Bovine collagen composition with antioxidant activity and preparation method thereof

Abstract

The invention relates to the field of collagen peptides, in particular to a bovine collagen composition with antioxidant activity and a preparation method of the bovine collagen composition, and solves the problems that collagen is easily affected by the environment in the process of being prepared into dry powder and is reduced, oxidative substances are broken, disulfide bonds are mistakenly connected, and the stability of the collagen is poor. According to the present invention, the problems that the three-helix structure of the collagen is damaged due to the disruption of the collagen and the biological activity of the collagen is reduced in the prior art are solved, and the collagen composition comprises the following components in parts by weight: 100-200 parts of a collagen precipitation body, 10-20 parts of trehalose, 5-10 parts of oxidized glutathione, and 20-40 parts of an iodoacetamide sustained-release body, the iodoacetamide sustained-release body comprises iodoacetamide and a sustained-release carrier, and the weight ratio of the iodoacetamide to the sustained-release carrier is (1-2): 10; the slow-release carrier is one of chitosan, lipidosome or sodium alginate. The collagen with high biological activity can be obtained, has extremely high oxidation resistance, and is few in misfolding and high in activity in an unsuitable environment.

Description

Technical Field

[0001] The present invention relates to the technical field of collagen peptides, and specifically relates to a bovine collagen composition with antioxidant activity and a preparation method thereof. Background Art

[0002] Collagen is a fibrous filamentous protein, and its specific shape is like a twisted rope. The biological activity of collagen highly depends on its triple helix structure, which endows it with unique physicochemical properties and biological functions. If this structure is lost, the mechanical properties of collagen will be greatly reduced and degraded into an aggregate of non-functional biological macromolecules. Therefore, in order to maintain this morphology, it is necessary to maintain the stability between protein chains. The key to maintaining the collagen morphology lies in the molecular nail-like structure clusters of sulfur amino acids. How to maintain these nail-like structure clusters is the key to ensuring the activity of collagen.

[0003] In an antioxidant collagen peptide and its preparation method with the patent publication number CN116035940B, it is disclosed that ascorbic acid or peroxidase reacts and binds with collagen peptide after mixing and grinding using deionized water, enhancing the antioxidant property of the collagen peptide and having the ability to inhibit free radicals. However, during the process of preparing the collagen dry powder, excessive exposure to a dry environment will exacerbate the oxidation of proteins, especially the oxidation of sulfhydryl groups (-SH), resulting in the destruction of the spatial structure and the loss of the original activity and binding ability. Moreover, ascorbic acid or peroxidase will also lose its original catalytic activity and binding ability during the binding process. Therefore, the operator needs to increase the addition amount of ascorbic acid or peroxidase. However, increasing the usage amount will cause damage to the functional groups of the protein, and the same amount of increased oxidant will trigger an oxidative stress response in cells, leading to the loss of protein activity.

[0004] For other substances that maintain disulfide bonds, such as glutathione, it can act as a reducing agent to maintain the form of disulfide bonds through glutathione reductase. However, it is easily oxidized in an aerobic environment, resulting in the inability to maintain the ability to maintain disulfide bonds for a long time. Although the introduction of exogenous disulfide isomerase can maintain the morphology of collagen, when used, it will cause an immune response in the organism by exogenous enzyme bodies, leading to the production of antibodies, and the operation difficulty is relatively large, making it difficult to maintain the triple helix spatial structure of collagen for a long time. Summary of the Invention

[0005] In order to solve the problem that collagen is easily affected by the environment during the preparation of dry powder, is reduced, and the disulfide bonds are broken and misconnected by oxidizing substances, resulting in the destruction of the triple helix structure of collagen and the reduction of its biological activity, the present invention provides a bovine collagen composition with antioxidant activity and a preparation method thereof. The technical solution adopted by the present invention is as follows:

[0006] First, the bovine collagen composition with antioxidant activity prepared in this application includes the following components in parts by weight: 100-200 parts of collagen precipitate, 10-20 parts of trehalose, 5-10 parts of oxidized glutathione, and 20-40 parts of iodoacetamide sustained-release body;

[0007] The iodoacetamide sustained-release body includes iodoacetamide and a sustained-release carrier, and the weight ratio of iodoacetamide to the sustained-release carrier is 1-2:10;

[0008] The sustained-release carrier is one of chitosan, liposome or sodium alginate.

[0009] In this application, oxidized glutathione is used to combine with the collagen precipitate, so that the native triple helix of collagen is affected by oxidized glutathione to maintain the oxidized state of protein disulfide bonds. Oxidized glutathione participates in the formation of protein disulfide bonds through a thiol-disulfide exchange reaction, and can effectively convert the reduced disulfide bonds into disulfide bonds again; while trehalose is used as a protein stabilizer, which can form a vitrified state after the subsequent drying process, form hydrogen bonds with the protein surface, reduce the interaction between protein molecules, can effectively inhibit the aggregation and denaturation of collagen, and can maintain the original secondary and tertiary structures of collagen, and will not affect glutathione either. Trehalose is a non-reducing sugar and will not affect the effect of oxidized glutathione on maintaining disulfide bonds.

[0010] This application also uses an iodoacetamide sustained-release body as an isolating substance outside the collagen to prevent the misbinding of sulfhydryl groups in the collagen triple helix structure. When a reducing substance breaks through the protective layers of trehalose and oxidized glutathione, the formed free sulfhydryl groups will be connected after misfolding or molecular movement, thus destroying the triple helix structure of collagen. Therefore, an iodoacetamide sustained-release body is used externally as an isolation system to prevent the miscontact and connection of sulfhydryl groups. In order to reduce the reduction and damage of the disulfide bonds on the protein by iodoacetamide itself, a sustained-release carrier is used for slow and long-term release, which can effectively preserve the protein for a long time, ensure the triple helix spatial structure of the protein, thus having antioxidant properties, reducing misfolding, and enhancing the activity of collagen.

[0011] Preferably, the preparation steps of the collagen precipitate are as follows:

[0012] S1. Pretreat the collagen raw material by degreasing and acid leaching to obtain a crude collagen extract;

[0013] S2. Hydrolyze and extract the crude collagen extract with a composite enzyme, the hydrolysis temperature is 50-55 °C, the hydrolysis time is 3-6 h, and the hydrolysis pH is 7.5-8 to obtain a crude collagen hydrolysis product;

[0014] S3. Centrifuge the crude collagen hydrolysate, then add the salting-out solution, let it stand to obtain precipitated collagen, and finally perform dialysis to obtain the collagen precipitate.

[0015] The collagen raw materials used in this application are raw materials such as cowhide, beef tendon, and cow bone.

[0016] Preferably, the solution used for degreasing in step S1 is a sodium carbonate solution with a mass fraction of 5-10%, and it is heated to 63-92°C for treatment. The solution used for acid leaching is a hydrochloric acid solution with a mass fraction of 0.5%.

[0017] Preferably, the complex enzyme in step S2 is a composition of papain, trypsin, and thymol. The weight ratio of papain, trypsin, and thymol is 1-2:0.5:0.5.

[0018] The complex enzyme used itself will not have a great destructive effect on disulfide bonds and can perfectly retain the triple helix spatial structure of collagen.

[0019] This application also provides a preparation method of a bovine collagen composition with antioxidant activity. The specific preparation steps are as follows:

[0020] A1. Place the collagen precipitate and oxidized glutathione in deionized water. The solid-liquid ratio of the collagen precipitate and oxidized glutathione in deionized water is 1-5:10, and stir to obtain a mixed solution.

[0021] A2. Ultrasonically treat the mixed solution prepared in A1 with an ultrasonic energy of 8 kJ / mL to form a composite solution A, then heat it to 45-60°C, slowly add trehalose to form a composite solution B, and perform freeze-drying under vacuum on the composite solution B to form an active protein complex.

[0022] A3. Then perform a loading process on iodoacetamide and the sustained-release carrier to obtain an iodoacetamide sustained-release body. Mix the active protein complex in step A2 with the iodoacetamide sustained-release body and perform freeze-drying to obtain the antioxidant collagen composition.

[0023] In the process of preparing the collagen composition of the present application, first, the connection between the collagen precipitate and oxidized glutathione is constructed. The present application uses ultrasonic treatment to enable the preliminary anchoring connection between the collagen precipitate and oxidized glutathione, promoting the connection between oxidized glutathione and collagen and the formation of disulfide bonds. Thereby, the contact effect between collagen and oxidized glutathione is improved, and collagen and glutathione will not be isolated by trehalose. As can be seen from the experimental data in the example part, when trehalose is added to collagen and glutathione without ultrasonic treatment, it will be difficult for glutathione to act effectively on collagen, and thus the triple helix structure of collagen cannot be maintained.

[0024] Then, after trehalose coats the collagen, a loading process is used to confine iodoacetamide in the sustained-release carrier and mix it with collagen. As a matrix, iodoacetamide that can react with free sulfhydryl groups is slowly released to form thioether bonds, avoiding the misfolding of collagen chains.

[0025] Preferably, the loading process in step A3 is specifically as follows:

[0026] Chitosan is placed in deionized water to form a solution with a concentration of 1-2% wt. Then, iodoacetamide is added to the chitosan solution, and a sodium tripolyphosphate solution is added dropwise for crosslinking to obtain an iodoacetamide sustained-release body. The concentration of the sodium tripolyphosphate solution is 0.1-0.5% wt, and the pH value of the solution is 4.5-5.5.

[0027] Preferably, the loading process in step A3 is specifically as follows:

[0028] Liposomes and iodoacetamide are placed in an organic solvent, and a thin film is formed by rotary evaporation. Then, after hydration, ultrasonic dispersion is carried out to obtain an iodoacetamide sustained-release body. The power of the ultrasonic treatment is 550 W, and the ultrasonic treatment time is 5-10 min.

[0029] Preferably, the liposomes are phospholipids or cholesterol.

[0030] Preferably, the loading process in step A3 is specifically as follows:

[0031] Sodium alginate is configured into a solution with a concentration of 1-3% wt using deionized water. Then, iodoacetamide is added to the sodium alginate solution for mixing, and a calcium chloride solution is added dropwise for gelation to obtain an iodoacetamide sustained-release body. The concentration of calcium chloride is 0.1-0.5 mol / L.

[0032] The preparation method of the bovine collagen composition with antioxidant activity of the present application can be applied in the preparation of antioxidant high-quality protein peptides.

[0033] The beneficial effects of the present invention are:

[0034] In the present application, oxidized glutathione is bound to the outside of the collagen triple helix chain to maintain the dynamic balance of disulfide bonds, enabling the sulfhydryl groups formed by broken disulfide bonds to be oxidized again. Through ultrasonic treatment, they will not be isolated by trehalose. Then, the glassy matrix formed after drying with trehalose further protects the protein form and improves the protein's resistance. Secondly, iodoacetamide, a substance that can protect sulfhydryl groups, is used as an isolation environment for sulfhydryl groups on the outside, which can effectively prevent the misfolding of collagen caused by the incorrect connection of sulfhydryl groups, indirectly improving the biological activity of collagen and having strong antioxidant capacity. Description of the Drawings

[0035] Figure 1 It is a line graph of the experimental data of the embodiments and comparative examples of the present invention.

[0036] Figure 2 It is a line graph of the experimental data of the embodiments and comparative examples of the present invention. Detailed Description of the Embodiments

[0037] The following will refer to the reference drawings Figures 1 to 2 to detail the embodiments of the present invention. Those skilled in the art should understand that these embodiments are only used to explain the technical principle of the present invention and are not intended to limit the protection scope of the present invention.

[0038] Preparation Example 1 of Collagen Precipitate

[0039] S1. Clean the cowhide and beef tendon by removing hair and washing, and place the cowhide and beef tendon in a large reaction kettle. Pour a sodium carbonate solution with a mass fraction of 7.5% to soak and heat to 72°C for 2 hours to complete degreasing. Then, repeatedly wash with alcohol and water, and perform an acid leaching operation with a hydrochloric acid solution with a mass fraction of 0.5% for 12 hours to obtain a crude collagen extract;

[0040] S2. Weigh papain, pancreatin, and thymol and compound them to obtain a compound enzyme. The weight ratio of papain, pancreatin, and thymol is 1.5:0.5:0.5. Add the compound enzyme to the crude collagen extract prepared in step S1 for hydrolysis extraction. The usage amount of the compound enzyme is 0.5%, the hydrolysis temperature is 53°C, the hydrolysis time is 5 hours, and the hydrolysis pH is 7.5 to obtain a crude collagen hydrolysis product;

[0041] S3. Centrifuge the crude collagen hydrolysis product obtained in step S2 at 4000 rpm / min for 20 minutes, then add a 1 mol / L NaCl salting-out solution, stand for 12 hours to obtain precipitated collagen, and finally perform dialysis using a macromolecular dialysis membrane to obtain a collagen precipitate.

[0042] Preparation Example 2 of Collagen Precipitate

[0043] S1. Clean the cowhide and beef tendon by removing hair and washing, etc. Place the cowhide and beef tendon in a large reaction kettle, pour in a sodium carbonate solution with a mass fraction of 5% for soaking, heat up to 63°C and keep it for 2 hours to complete degreasing. Then wash repeatedly with alcohol and water, and then perform acid leaching operation with a hydrochloric acid solution with a mass fraction of 0.5% for 12 hours to obtain a crude collagen extract;

[0044] S2. Weigh papain, trypsin and thymol for compounding to obtain a compound enzyme. The weight ratio of papain, trypsin and thymol is 1:0.5:0.5. Add the compound enzyme to the crude collagen extract prepared in step S1 for hydrolysis extraction. The usage amount of the compound enzyme is 0.5%, the hydrolysis temperature is 50°C, the hydrolysis time is 6 hours, and the hydrolysis pH is 8 to obtain a crude collagen hydrolysis product;

[0045] S3. Centrifuge the crude collagen hydrolysis product obtained in step S2 at 4000 rpm / min for 20 minutes, then add a 1 mol / L NaCl salting-out solution, stand for 12 hours to obtain precipitated collagen, and finally perform dialysis using a macromolecular dialysis membrane to obtain a collagen precipitate.

[0046] Preparation Example 3 of Collagen Precipitate

[0047] S1. Clean the cowhide and beef tendon by removing hair and washing, etc. Place the cowhide and beef tendon in a large reaction kettle, pour in a sodium carbonate solution with a mass fraction of 10% for soaking, heat up to 92°C and keep it for 2 hours to complete degreasing. Then wash repeatedly with alcohol and water, and then perform acid leaching operation with a hydrochloric acid solution with a mass fraction of 0.5% for 12 hours to obtain a crude collagen extract;

[0048] S2. Weigh papain, trypsin and thymol for compounding to obtain a compound enzyme. The weight ratio of papain, trypsin and thymol is 2:0.5:0.5. Add the compound enzyme to the crude collagen extract prepared in step S1 for hydrolysis extraction. The usage amount of the compound enzyme is 0.5%, the hydrolysis temperature is 55°C, the hydrolysis time is 3 hours, and the hydrolysis pH is 7.5 to obtain a crude collagen hydrolysis product;

[0049] S3. Centrifuge the crude collagen hydrolysis product obtained in step S2 at 4000 rpm / min for 20 minutes, then add a 1 mol / L NaCl salting-out solution, stand for 12 hours to obtain precipitated collagen, and finally perform dialysis using a macromolecular dialysis membrane to obtain a collagen precipitate.

[0050] Preparation Example 1 of Iodoacetamide Sustained Release System

[0051] Weigh 1.5 parts of iodoacetamide and 10 parts of chitosan by weight. Place 10 parts of chitosan in deionized water to form a solution with a concentration of 1.5% wt, and then add iodoacetamide to the chitosan solution. Weigh 20 parts by weight of sodium tripolyphosphate, prepare a solution with a concentration of 0.3% wt, and add the sodium tripolyphosphate solution dropwise to the chitosan solution, maintaining the pH of the solution at 5 during the dropping process to form an iodoacetamide sustained-release body.

[0052] Preparation Example 2 of Iodoacetamide Sustained-Release Body

[0053] Weigh 1 part of iodoacetamide and 10 parts of phospholipid by weight and place them in a mixed solution of chloroform and methanol with a mass ratio of 1:3. Set the temperature of the rotary evaporator at 45 °C and the rotation speed at 120 rpm until the solvent completely evaporates to form a film, and then purge it with nitrogen. Add 200 parts of PBS buffer for hydration at a hydration temperature of 50 °C for 1 h. After the hydration is completed, use an ultrasonic instrument with a power of 550 W for ultrasonic dispersion for 10 min to obtain an iodoacetamide sustained-release body with uniform particle size, and then remove the hydration solution by centrifugation.

[0054] Preparation Example 3 of Iodoacetamide Sustained-Release Body

[0055] Weigh 2 parts of iodoacetamide and 10 parts of cholesterol by weight and place them in a mixed solution of chloroform and methanol with a mass ratio of 1:3. Set the temperature of the rotary evaporator at 45 °C and the rotation speed at 120 rpm until the solvent completely evaporates to form a film, and then purge it with nitrogen. Add 200 parts of PBS buffer for hydration at a hydration temperature of 50 °C for 1 h. After the hydration is completed, use an ultrasonic instrument with a power of 550 W for ultrasonic dispersion for 5 min to obtain an iodoacetamide sustained-release body with uniform particle size, and then remove the hydration solution by centrifugation.

[0056] Preparation Example 4 of Iodoacetamide Sustained-Release Body

[0057] Weigh 1 part of iodoacetamide and 10 parts of sodium alginate by weight, and use deionized water to prepare a sodium alginate solution with a concentration of 2% wt. Then add iodoacetamide to the sodium alginate solution for mixing, and dropwise add a calcium chloride solution with a concentration of 0.2 mol / L for gelation to obtain an iodoacetamide sustained-release body.

[0058] Example 1

[0059] Preparation of Antioxidant Collagen Composition

[0060] A1. Weigh 150 parts of the collagen precipitate obtained in Preparation Example 1 of the collagen precipitate and 8 parts of oxidized glutathione by weight, and place them in deionized water. The solid-liquid ratio of the collagen precipitate and oxidized glutathione in deionized water is 2:10, and stir to obtain a mixed solution.

[0061] A2. Ultrasonically treat the mixed solution obtained in A1 with an ultrasonic energy of 8 kJ / mL to form a composite solution A, then heat it to 50 °C, slowly add 15 parts of trehalose to form a composite solution B, and perform freeze-drying on the composite solution B at a freezing temperature of -50 °C to form an active protein complex.

[0062] A3. Then mix 30 parts of the iodoacetamide sustained-release body obtained in Preparation Example 1 of the iodoacetamide sustained-release body with the active protein complex in step A2, and perform freeze-drying to obtain an antioxidant collagen composition.

[0063] Example 2

[0064] Preparation of antioxidant collagen composition

[0065] A1. Weigh 100 parts of the collagen precipitate obtained in Preparation Example 2 of the collagen precipitate and 5 parts of oxidized glutathione by weight, and place them in deionized water. The solid-liquid ratio of the collagen precipitate and oxidized glutathione in deionized water is 1:10, and stir to obtain a mixed solution.

[0066] A2. Ultrasonically treat the mixed solution obtained in A1 with an ultrasonic energy of 8 kJ / mL to form a composite solution A, then heat it to 45 °C, slowly add 10 parts of trehalose to form a composite solution B, and perform freeze-drying on the composite solution B at a freezing temperature of -50 °C to form an active protein complex.

[0067] A3. Then mix 20 parts of the iodoacetamide sustained-release body obtained in Preparation Example 2 of the iodoacetamide sustained-release body with the active protein complex in step A2, and perform freeze-drying to obtain an antioxidant collagen composition.

[0068] Example 3

[0069] Preparation of antioxidant collagen composition

[0070] A1. Weigh 200 parts of the collagen precipitate obtained in Preparation Example 3 of the collagen precipitate and 10 parts of oxidized glutathione by weight, and place them in deionized water. The solid-liquid ratio of the collagen precipitate and oxidized glutathione in deionized water is 5:10, and stir to obtain a mixed solution.

[0071] A2. Ultrasonically treat the mixed solution obtained in A1 with an ultrasonic energy of 8 kJ / mL to form a composite solution A. Then, raise the temperature to 60 °C and slowly add 20 parts of trehalose to form a composite solution B. Subject the composite solution B to freeze-drying under vacuum at a freezing temperature of -50 °C to form an active protein complex;

[0072] A3. Then, mix 40 parts of the iodoacetamide sustained-release body prepared in Preparation Example 3 of the iodoacetamide sustained-release body with the active protein complex obtained in step A2 and perform freeze-drying to obtain an antioxidant collagen composition.

[0073] Example 4

[0074] Preparation of an antioxidant collagen composition

[0075] A1. Place 130 parts of the collagen precipitate prepared in Preparation Example 1 of the collagen precipitate and 6 parts of oxidized glutathione in deionized water according to parts by weight. The solid-liquid ratio of the collagen precipitate and oxidized glutathione in deionized water is 3:10, and stir to obtain a mixed solution;

[0076] A2. Ultrasonically treat the mixed solution obtained in A1 with an ultrasonic energy of 8 kJ / mL to form a composite solution A. Then, raise the temperature to 55 °C and slowly add 16 parts of trehalose to form a composite solution B. Subject the composite solution B to freeze-drying under vacuum at a freezing temperature of -50 °C to form an active protein complex;

[0077] A3. Then, mix 30 parts of the iodoacetamide sustained-release body prepared in Preparation Example 4 of the iodoacetamide sustained-release body with the active protein complex obtained in step A2 and perform freeze-drying to obtain an antioxidant collagen composition.

[0078] Comparative Example 1 - Oxidized glutathione was not used in combination with the collagen precipitate

[0079] Preparation of an antioxidant collagen composition

[0080] A1. Place 150 parts of the collagen precipitate prepared in Preparation Example 1 of the collagen precipitate and 8 parts of reduced glutathione in deionized water according to parts by weight. The solid-liquid ratio of the collagen precipitate and reduced glutathione in deionized water is 2:10, and stir to obtain a mixed solution;

[0081] A2. Ultrasonically treat the mixed solution obtained in A1 with an ultrasonic energy of 8 kJ / mL to form a composite solution A. Then, raise the temperature to 50 °C and slowly add 15 parts of trehalose to form a composite solution B. Subject the composite solution B to freeze-drying under vacuum at a freezing temperature of -50 °C to form an active protein complex;

[0082] A3. Then, mix 25 parts of the iodoacetamide sustained-release body prepared in Preparation Example 1 of the iodoacetamide sustained-release body with the active protein complex in Step A2, and perform freeze-drying to obtain the antioxidant collagen composition.

[0083] Comparative Example 2 - The mixed solution obtained in A1 was not subjected to ultrasonic treatment.

[0084] Preparation of the antioxidant collagen composition

[0085] A1. Place 150 parts of the collagen precipitate prepared in Preparation Example 1 of the collagen precipitate and 8 parts of oxidized glutathione by weight in deionized water. The solid-liquid ratio of the collagen precipitate and oxidized glutathione in deionized water is 2:10, and stir to obtain a mixed solution.

[0086] A2. Heat the mixed solution obtained in A1 to 50°C, slowly add 15 parts of trehalose to form a composite solution B, and perform freeze-vacuum drying on the composite solution B. The freezing temperature is -50°C to form an active protein complex.

[0087] A3. Then, mix 35 parts of the iodoacetamide sustained-release body prepared in Preparation Example 1 of the iodoacetamide sustained-release body with the active protein complex in Step A2, and perform freeze-drying to obtain the antioxidant collagen composition.

[0088] Comparative Example 3 - Trehalose was not used for coating.

[0089] Preparation of the antioxidant collagen composition

[0090] A1. Place 150 parts of the collagen precipitate prepared in Preparation Example 1 of the collagen precipitate and 8 parts of oxidized glutathione by weight in deionized water. The solid-liquid ratio of the collagen precipitate and oxidized glutathione in deionized water is 2:10, and stir to obtain a mixed solution.

[0091] A2. Perform ultrasonic treatment on the mixed solution obtained in A1 with an ultrasonic energy of 8 kJ / mL to form a composite solution A, then heat it to 50°C to form a composite solution B, and perform freeze-vacuum drying on the composite solution B. The freezing temperature is -50°C to form an active protein complex.

[0092] A3. Then, mix 30 parts of the iodoacetamide sustained-release body prepared in Preparation Example 1 of the iodoacetamide sustained-release body with the active protein complex in Step A2, and perform freeze-drying to obtain the antioxidant collagen composition.

[0093] Comparative Example 4 - The iodoacetamide was not subjected to the loading process.

[0094] Preparation of the antioxidant collagen composition

[0095] A1. Put 150 parts of the collagen precipitate prepared in Preparation Example 1 of the collagen precipitate and 8 parts of oxidized glutathione into deionized water. The solid-liquid ratio of the collagen precipitate and oxidized glutathione in deionized water is 2:10, and stir to obtain a mixed solution;

[0096] A2. Ultrasonically treat the mixed solution prepared in A1 with an ultrasonic energy of 8 kJ / mL to form a composite solution A. Then heat it to 50°C and slowly add 15 parts of trehalose to form a composite solution B. Subject the composite solution B to freeze-drying under vacuum at a freezing temperature of -50°C to form an active protein complex;

[0097] A3. Then mix 20 parts by weight of iodoacetamide with the active protein complex obtained in step A2 and perform freeze-drying to obtain an antioxidant collagen composition.

[0098] Experiments and Data

[0099] Perform stability tests, biological activity tests, and determination of disulfide bond content on the antioxidant collagen compositions prepared in the above-mentioned examples and comparative examples. The specific test methods are as follows:

[0100] Stability test: Prepare the antioxidant collagen composition into a 10 wt% solution, stir and filter it until it is in a clear state, and store it at room temperature for 12, 24, and 36 days, and observe the liquid state.

[0101] Biological activity test: Prepare a 0.04 mg / mL DPPH solution, prepare the antioxidant collagen composition into solutions of 2, 4, 6, and 8 mg / mL using distilled water, set up blank control, sample control, and positive control groups. After reacting for 30 min, use a spectrophotometer to measure the absorbance values A0, A1, and A2 of each group.

[0102] Scavenging rate = (1 - ((A1 - A2) / A0)) × 100%

[0103] A0: Absorbance value of the blank control

[0104] A1: Absorbance value after the sample reacts with DPPH

[0105] A2: Absorbance value of the sample solution (without adding DPPH)

[0106] Determination of disulfide bond content: Mix 0.5 mL of the sample to be tested and 2 mL of buffer, then add 50 μL of mercaptoethanol, react at room temperature for 1 h, add 250 μL of 4 mg / mL DTNB, and measure the light absorption value at a wavelength of 412 nm after the reaction.

[0107] Content of disulfide bond = ((73.53 × measured absorbance value × (dilution factor / sample concentration)) - free sulfhydryl content) / 2

[0108] Free sulfhydryl content = (dilution factor × measured absorbance value × 73.53) / sample concentration Unit: μmol / g

[0109] The buffer solution is obtained by slowly mixing 86 mmol / L Tris, 90 mmol / L Gly, 8 M urea and 0.5% SDS.

[0110] The experimental data of the stability test are shown in Table 1 below:

[0111]

[0112]

[0113] The experimental data of the bioactivity test and the determination of the disulfide bond content are shown in Table 2 below:

[0114] Clearance rate Disulfide bond content Example 1 97.8 26.10 Example 2 97.2 25.54 Example 3 96.9 26.20 Example 4 95.7 25.45 Comparative example 1 73.0 12.35 Comparative example 2 68.5 10.96 Comparative example 3 51.0 9.21 Comparative example 4 36.0 6.25

[0115] Analysis

[0116] According to the experimental data in Table 1 and Table 2, it can be seen that the antioxidant collagen compositions prepared in Example 1, Example 2, Example 3 and Example 4 show excellent stability, bioactivity and relatively high disulfide bond content after long-term storage, proving that the antioxidant collagen compositions prepared in this application can have good antioxidant performance and bioactivity, can maintain the spatial triple helix structure and keep the activity.

[0117] According to the experimental data in Table 1 and Table 2, it can be seen that after storage, turbidity and precipitation phenomena occurred in Comparative Example 1, and the scavenging rate of Comparative Example 1 decreased to a certain extent, and the disulfide bond content was also relatively low. The difference between Comparative Example 1 and the examples is that oxidized glutathione was not used to combine with the collagen precipitate, which proves that oxidized glutathione has a certain effect on maintaining the disulfide bonds on the collagen triple helix, can reduce the breakage of the disulfide bonds of the triple helix structure collagen, and thus improve the stability and activity of collagen.

[0118] It can be seen from the experimental data in Table 1 and Table 2 that after standing, turbidity and precipitation occurred in Comparative Example 2, and the clearance rate of Comparative Example 2 decreased to a certain extent, and the disulfide bond content was also low. The difference between Comparative Example 2 and the Example is that the mixed solution prepared in Step A1 was not ultrasonically treated during subsequent use, which proves that ultrasonic treatment can effectively connect oxidized glutathione and collagen, so that the two are not isolated by trehalose, thereby improving the effect of oxidized glutathione on the collagen peptide chain, protecting the disulfide bond and thus enhancing the activity and stability of collagen.

[0119] It can be seen from the experimental data in Table 1 and Table 2 that after standing, turbidity and precipitation occurred in Comparative Example 3, and the clearance rate of Comparative Example 3 decreased significantly, and the disulfide bond content was also very low. The difference between Comparative Example 3 and the Example is that trehalose was not used to coat collagen and oxidized glutathione, which proves that trehalose can effectively isolate the direct and rapid contact between collagen and the externally strong reducing iodoacetamide, so that the locally broken disulfide bonds of collagen can re-establish a connection relationship and will not be directly reduced to thioether bonds by the reaction of iodoacetamide, thereby maintaining the integrity of the collagen triple helix structure, and further improving the biological activity and the number of disulfide bonds of collagen.

[0120] It can be seen from the experimental data in Table 1 and Table 2 that after standing, turbidity and precipitation occurred in Comparative Example 4, and the clearance rate of Comparative Example 4 decreased the most, and the disulfide bond content was also the lowest. The difference between Comparative Example 4 and the Example is that the iodoacetamide was not subjected to the loading process, which proves that under the slow-release action of iodoacetamide, a relatively stable environment can be maintained for a long time and effectively, preventing the contact between different chains of collagen, avoiding misconnection, forming a correct spatial conformation, and the appropriate concentration of iodoacetamide ensures that collagen will not be greatly affected by reducibility, further promoting the reformation of broken sulfhydryl groups into disulfide bonds and improving the activity and stability of collagen.

[0121] So far, the technical solution of the present invention has been described in conjunction with the preferred embodiments shown in the drawings. However, it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the protection scope of the present invention.

Claims

1. A bovine collagen composition having antioxidant activity, characterized in that, It comprises the following components in parts by weight: 100 - 200 parts of collagen precipitate, 10 - 20 parts of trehalose, 5 - 10 parts of oxidized glutathione, and 20 - 40 parts of iodoacetamide sustained-release body; The iodoacetamide sustained-release body comprises iodoacetamide and a sustained-release carrier, and the weight ratio of iodoacetamide to the sustained-release carrier is 1 - 2:10; The sustained-release carrier is one of chitosan, liposome or sodium alginate.

2. The bovine collagen composition with antioxidant activity according to claim 1, characterized in that, The preparation steps of the collagen precipitate are as follows: S1. Perform degreasing and acid leaching pretreatment on the collagen raw material to obtain a crude collagen extract; S2. Hydrolyze and extract the crude collagen extract with a composite enzyme at a hydrolysis temperature of 50 - 55 °C, a hydrolysis time of 3 - 6 h, and a hydrolysis pH of 7.5 - 8 to obtain a crude collagen hydrolysis product; S3. Centrifuge the crude collagen hydrolysis product, then add a salting-out solution, stand to obtain precipitated collagen, and finally perform dialysis to obtain a collagen precipitate.

3. The bovine collagen composition with antioxidant activity according to claim 2, characterized in that, In step S1, the solution used for degreasing is a sodium carbonate solution with a mass fraction of 5 - 10%, and it is heated to 63 - 92 °C for treatment. The solution used for acid leaching is a hydrochloric acid solution with a mass fraction of 0.5%.

4. A bovine collagen composition having antioxidant activity according to claim 2, characterized in that, In step S2, the composite enzyme is a composition of papain, pancreatin, and thymol, and the weight ratio of papain, pancreatin, and thymol is 1 - 2:0.5:0.

5.

5. The preparation method of a bovine collagen composition with antioxidant activity according to claims 1-4, characterized in that, The specific preparation steps are as follows: A1. Place the collagen precipitate and oxidized glutathione in deionized water. The solid-liquid ratio of the collagen precipitate and oxidized glutathione in deionized water is 1 - 5:10, and stir to obtain a mixed solution; A2. Ultrasonically treat the mixed solution prepared in A1 with an ultrasonic energy of 8 kJ / mL to form a composite solution A, then heat it to 45 - 60 °C, slowly add trehalose to form a composite solution B, and perform freeze-drying under vacuum on the composite solution B to form an active protein complex; A3. Then perform a loading process on iodoacetamide and the sustained-release carrier to obtain an iodoacetamide sustained-release body. Mix the active protein complex in step A2 with the iodoacetamide sustained-release body and perform freeze-drying to obtain an antioxidant collagen composition.

6. The preparation method of a bovine collagen composition with antioxidant activity according to claim 5, characterized in that, The loading process in step A3 is specifically as follows: Place chitosan in deionized water to form a solution with a concentration of 1 - 2% wt, then add iodoacetamide to the chitosan solution, and dropwise add a sodium tripolyphosphate solution for cross-linking to obtain an iodoacetamide sustained-release body. The concentration of the sodium tripolyphosphate solution is 0.1 - 0.5% wt, and the pH value of the solution is 4.5 - 5.

5.

7. A method for preparing a bovine collagen composition having antioxidant activity according to claim 5, characterized in that, The loading process in step A3 is specifically as follows: Place liposome and iodoacetamide in an organic solvent, form a thin film by rotary evaporation, and then perform hydration and ultrasonic dispersion to obtain an iodoacetamide sustained-release body. The power of the ultrasonic treatment is 550 W, and the ultrasonic treatment time is 5 - 10 min.

8. The preparation method of a bovine collagen composition with antioxidant activity according to claim 7, characterized in that, The liposome is phospholipid or cholesterol.

9. The preparation method of a bovine collagen composition with antioxidant activity according to claim 8, characterized in that, The loading process in step A3 is specifically as follows: Sodium alginate was dissolved in deionized water to form a solution with a concentration of 1-3% wt, and then iodoacetamide was added to the sodium alginate solution and mixed. A calcium chloride solution was added dropwise for gelation to obtain an iodoacetamide sustained-release body, and the concentration of the calcium chloride was 0.1-0.5 mol / L.

10. Use of the preparation method of a bovine collagen composition with antioxidant activity as described in claims 5-9 in the preparation of antioxidant protein peptides.

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