A collagen composition and a method for preparing the same

Abstract

The present application belongs to the technical field of protein composition, and relates to a collagen composition and a preparation method thereof. The present application provides a preparation method of a collagen composition, which comprises the following steps: preparing a collagen solution by treating raw hide which has not been subjected to chemical cross-linking treatment; adding a cross-linking promoter, an isothiocyanate and alpha-mammeigin to the collagen solution; and finally obtaining the collagen composition by multiple short-time microwave treatment. The collagen composition provided by the present application avoids the use of a cross-linking agent, has the advantages of no cytotoxicity, high cross-linking degree and good stability, and exhibits excellent antibacterial and antioxidant functions.

Description

Technical Field

[0001] This invention belongs to the field of protein composition technology, and relates to a collagen composition and its preparation method. Background Technology

[0002] Collagen, the most abundant structural protein in the human body, is widely distributed in connective tissues such as skin, bones, tendons, and blood vessels, forming the basic framework for maintaining tissue shape and function. Its unique molecular structure consists of three α-chains intertwined by hydrogen bonds and van der Waals forces to form a triple helix conformation. This highly ordered arrangement endows collagen with excellent mechanical strength and biocompatibility. However, when collagen is extracted from natural tissues and applied in fields such as biomedical engineering, tissue repair, or cosmetic medicine, its inherent physicochemical properties become a bottleneck restricting practical applications—unmodified natural collagen is easily degraded by proteases in the in vitro environment, lacks mechanical stability, and rapidly absorbs water and swells under physiological conditions, leading to structural collapse. Therefore, the intervention of cross-linking agents has become a necessary technical path for the functional modification of collagen.

[0003] Cross-linking technology introduces chemical bonds or physical forces to establish a stable bridging network between collagen molecular chains, thereby reshaping its three-dimensional structure. Chemical cross-linking agents, such as glutaraldehyde, carbodiimide (EDC / NHS system), and epoxy compounds, react with the ε-amino groups of lysine and hydroxylysine residues in collagen molecules through covalent bonds, forming intermolecular cross-links. CN118241482A discloses a cross-linked collagen fiber for filling, which is made by cross-linking collagen fibers with glutaraldehyde as a cross-linking agent. Glutaraldehyde is an irritating chemical reagent. It can link with the amino and peptide bonds of protein molecules to form cross-links, thereby enhancing the protein structure. However, this cross-linking process can also cause protein coagulation, causing it to lose some activity. In addition, while cross-linking agents can also enhance molecular structure, their use can increase the risk of allergies. The use of cross-linking agents can also prolong the skin's metabolic cycle, making collagen more difficult for the skin to absorb and degrade. Therefore, there is an urgent need in this field to develop a green modification technology that can precisely control the degree of collagen crosslinking without introducing chemical crosslinking agents, and to prepare novel collagen-based functional materials that combine high stability, controllable degradation and complete biocompatibility, so as to overcome the contradiction between biosafety and material performance in existing crosslinking systems. Summary of the Invention

[0004] The purpose of this invention is to solve the technical problem of relying on chemical cross-linking agents to improve cross-linking strength in the preparation of existing collagen materials. To this end, this invention provides a collagen composition and its preparation method to solve the aforementioned technical problem.

[0005] This invention relates to a method for preparing a collagen composition, comprising: processing raw hide that has not undergone chemical cross-linking treatment to obtain a collagen solution; adding a cross-linking agent, isothiocyanate and α-dextrin to the collagen solution; and finally subjecting the solution to multiple short-time microwave treatments to obtain the collagen composition.

[0006] Furthermore, in the preparation method of the collagen composition provided by the present invention, the composition comprises, by weight: 100-200 parts of collagen solution, 4-8 parts of cross-linking agent, 4-6 parts of isothiocyanate and 1-3 parts of α-dextrin.

[0007] The concentration of collagen in the collagen solution is 0.1~8wt%.

[0008] Furthermore, in the preparation method of the collagen composition provided by the present invention, the cross-linking agent is selected from at least one of ethylene glycol, diethylene glycol, and glycerol.

[0009] Furthermore, in the preparation method of the collagen composition provided by the present invention, the number of short microwave cycles is 5 to 10, the total microwave duration is 40 to 120 seconds, and the microwave power is 400 to 600 W.

[0010] Furthermore, in the preparation method of the collagen composition provided by the present invention, the raw hide is sheepskin.

[0011] Furthermore, the preparation method of the collagen composition provided by the present invention includes: soaking uncrosslinked sheepskin to remove salt-soluble proteins and other soluble impurities, purifying it by defatting and acid-enzyme extraction to obtain collagen, and dispersing the collagen in deionized water to obtain a collagen solution.

[0012] Furthermore, in the preparation method of the collagen composition provided by the present invention, in the acid-enzyme combination method, pepsin and ternary organic acids are used for extraction.

[0013] Furthermore, in the preparation method of the collagen composition provided by the present invention, the ternary organic acid is selected from one of citric acid, mesitylene benzoic acid, and cyclohexanetriic acid.

[0014] This invention relates to a collagen composition prepared by the method described above.

[0015] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0016] This invention utilizes the synergistic effect of a cross-linking agent, isothiocyanate, and α-thujone, combined with multiple short-duration microwave treatments, to construct a composite network of non-covalent and covalent cross-links between collagen molecules. α-thujone binds to isothiocyanate through π-π stacking and hydrophobic interactions, while simultaneously binding to collagen via hydrogen bonds, forming a ternary complex. Furthermore, the use of a cross-linking agent and multiple short-duration microwave treatments solves the technical problem of relying on chemical cross-linking agents to improve cross-linking strength in existing collagen material preparations. The collagen composition provided by this invention, while maintaining good cross-linking degree, exhibits good antioxidant and antibacterial capabilities. Isothiocyanate undergoes a Michael addition reaction with the ε-amino group of collagen through active thioester groups, forming a stable thiocarbamate cross-linked structure. α-Retropine interacts with aromatic residues in the collagen triple helix structure through a π-π stacking effect, enhancing intermolecular hydrogen bonds and hydrophobic interactions. Microwave treatment utilizes short-duration, localized, instantaneous high temperatures to promote the cross-linking reaction kinetics and avoid degradation of isothiocyanate and α-retropine due to heat accumulation. Finally, the addition of α-retropine effectively improves the stability of the collagen composition, avoiding the problem of easy storage failure caused by the addition of isothiocyanate. Detailed Implementation

[0017] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental and detection methods described in each embodiment are conventional methods; the reagents and materials described are commercially available unless otherwise specified. Unless otherwise specified, all percentages in the following embodiments refer to mass percentages. Unless otherwise specified, all proportions in the following embodiments refer to mass ratios. Example 1

[0018] This embodiment provides a process for preparing collagen.

[0019] Step 1: Add 5% food-grade sodium chloride and 0.1% potassium sorbate preservative to the raw hides derived from sheep, soak in the pretreatment solution (20±1℃) for 48 hours, change the isothermal sterile water every 10 hours and keep stirring at a constant speed of 100 rpm.

[0020] Step 2: Mix quicklime and sodium sulfide in a 3:1 mass ratio, add an appropriate amount of purified water to make a viscous paste, apply the hair removal agent evenly to the epidermal layer, control the coating thickness to 2~3mm, react in a 35℃ constant temperature oven for 3 hours, then mechanically scrape off the hair and remove the subcutaneous fat.

[0021] Step 3: Wash the sheepskin with room temperature water, then deash it with NH4Cl. After deashing, remove the subcutaneous fat tissue, cut it into even pieces, and freeze it for storage.

[0022] Step 4: Thaw the shredded skin and soak it in a 10% NaCl solution overnight to remove salt-soluble proteins and other soluble impurities;

[0023] Step 5: Rinse the dermis after hair removal with pre-cooled saline until pH ≤ 8.5; perform initial degreasing with a mixture of 6% sodium carbonate and 1% Triton X-114 (solid-liquid ratio 1:10), and perform ultrasonic-assisted degreasing at 30℃ for 1 hour; repeat the degreasing solution formula and cycle it 3 times using pulse stirring (200rpm×10min / 5min standing).

[0024] Step 6: At 4°C, place the extract mixed with sheepskin (0.05 mol / L citric acid and 2% pepsin) in a 4°C refrigerator, stirring slowly during the process. After coarse filtration with double-layer medical gauze, gradually adjust the pH of the filtrate to 10.0 with 1M NaOH and maintain for 24 hours to ensure complete enzyme inactivation.

[0025] Step 7: Adjust the pH of the solution to neutral, add NaCl in batches to a final concentration of 2.5M, and let it stand at 4℃ for 24 hours to form a complex precipitate; separate the precipitate using a pre-cooled centrifuge at 4℃ (14000xg, 30min) to obtain collagen precipitate;

[0026] Step 8: Dissolve the precipitate in 0.02M citrate buffer and use a dialysis bag with a molecular weight cutoff of 8~14kDa; perform dynamic dialysis with 0.02M Na2HPO4 (pH 8.6) for 48h (flow rate 200mL / min); perform dialysis with double-distilled water for 24h, changing the dialysis solution every 5h with magnetic stirring (500rpm×10min / h); perform vacuum freeze-drying at a cold trap temperature of -55℃ and a vacuum degree of 10Pa, with a main drying period of 24h and a desorption drying period of 6h, then seal the package and store at -80℃. Example 2

[0027] This embodiment provides a process for preparing a collagen composition.

[0028] Step 1: Add the collagen obtained in Example 1 to deionized water and stir slowly at 45°C to obtain a 0.1~8wt% collagen solution;

[0029] Step 2: By weight, take 100-200 parts of collagen solution, 4-8 parts of cross-linking agent, 4-6 parts of isothiocyanate and 1-3 parts of α-dextrin;

[0030] Step 3: Add the cross-linking agent, isothiocyanate and α-retropine to the collagen solution respectively, and mix them evenly to obtain a semi-finished solution.

[0031] Step 4: Expose the semi-finished solution obtained in Step 3 to microwave for multiple short periods of time. The number of short microwave cycles is 5 to 10, the total microwave duration is 40 to 120 seconds, and the microwave power is 400 to 600 W. Example 3

[0032] This embodiment provides a collagen composition.

[0033] This embodiment refers to the preparation process of Example 2, and its specific parameters are as follows:

[0034] In step 1, a 0.1 wt% collagen solution is used;

[0035] In step 2, by weight, take 100 parts of collagen solution, 4 parts of ethylene glycol, 4 parts of isothiocyanate and 1 part of α-dextrin;

[0036] In step 4, the number of short microwave pulses is 5, the total microwave duration is 40s, and the microwave power is 400W. Example 4

[0037] This embodiment provides a collagen composition.

[0038] This embodiment refers to the preparation process of Example 2, and its specific parameters are as follows:

[0039] In step 1, a 3wt% collagen solution is used;

[0040] In step 2, by weight, take 120 parts of collagen solution, 6 parts of diethylene glycol, 5 parts of isothiocyanate and 2 parts of α-dextrin;

[0041] In step 4, the number of short microwave cycles is 6, the total microwave duration is 60s, and the microwave power is 500W. Example 5

[0042] This embodiment provides a collagen composition.

[0043] This embodiment refers to the preparation process of Example 2, and its specific parameters are as follows:

[0044] In step 1, a 4wt% collagen solution is used;

[0045] In step 2, by weight, take 150 parts of collagen solution, 6 parts of diethylene glycol, 5 parts of isothiocyanate and 2 parts of α-dextrin;

[0046] In step 4, the number of short microwave cycles is 8, the total microwave duration is 80s, and the microwave power is 500W. Example 6

[0047] This embodiment provides a collagen composition.

[0048] This embodiment refers to the preparation process of Example 2, and its specific parameters are as follows:

[0049] In step 1, a 5wt% collagen solution is used;

[0050] In step 2, by weight, take 180 parts of collagen solution, 6 parts of diethylene glycol, 5 parts of isothiocyanate and 2 parts of α-dextrin;

[0051] In step 4, the number of short microwave cycles is 9, the total microwave duration is 90s, and the microwave power is 500W. Example 7

[0052] This embodiment provides a collagen composition.

[0053] This embodiment refers to the preparation process of Example 2, and its specific parameters are as follows:

[0054] In step 1, an 8wt% collagen solution is used;

[0055] In step 2, by weight, take 200 parts of collagen solution, 8 parts of glycerol, 6 parts of isothiocyanate and 3 parts of α-dextrin;

[0056] In step 4, the number of short microwave cycles is 10, the total microwave duration is 120s, and the microwave power is 600W.

[0057] Comparative Example 1

[0058] This comparative example is the same as Example 7, except that α-dextrin was not added.

[0059] Comparative Example 2

[0060] This comparative example is the same as Example 7, except that α-dextrin is replaced with psoralen.

[0061] Comparative Example 3

[0062] This comparative example is the same as Example 7, except that isothiocyanate was not added.

[0063] Comparative Example 4

[0064] This comparative example is the same as Example 7, except that in step 4, a single continuous microwave is used, with a total microwave duration of 120s and a microwave power of 600W.

[0065] Test Example 1

[0066] This test case provides information on the antioxidant capacity, antibacterial capacity, and cross-linking degree of the collagen composition.

[0067] This test example uses DPPH free radical scavenging rate to measure antioxidant capacity. The higher the DPPH free radical scavenging rate, the stronger the antioxidant capacity of the product. The test method for DPPH free radical scavenging rate refers to T / SHRH 006-2018 Cosmetics - Experimental Method for Free Radical (DPPH) Scavenging. This test example uses WS / T 650-2019 Evaluation Method for Antibacterial and Bacteriostatic Effects to measure antibacterial capacity.

[0068] This test example uses the TNBS method to evaluate the degree of crosslinking of collagen compositions. 0.5% of 2,4,6-trinitrobenzenesulfonic acid (TNBS) is added to the semi-finished product or collagen composition to react with amino acid residues. After reacting at 40°C for 4 hours, 6 mol / L HCl is added, and after reacting at 60°C for 1.5 hours, an appropriate amount of anhydrous diethyl ether is added to remove unreacted TNBS. The UV absorbance at 345 nm is then measured, and the degree of crosslinking is calculated.

[0069] The formula for calculating the degree of crosslinking is: α = [(A2 - A0) - (A1 - A0)] / (A2 - A0) × 100%;

[0070] Where α is the degree of crosslinking, A2 is the absorbance of the semi-finished product, A1 is the absorbance of the finished product, and A0 is the absorbance of the background.

[0071] The test results are shown in Table 1.

[0072] Table 1 Antioxidant capacity of collagen compositions

[0073]

[0074] As shown in Table 1, this invention utilizes α-retropine to bind with isothiocyanate through π-π stacking and hydrophobic interactions, while simultaneously binding with collagen via hydrogen bonds to form a ternary complex. Furthermore, by employing a cross-linking agent and multiple short-duration microwave treatments, the technical problem of relying on chemical cross-linking agents to improve cross-linking strength in existing collagen material preparations is solved. The collagen composition provided by this invention maintains a good degree of cross-linking (the cross-linking degree of the sample cross-linked with the traditional chemical cross-linking agent formaldehyde is 65%), while also exhibiting good antioxidant and antibacterial capabilities. Comparative Example 1 shows that the addition of α-retropine effectively enhances the antioxidant and antibacterial effects of the collagen composition and also improves the degree of cross-linking. Comparative Example 2 shows that replacing α-retropine with a similar small biological molecule, psoralen, yields test results similar to Comparative Example 1, indicating that α-retropine cannot be replaced by conventional small biological molecules. Comparative Example 3 shows that the addition of isothiocyanate can effectively enhance the antioxidant capacity and antibacterial effect of the collagen composition, but has little effect on improving the degree of cross-linking. Comparative Example 4 shows that although a single continuous microwave treatment can effectively improve the degree of cross-linking of collagen compared to multiple short microwave treatments, it will disrupt the complex structure of α-dextrin, isothiocyanate, and collagen, leading to a loss of antioxidant capacity.

[0075] Test Example 2

[0076] This test example provides information on the stability of collagen compositions.

[0077] After storing the collagen composition in a refrigerator at 4°C for 3 months, the antioxidant capacity of the collagen composition was re-determined according to Test Example 1. The test results are shown in Table 2.

[0078] Table 2. Changes in the antioxidant capacity of collagen compositions

[0079]

[0080] As shown in Table 2, the present invention effectively improves the stability of collagen composition by adding α-dextrin, avoiding the problem of easy storage failure of collagen composition due to the addition of isothiocyanate.

[0081] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A method for preparing a collagen composition, characterized by, The collagen composition is prepared by the following steps: The collagen solution is prepared by treating the raw hide which is not treated by chemical cross-linking, adding a cross-linking promoter, isothiocyanate and α-physalin into the collagen solution, and finally treating the collagen solution by multiple short-time microwave treatment; The collagen solution is prepared by treating the raw hide which is not treated by chemical cross-linking, adding a cross-linking promoter, isothiocyanate and α-physalin into the collagen solution, and finally treating the collagen solution by multiple short-time microwave treatment; The concentration of the collagen in the collagen solution is 0.1-8wt%. The cross-linking promoter is at least one of ethylene glycol, diethylene glycol or glycerol. The microwave times of the multiple short-time microwave treatment are 5-10 times, the total microwave time is 40-120s, and the microwave power is 400-600W.

2. The method of claim 1, wherein the collagen composition is prepared by the steps of: The raw hide is sheepskin.

3. The method of claim 2, wherein the collagen composition is prepared by, The collagen solution is prepared by treating the raw hide which is not treated by chemical cross-linking, adding a cross-linking promoter, isothiocyanate and α-physalin into the collagen solution, and finally treating the collagen solution by multiple short-time microwave treatment; In the acid-enzyme combined method, pepsin and a ternary organic acid are used for combined dissolution.

4. The method of claim 3, wherein the collagen composition is prepared by, The ternary organic acid is one of citric acid, trimesic acid or cyclohexane tricarboxylic acid.

5. The method of claim 4, wherein the collagen composition is prepared by the steps of: The collagen composition is prepared by the method of any one of claims 1-5.

6. A collagen composition, characterized by, ​

Images