gavage. During day 10 to day 12 of gavage, mice in Group I were intraperitoneally injected with 200 μL of cyclophosphamide (80 mg / kg·bw) daily.

[0219] Group II: From day 1 to day 12, mice in Group I were orally administered the fish scale active peptide II solution prepared in step 1 of Example 2 via gavage. The prepared solution was freeze-dried and then dissolved in physiological saline. The dosage was 1.2 g / kg·bw, and 200 μL was administered via gavage. From day 10 to day 12 after gavage, mice in Group II were intraperitoneally injected with 200 μL of cyclophosphamide (80 mg / kg·bw) daily.

[0220] Group III: From day 1 to day 12, mice in Group I were orally administered the sheepskin collagen active peptide III solution prepared in step 1 of Example 3 via gavage. The prepared solution was freeze-dried and then dissolved in physiological saline. The dosage was 1.2 g / kg·bw, and 200 μL was administered via gavage. From day 10 to day 12 after gavage, mice in Group III were intraperitoneally injected with 200 μL of cyclophosphamide (80 mg / kg·bw) daily.

[0221] After 12 days of continuous administration, the mice were fasted but allowed free access to water overnight. Their weight was recorded, and they were then anesthetized by inhaling isoflurane. Whole blood was collected from the mice's eyes for analysis of blood cell counts and biochemical parameters.

[0222] After blood collection, the mice were soaked in 75% ethanol, removed and placed on a sterile operating table. The spleens of the mice were removed and prepared into a suspension of mouse spleen cells for flow cytometry analysis of mouse spleen lymphocytes, mouse spleen lymphocyte proliferation experiment and mouse NK cell activity experiment.

[0223] 2. Experimental Results:

[0224] Contains EX n The immunomodulatory effects of animal-derived collagen bioactive peptides with the -P short peptide sequence on immunocompromised mice are shown in Tables 10, 11, and 12; the antioxidant effects are shown in Table 13; the anti-inflammatory effects are shown in Table 14; and the organ iron ion results are shown in Table 15. Figure 1 .

[0225] (1) The results of blood count indicators of mice in each group are shown in Table 10.

[0226] Table 10 Effects of bioactive peptides I, II, and III on blood count parameters in immunocompromised mice

[0227]

[0228] The results, as shown in Table 10, indicated that the cyclophosphamide immunodeficiency model group showed significant differences in white blood cell count, lymphocyte ratio, and basophil ratio compared to normal mice (p<0.05). Animal-derived collagen active peptides I, II, and III prepared in different forms significantly increased the white blood cell count, lymphocyte ratio, and basophil ratio in immunodeficiency mice compared to the immunodeficiency model group (p<0.05).

[0229] (2) The proliferation of splenic lymphocytes is a key indicator of immune enhancement and can reflect the body's cellular immune capacity. The proliferation of splenic lymphocytes in mice is shown in Table 11.

[0230] Table 11 Effects of animal-derived collagen bioactive peptides I, II, and III on the proliferation of mouse spleen lymphocytes

[0231]

[0232] The results, as shown in Table 11, indicate that the lymphocyte stimulation index was significantly lower in the immunocompromised model group compared to normal mice (p<0.05). Different preparation methods yielded EX... n Compared with the immunodeficiency model group, the animal-derived collagen active peptides I, II, and III with the -P short peptide sequence significantly increased the spleen lymphocyte stimulation index in mice (p<0.05).

[0233] (3) Splenic lymphocyte populations are an important part of the immune system. CD3 + It is a marker of T cells, CD4 + It is a helper T cell that plays a key role in the immune response, CD8 + These are cytotoxic T cells that can directly kill virus-infected cells, CD4. + / CD8 + The ratio is an important indicator for assessing the body's immune status, reflecting the balance between helper T cells and cytotoxic T cells. D45R, also known as B220, is a marker of B cells. (The last part, "containing EX," appears to be an unrelated fragment and is omitted from the translation.) n The effects of animal-derived collagen bioactive peptides with the -P short peptide sequence on the level of splenic lymphocytes in immunocompromised mice are shown in Table 12.

[0234] Table 12 Effects of animal-derived collagen bioactive peptides I, II, and III on splenic lymphocyte levels in immunocompromised mice.

[0235]

[0236]

[0237] Table 12 shows that, compared with normal mice, the phosphamide immunodeficiency model group had lower CD3 levels. +T cell subsets, CD4 + T cell subsets, CD4 + / CD8 + Subpopulation ratios and CD45R B cell subset numbers were significantly decreased (p<0.05). Animal-derived collagen peptides I, II, and III prepared in different forms significantly increased the number of CD3+ T cell subsets, CD4+ T cell subsets, CD4+ / CD8+ subset ratios, and CD45R B cell subset numbers in immunocompromised mice compared to the immunocompromised model group (p<0.05). This indicates that animal-derived collagen active peptides I–III containing the E-Xn-P short peptide sequence can effectively alleviate cyclophosphamide-induced immune damage to the splenic lymphocytes in mice.

[0238] (4) Serum malondialdehyde (MDA) levels and superoxide dismutase (SOD) levels in mice are important biomarkers for assessing oxidative stress and antioxidant capacity. Elevated MDA levels are generally associated with oxidative damage, while changes in SOD levels reflect the activity status of the body's antioxidant system. (Containing EX) n The effects of animal-derived collagen bioactive peptides with -P short peptide sequences on serum antioxidant indices in immunocompromised mice are shown in Table 13.

[0239] Table 13 Effects of animal-derived collagen bioactive peptides I, II, and III on serum antioxidant markers in immunocompromised mice

[0240]

[0241] Table 13 shows that, compared with normal mice, the serum malondialdehyde level in the phosphoramide immunodeficiency model group was significantly increased, and the superoxide dismutase level was significantly decreased (p<0.05). EX prepared in different forms... n Animal-derived collagen bioactive peptides with the -P short peptide sequence significantly reduced serum malondialdehyde (MDA) levels and increased superoxide dismutase (SOD) levels in immunocompromised mice compared to the immunocompromised model group (p<0.05). This indicates that the presence of EX... n Animal-derived collagen bioactive peptides I-III with short P-peptide sequences exhibit different levels of antioxidant activity.

[0242] (5) Inflammatory factors, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), play crucial roles in regulating immune responses and inflammation. Their levels vary across various pathological states and are closely related to disease severity and progression. (Containing EX) n The effects of animal-derived collagen bioactive peptides with -P short peptide sequences on serum anti-inflammatory markers in immunocompromised mice are shown in Table 14.

[0243] Table 14 Effects of animal-derived collagen bioactive peptides I, II, and III on serum anti-inflammatory markers in immunocompromised mice

[0244]

[0245]

[0246] Table 14 shows that the model group exhibited a significant inflammatory response, characterized by a significant increase in TNF-α levels and a decrease in IL-6 levels. Treatments in groups I, II, and III showed varying degrees of anti-inflammatory effects. Group III, in particular, showed a better anti-inflammatory effect, with IL-6 and TNF-α levels approaching or returning to normal levels. This indicates that treatments containing EX... n Animal-derived collagen bioactive peptides I-III with short P-peptide sequences exhibit different levels of anti-inflammatory activity.

[0247] (6) Cyclophosphamide may cause immune loss in mice and oxidative stress that leads to an imbalance in iron homeostasis. Figure 1 This is an image of a Prussian blue-stained section of a mouse spleen showing iron levels. Figure 1 It was found that the spleen iron content was significantly increased in the phosphoramide-induced immunodeficiency model group compared to normal mice. The spleen iron content was significantly decreased in the animal-derived collagen active peptides I, II, and III groups compared to the immunodeficiency model group, and showed a trend of reversion towards the normal group. This indicates that the presence of EX... n Animal-derived collagen active peptides I-III with short P-peptide sequences can effectively alleviate the iron homeostasis imbalance caused by cyclophosphamide in the body.

[0248] In summary, considering the presence of EX n Amino acid composition analysis of animal-derived collagen bioactive peptides containing the -P short peptide sequence, including EX... n -P short peptide sequences account for 55-70% of peptides with high matching degree, exceeding half, and have a higher detection abundance compared to other peptide sequences. Therefore, it is believed that they contain EX. n Animal-derived collagen bioactive peptides with -P short peptide sequences possess immunomodulatory, antioxidant, and anti-inflammatory activities. n -P short peptide sequence is one of the active components of its animal-derived collagen peptide.

[0249] Experimental Example 6: EX n Antioxidant capacity of the -P short peptide sequence (EGGGAEVPEGQR peptide)

[0250] Based on the results of in vitro DPPH free radical scavenging, Example 1, which contains EX, was selected because it has strong in vitro antioxidant capacity. nThe EGGGAEVPEGQR peptide (peptide IV) sequence, a highly abundant active peptide I derived from animal-derived collagen, was synthesized. Cell viability is an important indicator of cell survival or death after exposure to toxic substances. The effect of peptide IV on the antioxidant capacity of Caco-2 cells induced by 0.2 μmol / L H2O2 was investigated using the CCK-8 assay.

[0251] The specific method is as follows:

[0252] (1) Caco-2 cells were cultured in DMEM high-glucose cell culture medium (containing 20% ​​FBS and 1% penicillin antibiotics) at 37°C in a 5% CO2 incubator, and the culture medium was changed every 1-2 days. When the cells reached 80% confluence, they were passaged.

[0253] (2) The cells obtained in step (1) were seeded into 6 wells (1×10⁻⁶ cells per well). 5 Caco-2 cells were cultured in DMEM cell culture medium (containing 200 μmol / L H2O2) for 4 hours to establish an H2O2-induced Caco-2 cell oxidative stress model. The Caco-2 cells were divided into four groups: a model group (no additional treatment), a low-dose group (containing 200 μL of 300 mg / L peptide IV solution), a medium-dose group (containing 200 μL of 600 mg / L peptide IV solution), a high-dose group (containing 200 μL of 900 mg / L peptide IV solution), and a normal group (no additional treatment). Finally, CCK-8 reagent was added and incubated for 2 hours. Absorbance at 450 nm was measured using a microplate reader. Cell viability is an important indicator of cell survival or death after exposure to toxic substances; the results are shown in Table 15.

[0254] Table 15 Protective effect of EGGGAEVPEGQR peptide against H2O2-induced oxidative damage in Caco-2 cells

[0255]

[0256] Table 14 shows that cell survival rate was significantly reduced after exposure to H2O2, but significantly increased after treatment with peptide IV (p<0.05), and the increase in cell survival rate was dose-dependent. These results indicate that peptide IV can protect Caco-2 cells from H2O2-induced oxidative damage.

[0257] Experimental Example 9: EX n Evaluation of the anti-inflammatory activity of the -P short peptide sequence

[0258] Preparation of mixed short peptides: The synthesized EEP, ERP and EGTP were mixed in a mass ratio of 1:1:1 to obtain mixed short peptides. The CCK8 assay was used to investigate the anti-inflammatory effect of the mixed short peptides on lipopolysaccharide (LPS) induced Caco-2 cells. The specific steps are as follows.

[0259] Caco-2 cells were cultured in high-glucose DMEM medium containing 20% ​​FBS and 1% penicillin antibiotics at 37°C for 24 h. LPS was prepared at a concentration of 1.0 μg / mL, and Caco-2 cells were divided into five groups: normal group, model group, low-dose group, medium-dose group, and high-dose group. 2 mL of cell suspension (concentration 1 × 10⁻⁶) was added to each well of a 6-well plate. 5 Cells / mL were added to each well of all groups except the control group. After 4 hours of treatment, 0.25 mL of LPS solution was added to each well of the low-dose group, 200 μL of 300 mg / L mixed short peptide solution was added to the medium-dose group, 200 μL of 600 mg / L mixed short peptide solution was added to the medium-dose group, and 200 μL of 900 mg / L mixed short peptide solution was added to the high-dose group. No additional treatment was given to the normal group. After 24 hours of culture, the cell supernatant was collected for the determination of relevant indicators of inflammatory cytokines.

[0260] Table 16 shows that LPS stimulation significantly increased the expression of inflammatory cytokines IL-6 and TNF-α, while the mixed short peptide intervention significantly reduced the expression of LPS-induced inflammatory cytokines. These results indicate that the mixed short peptides possess certain anti-inflammatory capabilities.

[0261] Table 16 Effects of mixed short peptides on lipopolysaccharide-induced anti-inflammatory activity of Caco-2 cells

[0262]

[0263] The above experiments show that the EX product prepared by the present invention using enzymatic hydrolysis and chelation followed by dissociation and separation has... n Animal-derived collagen peptides with a short P-peptide sequence can significantly improve the body's immune status. This is manifested in their ability to significantly positively regulate leukocytes, lymphocytes, and basophils (p<0.05); and to stimulate the proliferation of splenic lymphocytes (p<0.05). Furthermore, in terms of cellular immunity, they can effectively alleviate immune damage to T and B lymphocytes in mouse spleen cells (p<0.05). In addition, the prepared animal-derived collagen peptides also possess strong ferrous ion chelating ability and in vitro antioxidant capacity. They also exhibit strong antioxidant and anti-inflammatory effects. They can be applied in the production of food, health products, pharmaceuticals, and skincare products.

[0264] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An E-Xn-P short peptide, characterized in that, The E-Xn-P short peptide sequence is EGGGAEVPEGQR.

2. An active peptide-metal chelate, characterized in that, The active peptide metal chelate is obtained by chelating the E-Xn-P short peptide described in claim 1 with divalent iron ions.

3. The active peptide-metal chelate according to claim 2, characterized in that, The chelation reaction is as follows: the mass ratio of the short peptide to divalent iron ions is adjusted to 2:1 to 4:1, the pH is adjusted to 7.0 to 9.0, the temperature is 30 to 50 ℃, the shaking speed is 150 to 200 rpm, the chelation reaction time is 30 to 60 min, and after filtration, the product is freeze-dried to obtain the active peptide metal chelate.

4. The use of the active peptide metal chelate according to claim 2 or 3 in the preparation of products for improving iron deficiency anemia, characterized in that, The product contains an effective dose of active peptide metal chelate, and the product is a drug or health product.

5. The application according to claim 4, characterized in that, The health products also contain acceptable excipients.

6. The application according to claim 4, characterized in that, The drug is in the form of a liquid or solid formulation.

7. The application according to claim 6, characterized in that, The dosage form of the drug is granules, capsules, tablets, pills, or oral liquid.

8. The application according to claim 7, characterized in that, The drug also includes pharmaceutically acceptable excipients.

9. The application according to claim 8, characterized in that, The excipients include any one or more of the following: solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, buffers, plasticizers, defoamers, thickeners, humectants, filter aids, and release inhibitors.

10. The application of the E-Xn-P short peptide according to claim 1 in the preparation of antioxidant products, characterized in that, The product contains an effective dose of E-Xn-P short peptide, and the product is a drug.

11. The application according to claim 10, characterized in that, The drug is in the form of a liquid or solid formulation.

12. The application according to claim 11, characterized in that, The dosage form of the drug is granules, capsules, tablets, pills, or oral liquid.

13. The application according to claim 12, characterized in that, The drug also includes pharmaceutically acceptable excipients.

14. The application according to claim 13, characterized in that, The excipients include any one or more of the following: solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, buffers, plasticizers, defoamers, thickeners, humectants, filter aids, and release inhibitors.

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