A kind of donkey-hide gelatin peptide for delaying aging and its preparation method and application

Abstract

The present invention provides a kind of donkey-hide gelatin peptide for delaying aging and its preparation method and application, belonging to the field of biotechnology. The preparation method of the donkey-hide gelatin peptide for delaying aging of the present invention comprises: mixing the ultrafine grinding of donkey-hide gelatin with a citric acid aqueous solution, treating it for 60min-90min under a boiling water state, adding a composite enzyme enzymolysis, and drying the obtained filtrate after centrifugation and ultrafiltration to obtain the donkey-hide gelatin peptide. Ultrafine grinding increases the reaction surface area of ​​donkey-hide gelatin; the citric acid aqueous solution can break the peptide bond of the donkey-hide gelatin protein molecule and promote the hydrolysis reaction; the boiling water state puts the reaction system in a vibrating state and shortens the reaction time; the composite enzyme enzymolysis accurately controls the degree of enzymolysis to obtain the donkey-hide gelatin peptide for delaying aging. The experimental results show that the donkey-hide gelatin peptide of the present invention has excellent antioxidant properties, can increase the content of SOD, GSH-Px, and CAT in the plasma of D-galactose-induced aging mice, reduce the content of MDA, and play a role in delaying aging.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an anti-aging donkey-hide gelatin peptide and a preparation method and application thereof. Background Art

[0002] Body aging is a comprehensive manifestation of the decline and disorder of physiological functions during the degeneration period. It is an irreversible life process affected by various internal and external factors. Under normal physiological conditions, the body's own defense system can promptly remove oxygen free radicals produced during energy metabolism, so that the generation and removal of free radicals are in a state of dynamic balance. In the process of body aging, the body's antioxidant capacity continues to decline, which breaks this balance and weakens the antioxidant defense system, leading to the occurrence of various diseases.

[0003] Donkey hide gelatin is a solid gelatin made by boiling and concentrating the dried or fresh skin of donkeys, an equine animal. It is known as a "national treasure for nourishing and blood-replenishing medicine". Its main chemical components are amino acids, polypeptides and proteins, which have the effects of nourishing blood, stopping bleeding, nourishing yin and moistening dryness. However, due to the large molecular weight of the protein in donkey hide gelatin, it affects the absorption of the human body and its bioavailability is affected.

[0004] At present, enzymatic hydrolysis and fermentation methods are usually used to decompose the proteins in donkey-hide gelatin into small molecular peptides to improve bioavailability. However, the preparation method of donkey-hide gelatin peptides is time-consuming, and its main efficacy is still to nourish blood and nourish. For example, in the patent with application number 202311437501.1, donkey-hide gelatin peptides are used to treat aplastic anemia; in the patent with application number 202211696943.3, donkey-hide gelatin oligopeptides are used to replenish qi, nourish blood, and stabilize the fetus; in the patent with application number 202311131900.5, donkey-hide gelatin characteristic peptides have a nourishing effect before pregnancy and can be used as a raw material for preparing pre-pregnancy conditioning pastes. However, there is no relevant report on the role of donkey-hide gelatin peptides in delaying the aging of the body. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-aging donkey-hide gelatin peptide and its preparation method and application. The preparation method of the present invention is simple and time-saving. The prepared donkey-hide gelatin peptide has excellent antioxidant properties and has an effect of delaying body aging.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a method for preparing donkey-hide gelatin peptide for delaying aging, comprising: ultrafinely grinding donkey-hide gelatin, sieving to obtain donkey-hide gelatin powder, then mixing with citric acid aqueous solution, treating in a boiling state for 60 minutes to 90 minutes, adding composite enzyme for enzymolysis, centrifuging and ultrafiltration, and drying the obtained filtrate to obtain the donkey-hide gelatin peptide; the composite enzyme is composed of collagenase and papain in a weight ratio of (2-5):(1-3).

[0008] Preferably, the sieve mesh number is 400-500 mesh.

[0009] Preferably, the pH value of the citric acid aqueous solution is 2-4.

[0010] Preferably, the mass ratio of the donkey-hide gelatin powder to the citric acid aqueous solution is 1:(25-35).

[0011] Preferably, the amount of the complex enzyme added in the enzymatic hydrolysis is 2%-5% of the mass of the donkey-hide gelatin powder, the temperature of the enzymatic hydrolysis is 30°C-40°C, the pH value of the enzymatic hydrolysis is 6-8, and the time of the enzymatic hydrolysis is 2h-3h.

[0012] Preferably, the centrifugal speed is 8000rpm-12000rpm, and the time is 3min-8min.

[0013] The present invention also provides donkey-hide gelatin peptide obtained by the above preparation method.

[0014] Preferably, the molecular weight of the donkey-hide gelatin peptide is 500Da-1000Da.

[0015] The present invention also provides the use of the donkey-hide gelatin peptide in the preparation of anti-aging products.

[0016] Preferably, the aging is aging of the body.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The invention provides a method for preparing donkey-hide gelatin peptide for delaying aging, comprising: ultrafinely grinding donkey-hide gelatin and mixing it with a citric acid aqueous solution, treating it in a boiling water state for 60 minutes to 90 minutes, adding a composite enzyme for enzymolysis, centrifuging and ultrafiltration, and drying the obtained filtrate to obtain the donkey-hide gelatin peptide; the composite enzyme is composed of collagenase and papain. The present invention ultrafinely grinds donkey-hide gelatin, increases the reaction surface area of ​​donkey-hide gelatin, improves its solubility, and increases the generation efficiency of donkey-hide gelatin peptides; the citric acid aqueous solution can break the peptide bonds of donkey-hide gelatin protein molecules, thereby destroying the higher-order structure of donkey-hide gelatin protein, allowing the protein molecules to unfold and expose more peptide bonds, thereby promoting the hydrolysis reaction; the boiling water state causes the reaction system to be at a higher temperature, denatures the protein in donkey-hide gelatin, unfolds the protein molecular structure, exposes more sites for reaction, is conducive to the subsequent enzymatic hydrolysis reaction, and increases the generation efficiency of donkey-hide gelatin peptides, and the boiling water state causes the reaction system to be in a vibrating state, which can increase the contact between the ultrafinely grinded donkey-hide gelatin surface and the citric acid aqueous solution and shorten the reaction time; the composite enzyme has a synergistic effect, hydrolyzes the protein in donkey-hide gelatin from multiple angles, thereby more efficiently decomposing macromolecular proteins into small-molecule donkey-hide gelatin peptides, and by selecting a suitable composite enzyme combination and reaction conditions, the degree of enzymatic hydrolysis is accurately controlled to obtain donkey-hide gelatin peptides that delay aging. The preparation method of the present invention can significantly shorten the time for preparing donkey-hide gelatin peptides.

[0019] The experimental results show that the donkey-hide gelatin peptide prepared by the present invention has excellent antioxidant properties, improves the activity of SOD, GSH-Px, and CAT in the plasma of aging mice induced by D-galactose, and inhibits the content of MDA, thus playing an anti-aging role. DETAILED DESCRIPTION

[0020] The invention provides a method for preparing donkey-hide gelatin peptide for delaying aging, comprising: ultrafinely grinding donkey-hide gelatin, sieving to obtain powder, then mixing with citric acid aqueous solution, treating in a boiling water state for 60min-90min, adding composite enzyme for enzymolysis, centrifuging and ultrafiltration, and drying the obtained filtrate to obtain the donkey-hide gelatin peptide; the composite enzyme is composed of collagenase and papain in a weight ratio of (2-5):(1-3).

[0021] The mesh size of the sieve after ultrafine grinding of the present invention is preferably 400-500 mesh, more preferably 450 mesh. Ultrafine grinding can make the donkey-hide gelatin particles extremely fine, thereby greatly increasing the contact area between donkey-hide gelatin, citric acid aqueous solution and complex enzyme, which helps to accelerate the decomposition of donkey-hide gelatin, accelerate the cleavage of peptide bonds and the generation of peptides, and improve the generation efficiency of donkey-hide gelatin peptides; the fine donkey-hide gelatin powder particles can be more evenly dispersed in the solution, which makes the reaction system more uniform, improves solubility, and is conducive to the reaction; the effective ingredients in donkey-hide gelatin are more fully extracted, the waste of raw materials is reduced, and the extraction efficiency is improved; and the ultrafine grinding of donkey-hide gelatin is more easily absorbed and utilized by the human body, thereby improving the biological activity and efficacy of donkey-hide gelatin peptides.

[0022] The pH value of the citric acid aqueous solution of the present invention is preferably 2-4, more preferably 3; the mass ratio of the donkey-hide gelatin powder to the citric acid aqueous solution is preferably 1:(25-35), more preferably 1:30.

[0023] The hydrogen ions in the citric acid aqueous solution of the present invention can attack the peptide bonds in the donkey-hide gelatin protein molecules, causing the peptide bonds to break, destroying the higher-order structure of the donkey-hide gelatin protein, unfolding the protein molecules, exposing more peptide bonds, and thus promoting the hydrolysis reaction.

[0024] The boiling state of the water in the present invention makes the reaction system at a higher temperature, denatures the protein in the donkey-hide gelatin, unfolds the protein molecular structure, exposes more sites available for reaction, is conducive to the subsequent enzymatic hydrolysis reaction, and improves the generation efficiency of donkey-hide gelatin peptides. The boiling state of the water makes the reaction system in a vibrating state, which can increase the contact between the surface of the ultrafinely crushed donkey-hide gelatin and the citric acid aqueous solution, shortening the reaction time. The present invention combines the treatment of donkey-hide gelatin with the citric acid aqueous solution and the treatment of donkey-hide gelatin in the boiling state, greatly shortening the time for preparing donkey-hide gelatin peptides.

[0025] The amount of the composite enzyme added in the enzymolysis of the present invention is preferably 2%-5% of the mass of the donkey-hide gelatin powder, more preferably 3%, the temperature of the enzymolysis is preferably 30°C-40°C, more preferably 35°C, the pH value of the enzymolysis is preferably 6-8, more preferably 7, and the time of the enzymolysis is preferably 2h-3h, more preferably 2.5h. The composite enzyme collagenase and papain of the present invention have a synergistic effect, and can hydrolyze the protein in donkey-hide gelatin from multiple angles, thereby more efficiently decomposing the macromolecular protein into small molecular donkey-hide gelatin peptides, and the degree of enzymolysis can be accurately controlled by selecting a suitable composite enzyme combination and reaction conditions.

[0026] The centrifugal speed of the present invention is preferably 8000 rpm-12000 rpm, more preferably 10000 rpm, and the time is preferably 3 min-8 min, more preferably 5 min.

[0027] The ultrafiltration membrane used in the ultrafiltration of the present invention is an ultrafiltration membrane with a molecular weight cutoff of 1000Da.

[0028] The drying method of the present invention is preferably vacuum freeze drying, the temperature of the vacuum freeze drying is preferably -40°C, the pressure is preferably 50Pa, and the time is preferably 24h.

[0029] The present invention also provides donkey-hide gelatin peptide obtained by the above preparation method.

[0030] The molecular weight of the donkey-hide gelatin peptide of the present invention is preferably 500Da-1000Da, which is more easily absorbed by the human body.

[0031] The present invention also provides the use of the donkey-hide gelatin peptide in the preparation of anti-aging products.

[0032] The aging described in the present invention refers to aging of the body.

[0033] The dosage form of the product of the present invention is preferably an injection, capsule, tablet, oral preparation or microcapsule preparation.

[0034] The product of the present invention preferably comprises the donkey-hide gelatin prepared as described above and auxiliary materials; the auxiliary material components are substances that are suitable for humans and / or animals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), that is, substances with a reasonable benefit / risk ratio, and can be selected according to the dosage form, such as dispersants, granulating agents, surfactants, emulsifiers, disintegrants, adhesives, preservatives, lubricants, colorants, coating agents, sweeteners and flavoring agents.

[0035] The product of the present invention can be prepared by methods known in the art, including combining the donkey-hide gelatin peptide with auxiliary ingredients, and if necessary and / or desired, molding and / or packaging the product into desired single-dose or multi-dose units.

[0036] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0037] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] Example 1

[0039] Preparation of donkey-hide gelatin peptide for delaying aging

[0040] (1) Boiling treatment with citric acid water

[0041] Ultrafinely grind donkey-hide gelatin and pass it through a 450-mesh sieve to obtain donkey-hide gelatin powder; mix 200 g donkey-hide gelatin powder with 6 L of citric acid aqueous solution with a pH value of 3, heat to boiling and keep for 75 minutes;

[0042] (2) Enzymatic hydrolysis

[0043] The temperature was lowered to 35°C, 6 g of compound enzyme (composed of 3.6 g of collagenase and 2.4 g of papain) was added, and enzymolysis was performed at 35°C and pH 7 for 2.5 h to obtain an enzymolysis solution;

[0044] (3) Centrifugation, filtration, and drying

[0045] The enzymatic hydrolysate was centrifuged at 10000 rpm for 5 min, the enzymatic hydrolysate supernatant was filtered with an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and the filtrate was vacuum freeze-dried at -40°C and 50 Pa for 24 h to obtain the donkey-hide gelatin peptide.

[0046] Example 2

[0047] Preparation of donkey-hide gelatin peptide for delaying aging

[0048] (1) Boiling treatment with citric acid water

[0049] Ultrafinely grind donkey-hide gelatin and pass it through a 400-mesh sieve to obtain donkey-hide gelatin powder; mix 200 g donkey-hide gelatin powder with 5 L pH 3 citric acid aqueous solution, heat to boiling and keep for 60 min;

[0050] (2) Enzymatic hydrolysis

[0051] The temperature was lowered to 30°C, 4 g of compound enzyme (composed of 2.7 g of collagenase and 1.3 g of papain) was added, and enzymolysis was performed at 30°C and pH 6 for 3 h to obtain an enzymolysis solution;

[0052] (3) Centrifugation, filtration, and drying

[0053] The enzymatic hydrolysate was centrifuged at 8000 rpm for 8 minutes, the enzymatic hydrolysate supernatant was filtered with an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and the filtrate was vacuum freeze-dried at -40°C and 50 Pa for 24 hours to obtain the donkey-hide gelatin peptide.

[0054] Example 3

[0055] Preparation of donkey-hide gelatin peptide for delaying aging

[0056] (1) Boiling treatment with citric acid water

[0057] Grind the donkey-hide gelatin ultrafinely and pass it through a 500-mesh sieve to obtain donkey-hide gelatin powder; mix 200 g of donkey-hide gelatin powder with 7 L of pH 4 citric acid aqueous solution, heat to boiling and keep for 90 min;

[0058] (2) Enzymatic hydrolysis

[0059] The temperature was lowered to 40°C, 10 g of compound enzyme (composed of 6.25 g of collagenase and 3.75 g of papain) was added, and enzymolysis was performed at 40°C and pH 8 for 2 h to obtain an enzymolysis solution;

[0060] (3) Centrifugation, filtration, and drying

[0061] The enzymatic hydrolysate was centrifuged at 12000 rpm for 3 min, the enzymatic hydrolysate supernatant was filtered with an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and the filtrate was vacuum freeze-dried at -40°C and 50 Pa for 24 h to obtain the donkey-hide gelatin peptide.

[0062] The molecular weight of the donkey-hide gelatin peptides obtained in Examples 1-3 was determined to be 500Da-1000Da by high performance liquid chromatography.

[0063] Comparative Example 1

[0064] The specific implementation is the same as that of Example 1, except that the citric acid aqueous solution with a pH value of 3 is replaced by deionized water.

[0065] Comparative Example 2

[0066] The specific implementation is the same as that of Example 1, except that the citric acid aqueous solution with a pH value of 3 is replaced by a NaOH solution with a pH value of 7.

[0067] Comparative Example 3

[0068] The specific implementation is the same as that of Comparative Example 1, except that no water boiling treatment is performed.

[0069] Comparative Example 4

[0070] The specific implementation is the same as that of Example 1, except that the enzymatic hydrolysis step is not performed.

[0071] Comparative Example 5

[0072] The specific implementation is the same as that of Example 1, except that the complex enzyme is replaced by collagenase.

[0073] Comparative Example 6

[0074] The specific implementation is the same as that of Example 1, except that the complex enzyme is replaced by papain.

[0075] Comparative Example 7

[0076] The specific implementation method is the same as that of Example 1, except that the ultrafiltration membrane with a molecular weight cutoff of 3 kDa is used for filtration.

[0077] Experimental Example 1

[0078] In vitro experiments

[0079] (1) Determination of hydrolysis degree and protein recovery rate

[0080] The hydrolysis degree and protein recovery rate of the donkey-hide gelatin peptides obtained in Examples 1-3 and Comparative Examples 1-7 were tested respectively. The specific results are shown in Table 1.

[0081] The hydrolysis degree of donkey-hide gelatin peptide was detected by OPA method.

[0082] The protein content of donkey-hide gelatin and enzymatic hydrolysis supernatant was determined by the Kjeldahl method of GB 5009.5-2016.

[0083] Protein recovery rate (%) = protein content of enzymatic supernatant / protein content of donkey-hide gelatin × 100%.

[0084] Table 1 Hydrolysis degree and protein recovery rate of donkey-hide gelatin peptides treated with different methods

[0085]

[0086] It can be seen from the data in Table 1 that, compared with Comparative Examples 1-7, the donkey-hide gelatin peptides prepared by the methods of Examples 1-3 of the present invention have higher hydrolysis degrees and protein recovery rates.

[0087] (2) Determination of antioxidant capacity

[0088] Donkey-hide gelatin solutions of Examples 1-3 and Comparative Examples 1-7 were prepared at different concentrations of 0.15, 0.2, and 0.3 mg / mL. The DPPH free radical scavenging ability, superoxide anion scavenging ability, and hydroxyl free radical scavenging ability of the donkey-hide gelatin solutions at the above concentrations were tested respectively. The specific results are shown in Table 2.

[0089] Prepare donkey-hide gelatin solutions of Examples 1-3 and Comparative Examples 1-7 with different concentrations of 10, 50, and 100 μg / mL. ABTS of donkey-hide gelatin solutions at the above concentrations were detected respectively. + The free radical scavenging ability is shown in Table 2.

[0090] DPPH free radical scavenging rate, hydroxyl free radical scavenging rate and ABTS + The free radical scavenging rate was detected by spectrophotometry; the superoxide free radical scavenging rate was detected by pyrogallol method.

[0091] Table 2 Antioxidant capacity of donkey-hide gelatin peptides of Examples 1-3 and Comparative Examples 1-7

[0092]

[0093] From the results in Table 2, it can be seen that as the concentration of donkey-hide gelatin solution increases, the antioxidant capacity gradually increases. Compared with comparative examples 1-7, examples 1-3 of the present invention are superior in DPPH free radical scavenging capacity, superoxide anion scavenging capacity, hydroxyl free radical scavenging capacity and ABTS + The free radical scavenging ability is stronger. This shows that the preparation method of the present invention has various steps that are closely linked to each other, which improves the antioxidant ability of donkey-hide gelatin peptide and has the potential for anti-aging.

[0094] Experimental Example 2

[0095] Animal experiments

[0096] Using PBS as solvent, 3 mg / mL, 10 mg / mL, and 30 mg / mL donkey-hide gelatin solutions of Example 1 were prepared.

[0097] One hundred 8-week-old C57BL / 6J mice were equally divided into 5 groups:

[0098] Control group: subcutaneous injection of 500 mg / kg / day of normal saline every day for 6 consecutive weeks, and tail vein injection of 1 mL of PBS every week;

[0099] Model group: D-galactose was subcutaneously injected at 500 mg / kg / day for 6 consecutive weeks to construct an aging model;

[0100] Example 1 low, medium and high dose groups of donkey-hide gelatin peptide: D-galactose was subcutaneously injected at 500 mg / kg / day for 6 consecutive weeks, and 1 mL of 3 mg / mL, 10 mg / mL, 30 mg / mL of donkey-hide gelatin peptide solution of Example 1 was injected weekly for 6 consecutive weeks.

[0101] After 6 weeks, the plasma of mice was collected and the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and malondialdehyde (MDA) in the plasma were detected using kits. The specific results are shown in Table 3.

[0102] Table 3 Effects of different groups on plasma parameters

[0103]

[0104] From the data in Table 3, it can be seen that the content of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) in the plasma of mice in the model group was significantly reduced compared with the control group, indicating that the D-galactose-induced aging model mice were successfully modeled. Compared with the model group, the low, medium and high dose groups of Example 1 can increase the activity of SOD, GSH-Px and CAT in the plasma of aging mice induced by D-galactose, and inhibit the increase of MDA, thus playing an anti-aging role.

[0105] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing donkey-hide gelatin peptide, characterized in that: include: Ultrafinely pulverizing donkey-hide gelatin, sieving to obtain donkey-hide gelatin powder, then mixing with citric acid aqueous solution, treating in boiling water for 60-90 minutes, adding complex enzyme for enzymolysis, centrifuging and ultrafiltration, and drying the obtained filtrate to obtain the donkey-hide gelatin peptide; The complex enzyme is composed of collagenase and papain in a weight ratio of (2-5):(1-3); The pH value of the citric acid aqueous solution is 2-4; The mass ratio of the donkey-hide gelatin powder to the citric acid aqueous solution is 1:(25-35); The amount of the complex enzyme added in the enzymatic hydrolysis is 2%-5% of the mass of the donkey-hide gelatin powder, the enzymatic hydrolysis temperature is 30°C-40°C, the enzymatic hydrolysis pH value is 6-8, and the enzymatic hydrolysis time is 2-3h.

2. The preparation method according to claim 1, characterized in that: The sieving mesh number is 400-500 mesh.

3. The preparation method according to claim 1, characterized in that: The centrifugal speed is 8000rpm-12000rpm, and the time is 3min-8min.

4. The donkey-hide gelatin peptide obtained by the preparation method according to any one of claims 1 to 3.

5. The donkey-hide gelatin peptide according to claim 4, characterized in that: The molecular weight of the donkey-hide gelatin peptide is 500Da-1000Da.

6. Use of the donkey-hide gelatin peptide according to claim 5 in the preparation of antioxidant products.

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