Collagen composition and preparation method thereof

Abstract

The invention belongs to the technical field of protein compositions, and relates to a collagen composition and a preparation method thereof. The invention provides a preparation method of a collagen composition, which comprises the following steps: treating raw hide which is not subjected to chemical cross-linking treatment to obtain a collagen solution, adding a cross-linking promoting agent, isothiocyanate and alpha-mangostin into the collagen solution, and finally performing short-time microwave treatment for multiple times to obtain the collagen composition. The collagen composition provided by the invention avoids the use of a cross-linking agent, has the advantages of no cytotoxicity, high cross-linking degree and good stability, and shows excellent bacteriostatic and antioxidant functions.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein compositions and relates to a collagen composition and a preparation method thereof. Background Art

[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 fundamental framework for maintaining tissue morphology and function. Its unique molecular structure consists of three α-chains entangled through hydrogen bonds and van der Waals forces to form a triple helical 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 physical and chemical properties become a bottleneck restricting practical application. Unmodified natural collagen is susceptible to protease degradation in vitro, lacks mechanical stability, and rapidly absorbs water and swells under physiological conditions, leading to structural collapse. This makes the intervention of crosslinking agents an essential technical path for the functionalization of collagen.

[0003] Cross-linking technology creates a stable bridge network between collagen chains by introducing chemical bonds or physical forces, thereby reshaping its three-dimensional structure. Chemical cross-linking agents such as glutaraldehyde, carbodiimide (EDC / NHS system), and epoxy compounds react covalently with the ε-amino groups of lysine and hydroxylysine residues in collagen molecules, forming intermolecular cross-links. CN118241482A discloses cross-linked collagen fibers for filling. These fibers are formed by cross-linking collagen fibers using glutaraldehyde as a cross-linking agent. Glutaraldehyde is an irritating chemical reagent. It can form cross-links with amino groups and peptide bonds in protein molecules, thereby strengthening the protein structure. However, this cross-linking process also causes protein coagulation, partially deactivating it. Cross-linking agents can also strengthen the molecular structure, but their use can also increase the risk of allergies. The use of cross-linking agents also prolongs the skin's metabolic cycle, making collagen less easily absorbed and degraded. Therefore, this field urgently needs to develop a green modification technology that can precisely regulate the cross-linking degree of collagen without introducing chemical cross-linking agents, and to prepare new collagen-based functional materials with high stability, controllable degradability and complete biocompatibility, so as to break the contradiction between biosafety and material performance of the existing cross-linking system. Summary of the Invention

[0004] The present invention aims to solve the technical problem of relying on chemical crosslinking agents to increase crosslinking strength in the preparation of existing collagen materials. To this end, the present invention provides a collagen composition and a preparation method thereof to solve the above technical problem.

[0005] The application relates to a preparation method of a collagen composition, which comprises the following steps: treating raw hide without chemical cross-linking treatment to obtain a collagen solution, adding a cross-linking promoter, isothiocyanate and alpha-mangosteen to the collagen solution, and finally obtaining the collagen composition through multiple short-time microwave treatments.

[0006] Further, in the preparation method of the collagen composition, the collagen solution is 100-200 parts, the cross-linking promoter is 4-8 parts, the isothiocyanate is 4-6 parts, and the alpha-mangosteen is 1-3 parts by weight. The concentration of the collagen in the collagen solution is 0.1-8 wt%.

[0007] Further, in the preparation method of the collagen composition, the cross-linking promoter is at least one selected from ethylene glycol, diethylene glycol and glycerol.

[0008] Further, in the preparation method of the collagen composition, the microwave times of the multiple short-time microwave treatments are 5-10 times, the total microwave time is 40-120 s, and the microwave power is 400-600 W.

[0009] Further, in the preparation method of the collagen composition, the raw hide is sheepskin.

[0010] Further, in the preparation method of the collagen composition, the following steps are further included: the sheepskin without chemical cross-linking treatment is soaked to remove impurities and remove salt-soluble proteins and other soluble impurities, and the collagen is prepared through degreasing and acid-enzyme combined extraction and purification, and the collagen is dispersed in deionized water to obtain the collagen solution.

[0011] Further, in the preparation method of the collagen composition, in the acid-enzyme combined method, pepsin and ternary organic acid are combined for dissolution and extraction.

[0012] Further, in the preparation method of the collagen composition, the ternary organic acid is one selected from citric acid, trimesic acid and cyclohexane tricarboxylic acid.

[0013] The application relates to a collagen composition prepared by the preparation method of the collagen composition.

[0014] Compared with the prior art, the technical scheme provided by the application has at least the following beneficial effects or advantages: The present invention uses the synergistic effect of a cross-linking agent, isothiocyanate, and α-mangostin, combined with multiple short-time microwaves, to achieve the construction of a composite network of non-covalent cross-linking and covalent cross-linking between collagen molecules. The present invention uses α-mangostin to combine with isothiocyanate through π-π stacking and hydrophobic interaction, and simultaneously combines with collagen through hydrogen bonds to form a ternary complex. In addition, the cross-linking agent and multiple short-time microwaves solve the technical problem of relying on chemical cross-linking agents to increase cross-linking strength in the preparation of existing collagen materials. The collagen composition provided by the present invention has good antioxidant and antibacterial capabilities while maintaining a good degree of cross-linking. ; Isothiocyanate undergoes Michael addition reaction with the ε-amino group of collagen through active thioester group to form a stable thiocarbamate cross-linking structure, and α-mangostin interacts with the aromatic residues in the collagen triple helix structure through the π-π stacking effect, thereby enhancing the intermolecular hydrogen bonds and hydrophobic forces; microwave treatment utilizes short-term local instantaneous high temperature to promote the kinetic process of the cross-linking reaction and avoid the degradation of isothiocyanate and α-mangostin caused by heat accumulation; finally, the present invention effectively improves the stability of the collagen composition by adding α-mangostin, thereby avoiding the problem of easy storage failure of the collagen composition due to the addition of isothiocyanate. DETAILED DESCRIPTION

[0015] The technical solutions of the present invention are described below with reference to the following examples. However, the present invention is not limited to the following examples. The experimental methods and detection methods described in each example are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified. The percentages in the following examples are percentages by mass unless otherwise specified. The ratios in the following examples are ratios by mass unless otherwise specified. Example 1

[0016] This example provides a process for preparing collagen.

[0017] Step 1: Add 5% food-grade sodium chloride and 0.1% potassium sorbate as preservatives to the raw hides from sheep and soak them in a pretreatment solution (20±1°C) for 48 hours. Replace the isothermal sterile water every 10 hours and maintain a constant stirring speed of 100 rpm. Step 2: Mix quicklime and sodium sulfide in a mass ratio of 3:1, add an appropriate amount of purified water to prepare a viscous paste, evenly apply the depilatory agent to the epidermis, control the coating thickness to 2-3mm, react in a 35℃ constant temperature box for 3 hours, then mechanically shave off the hair and remove subcutaneous fat; Step 3: Wash the sheepskin with water at room temperature, then decalcify with NH4Cl. After decalcification, remove the subcutaneous fat tissue, cut it into even pieces, and freeze it for storage; Step 4: Thaw the chopped skin and soak it in 10% NaCl solution overnight to remove salt-soluble proteins and other soluble impurities; Step 5: Rinse the depilated dermis with pre-cooled saline to a pH of 8.5 or less; perform initial degreasing with a mixture of 6% sodium carbonate and 1% Triton X-114 (solid-liquid ratio 1:10) and ultrasonically degrease at 30°C for 1 hour; repeat the degreasing solution formula and use pulsed stirring (200 rpm × 10 minutes / standing for 5 minutes) for 3 cycles.

[0018] Step 6: Place the sheepskin extract (0.05 mol / L citric acid and 2% pepsin) in a 4°C refrigerator with slow stirring. Filter through a double layer of medical gauze and gradually adjust the filtrate to pH 10.0 with 1 M NaOH for 24 hours to ensure complete enzyme inactivation. Step 7: Adjust the pH of the solution to neutral, add NaCl in batches to a final concentration of 2.5 M, and let it stand at 4°C for 24 hours to form a composite precipitate; use a 4°C pre-cooled centrifuge (14,000 x g, 30 minutes) to separate the precipitate to obtain collagen precipitate; Step 8: The precipitate was dissolved in 0.02 M citric acid buffer using a dialysis bag with a molecular weight cutoff of 8-14 kDa. Dynamic dialysis was performed against 0.02 M Na2HPO4 (pH 8.6) for 48 h (flow rate 200 mL / min). The product was then dialyzed against double-distilled water for 24 h, with the dialysate replaced every 5 h and magnetic stirring applied (500 rpm × 10 min / h). The product was then freeze-dried at a cold trap temperature of -55°C, a vacuum degree of 10 Pa, a main drying period of 24 h, a desorption drying period of 6 h, and sealed for storage at -80°C. Example 2

[0019] This example provides a preparation process of a collagen composition.

[0020] Step 1: Add the collagen prepared in Example 1 to deionized water and slowly stir at 45° C. to obtain a 0.1-8 wt % collagen solution; Step 2: In parts 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-mangostin; Step 3: Add the cross-linking agent, isothiocyanate and α-mangostin to the collagen solution respectively, mix them evenly to obtain a semi-finished product solution.

[0021] Step 4: subjecting the semi-finished product solution obtained in step 3 to multiple short-time microwaves, wherein the number of microwaves in the multiple short-time microwaves is 5 to 10 times, the total microwave duration is 40 to 120 seconds, and the microwave power is 400 to 600 W. Example 3

[0022] This embodiment provides a collagen composition.

[0023] This example is prepared with reference to Example 2, and its specific parameters are as follows: In step 1, a 0.1 wt% collagen solution was used; In step 2, 100 parts of collagen solution, 4 parts of ethylene glycol, 4 parts of isothiocyanate and 1 part of α-mangostin are taken by weight; In step 4, the number of microwaves of the multiple short-time microwaves is 5 times, the total microwave duration is 40 seconds, and the microwave power is 400W. Example 4

[0024] This embodiment provides a collagen composition.

[0025] This example is prepared with reference to Example 2, and its specific parameters are as follows: In step 1, a 3 wt% collagen solution was used; In step 2, 120 parts of collagen solution, 6 parts of diethylene glycol, 5 parts of isothiocyanate and 2 parts of α-mangostin are taken in parts by weight; In step 4, the number of microwaves of the multiple short-time microwaves is 6 times, the total microwave duration is 60 seconds, and the microwave power is 500W. Example 5

[0026] This embodiment provides a collagen composition.

[0027] This example is prepared with reference to Example 2, and its specific parameters are as follows: In step 1, a 4 wt% collagen solution was used; In step 2, 150 parts of collagen solution, 6 parts of diethylene glycol, 5 parts of isothiocyanate and 2 parts of α-mangostin are taken in parts by weight; In step 4, the number of microwaves of the multiple short-time microwaves is 8 times, the total microwave duration is 80 seconds, and the microwave power is 500W. Example 6

[0028] This embodiment provides a collagen composition.

[0029] This example is prepared with reference to Example 2, and its specific parameters are as follows: In step 1, a 5 wt% collagen solution was used; In step 2, 180 parts of collagen solution, 6 parts of diethylene glycol, 5 parts of isothiocyanate and 2 parts of α-mangostin are taken in parts by weight; In step 4, the number of microwaves of the multiple short-time microwaves is 9 times, the total microwave duration is 90 seconds, and the microwave power is 500W. Example 7

[0030] This embodiment provides a collagen composition.

[0031] This example is prepared with reference to Example 2, and its specific parameters are as follows: In step 1, an 8 wt% collagen solution was used; In step 2, 200 parts of collagen solution, 8 parts of glycerol, 6 parts of isothiocyanate and 3 parts of α-mangostin are taken in parts by weight; In step 4, the number of microwave times of the multiple short-time microwaves is 10 times, the total microwave duration is 120 seconds, and the microwave power is 600W.

[0032] Comparative Example 1 This comparative example is the same as Example 7, except that α-mangostin is not added.

[0033] Comparative Example 2 This comparative example is the same as Example 7, except that α-mangostin is replaced by psoralen.

[0034] Comparative Example 3 This comparative example is the same as Example 7, except that no isothiocyanate is added.

[0035] Comparative Example 4 This comparative example is the same as Example 7, except that, in step 4, a single continuous microwave is used, the total microwave duration is 120 s, and the microwave power is 600 W.

[0036] Test Example 1 This test example provides the antioxidant capacity, antibacterial capacity and cross-linking degree of the collagen composition.

[0037] 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 - Free Radical (DPPH) Scavenging Test Method. This test example uses WS / T 650-2019 Antibacterial and Antibacterial Effect Evaluation Method to measure antibacterial ability.

[0038] This test example uses the TNBS method to evaluate the degree of cross-linking of the collagen composition. 0.5% 2,4,6-trinitrobenzenesulfonic acid (TNBS) is added to the semi-finished product or collagen composition to react with the amino acid residues. After reacting at 40°C for 4 hours, 6 mol / L HCl is added. After reacting at 60°C for 1.5 hours, an appropriate amount of anhydrous ether is added to remove unreacted TNBS. The UV absorbance at 345 nm is then measured to calculate the degree of cross-linking.

[0039] The cross-linking degree calculation formula is: a = [(A2-A0)-(A1-A0)] / (A2-A0) x 100%; Wherein, a is the cross-linking degree, 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.

[0040] The test results are shown in Table 1.

[0041] Table 1 Antioxidant capacity of collagen protein composition

[0042] As shown in Table 1, the alpha-ambrettolide in the application is combined with isothiocyanate through pi-pi stacking and hydrophobic interaction, and is combined with collagen protein through hydrogen bond to form a ternary complex. In addition, by using a cross-linking promoter and multiple short-time microwaves, the technical problem of relying on a chemical cross-linking agent to improve the cross-linking strength in the preparation of existing collagen protein materials is solved. The collagen protein composition provided by the application has good antioxidant capacity and antibacterial capacity while maintaining a good cross-linking degree (the cross-linking degree of a sample cross-linked by a traditional chemical cross-linking agent formaldehyde is 65%). As shown in Comparative Example 1, the addition of alpha-ambrettolide effectively improves the antioxidant capacity and antibacterial effect of the collagen protein composition, and also has a certain effect on improving the cross-linking degree. As shown in Comparative Example 2, alpha-ambrettolide is replaced by a similar biological small molecule, psoralen, and the test results are similar to those of Comparative Example 1, indicating that alpha-ambrettolide cannot be replaced by a conventional biological small molecule. As shown in Comparative Example 3, the addition of isothiocyanate can effectively improve the antioxidant capacity and antibacterial effect of the collagen protein composition, but has little effect on improving the cross-linking degree. As shown in Comparative Example 4, compared with multiple short-time microwaves, continuous microwave can effectively improve the cross-linking degree of collagen protein, but can destroy the complex structure of alpha-ambrettolide, isothiocyanate and collagen protein, resulting in a loss of antioxidant capacity.

[0043] Test Example 2 The present test example provides the stability of the collagen protein composition.

[0044] After the collagen protein composition is stored in a refrigerator at 4℃ for 3 months, the antioxidant capacity of the collagen protein composition is re-determined according to Test Example 1, and the test results are shown in Table 2.

[0045] Table 2 Change in antioxidant capacity of collagen protein composition

[0046] As shown in Table 2, the addition of alpha-ambrettolide effectively improves the stability of the collagen protein composition, and avoids the problem of storage failure of the collagen protein composition caused by the addition of isothiocyanate.

[0047] As described above, the basic principles, main features and advantages of the present invention are well described. The above embodiments and descriptions 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 scope of protection determined by the present invention.

Claims

1. A method for preparing a collagen composition, characterized in that: include: The collagen solution is prepared by treating raw hides that have not been chemically cross-linked, a cross-linking agent, isothiocyanate and α-mangostin are added to the collagen solution, and finally the collagen composition is obtained after multiple short-time microwave treatments.

2. The method for preparing a collagen composition according to claim 1, wherein: The preparation comprises, in parts 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-mangostin; The concentration of collagen in the collagen solution is 0.1~8wt%.

3. The method for preparing a collagen composition according to claim 1, wherein: The cross-linking accelerator is selected from at least one of ethylene glycol, diethylene glycol and glycerol.

4. The method for preparing a collagen composition according to claim 1, wherein: The number of microwave times of multiple short-time microwaves is 5 to 10 times, the total microwave time is 40 to 120 seconds, and the microwave power is 400 to 600W.

5. The method for preparing the collagen composition according to claim 1, wherein The raw hide is sheepskin.

6. The method for preparing a collagen composition according to claim 5, characterized in that: include: The sheepskin which has not been chemically cross-linked is soaked to remove salt-soluble proteins and other soluble impurities, and then purified by defatting and acid-enzyme combined extraction to obtain collagen, which is then dispersed in deionized water to obtain a collagen solution.

7. The method for preparing a collagen composition according to claim 6, characterized in that: In the acid-enzyme combination method, pepsin and ternary organic acid are used for extraction.

8. The method for preparing a collagen composition according to claim 7, characterized in that: The ternary organic acid is selected from one of citric acid, mesitylene tribenzoic acid and cyclohexanetrilic acid.

9. A collagen composition, characterized in that The collagen composition is prepared by the preparation method of any one of claims 1 to 8.

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