Preparation and application of animal-derived collagen peptide containing E-Xn-P oligopeptide sequence

Abstract

The invention discloses preparation and application of animal-derived collagen peptide containing an E-Xn-P oligopeptide sequence, and belongs to the field of preparation technology and application of bioactive peptide. The animal-derived collagen active peptide containing an E-Xn-P oligopeptide sequence is obtained through separation by adopting a method of enzymolysis and chelating and then dissociation, E represents glutamic acid, P represents proline, X is natural L-amino acid, n is 0-4, and the animal-derived collagen active peptide has strong divalent metal ion chelating capacity, metal ion absorption promoting capacity, immunoregulatory activity and antioxidant and anti-inflammatory capacity. The animal source collagen active peptide containing the E-Xn-P oligopeptide sequence prepared by the invention can be used for developing novel health-care foods, medicines, skin care products and the like, has the characteristics of high biological activity and rich nutritional value, makes a contribution to the product utilization rate of collagen, and also provides a thought and a way for developing functional health-care foods, medicines and skin care products.

Description

Technical Field

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

[0002] As people pay more and more attention to health and beauty, collagen as an important biomacromolecule has received widespread attention. Collagen is an important structural protein that is widely present in connective tissue and participates in maintaining the structure and function of multiple biological systems such as skin, bones, and joints. With the increase of age, the synthesis of collagen gradually decreases, resulting in sagging skin, wrinkles, and decreased joint function. Therefore, collagen peptides, as low-molecular products after collagen hydrolysis, are more easily absorbed by the human body and are widely used in anti-aging, joint health, and wound healing. As a natural ingredient with significant biological activity, collagen peptides have become a hot product on the market. The structural characteristics of collagen peptides determine that they have multiple 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 active peptides have become important topics in the field of biological products.

[0003] Traditional animal-derived collagen peptides (such as fish scales, mammal skin and bones, etc.) have been widely used in food, cosmetics and medical and health products. However, existing collagen peptide products have certain limitations in the precise regulation of active ingredients and functional enhancement, such as single function and weak structural optimization, which restricts the efficacy and market competitiveness of the products.

[0004] Collagen peptides are usually obtained by hydrolyzing collagen or gelatin, and the hydrolysis process usually uses acids, alkalis or enzymes as catalysts. Enzymatic hydrolysis, as an efficient and highly specific hydrolysis method, has been widely used in recent years. However, in the traditional collagen hydrolysis process, the peptide chains obtained by enzymatic hydrolysis are of various types, and their biological activities vary greatly, making it difficult to effectively control the generation of specific active peptides. This makes how to screen and separate active peptides with specific biological activities, such as anti-aging, antioxidant, anti-inflammatory and other functions, from collagen peptides a current research problem.

[0005] At present, in the art, collagen peptides are mostly treated with enzymatic treatment, and the molecular weight of the peptides after enzymatic hydrolysis is measured, that is, collagen is hydrolyzed into small molecule peptides to facilitate digestion and absorption, but the sequence and functionality of the obtained peptides are relatively specific, and the molecular weight of the peptides obtained by enzymatic hydrolysis is mostly below 10000Da. Patent CN118879814 A provides a method for preparing anti-aging fish collagen peptides, which only directly enzymatically hydrolyzes collagen after pre-treatment, and the molecular weight of the obtained peptides is relatively large, and the function is relatively single. CN111635920 A discloses a method for preparing donkey bone collagen peptide powder by secondary biomimetic enzymatic hydrolysis technology, which provides a method for producing donkey bone collagen peptides that are more in line with human absorption and biological effects by using a secondary biological enzymatic hydrolysis production process under simulated human stomach and intestinal physiological environment conditions. Although more efficient collagen peptides can be obtained, the functionality is not clear enough. Summary of the invention

[0006] The present invention aims to solve the deficiencies in the prior art. By experimentally decomposing and optimizing collagen peptides from different sources, a new type of short peptide sequence containing an E-Xn-P active structure is found. This peptide segment exhibits significant biological activity, and the active peptide is not directly included in the product obtained by conventional enzymatic hydrolysis in the existing enzymatic hydrolysis system. Specifically, the E-Xn-P short peptide sequence is E (glutamic acid), Xn (natural L-amino acid sequence) and P (proline), where n is 0 to 4. The peptide segment exhibits unique functionality in a variety of physiological activity tests, such as improving the body's immunity, improving the body's anti-inflammatory and antioxidant abilities, etc. The E-Xn-P short peptide is a new collagen-derived active peptide, and its unique amino acid arrangement structure and activity characteristics provide a new direction for the study of collagen peptides. Secondly, the particularity of the peptide segment produced when hydrolyzing collagen provides a new idea for the preparation of collagen peptides with higher activity. In the traditional enzymatic hydrolysis process, common active peptides are mostly amino acid chains with relatively simple structures, while the special structure of E-Xn-P short peptide makes it show more potential in the application of collagen peptides.

[0007] In view of the existing problems, this patent adopts the method of enzymatic hydrolysis and chelation and then dissociation to separate the EX n -P short peptide sequence of animal-derived collagen active peptide, wherein E represents glutamic acid, P represents proline, and X represents natural L-amino acid, wherein n is 0 to 4, and has strong divalent metal ion chelating ability and the ability to promote divalent metal ion absorption.

[0008] The object of the present invention is to provide a n -P short peptide sequence of animal-derived collagen active peptides, containing EX n-P short peptide sequence of animal-derived collagen active peptides and their application in improving the body's immunity, anti-inflammatory and antioxidant capabilities. It provides theoretical support for the rational use of collagen resources and the development of new dietary supplements and skin care products.

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

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

[0011] (1) Enzymatic hydrolysis: adding an enzyme preparation to a system containing animal-derived collagen peptides to enzymatically hydrolyze the protein to obtain an animal-derived collagen peptide enzymatic hydrolyzate;

[0012] (2) Chelation: Chelating the animal-derived collagen peptide hydrolysate obtained in step (1) with metal ions to obtain an animal-derived collagen peptide metal chelate;

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

[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 enzymes are papain and alkaline protease, a step-by-step enzymolysis is adopted: the concentration of the enzymolysis substrate is 5-10%, 1500-2500 U / mL of papain is first added to the enzymolysis system, the pH is maintained at 7.0-9.0, the temperature is 40-50°C, and the enzymolysis is performed for 1.5-3.0 hours; after the first step of enzymolysis is completed, the pH is adjusted to be maintained at 11.0-12.0, and 3000-5000 U / mL of alkaline protease is added to the enzymolysis system, and the pH is adjusted to be maintained at 11.0-12.0, the temperature is 40-50°C, and the enzymolysis is performed for 4.0-6.0 hours;

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

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

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

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

[0020] In one embodiment of the present invention, in step (3), the ethanol graded precipitation is to add pre-cooled anhydrous ethanol to the animal-derived collagen peptide metal chelate, control the ethanol volume fraction in the final system to be 35-85%, stir and mix, let stand for 0.5-2h at 4-25°C, collect the precipitate by centrifugation, repeat the above operation 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, and place the precipitate obtained by the last washing in a 40-60°C water bath to cause the ethanol to evaporate to dryness;

[0021] In one embodiment of the present invention, in step (3), the acidic dissociation is to redissolve the animal-derived collagen peptide metal chelate after ethanol graded precipitation, adjust the pH to 3.0-5.0, and then put it into microwave heating treatment, the temperature is controlled at 40-60°C, the power is controlled at 200-400W, and the heating time is controlled at 3-10min; after the reaction is completed, centrifuge to obtain the supernatant, adjust the pH to 7.0-9.0, let it stand for 10-30min, and then adjust the solution pH to 3.0-5.0, and perform microwave heating treatment (temperature controlled at 40-60°C, power controlled at 200-400W, heating time controlled at 3-10min;), after the reaction is completed and cooled, centrifuge to obtain the supernatant, repeat the above operation 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, and its chelating ability is 20 to 60 mg / mL, and can promote the absorption of divalent iron, calcium, and zinc ions.

[0023] The present 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: animal-derived collagen is selected, and one or more enzyme preparations of hydrolyzing protease, papain, alkaline protease, neutral protease and flavor protease are added to hydrolyze the protein, inactivate the enzyme, and centrifuge to obtain the supernatant, which is the animal-derived collagen peptide enzymatic hydrolyzate;

[0025] (2) Chelation and dissociation: The animal-derived collagen peptide is chelated with metal ions by vibration, ethanol graded precipitation, acid dissociation, and freeze-drying to obtain an active peptide containing the E-Xn-P short peptide sequence.

[0026] In one embodiment of the present invention, the animal-derived collagen described in step (1) is derived from skin and bone collagen, connective tissue or collagen products of bulk 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 enzymes are papain and alkaline protease, step-by-step enzymolysis is adopted: the concentration of the enzymolysis substrate is 5-10%, 1500-2500U / mL of papain is first added to the enzymolysis system, the pH is maintained at 7.0-9.0, the temperature is 40-50°C, the enzymolysis is carried out for 1.5-3.0h, the pH is adjusted to be maintained at 11.0-12.0, and then 3000-5000U / mL of alkaline protease is added to the enzymolysis system, and the pH is adjusted to be maintained at 11.0-12.0 again, the temperature is 40-50°C, and the enzymolysis is carried out for 4.0-6.0h.

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

[0029] In one embodiment of the present invention, when the enzyme is flavor protease, a single enzymatic hydrolysis is adopted, the concentration of the enzymatic substrate is 5-10%, 2000-3000U / mL of flavor protease is first added to the enzymatic hydrolysis system, the pH is maintained at 5.5-7.0, the temperature is 45-60°C, the enzymatic hydrolysis is performed for 3.0-6.0h, and the pH is adjusted to be maintained 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 be 15-30 min; after the enzyme is inactivated, the sample is placed at room temperature until it reaches room temperature, and the centrifuge speed is set to 4000-6000 r / min, and the centrifugation time is 15-30 min.

[0031] In one embodiment of the present invention, the divalent metal ion described 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 chelating reaction time is 30 to 60 min.

[0032] In one embodiment of the present invention, the anhydrous ethanol described in step (2) should be precooled at 4°C in advance, and the precooled anhydrous ethanol is added to the supernatant to control the ethanol volume fraction in the final system to be 35-85%, and the mixture is stirred and mixed. After standing for 0.5-2h at 4°C, the centrifuge speed is set to 4500-6000r / min, and the centrifugation time is 15-30min. After centrifugation, a precipitate is obtained; after the precipitate is re-dissolved in deionized water, anhydrous ethanol is added again to control the ethanol volume fraction in the final system to be 35-85%, and after standing for 20-40min, the centrifuge speed is set to 3000-5000r / min, and the centrifugation time is 15-30min. 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, and the precipitate obtained by the last washing is placed in a 40-60°C water bath to cause the ethanol to evaporate to dryness.

[0033] In one embodiment, the acidic dissociation described in step (2) is used to redissolve the animal-derived collagen peptide metal chelate after ethanol graded precipitation, and then the pH is adjusted to 3.0-5.0 with a 2 mol / L HCl solution. The collagen peptide metal chelate solution is placed in a microwave reaction bottle 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 3-10min. After the reaction is completed and cooled, the centrifuge speed is set to 4500-6000 r / min, the centrifugation time is 10-20 min, the supernatant is taken after centrifugation, and the pH is adjusted to 7.0-9.0 with 0.5 mol / L NaOH solution, and then the solution pH is adjusted to 3.0-5.0 after standing for 10-30 min, and microwave-assisted dissociation (temperature is controlled at 40-60° C., power is controlled at 200-400 W, and heating time is controlled at 3-10 min). After the reaction is completed and cooled, the supernatant is taken after centrifugation, and the above operation is repeated 3-5 times to obtain an active peptide solution containing the E-Xn-P short peptide sequence.

[0034] The present invention provides the use of the active peptide containing the E-Xn-P short peptide sequence and the product thereof in preparing products for improving the body's immunity and enhancing the body's antioxidant and anti-inflammatory abilities.

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

[0036] The present invention provides the use of the active peptide containing the E-Xn-P short peptide sequence in the preparation of products for improving body immunity, enhancing body antioxidant and anti-inflammatory properties and promoting the absorption of divalent iron, calcium and zinc ions.

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

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

[0039] The present invention also provides the use of the above active peptide in preparing products having any one or more of the following effects:

[0040] (a) Increase the number of white blood cells and lymphocytes in immunocompromised individuals, reduce the proportion of basophils in immunocompromised individuals; and stimulate the proliferation of splenic lymphocytes;

[0041] (b) Alleviate the immune damage of individual spleen cell T lymphocyte group and B lymphocyte group; Alleviate the immune damage of individual spleen lymphocyte group caused by cyclophosphamide;

[0042] (c) improving the body's antioxidant capacity, including but not limited to increasing the scavenging rate of DPPH free radicals, 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) enhancing the body's anti-inflammatory ability, including but not limited to increasing the expression of interleukin IL-6 and reducing the expression of TNF-α;

[0044] (e) Improve the body's immune capacity; alleviate the imbalance of iron homeostasis caused by cyclophosphamide;

[0045] (f) As biomarkers 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 medicines, health foods, health products, foods for special medical purposes, foods, functional foods, dietary supplements, feeds, feed additives or pet foods.

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

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

[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 present invention, the drug further comprises a pharmaceutically acceptable excipient.

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

[0052] In one embodiment of the present invention, the food includes but is not limited to cereal 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 further comprises special dietary food.

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

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

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

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

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

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

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

[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 present invention, the drug further comprises a pharmaceutically acceptable excipient.

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

[0064] In one embodiment of the present invention, the food includes but is not limited to cereal 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 further comprises special dietary food.

[0066] In one embodiment of the present invention, the health care product further 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 (SEQID NO.1), that is, Glu-Gly-Gly-Gly-Ala-Glu-Val-Pro-Glu-Gly-Gln-Arg.

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

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

[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 medicines, health foods, health products, foods for special medical purposes, foods, functional foods, dietary supplements, feeds, feed additives or pet foods.

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

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

[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 present invention, the drug further comprises a pharmaceutically acceptable excipient.

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

[0076] In one embodiment of the present invention, the food includes but is not limited to cereal 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 further comprises special dietary food.

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

[0079] Beneficial Effects

[0080] (1) The present invention provides a method comprising n -P short peptide sequence of animal-derived collagen active peptides, through amino acid sequence analysis, in the animal-derived collagen peptides, contains a unique EX n -P amino acid sequence, where E represents glutamic acid, P represents proline, and X is a natural L-amino acid, where n is 0 to 4. According to the amino acid composition analysis, the acidic amino acids aspartic acid and glutamic acid account for 25 to 40% of the total amino acid content. The molecular weight distribution is 0 to 2000Da, and it has strong divalent metal ion chelating ability and iron absorption promoting ability.

[0081] (2) The present invention provides EX n -P short peptide sequence of animal collagen active peptides, can activate lymphocytes and other immune cells, promote the secretion of cytokines such as IL-6, and thus enhance the body's immune response. Compared with the existing technology, this active peptide showed stronger immunomodulatory ability in in vitro experiments, providing the possibility for the development of new immunomodulators.

[0082] (3) The present invention provides EX nThe active peptide of the -P short peptide sequence has significant antioxidant properties, increases the activity of superoxide dismutase (SOD), reduces the content of lipid peroxidation products (MDA), thereby reducing cell damage caused by oxidative stress, showing its potential for application in antioxidants. It also shows a strong effect in anti-inflammatory aspects and can effectively reduce inflammatory responses. The active peptide contains a specific amino acid sequence and can form stable complexes with a variety of metal ions such as iron, copper, zinc, etc., thereby reducing the harmful accumulation of these metal ions in the body. This feature makes it potentially useful 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 an immunomodulatory effect, 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. In view of the immunomodulatory, antioxidant, anti-inflammatory and metal ion chelating functions of the active peptide, it has broad application prospects in the medical field. It can be used as an adjuvant therapeutic agent to enhance the immunity of cancer patients and reduce the side effects caused by chemotherapy. In addition, it can also 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 skin care and beauty products.

[0084] (5) Since the active peptide is derived from animal collagen, its production process does not involve the use of any harmful chemicals, and the by-products are biodegradable and environmentally friendly. This is in line with the current trend of sustainable development and green chemistry. The production process of the active peptide is simple, low-cost, and the raw materials are widely available, so it has the potential for large-scale production. Its application in the fields of medicine and functional food is expected to bring significant economic benefits to enterprises. The promotion and use of this active peptide will help improve the public health level and reduce medical expenses, especially in immunomodulation and anti-inflammatory treatment. In addition, its application in metal ion chelation will help reduce environmental pollution and improve food safety.

[0085] (6) The use of collagen peptides containing E-Xn-P short peptide sequences as biomarkers for quality control can more accurately evaluate and monitor product quality, thereby ensuring that the product meets predetermined quality standards. By detecting the presence and content of the E-Xn-P short peptide sequence in the product, the quality and source of the product can be traced, providing consumers with more transparent product information. The method of the present invention can classify products into different quality grades based on the characteristics of the E-Xn-P short peptide sequence in the collagen peptide, thereby meeting the market demand for products of different qualities and contributing to the standardization and normalization of the entire industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1: Prussian blue-stained sections of mouse spleen iron levels. DETAILED DESCRIPTION

[0087] The following embodiments are used to better illustrate 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 hydrolyzing protease was purchased from Novozymes.

[0089] The mouse model involved in the following embodiments is: Cyclophosphamide mouse model, which is a commonly used immunosuppression model and is widely used to study the mechanism of immunomodulators, anti-tumor drugs and immune-related diseases. Cyclophosphamide (CTX) is an alkylating agent that is commonly used in anti-cancer treatment and the treatment of autoimmune diseases. In the study of autoimmune diseases and cancer immunotherapy, the cyclophosphamide mouse model is a commonly used experimental model;

[0090] Cyclophosphamide inhibits DNA synthesis and interferes with cell division, leading to decreased immune cell function, thereby inducing immunodeficiency in mice. Studies have shown that continuous injection of 80 mg / kg for 3 days can lead to thymic atrophy, spleen enlargement and a decrease in the proportion of immune cells.

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

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

[0093] (1) Enzymatic hydrolysis:

[0094] 1) First step: enzymatic hydrolysis

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

[0096] Before enzymatic hydrolysis, 0.5% nicotinamide relative to the mass of the substrate was dissolved in 2 mL of deionized water, and then added to the above collagen solution. After mixing, the pH of the above solution was adjusted to 9.0 with 0.5 mol / L NaOH solution, and papain (enzyme addition amount was 2000 U / mL) was added to a desktop constant temperature oscillator at 40°C and 200 rpm for constant temperature reaction for 2 h. During the reaction, the pH was checked every 30 min to always control the pH at 9.0.

[0097] 2) Second step enzymatic hydrolysis

[0098] After the first step of enzymatic hydrolysis, the system was taken out, and the pH of the solution was adjusted to 12.0 with 0.5 mol / L NaOH solution again. Alkaline protease (enzyme addition amount was 3000 U / mL) was added to the tabletop constant temperature oscillator at 40°C and 200 rpm for constant temperature oscillation reaction for 5 hours. During the reaction, the pH was checked every 30 minutes, and the pH was always controlled to be 12.0. After the end, the enzyme was inactivated at 95°C for 15 minutes, cooled to room temperature, the centrifuge speed was set to 4500 r / min, centrifuged for 20 minutes, and the supernatant was taken, which was the donkey-hide gelatin polypeptide enzymatic hydrolyzate.

[0099] (2) Oscillating chelation:

[0100] The donkey-hide gelatin polypeptide hydrolysate obtained in step (1) was mixed with a 0.2 mol / L FeSO4 solution, the mass ratio of the donkey-hide gelatin polypeptide hydrolysate to the FeSO4 solution was controlled to be 4:1, the pH was adjusted to 7.5, and the mixture was oscillated in a desktop constant temperature oscillator at 37°C and 200 rpm for 30 min. The supernatant was filtered to obtain a donkey-hide gelatin polypeptide iron chelate solution.

[0101] (3) Low temperature ethanol graded precipitation:

[0102] Take the donkey-hide gelatin 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, place it at 4°C for 120 minutes, then set the centrifuge speed to 4500r / min, the centrifugation time to 20min, and collect the precipitate by centrifugation;

[0103] Collect the obtained precipitate and redissolve it: add anhydrous ethanol again to make the volume fraction of anhydrous ethanol in the system 50%, and the remaining steps are consistent with the above steps. When washing for the fourth time (the volume fraction of anhydrous ethanol is 50% each time), take the supernatant after centrifugation and add sodium sulfide solution to observe whether the solution turns black. If not, stop washing, take the precipitate after centrifugation and place it in a 40°C water bath to make the ethanol evaporate to dryness, and obtain donkey-hide gelatin iron chelate. If the solution turns black, continue washing until the solution does not change color after reacting with the sodium sulfide solution, and the washing is completed.

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

[0105] The dried donkey-hide gelatin iron chelate was added with deionized water to re-dissolve so that the concentration of the chelate was 0.2 g / mL, and 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 bottle for heating treatment, with the temperature controlled at 55° C., the power controlled at 400 W, and the heating time controlled at 3 min.

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

[0108] 2. The obtained n -P short peptide sequence in the donkey-hide gelatin active peptide I solution amino acid sequence analysis.

[0109] The amino acid arrangement of the peptide will also affect its biological activity. n After freeze-drying the donkey-hide gelatin active peptide I solution containing a -P short peptide sequence, the peptide chain amino acid sequence in the peptide mixture was sequenced and identified using liquid chromatography-mass spectrometry and proteomics methods. The donkey-hide gelatin active peptide I solution 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 volume 5.0 μL, elution flow rate 300.0 nL / min. Mass spectrometry ionization mode is ESI+, positive charge spray voltage is 2.0 kV.

[0111] A fish protein database was established from the Uniprot database, and then database comparison, De novo sequencing and amino acid sequence analysis were performed using PEAKS Studio software. The results are shown in Table 1:

[0112] Table 1 contains EX n -P short peptide sequence has a high matching degree with donkey-hide gelatin active peptide I. Among the top 20 peptide sequences, 12 peptide sequences with higher concentrations are included. Among these peptide sequences, 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. n The -P sequence structure is a conserved feature in each peptide chain, and the short sequence may play a role in protein or peptide binding ability, antioxidant activity, and cell signaling.

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

[0114]

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

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

[0117] (1) Enzymatic hydrolysis:

[0118] 1) First step: enzymatic hydrolysis

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

[0120] Before enzymatic hydrolysis, 0.8% nicotinamide relative to the substrate mass was dissolved in 2 mL of deionized water, and then added to the above collagen solution to ensure uniform distribution. The pH of the above solution was adjusted to 7.0 with 0.5 mol / L NaOH solution, and neutral protease (enzyme addition amount was 1500 U / mL) was added to a desktop constant temperature oscillator at 40°C and 200 rpm for 2 hours. During the reaction, the pH was checked every 30 minutes to always control the pH at 7.0.

[0121] 2) Second step enzymatic hydrolysis

[0122] After the first step of enzymatic hydrolysis, the system was taken out, and the solution pH was adjusted to 9.0 with 0.5mol / L NaOH solution again, and the hydrolyzing protease (enzyme addition amount was 3000U / mL) was added to the desktop constant temperature oscillator at 50°C and 200rpm for 6h. During the reaction, the pH was checked every 30min, and the pH was always controlled to be 9.0. After the end, the enzyme was inactivated at 95°C for 20min, cooled to room temperature, the centrifuge speed was set to 6000r / min, centrifuged for 15min, and the supernatant was taken as the fish scale polypeptide enzymatic hydrolyzate.

[0123] (2) Oscillating chelation:

[0124] The fish scale polypeptide enzymatic hydrolyzate obtained in step (1) was mixed with a 0.25 mol / L FeSO4 solution, the mass ratio of the fish scale polypeptide enzymatic hydrolyzate to the FeSO4 solution was controlled to be 3:1, the pH was adjusted to 8.0, and the reaction was carried out in a desktop constant temperature oscillator at 45°C and 180 rpm for 45 minutes, and the supernatant was filtered to obtain a fish scale polypeptide iron chelate solution.

[0125] (3) Low temperature ethanol graded precipitation:

[0126] The ichthyopeptide iron chelate solution obtained in step (2) was added with 1.5 times the volume of the pre-cooled anhydrous ethanol solution to obtain a reaction system with an ethanol concentration of 60%, stirred and mixed, and placed at 4°C for 60 minutes, then the centrifuge speed was set to 5000 r / min, the centrifugation time was 15 minutes, and the precipitate was collected by centrifugation;

[0127] Collect the obtained precipitate and redissolve it: add anhydrous ethanol again to make the volume fraction of anhydrous ethanol in the system 60%, and the remaining steps are consistent with the above steps. When washing for the fourth time (the volume fraction of anhydrous ethanol is 60% each time), take the supernatant after centrifugation and add sodium sulfide solution to observe whether the solution turns black. If it does not turn black, stop washing, take the precipitate after centrifugation and place it in a 50°C water bath to make the ethanol evaporate to dryness, and obtain fish scale peptide iron chelate. If the solution turns black, continue washing until the solution reacts with the sodium sulfide solution without changing color, and the washing is completed.

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

[0129] The dried fish scale peptide iron chelate was added with deionized water to re-dissolve the chelate to a concentration of 0.5 g / mL, and the pH was adjusted to 3.5 with a 2 mol / L HCl solution to obtain a collagen peptide metal chelate solution;

[0130] The obtained fish scale peptide metal chelate solution was placed in a microwave reaction bottle for heating treatment, with the temperature controlled at 45° C., the power controlled at 300 W, and the heating time controlled at 8 min.

[0131] After the reaction is finished and cooled, the centrifuge speed is set to 5000r / min, centrifuged for 15min, and the supernatant is taken after centrifugation. The pH of the supernatant is adjusted to 7.5 with 0.5mol / LNaOH solution, and then the solution pH is adjusted to 3.5 after standing for 15min. Microwave-assisted dissociation is performed for 8min (conditions: temperature is controlled at 45℃, power is controlled at 300W). After the reaction is finished and cooled, the supernatant is taken after centrifugation. Repeat the above operation 5 times. The supernatant taken from the last centrifugation is the supernatant containing EX n -P short peptide sequence of fish scale active peptide II solution.

[0132] 2. The obtained n -P short peptide sequence amino acid sequence analysis of fish scale active peptide II solution.

[0133] The amino acid arrangement of the peptide will also affect its biological activity. n After freeze-drying the fish scale active peptide II solution with a -P short peptide sequence, the peptide chain amino acid sequence in the peptide mixture was sequenced and identified using liquid chromatography-mass spectrometry and proteomics methods. 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 volume 5.0 μL, elution flow rate 300.0 nL / min. Mass spectrometry ionization mode is ESI+, positive charge spray voltage is 2.0 kV.

[0135] A fish protein database was established from the Uniprot database, and then database comparison, De novo sequencing and amino acid sequence analysis were performed using PEAKS Studio software. The results are shown in Table 2:

[0136] Table 2 contains EX n -P short peptide sequence has a high matching degree with fish scale active peptide II. Among the top 18 peptide sequences, 12 peptide sequences with higher concentrations are included. Among these peptide sequences, 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 3. n The -P sequence structure is a conserved feature in each peptide chain, and the short sequence may play a role in protein or peptide binding ability, antioxidant activity, and cell signaling.

[0137] Table 2: Containing EX n -P short peptide sequence with a high matching degree of fish scale active peptide II

[0138]

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

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

[0141] (1) Enzymatic hydrolysis:

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

[0143] Before enzymatic hydrolysis, 1.0% nicotinamide relative to the substrate mass was dissolved in 2 mL of deionized water, and then added to the above collagen solution to ensure uniform distribution. The pH of the above solution was adjusted to 6.0 with 1.0 mol / L NaOH solution, and flavor protease (enzyme amount was 2500 U / mL) was added to a desktop constant temperature oscillator at 45°C and 200 rpm for 5 hours. During the reaction, the pH was checked every 30 minutes to always control the pH at 6.0.

[0144] After the end, the enzyme was inactivated at 95°C for 15 minutes, cooled to room temperature, the centrifuge speed was set to 4500r / min, centrifuged for 20 minutes, and the supernatant was taken as the sheepskin polypeptide enzymatic hydrolysate.

[0145] (2) Oscillating chelation:

[0146] The sheepskin polypeptide hydrolysate obtained in step (1) was mixed with a 0.3 mol / L FeSO4 solution, the mass ratio of the sheepskin polypeptide hydrolysate to the FeSO4 solution was controlled to be 2:1, the pH was adjusted to 9.0, and the reaction was carried out in a desktop constant temperature oscillator at 37°C and 150 rpm for 60 min. The supernatant was filtered to obtain a sheepskin peptide iron chelate solution.

[0147] (3) Low temperature ethanol graded precipitation:

[0148] Take the sheepskin 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, place it at 4°C for 100 minutes, then set the centrifuge speed to 6000r / min, the centrifugation time to 15min, and collect the precipitate by centrifugation;

[0149] Collect the obtained precipitate and redissolve it: add anhydrous ethanol again to make the volume fraction of anhydrous ethanol in the system 80%, and the remaining steps are consistent with the above steps. When washing for the fourth time (the volume fraction of anhydrous ethanol is 80% each time), take the supernatant after centrifugation and add sodium sulfide solution to observe whether the solution turns black. If it does not turn black, stop washing, take the precipitate after centrifugation and place it in a 60°C water bath to make the ethanol evaporate to dryness, and obtain sheepskin peptide iron chelate. If the solution turns black, continue washing until the solution does not change color after reacting with the sodium sulfide solution, and the washing is completed.

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

[0151] The dried sheepskin peptide iron chelate was taken and added with deionized water to re-dissolve 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 bottle for heating treatment, with the temperature controlled at 40° C., the power controlled at 200 W, and the heating time controlled at 10 min.

[0153] After the reaction is finished, cool the centrifuge and set the speed of the centrifuge to 5000r / min. Centrifuge for 15 minutes. Take the supernatant after centrifugation. Adjust the pH of the supernatant to 8.5 with 0.5mol / LNaOH solution. After standing for 15 minutes, adjust the pH of the solution to 3.5. Perform microwave-assisted dissociation for 10 minutes (conditions: temperature controlled at 40°C, power controlled at 200W). Centrifuge and take the supernatant. Repeat the above operation 4 times. The supernatant taken from the last centrifugation is the supernatant containing EX n -P short peptide sequence sheepskin collagen active peptide III solution.

[0154] 2. The obtained n -P short peptide sequence in sheepskin active peptide III solution amino acid sequence analysis.

[0155] The amino acid arrangement of the peptide will also affect its biological activity. n After the sheepskin active peptide III solution with a -P short peptide sequence was freeze-dried, the peptide chain amino acid sequence in the peptide mixture was sequenced and identified using liquid chromatography-mass spectrometry and proteomics methods. 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 volume 5.0 μL, elution flow rate 300.0 nL / min. Mass spectrometry ionization mode is ESI+, positive charge spray voltage is 2.0 kV.

[0157] A fish protein database was established from the Uniprot database, and then database comparison, De novo sequencing and amino acid sequence analysis were performed using PEAKS Studio software. The results are shown in Table 3:

[0158] Table 3 contains EX n -P short peptide sequence has a high matching degree with sheepskin active peptide III. Among the top 20 peptide sequences, 12 peptide sequences with higher concentrations are included. Among these peptide sequences, 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 4. nThe -P sequence structure is a conserved feature in each peptide chain, and the short sequence may play a role in protein or peptide binding ability, antioxidant activity, and cell signaling.

[0159] Table 3: Containing EX n -P short peptide sequence with a high matching degree of sheepskin active peptide III

[0160]

[0161] Example 4: EX n -P short peptide sequence analysis of animal-derived collagen active peptides

[0162] EX obtained in Examples 1 to 3 n -P short peptide sequence of animal-derived collagen active peptides (donkey-hide gelatin active peptide I, fish scale active peptide II, sheepskin active peptide III, named as active peptide I, active peptide II, active peptide III respectively), as follows:

[0163] 1. Contains EX n -P short peptide sequence of animal-derived collagen active peptide relative molecular weight distribution

[0164] High performance liquid chromatography was used to determine its molecular weight distribution. The specific steps are as follows: the prepared active peptides I, II, and III were freeze-dried, dissolved in ultrapure water, and prepared into a clear solution of 1.0 mg / mL, and the relative molecular weight distribution was determined by high performance liquid chromatography. The chromatographic column was TSK gel G2000 SWXL (7.8 mm × 300 mm), 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 and then injected, the sample volume was 10 μL, the flow rate was 0.5 mL / min, and the ultraviolet detection wavelength was 220 nm. The results are shown in Table 4.

[0165] Table 4 contains EX n -P short peptide sequence of animal-derived collagen active peptide relative molecular weight distribution

[0166]

[0167] As shown in Table 4, the n -P short peptide sequence of animal collagen active peptides relative molecular weight distribution is mainly concentrated in 0 ~ 2000Da, accounting for 80 ~ 95% of the total peptide content. After defatting enzymatic hydrolysis, ethanol graded precipitation and acid dissociation, more low molecular weight peptides are retained. These retained low molecular weight peptides can have a strong chelation effect with metal ions, are more stable and have higher biological nutritional value.

[0168] 2. Contains EXn -P short peptide sequence of animal-derived collagen active peptide amino acid composition analysis

[0169] The amino acid composition was analyzed by a high performance liquid chromatograph for hydrolysis amino acid analysis. The specific steps are as follows: After freeze-drying the prepared active peptides I, II, and III, 120.0 mg of active peptides I, II, and III samples were accurately weighed in a hydrolysis tube, 8 mL of 6 mol / L HCl solution was added, nitrogen was blown for 3 min, and hydrolyzed at 120 ° C for 22 h, and then transferred to a 25 mL volumetric flask after cooling, 4.80 mL of NaOH solution (10 mol / L) was added to neutralize and distilled water was made up to 25 mL, 1 mL of the filtrate was taken after mixing and filtering, and centrifuged at 10000 r / min for 10 min. 400 μL of the supernatant was placed in a liquid phase vial and analyzed for amino acid composition using a high performance liquid chromatograph for hydrolysis amino acid analysis. The results are shown in Table 5.

[0170] Table 5: Containing EX n -P short peptide sequence of animal-derived collagen active peptide amino acid composition

[0171]

[0172] As shown in Table 5, the total content of aspartic acid and glutamic acid accounts for a high proportion of the total amino acid composition, accounting for 25-40%. This shows that acidic amino acids are n -P short peptide sequence occupies a large proportion of animal-derived collagen active peptides. A high content of acidic amino acids helps to enhance the hydrophilicity and charge properties of collagen, and helps to maintain the elasticity and stability of collagen. Especially in animal-derived collagen active peptides, the abundance of acidic amino acids may enhance its activity in vivo. This shows that the collagen peptide may have strong structural stability and good biological activity, especially playing a key role in enhancing the functionality of collagen.

[0173] 3. Contains EX n -P short peptide sequence of animal-derived collagen active peptide cell experiment detection of anti-inflammatory and antioxidant capacity

[0174] Caco-2 cells were placed in high-glucose DMEM culture medium containing 20% ​​FBS and 1% double antibody, and cultured in a 37°C, 5% CO2 incubator for 24 hours. The concentration of LPS was configured to 1.0 μg / mL, and Caco-2 cells were divided into five groups, namely normal group, model group, active peptide I group, active peptide II group, and active peptide III group.

[0175] Normal group: 2 mL of cell suspension (concentration of 1×10 5Each well was treated 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, and the cell supernatant was collected.

[0176] Model group: 2 mL of cell suspension (concentration of 1×10 5 Each well was treated 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, and the cell supernatant was collected;

[0177] Active peptide group I: 2 mL of cell suspension (concentration of 1×10 5 / mL), add 0.25mL of LPS solution (1.0μg / mL) to each well for 4h, then add 200μL of 600mg / L active peptide I solution, culture at 37℃, 5% CO2 for 24h, and collect the cell supernatant;

[0178] Active peptide II group: 2 mL of cell suspension (concentration of 1×10 5 / mL), add 0.25mL of LPS solution (1.0μg / mL) to each well for 4h, then add 200μL of 600mg / L active peptide II solution, culture at 37℃, 5% CO2 for 24h, and collect the cell supernatant;

[0179] Active peptide III group: 2 mL of cell suspension (concentration of 1×10 5 / mL), add 0.25mL of LPS solution (1.0μg / mL) to each well for 4h, then add 200μL of 600mg / L active peptide III solution, culture at 37℃, 5% CO2 for 24h, and collect the cell supernatant;

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

[0181] Table 6: Containing EX n Effects of -P short peptide sequence on anti-inflammatory ability of Caco-2 cells induced by lipopolysaccharide

[0182]

[0183] As shown in Table 6, LPS stimulation can significantly increase the expression of inflammatory factors IL-6 and TNF-α, and active peptide I to III groups can significantly reduce the expression of LPS-induced inflammatory factors. nAnimal-derived collagen active peptides with -P short peptide sequence have certain anti-inflammatory ability.

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

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

[0186] (1) Drawing of standard curve

[0187] 10mg / mL Fe 2+ As the standard solution, the gradient concentration was set to 0, 0.40, 0.80, 1.20, 1.60, and 2.00 μg / mL. 2+ 1 mL of 0.2 mol / L acetic acid-sodium acetate buffer (pH 5.0) and 200 μL of 20 mg / mL ascorbic acid were added to the solution in sequence. After thorough mixing, 400 μL of 5 mg / mL o-phenanthroline solution was added to prepare the working color solution. The reaction was allowed to stand for 15 min. Ultrapure water was used as a blank control. The absorbance of the reaction solution at 510 nm was measured using a 96-well plate and an ELISA reader. 2+ Concentration and absorbance values ​​were plotted against each other.

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

[0189] The preparation method of the control group (donkey-hide gelatin peptide) is as follows: the same as step (1) in step 1 of Example 1, to obtain donkey-hide gelatin polypeptide hydrolysate, inactivate the enzyme at 95°C for 15 min, cool to room temperature, set the centrifuge speed to 4500 r / min, centrifuge for 20 min, take the supernatant, and freeze-dry to obtain donkey-hide gelatin peptide.

[0190] The results are shown in Table 7.

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

[0192]

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

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

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

[0196] The Ussing chamber system was used to evaluate the iron absorption capacity of the fish scale active peptide enzymatic hydrolysis products and the separated and purified products after enzymatic hydrolysis. The specific method is as follows:

[0197] (1) Preparation method of enzymatic peptide only

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

[0199] (2) The enzymatic peptide and fish scale active peptide II were prepared into 0.5 mg / mL solutions for experimental use.

[0200] Healthy SD rats were selected and anesthetized with ether after fasting for 12 hours. The duodenum was quickly isolated and cut into 2.0 cm segments. The serosal membrane and part of the muscle layer were removed with forceps. The prepared intestinal mucosa was installed in the perfusion chamber of the Ussing chamber with an effective permeability area of ​​0.5 cm. 2 . Add 5 mL of 37°C preheated buffer to both sides of the chamber, and continue to introduce a mixed gas of 95% O2 and 5% CO2 by volume. Keep the system temperature at 37°C and balance for 30 minutes to allow the intestinal mucosa to reach a stable state.

[0201] 5.0 mL of enzymatic 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 was added to the serosal side. After the absorption experiment started, 1.0 mL of serosal samples were collected at 0, 15, 30, 60, 90, and 120 minutes to determine the iron permeability, and an equal amount of buffer was added to the serosal side of each sampling point. The solution on the mucosal side was collected, and the cumulative iron absorption rate was determined according to the above method. According to The cumulative permeability and apparent permeability coefficient of iron to the serosal membrane were calculated by the method of et al. The cumulative permeability of iron was measured to evaluate the iron absorption capacity of the enzymatic hydrolyzed products of fish scale active peptides and the products separated and purified after enzymatic hydrolysis. The results are shown in Table 8.

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

[0203]

[0204] As can be seen from Table 8, compared with FeSO4·7H2O and the poor iron absorption rate of the peptide only hydrolyzed by enzyme, after separation, the iron absorption capacity of fish scale active peptide II was 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 active peptides with -P short peptide sequences

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

[0207] The specific method is as follows: freeze-dry the animal collagen active peptide I, II, III solution to obtain active peptide I, II, III powder. Use anhydrous ethanol to dissolve DPPH to prepare a 0.2mmol / L solution, use deionized water to dissolve active peptide I, II, III powders, and prepare a 2.0mg / mL sample solution. Take 2mL of DPPH solution and place it in a test tube, add 2mL of active peptide I, II, III solution respectively, shake and mix, place it at room temperature away from light for 30 minutes, and measure the absorbance of each sample at a wavelength of 517nm. Calculate the free radical scavenging rate according to the formula. A0 is the absorbance value of the blank control group, and A1 is the absorbance value of the sample solution. The results are shown in Table 9.

[0208] Table 9 In vitro antioxidant capacity of animal collagen active peptides I, II, and III

[0209]

[0210]

[0211] It can be seen from Table 9 that EX n -P short peptide sequence of animal collagen active peptides DPPH free radical scavenging rate reached more than 73%, containing EX n The free radical scavenging rate of animal-derived collagen active peptide I with -P short peptide sequence is close to 80%, indicating that they all have strong in vitro antioxidant capacity.

[0212] Experimental Example 5: Containing EX n -P short peptide sequence of animal-derived collagen active peptides improves the immune status of mice

[0213] 1. Animal experiments

[0214] The EX n -P short peptide sequence of animal collagen active peptides I, II, III (donkey-hide gelatin active peptide I, fish scale active peptide II, sheep skin active peptide III, named as active peptide I, active peptide II, active peptide III respectively) was applied to cyclophosphamide (CTX)-induced immunosuppressed mice to explore its immunomodulatory effect. The experimental methods and results are as follows:

[0215] Experimental method: The mice were pre-fed for 7 days before the formal experiment to adapt to the environment. After one week of feeding, the mice were randomly divided into 5 groups, namely normal control group (CON group), cyclophosphamide immunodeficiency group (CTX group), active peptide I group (I group), active peptide II group (II group), and active peptide III group (III group), with 10 mice in each group. After the pre-feeding, the treatment methods of the specific groups are as follows:

[0216] Normal control group (CON group): On days 1 to 12, mice in the CON group were orally gavaged with 200 μL of normal saline every day; on days 10 to 12 of gavage, mice in the CON group received intraperitoneal injection of 200 μL of normal saline every day;

[0217] Cyclophosphamide immunodeficiency group (CTX group): On days 1 to 12, mice in the CTX group were orally gavaged with 200 μL of normal saline every day; on days 10 to 12 of gavage, mice in the CTX group were intraperitoneally injected with 200 μL of cyclophosphamide (80 mg / kg·bw) every day;

[0218] Group I: On the 1st to 12th day, the mice in Group I were orally gavaged with the donkey-hide gelatin active peptide I solution prepared in step 1 of Example 1 every day. The prepared solution was freeze-dried and dissolved in physiological saline. The dose was 1.2 g / kg·bw, and 200 μL was gavaged. Among them, on the 10th to 12th day of gavage, 200 μL of cyclophosphamide (80 mg / kg·bw) was injected intraperitoneally into the mice in Group I every day;

[0219] Group II: On the 1st to 12th day, the fish scale active peptide II solution prepared in step 1 of Example 2 was orally administered to the mice in Group I every day. The prepared solution was freeze-dried and dissolved in physiological saline. The dose was 1.2 g / kg·bw, and 200 μL was administered by oral administration. Among them, on the 10th to 12th day of oral administration, 200 μL of cyclophosphamide (80 mg / kg·bw) was injected intraperitoneally into the mice in Group II every day;

[0220] Group III: On the 1st to 12th day, the sheepskin collagen active peptide III solution prepared in step 1 of Example 3 was orally administered to the mice in Group I every day. The prepared solution was freeze-dried and dissolved in physiological saline. The dose was 1.2 g / kg·bw, and 200 μL was administered by oral administration. Among them, on the 10th to 12th day of oral administration, 200 μL of cyclophosphamide (80 mg / kg·bw) was injected intraperitoneally into the mice in Group III every day;

[0221] After 12 consecutive days of drug administration, the mice were fasted but not watered overnight, and their weights were recorded before being anesthetized by inhalation of isoflurane. The eyeballs of the mice were removed to obtain whole blood, and the blood was collected to analyze the hemogram and biochemical indices.

[0222] After blood collection, the mice were soaked in 75% ethanol, the mice were taken out and placed on a sterile operating table, and the spleens of the mice were taken out to prepare mouse spleen cell suspensions for flow cytometry analysis of mouse spleen lymphocytes, mouse spleen lymphocyte proliferation experiments, and mouse NK cell activity experiments.

[0223] 2. Experimental results:

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

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

[0226] Table 10 Effects of active peptides I, II, and III on blood indicators of immunocompromised mice

[0227]

[0228] The results showed that, as shown in Table 10, there were significant differences in the number of white blood cells, lymphocyte ratio, and basophil ratio between the cyclophosphamide immunosuppression model group and normal mice (p<0.05). The animal-derived collagen active peptides I, II, and III groups prepared in different forms can significantly increase the number of white blood cells, lymphocyte ratio, and basophil ratio of immunosuppressed mice compared with the immunosuppressive model group (p<0.05).

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

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

[0231]

[0232] The results show that, as shown in Table 11, the lymphocyte stimulation index of the immunosuppressed model group was significantly lower than that of the normal mice (p<0.05). n Compared with the immunodeficiency model group, animal-derived collagen active peptides I, II, and III with -P short peptide sequence can significantly increase the spleen lymphocyte stimulation index of mice (p<0.05).

[0233] (3) Splenic lymphocytes are an important part of the immune system. CD3 + It is a marker of T cells, CD4 + CD8 is a helper T cell that plays a key role in immune response. + It is a cytotoxic T cell that can directly kill virus-infected cells. + / CD8 + The ratio is an important indicator for evaluating the body's immune status, which reflects the balance between helper T cells and cytotoxic T cells. D45R, also known as B220, is a marker for B cells. n The effects of animal-derived collagen active peptides with -P short peptide sequence on the level of spleen lymphocytes in immunocompromised mice are shown in Table 12.

[0234] Table 12 Effects of animal collagen active peptides I, II, and III on the level of spleen lymphocytes in immunocompromised mice

[0235]

[0236]

[0237] As shown in Table 12, the CD3 +T cell subsets, CD4 + T cell subsets, CD4 + / CD8 + The number of subpopulation ratios and CD45R B cell subpopulations were significantly reduced (p<0.05). Compared with the immunosuppressive model group, the animal-derived collagen peptides I, II, and III groups prepared in different forms can significantly increase the number of CD3+T cell subpopulations, CD4+T cell subpopulations, CD4+ / CD8+ subpopulation ratios, and CD45R B cell subpopulations in immunosuppressive mice (p<0.05). This indicates that animal-derived collagen active peptides I to III containing the E-Xn-P short peptide sequence can effectively alleviate the immune damage of the mouse spleen lymphoid group caused by cyclophosphamide.

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

[0239] Table 13 Effects of animal collagen active peptides I, II, and III on serum antioxidant indexes of immunocompromised mice

[0240]

[0241] As shown in Table 13, 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). n -P short peptide sequence of animal collagen active peptides can significantly reduce the serum malondialdehyde level of immunosuppressed mice and increase the superoxide dismutase level of immunosuppressed mice compared with the immunosuppressed model group (p<0.05). n -P short peptide sequence of animal-derived collagen active peptides I to III have different levels of antioxidant effects.

[0242] (5) Inflammatory factors, such as interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α), play a key role in regulating immune responses and inflammatory processes. Their level changes in various pathological conditions are closely related to the severity and progression of the disease. n The effects of animal-derived collagen active peptides with -P short peptide sequence on serum anti-inflammatory indicators of immunocompromised mice are shown in Table 14.

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

[0244]

[0245]

[0246] As shown in Table 14, the model group showed obvious inflammatory response, which was manifested by a significant increase in TNF-α level and a decrease in IL-6 level. The treatment of groups I, II, and III showed different degrees of anti-inflammatory effects, especially in group III, where the levels of IL-6 and TNF-α were close to or restored to the levels of the normal group, showing a good anti-inflammatory effect, indicating that the EX n -P short peptide sequence of animal-derived collagen active peptides I to III have different levels of anti-inflammatory effects.

[0247] (6) Cyclophosphamide may cause immune damage in mice and oxidative stress, leading to an imbalance in the body's iron homeostasis. Figure 1 This is a Prussian blue-stained section of mouse spleen iron levels. Figure 1 It can be seen that the spleen iron content of the phosphamide immunosuppression model group was significantly increased compared with the normal mice. The spleen iron content of the animal-derived collagen active peptide I, II, and III groups was significantly reduced compared with the immunosuppression model group, and there was a trend of callback to the normal group. This indicates that EX n -P short peptide sequence of animal-derived collagen active peptides I to III can effectively alleviate the iron homeostasis imbalance caused by cyclophosphamide in the body.

[0248] In summary, combined with EX n -P short peptide sequence of animal-derived collagen active peptide amino acid composition analysis, containing EX n -P short peptide sequences account for 55-70% of the peptides with higher matching degrees, accounting for more than half, and the abundance is higher than that of other peptide sequences, so it is believed that EX n -P short peptide sequence of animal-derived collagen active peptides has immunomodulatory activity, antioxidant and anti-inflammatory activity, EX n -P short peptide sequence is one of the active ingredients of its animal-derived collagen peptide.

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

[0250] Referring to the results of in vitro DPPH free radical scavenging rate, the EX n-P short peptide sequence of animal collagen active peptide I with high abundance of EGGGAEVPEGQR peptide (peptide IV) sequence, the sequence was synthesized. Cell viability is an important indicator of cell survival or death after exposure to toxic substances. The CCK-8 method was used to explore the effect of peptide IV on the antioxidant capacity of Caco-2 cells induced by 0.2μmol / L H2O2.

[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% double antibody by volume) in a 37°C, 5% CO2 constant temperature cell culture incubator, with the culture medium replaced every 1-2 days. Subculture was performed when the cells grew to 80%.

[0253] (2) The cells cultured in step (1) were inoculated into 6 wells (1×10 5 The blank group was normal Caco-2 cells without any treatment; the oxidative damage model cells were cultured in DMEM cell culture medium (containing 200 μmol / L H2O2) for 4 hours to prepare the H2O2-induced Caco-2 cell oxidative damage stress model. The Caco-2 cell oxidative damage stress model cells were divided into four groups, one of which was the model group without additional treatment, the low-dose group was added with 200 μL of 300 mg / L peptide IV solution, the medium-dose group was added with 200 μL of 600 mg / L peptide IV solution, the high-dose group was added with 200 μL of 900 mg / L peptide IV solution, and the normal group was not treated with additional treatment. Finally, CCK-8 reagent was added and incubated for 2 hours, and the absorbance at 450 nm was detected by an enzyme marker. Cell viability is an important indicator of the survival or death of cells after exposure to toxic substances. The results are shown in Table 15.

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

[0255]

[0256] As shown in Table 14, after the cells were exposed to H2O2, the survival rate was significantly reduced. After the intervention of peptide IV, the survival rate of the cells was significantly increased (p<0.05), and the increase in cell survival rate was related to the dose of peptide IV. These results show that peptide IV can protect Caco-2 cells from oxidative damage induced by H2O2.

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

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

[0259] Caco-2 cells were placed in high-glucose DMEM culture medium containing 20% ​​FBS and 1% double antibody, and cultured in a 37°C, 5% CO2 incubator for 24 hours. The concentration of LPS was configured to 1.0 μg / mL, and Caco-2 cells were divided into five groups, namely normal group, model group, low-dose group, medium-dose group and high-dose group. 2 mL of cell suspension (concentration of 1×10 5 / mL), except for the blank group, each well of the other groups was treated with 0.25mL of LPS solution for 4 hours, and then 200μL of 300mg / L mixed short peptide solution was added to the low-dose group, 200μL of 600mg / L mixed short peptide solution was added to the medium-dose group, and 200μL of 900mg / L mixed short peptide solution was added to the high-dose group. The normal group did not receive additional treatment. After 24 hours of culture, the cell supernatant was collected for the determination of related indicators of inflammatory cytokines.

[0260] As shown in Table 16, LPS stimulation can significantly increase the expression of inflammatory factors IL-6 and TNF-α, and mixed short peptide intervention can significantly reduce the expression of LPS-induced inflammatory factors. The above results show that mixed short peptides have certain anti-inflammatory ability.

[0261] Table 16 Effect of mixed short peptides on the anti-inflammatory ability of Caco-2 cells induced by lipopolysaccharide

[0262]

[0263] The above experiments show that the EX n -P short peptide sequence of animal-derived collagen active peptide can significantly improve the body's immune status. It is manifested in that it can significantly positively regulate leukocytes, lymphocytes, and basophils (p<0.05); it can also stimulate the proliferation of spleen lymphocytes (p<0.05). In addition, in terms of cellular immunity, it can also effectively alleviate the immune damage of mouse spleen cell T lymphocyte group and B lymphocyte group (p<0.05). In addition, the prepared animal-derived collagen peptide also has a strong divalent iron ion chelating ability and in vitro antioxidant capacity. In addition, it also shows strong efficacy in anti-oxidation and anti-inflammatory aspects. It can be used in the production of food, health products, medicines, skin care products, etc.

[0264] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An active peptide containing a short peptide sequence of E-Xn-P, characterized in that: The EX n -P short peptide sequence accounts for at least 60% of the active peptides, where E represents glutamic acid, P represents proline, X represents a natural L-amino acid, and n is 0 to 4; The active peptide is prepared according to the following method: (1) Enzymatic hydrolysis: adding an enzyme preparation to a system containing animal-derived collagen peptides to enzymatically hydrolyze the protein to obtain an animal-derived collagen peptide enzymatic hydrolyzate; (2) Chelation: Chelating the animal-derived collagen peptide hydrolysate obtained in step (1) with metal ions to obtain an animal-derived collagen peptide metal chelate; (3) Dissociation: The animal-derived collagen peptide metal chelate obtained in step (2) is subjected to ethanol graded precipitation and acidic dissociation to obtain an active peptide containing an E-Xn-P short peptide sequence.

2. The active peptide containing the E-Xn-P short peptide sequence according to claim 1, characterized in that: In step (1), the animal-derived collagen peptides include but are not limited to donkey-hide gelatin, fish scales, and sheepskin; Preferably, in step (1), when the enzymes are papain and alkaline protease, a step-by-step enzymolysis is adopted: the concentration of the enzymolysis substrate is 5-10%, 1500-2500 U / mL of papain is first added to the enzymolysis system, the pH is maintained at 7.0-9.0, the temperature is 40-50°C, and the enzymolysis is performed for 1.5-3.0 hours; after the first step of enzymolysis is completed, the pH is adjusted to be maintained at 11.0-12.0, and 3000-5000 U / mL of alkaline protease is added to the enzymolysis system, and the pH is adjusted to be maintained at 11.0-12.0, the temperature is 40-50°C, and the enzymolysis is performed for 4.0-6.0 hours; Preferably, in step (1), when the enzyme is a neutral protease and a hydrolytic protease, a step-by-step enzymolysis is adopted: the concentration of the enzymatic substrate is 5-10%, 1250-2000U / mL of a neutral protease is first added to the enzymatic system, the pH is maintained at 5.5-7.5, the temperature is 30-60°C, and the enzymatic hydrolysis is performed for 1.5-3.0h; after the first step of enzymatic hydrolysis is completed, the pH is adjusted to be maintained at 7.5-10.0, and then 2000-5000U / mL of a hydrolytic protease is added to the enzymatic system, and then the pH is adjusted to be maintained at 7.5-10.0, the temperature is 40-50°C, and the enzymatic hydrolysis is performed for 4.0-6.0h; Preferably, in step (1), when the enzyme is flavor protease, a single enzymatic hydrolysis is adopted, the concentration of the enzymatic substrate is 5-10%, 2000-3000 U / mL of flavor protease is first added to the enzymatic hydrolysis system, the pH is maintained at 5.5-7.0, the temperature is 45-60° C., the enzymatic hydrolysis is carried out for 3.0-6.0 hours, and the pH is adjusted to be maintained at 5.5-7.0; Preferably, in step (2), the chelation reaction is to adjust the mass ratio of animal-derived collagen peptide hydrolysate to divalent metal ions to 2:1-4:1, adjust the pH to 7.0-9.0, the temperature to 30-50°C, the shaking speed to 150-200 rpm, the chelation reaction time to 30-60 min, and filter the supernatant to obtain the animal-derived collagen peptide metal chelate; Preferably, the divalent metal ions are one or more of iron, calcium and zinc; Preferably, in step (3), the ethanol graded precipitation method is to add pre-cooled anhydrous ethanol to the animal-derived collagen peptide metal chelate, control the ethanol volume fraction in the final system to be 35-85%, stir, let stand for 0.5-2h at 4-25°C, collect the precipitate by centrifugation, repeat the above operation 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, and evaporate the ethanol in the precipitate obtained from the last washing to dryness; Preferably, the acidic dissociation method is to redissolve the animal-derived collagen peptide metal chelate after ethanol graded precipitation, adjust the pH to 3.0-5.0, and then perform microwave heating treatment, with the temperature controlled at 40-60°C, the power controlled at 200-400W, and the heating time controlled at 3-10min; after the reaction is completed, centrifuge to obtain the supernatant, adjust the pH to 7.0-9.0, let it stand for 10-30min, then adjust the solution pH to 3.0-5.0, and treat it according to the above-mentioned microwave heating treatment conditions. After the reaction is completed and cooled, centrifuge to obtain the supernatant, repeat the above operation 3-5 times to obtain an active peptide solution containing the E-Xn-P short peptide sequence.

3. A method for preparing an active peptide containing a short peptide sequence of E-Xn-P, characterized in that: The EX n -P short peptide sequence accounts for at least 60% of the active peptide, wherein E represents glutamic acid, P represents proline, X is a natural L-amino acid, and n is 0 to 4; the method comprises the following steps: (1) Enzymatic hydrolysis: adding an enzyme preparation to a system containing animal-derived collagen peptides to enzymatically hydrolyze the protein to obtain an animal-derived collagen peptide enzymatic hydrolyzate; (2) Chelation: Chelating the animal-derived collagen peptide hydrolysate obtained in step (1) with metal ions to obtain an animal-derived collagen peptide metal chelate; (3) Dissociation: The animal-derived collagen peptide metal chelate obtained in step (2) is subjected to ethanol graded precipitation and acidic dissociation to obtain an active peptide containing an E-Xn-P short peptide sequence.

4. The preparation method according to claim 3, characterized in that: In step (1), the animal-derived collagen peptides include but are not limited to donkey-hide gelatin, fish scales, and sheepskin; Preferably, in step (1), when the enzymes are papain and alkaline protease, a step-by-step enzymolysis is adopted: the concentration of the enzymolysis substrate is 5-10%, 1500-2500 U / mL of papain is first added to the enzymolysis system, the pH is maintained at 7.0-9.0, the temperature is 40-50°C, and the enzymolysis is performed for 1.5-3.0 hours; after the first step of enzymolysis is completed, the pH is adjusted to be maintained at 11.0-12.0, and 3000-5000 U / mL of alkaline protease is added to the enzymolysis system, and the pH is adjusted to be maintained at 11.0-12.0, the temperature is 40-50°C, and the enzymolysis is performed for 4.0-6.0 hours; Preferably, in step (1), when the enzyme is a neutral protease and a hydrolytic protease, a step-by-step enzymolysis is adopted: the concentration of the enzymatic substrate is 5-10%, 1250-2000U / mL of a neutral protease is first added to the enzymatic system, the pH is maintained at 5.5-7.5, the temperature is 30-60°C, and the enzymatic hydrolysis is performed for 1.5-3.0h; after the first step of enzymatic hydrolysis is completed, the pH is adjusted to be maintained at 7.5-10.0, and then 2000-5000U / mL of a hydrolytic protease is added to the enzymatic system, and then the pH is adjusted to be maintained at 7.5-10.0, the temperature is 40-50°C, and the enzymatic hydrolysis is performed for 4.0-6.0h; Preferably, in step (1), when the enzyme is flavor protease, a single enzymatic hydrolysis is adopted, the concentration of the enzymatic substrate is 5-10%, 2000-3000 U / mL of flavor protease is first added to the enzymatic hydrolysis system, the pH is maintained at 5.5-7.0, the temperature is 45-60° C., the enzymatic hydrolysis is carried out for 3.0-6.0 hours, and the pH is adjusted to be maintained at 5.5-7.0; Preferably, in step (2), the chelation reaction is to adjust the mass ratio of animal-derived collagen peptide hydrolysate to divalent metal ions to 2:1-4:1, adjust the pH to 7.0-9.0, the temperature to 30-50°C, the shaking speed to 150-200 rpm, the chelation reaction time to 30-60 min, and filter the supernatant to obtain the animal-derived collagen peptide metal chelate; Preferably, the divalent metal ions are one or more of iron, calcium and zinc; Preferably, in step (3), the ethanol graded precipitation method is to add pre-cooled anhydrous ethanol to the animal-derived collagen peptide metal chelate, control the ethanol volume fraction in the final system to be 35-85%, stir, let stand for 0.5-2h at 4-25°C, collect the precipitate by centrifugation, repeat the above operation 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, and evaporate the ethanol in the precipitate obtained from the last washing to dryness; Preferably, the acidic dissociation method is to redissolve the animal-derived collagen peptide metal chelate after ethanol graded precipitation, adjust the pH to 3.0-5.0, and then perform microwave heating treatment, with the temperature controlled at 40-60°C, the power controlled at 200-400W, and the heating time controlled at 3-10min; after the reaction is completed, centrifuge to obtain the supernatant, adjust the pH to 7.0-9.0, let it stand for 10-30min, then adjust the solution pH to 3.0-5.0, and treat it according to the above-mentioned microwave heating treatment conditions. After the reaction is completed and cooled, centrifuge to obtain the supernatant, repeat the above operation 3-5 times to obtain an active peptide solution containing the E-Xn-P short peptide sequence.

5. Use of the active peptide containing the E-Xn-P short peptide sequence according to claim 1 or 2 in the preparation of products having any one or more of the following effects; (a) Increase the number of white blood cells and lymphocytes in immunocompromised individuals, reduce the proportion of basophils in immunocompromised individuals; and stimulate the proliferation of splenic lymphocytes; (b) alleviating the immune damage of the individual's spleen cell T lymphocyte group and B lymphocyte group or alleviating the immune damage of the individual's spleen lymphocyte group caused by cyclophosphamide; (c) improving the body's antioxidant capacity, including but not limited to increasing the scavenging rate of DPPH free radicals, 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; (d) enhancing the body's anti-inflammatory ability, including but not limited to increasing the expression of interleukin IL-6 and reducing the expression of TNF-α; (e) Improve the body's immune capacity; alleviate the imbalance of iron homeostasis caused by cyclophosphamide; (f) As biomarkers for quality control.

6. The use according to claim 5, characterized in that: The product contains an effective dose of an active peptide containing an E-Xn-P short peptide sequence, and the product includes but is not limited to one or more of a drug, a health food, a health product, a food for special medical purposes, a food, a functional food, a dietary supplement, a feed, a feed additive or a pet food; Preferably, the drug can alleviate body damage caused by immunosuppression; Preferably, the dosage form of the drug is a liquid preparation or a solid preparation; Preferably, the dosage form of the drug includes but is not limited to granules, capsules, tablets, pills or oral liquids; Preferably, the drug further comprises a pharmaceutically acceptable excipient; Preferably, the pharmaceutical excipients include: any one or more of solubilizers, emulsifiers, colorants, adhesives, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrities, pH regulators, buffers, plasticizers, defoamers, thickeners, humectants, filter aids and release retardants; Preferably, the food includes, but is not limited to, cereal products, vegetable products, fruit products, fermented foods, meat products, egg products, dairy products, drinks, and snacks; Preferably, the food also includes special dietary food; Preferably, the health care product further contains acceptable excipients.

7. An active peptide metal chelate, characterized in that: The active peptide metal chelate is obtained by chelating the active peptide containing the E-Xn-P short peptide sequence according to claim 1 or 2 with metal ions; Preferably, the chelation reaction is as follows: adjusting the mass ratio of the active peptide to the divalent metal ion to 2:1-4:1, adjusting the pH to 7.0-9.0, the temperature to 30-50°C, the shaking speed to 150-200rpm, the chelation reaction time to 30-60min, filtering the supernatant and freeze-drying to obtain the active peptide metal chelate; Preferably, the divalent metal ions are one or more of iron, calcium and zinc.

8. Use of the active peptide containing the E-Xn-P short peptide sequence according to claim 1 or 2, or the active peptide metal chelate according to claim 7, or a product containing the active peptide or the active peptide metal chelate in the preparation of a product for improving body immunity or iron deficiency anemia; characterized in that: The product contains an effective dose of active peptides, and the product includes but is not limited to one or more of medicines, health foods, health products, foods for special medical purposes, foods, functional foods, dietary supplements, feeds, feed additives or pet foods; Preferably, the drug can alleviate body damage caused by immunosuppression; Preferably, the dosage form of the drug is a liquid preparation or a solid preparation; Preferably, the dosage form of the drug includes but is not limited to granules, capsules, tablets, pills or oral liquids; Preferably, the drug further comprises a pharmaceutically acceptable excipient; Preferably, the pharmaceutical excipients include: any one or more of solubilizers, emulsifiers, colorants, adhesives, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrities, pH regulators, buffers, plasticizers, defoamers, thickeners, humectants, filter aids and release retardants; Preferably, the food includes, but is not limited to, cereal products, vegetable products, fruit products, fermented foods, meat products, egg products, dairy products, drinks, and snacks; Preferably, the food also includes special dietary food; Preferably, the health care product further contains acceptable excipients.

9. An E-Xn-P short peptide or its use in the preparation of an antioxidant or anti-inflammatory product, characterized in that: The E-Xn-P short peptide sequence is EGGGAEVPEGQR, or the E-Xn-P short peptide is a mixed short peptide obtained by mixing EEP, ERP, and EGTP in a mass ratio of 1:1:

1.

10. The use according to claim 9, characterized in that: 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 medicine, health food, health product, food for special medical purposes, food, functional food, dietary supplement, feed, feed additive or pet food; Preferably, the drug can alleviate body damage caused by immunosuppression; Preferably, the dosage form of the drug is a liquid preparation or a solid preparation; Preferably, the dosage form of the drug includes but is not limited to granules, capsules, tablets, pills or oral liquids; Preferably, the drug further comprises a pharmaceutically acceptable excipient; Preferably, the pharmaceutical excipients include: any one or more of solubilizers, emulsifiers, colorants, adhesives, disintegrants, fillers, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrities, pH regulators, buffers, plasticizers, defoamers, thickeners, humectants, filter aids and release retardants; Preferably, the food includes, but is not limited to, cereal products, vegetable products, fruit products, fermented foods, meat products, egg products, dairy products, drinks, and snacks; Preferably, the food also includes special dietary food; Preferably, the health care product further contains acceptable excipients.

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