Preparation method of low-viscosity collagen solution

Abstract

The invention discloses a preparation method of a low-viscosity collagen solution. The preparation method disclosed by the invention comprises the following steps: (1) pretreatment: taking fresh bovine achilles tendon, cleaning, dicing, sequentially soaking in an ether alcohol solution and a sodium hydroxide solution, fully cleaning until no peculiar smell exists, and crushing for later use; (2) enzymolysis: putting the pretreated bovine achilles tendons into an enzymatic hydrolysate, and carrying out enzymolysis treatment to obtain the enzymatic hydrolysate; (3) salting-out / acid process crude extraction: adding a sodium chloride solution into the enzymatic hydrolysate for salting-out, discarding supernatant to obtain precipitated collagen, and putting the collagen into a glacial acetic acid solution with the same volume as the discarded supernatant to obtain an acid sol stock solution; and (4) purification / filtration: carrying out ultrafiltration treatment on the salted acid sol stock solution through a 100kDa-150kDa ultrafiltration membrane to obtain a purified collagen solution, diluting the purified collagen solution, and filtering the diluted collagen solution through a 0.45 mu m filter element to obtain the low-viscosity collagen solution. The collagen solution prepared by the method is relatively low in viscosity, can be produced in a filtration sterilization mode, and is low in microorganism content and high in product stability and safety.

Description

Technical Field

[0001] The invention relates to the technical field of collagen preparation, and in particular to a method for preparing a low-viscosity collagen solution. Background Art

[0002] Collagen is the structural protein with the highest protein content and the widest distribution in human tissues. It plays an important role in protecting and connecting various tissues and supporting the structure of the human body. This has attracted widespread attention and become a research hotspot. At present, most collagen products on the market are of animal origin. The amino acid composition of mammals such as cattle and pigs is about 95% similar to that of humans, and the human utilization rate of collagen extracted from cattle exceeds 95%. In addition, the extracted collagen has a unique triple helix structure and diverse biological properties, including low immunogenicity and good biocompatibility, and is widely used in food, cosmetics, medical supplies, tissue engineering products and other fields. In addition, there are also a small number of aquatic products as collagen sources, including fish skin, bones, scales, etc., but the amino acid composition of edible fish is about 65% similar to that of humans, and the human utilization rate of extracted collagen is less than 65%. Most of them are used in food and cosmetics, and are basically not used in medical devices or tight tissue engineering products.

[0003] At present, the mainstream methods of collagen extraction include hot water extraction, acid method, alkali method, salt method, enzyme method, etc. The collagen products obtained are different due to different extraction conditions. Among them, hot water extraction can obtain gelatin or collagen polypeptides; acid method is often combined with enzyme method to extract active collagen; collagen extracted by alkali method has a complete structure, but the treatment process is prone to protein denaturation; salt method can precipitate different types of collagen. According to the characteristics of the extraction method, collagen for medical devices is mostly produced by combining acid method with enzyme method. However, collagen solution device products on the market will be sterilized by electron irradiation after using collagen solution to meet microbial limit control or sterility requirements, but this sterilization method is prone to protein denaturation and affect the stability of the product. For this reason, it is possible to consider using filtration sterilization to meet the microbial requirements of collagen solution medical devices. The extracted collagen solution is a transparent, clear, viscous liquid with high viscosity, and cannot completely pass through the 0.2μm sterilization filter element. The protein retention is large, and the protein content of the filtrate is also low, which cannot meet the product requirements.

[0004] Therefore, it is of great significance to develop a collagen preparation method that can balance collagen activity and reduce viscosity. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for preparing a low-viscosity collagen solution. The collagen solution obtained by the preparation method of the present invention has a low viscosity and can be produced by filtering and sterilizing, meeting the product microbial requirements and significantly improving the product stability and safety.

[0006] The technical solution of the present invention is as follows:

[0007] A method for preparing a low-viscosity collagen solution, the preparation comprising the following steps:

[0008] (1) Pretreatment: Take fresh beef Achilles tendon, wash it, cut it into pieces, soak it in ether alcohol solution and sodium hydroxide solution in sequence, wash it thoroughly until there is no odor, and grind it for later use;

[0009] (2) enzymolysis: putting the pretreated bovine Achilles tendon into an enzymolysis solution for enzymolysis to obtain an enzymolysis solution;

[0010] (3) Salting out / acid extraction: Add sodium chloride solution to the enzymatic hydrolyzate for salting out, discard the supernatant to obtain the precipitated collagen, and then place the collagen in an acid solution with an equal volume to the discarded supernatant to obtain an acid-soluble collagen stock solution;

[0011] (4) Purification / filtration: The acid-soluble collagen stock solution after salting out is ultrafiltered through a 100 kDa to 150 kDa ultrafiltration membrane to obtain a purified collagen solution, which is then diluted and filtered through a 0.45 μm filter element to obtain the low-viscosity collagen solution.

[0012] Furthermore, in step (1), the ether-alcohol solution is selected from any one of an ethanol solution and an ether solution, and the mass percentage concentration is 70% to 90%; the amount of the ether-alcohol solution is 5 to 7 times the mass of the bovine Achilles tendon, and the immersion time in the ether-alcohol solution is 1 to 2 hours.

[0013] Furthermore, in step (1), the mass percentage concentration of the sodium hydroxide solution is 3% to 6%; the amount of the sodium hydroxide solution used is 5 to 7 times the mass of the bovine Achilles tendon, and the immersion time in the sodium hydroxide solution is 10 to 20 minutes.

[0014] Furthermore, in step (2), the enzymatic hydrolysis solution is prepared by mixing glacial acetic acid and pepsin; the concentration of glacial acetic acid in the enzymatic hydrolysis solution is 0.3-0.6 mol / L, and the concentration of pepsin is 500-700 mg / L; the amount of the enzymatic hydrolysis solution is 25-35 times the mass of the bovine Achilles tendon; the temperature of the enzymatic hydrolysis is 4-25°C, and the time is 40-50 hours.

[0015] Furthermore, in step (3), the mass percentage concentration of the sodium chloride solution is 25% to 35%; the amount of the sodium chloride solution used is 8 to 10 times the mass of the bovine Achilles tendon; and the salting-out time is 12 to 16 hours.

[0016] Furthermore, in step (3), the acid solution is a glacial acetic acid solution or a phosphate buffer solution; the concentration of the glacial acetic acid solution is 0.3 to 0.6 mol / L; and the pH value of the phosphate buffer solution is 2.5 to 4.0.

[0017] Furthermore, in step (4), the ultrafiltration is performed 8 to 10 times in total; wherein the replenishing liquid for the 1st to 7th ultrafiltrations is acid solution, and the replenishing liquid for the 8th to 10th ultrafiltrations is a mixture of acid solution, glycerol and 1-ethyl-3-methylimidazolium acetate ionic liquid.

[0018] Furthermore, the acid solution is a glacial acetic acid solution or a phosphate buffer solution; the concentration of the glacial acetic acid solution is 0.3-0.6 mol / L; the pH value of the phosphate buffer solution is 2.5-4.0; in the 8th to 10th replenishing solution of the ultrafiltration, the volume ratio of the acid solution, glycerol and 1-ethyl-3-methylimidazolium acetate ion liquid is 100:0.3-1.2:5-15.

[0019] Furthermore, in step (4), the protein content of the diluted collagen solution is controlled at 1.5-3.5 mg / g; and the 0.45 μm filter element is filtered 2-3 times.

[0020] A low-viscosity collagen solution prepared by the preparation method.

[0021] The beneficial technical effects of the present invention are:

[0022] The preparation method of the present invention, combined with acid, enzyme and salt extraction methods, can further optimize the quality of collagen solution and obtain low-viscosity, high-purity collagen solution. The protein purity of the collagen solution prepared by the present invention can reach more than 95%; the low viscosity property enables it to be suitable for some filtered and sterilized products, without terminal sterilization, and directly carry out process aseptic production, better retaining the triple helix structure of collagen, improving product stability, and avoiding the gelation and collagen denaturation caused by the radiation sterilization treatment of conventional collagen solution, ensuring the safety of product use.

[0023] The preparation method provided by the present invention utilizes the characteristics of alkaline solution and acid solution to effectively control the microbial limit of the collagen solution and improve the safety of the collagen solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The present invention is a process flow chart for preparing the low-viscosity collagen solution. DETAILED DESCRIPTION

[0025] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0026] Example 1

[0027] A method for preparing a low-viscosity collagen solution comprises the following steps:

[0028] (1) Pretreatment: Take fresh beef Achilles tendon, wash and cut it into pieces, add ether solution (70wt%) 5 times the mass of the beef Achilles tendon into the small pieces of beef Achilles tendon, soak for 1.5 hours, and wash thoroughly until there is no odor; then add sodium hydroxide solution (3wt%) 6 times the mass of the beef Achilles tendon, soak for 20 minutes, wash thoroughly until there is no odor, and crush for later use.

[0029] (2) Enzymatic hydrolysis: The pretreated bovine Achilles tendon is placed in an enzymatic hydrolysis solution (in the enzymatic hydrolysis solution, 0.4 mol / L glacial acetic acid and 500 mg / L protease), the mixing mass ratio of the bovine Achilles tendon to the enzymatic hydrolysis solution is 1:25, the enzymatic hydrolysis time is controlled at 50 h, and the enzymatic hydrolysis temperature is controlled at 4-25° C. to obtain an enzymatic hydrolysis solution.

[0030] (3) Salting out / acid extraction: Add 8 times the mass of bovine Achilles tendon sodium chloride solution (30 wt%) to the enzymatic hydrolyzate and soak for 12 h to obtain white flocs precipitated. Centrifuge at 5500 r / min for 15 min, discard the supernatant, and then put it into a 0.4 mol / L glacial acetic acid solution with an equal volume to the discarded supernatant, stir evenly, and obtain an acid-soluble collagen stock solution.

[0031] (4) Purification / filtration: The acid-soluble collagen stock solution after salting out was ultrafiltered 7 times through a 100 kDa ultrafiltration membrane, and the supplementing liquid during ultrafiltration was 0.4 mol / L glacial acetic acid solution; then, a mixed acid solution (the volume ratio of 0.4 mol / L glacial acetic acid solution, glycerol and 1-ethyl-3-methylimidazolium acetate ionic liquid was 100:1.2:15) was prepared as a supplementing liquid for the 8th ultrafiltration to obtain a purified collagen solution, which was diluted to 3.5 mg / g using a mixed acid solution (the volume ratio of 0.4 mol / L glacial acetic acid solution, glycerol and ionic liquid was 100:1.2:15), and filtered through a 0.45 μm filter element for 3 times to obtain a low-viscosity collagen solution.

[0032] The prepared low-viscosity collagen solution was tested for appearance, identification, collagen purity, collagen content, hydroxyproline content, microbial limit, viscosity, 0.2 μm filter element filtration and other items. The specific data are shown in the table below:

[0033]

[0034]

[0035] Example 2

[0036] A method for preparing a low-viscosity collagen solution comprises the following steps:

[0037] (1) Pretreatment: Wash and cut fresh beef Achilles tendon into pieces, add 6 times the weight of the beef Achilles tendon in ethanol solution (90 wt%) to the small pieces of beef Achilles tendon, soak for 1 hour, and wash thoroughly until there is no odor. Then add 5 times the weight of the beef Achilles tendon in sodium hydroxide solution (4 wt%), soak for 15 minutes, wash thoroughly until there is no odor, and crush for later use.

[0038] (2) Enzymatic hydrolysis: The pretreated bovine Achilles tendon is placed in an enzymatic hydrolysis solution (in the enzymatic hydrolysis solution, 0.3 mol / L glacial acetic acid and 600 mg / L pepsin), the mixing mass ratio of the bovine Achilles tendon to the enzymatic hydrolysis solution is 1:30, the enzymatic hydrolysis time is controlled at 45 h, and the enzymatic hydrolysis temperature is controlled at 4-25° C. to obtain a collagen solution.

[0039] (3) Salting out / acid extraction: Add 9 times the mass of bovine Achilles tendon sodium chloride solution (25wt%) to the collagen solution after enzymatic hydrolysis and soak for 14h to obtain white flocs precipitated. Centrifuge at 5000r / min for 20min, discard the supernatant, and then add an equal volume of 0.6mol / L glacial acetic acid solution to the supernatant and stir evenly to obtain acid-soluble collagen solution.

[0040] (4) Purification / filtration: The acid-soluble collagen stock solution after salting out was ultrafiltered 7 times through a 100 kDa ultrafiltration membrane, and the supplementing liquid during ultrafiltration was 0.6 mol / L glacial acetic acid solution; then, a mixed acid solution (the volume ratio of 0.6 mol / L glacial acetic acid solution, glycerol and 1-ethyl-3-methylimidazolium acetate ionic liquid was 100:0.8:10) was prepared as a supplementing liquid and the ultrafiltration was repeated twice to obtain a purified collagen solution, which was diluted to 3.0 mg / g using a mixed acid solution (the volume ratio of 0.6 mol / L glacial acetic acid solution, glycerol and ionic liquid was 100:0.8:10), and filtered twice through a 0.45 μm filter element to obtain a low-viscosity collagen solution.

[0041] The prepared low-viscosity collagen solution was tested for appearance, identification, collagen purity, collagen content, hydroxyproline content, microbial limit, viscosity, 0.2 μm filter element filtration and other items. The specific data are shown in the table below:

[0042]

[0043] Example 3

[0044] A method for preparing a low-viscosity collagen solution comprises the following steps:

[0045] (1) Pretreatment: Wash and cut fresh beef Achilles tendon into pieces, add ethanol solution (80 wt%) 6 times the mass of the beef Achilles tendon into the small pieces of beef Achilles tendon, soak for 1 hour, and wash thoroughly until there is no odor. Then add sodium hydroxide solution (6 wt%) 6 times the mass of the beef Achilles tendon, soak for 10 minutes, wash thoroughly until there is no odor, and crush for later use.

[0046] (2) Enzymatic hydrolysis: The pretreated bovine Achilles tendon is placed in an enzymatic hydrolysis solution (in the enzymatic hydrolysis solution, glacial acetic acid 0.5 mol / L, pepsin 700 mg / L), the mixing mass ratio of bovine Achilles tendon to the enzymatic hydrolysis solution is 1:30, the enzymatic hydrolysis time is controlled at 40 h, and the enzymatic hydrolysis temperature is controlled at 4-25° C. to obtain a collagen solution.

[0047] (3) Salting out / acid extraction: Add 9 times the mass of bovine Achilles tendon sodium chloride solution (35wt%) to the collagen solution after enzymatic hydrolysis and soak for 14h to obtain white flocs precipitated. Centrifuge at 6000r / min for 10min, discard the supernatant, and then put it into a phosphate buffer solution with a pH value of 2.5 of an equal volume to the discarded supernatant, stir evenly, and obtain an acid-soluble collagen solution.

[0048] (4) Purification / filtration: The acid-soluble collagen stock solution after salting out was ultrafiltered 7 times through a 150 kDa ultrafiltration membrane, and the supplementing liquid during ultrafiltration was a phosphate buffer solution with a pH of 2.5; then, a mixed acid solution (a phosphate buffer solution with a pH of 2.5, glycerol and 1-ethyl-3-methylimidazolium acetate ionic liquid in a volume ratio of 100:0.5:5) was prepared as a supplementing liquid and the ultrafiltration was repeated 3 times to obtain a purified collagen solution, which was diluted to 2.0 mg / g using a mixed acid solution (a phosphate buffer solution with a pH of 2.5, glycerol and ionic liquid in a volume ratio of 100:0.5:5), and filtered twice through a 0.45 μm filter element to obtain a low-viscosity collagen solution.

[0049] The prepared low-viscosity collagen solution was tested for appearance, identification, collagen purity, collagen content, hydroxyproline content, microbial limit, viscosity, 0.2 μm filter element filtration and other items. The specific data are shown in the table below:

[0050]

[0051]

[0052] Example 4

[0053] A method for preparing a low-viscosity collagen solution comprises the following steps:

[0054] (1) Pretreatment: Take fresh beef Achilles tendon, wash and cut into pieces, add ether solution (80%wt) 7 times the mass of the beef Achilles tendon into the small pieces of beef Achilles tendon, soak for 2 hours, and wash thoroughly until there is no odor. Then add sodium hydroxide solution (3wt%) 7 times the mass of the beef Achilles tendon, soak for 20 minutes, wash thoroughly until there is no odor, and crush for later use.

[0055] (2) Enzymatic hydrolysis: The pretreated bovine Achilles tendon is placed in an enzymatic hydrolysis solution (in the enzymatic hydrolysis solution, glacial acetic acid 0.6 mol / L, pepsin 600 mg / L), the mixing mass ratio of bovine Achilles tendon to the enzymatic hydrolysis solution is 1:35, the enzymatic hydrolysis time is controlled at 45 h, and the enzymatic hydrolysis temperature is controlled at 4-25° C. to obtain a collagen solution.

[0056] (3) Salting out / acid extraction: Add 10 times the mass of bovine Achilles tendon sodium chloride solution (25 wt%) to the collagen solution after enzymatic hydrolysis and soak for 16 h to obtain white flocs precipitated. Centrifuge at 5500 r / min for 15 min, discard the supernatant, and then put it into a phosphate buffer solution of pH = 4.0 with an equal volume to the discarded supernatant, stir evenly, and obtain an acid-soluble collagen solution.

[0057] (4) Purification / filtration: The acid-soluble collagen stock solution after salting out was ultrafiltered 7 times through a 150 kDa ultrafiltration membrane. The replenishing liquid during ultrafiltration was a phosphate buffer solution with a pH of 4.0. Then, a mixed acid solution (a phosphate buffer solution with a pH of 4.0, glycerol and 1-ethyl-3-methylimidazolium acetate ionic liquid in a volume ratio of 100:0.3:10) was prepared as a replenishing liquid and the ultrafiltration was repeated twice to obtain a purified collagen solution. The solution was diluted to 1.5 mg / g using a mixed acid solution (a phosphate buffer solution with a pH of 4.0, glycerol and ionic liquid in a volume ratio of 100:0.3:10), and filtered twice through a 0.45 μm filter element to obtain a low-viscosity collagen solution.

[0058] The prepared low-viscosity collagen solution was tested for appearance, identification, collagen purity, collagen content, hydroxyproline content, microbial limit, viscosity, 0.2 μm filter element filtration and other items. The specific data are shown in the table below:

[0059]

[0060] The above is only a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and changes directly derived or associated by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included in the protection scope of the present invention.

Claims

1. A method for preparing a low-viscosity collagen solution, characterized in that: The preparation comprises the following steps: (1) Pretreatment: Take fresh beef Achilles tendon, wash it, cut it into pieces, soak it in ether alcohol solution and sodium hydroxide solution in sequence, wash it thoroughly until there is no odor, and grind it for later use; (2) enzymolysis: putting the pretreated bovine Achilles tendon into an enzymolysis solution for enzymolysis to obtain an enzymolysis solution; (3) Salting out / acid extraction: Add sodium chloride solution to the enzymatic hydrolyzate for salting out, discard the supernatant to obtain the precipitated collagen, and then place the collagen in an acid solution with an equal volume to the discarded supernatant to obtain an acid-soluble collagen stock solution; (4) Purification / filtration: The acid-soluble collagen stock solution after salting out is ultrafiltered through a 100 kDa to 150 kDa ultrafiltration membrane to obtain a purified collagen solution, which is then diluted and filtered through a 0.45 μm filter element to obtain the low-viscosity collagen solution.

2. The preparation method according to claim 1, characterized in that: In step (1), the ether alcohol solution is selected from any one of an ethanol solution and an ether solution, and the mass percentage concentration is 70% to 90%; the amount of the ether alcohol solution is 5 to 7 times the mass of the bovine Achilles tendon, and the immersion time in the ether alcohol solution is 1 to 2 hours.

3. The preparation method according to claim 1, characterized in that: In step (1), the mass percentage concentration of the sodium hydroxide solution is 3% to 6%; the amount of the sodium hydroxide solution used is 5 to 7 times the mass of the bovine Achilles tendon, and the immersion time in the sodium hydroxide solution is 10 to 20 minutes.

4. The preparation method according to claim 1, characterized in that: In step (2), the enzymatic hydrolysis solution is prepared by mixing glacial acetic acid and pepsin; the concentration of glacial acetic acid in the enzymatic hydrolysis solution is 0.3-0.6 mol / L, and the concentration of pepsin is 500-700 mg / L; the amount of the enzymatic hydrolysis solution is 25-35 times the mass of the bovine Achilles tendon; the temperature of the enzymatic hydrolysis is 4-25° C., and the time is 40-50 hours.

5. The preparation method according to claim 1, characterized in that: In step (3), the mass percentage concentration of the sodium chloride solution is 25% to 35%; the amount of the sodium chloride solution used is 8 to 10 times the mass of the bovine Achilles tendon; and the salting-out time is 12 to 16 hours.

6. The preparation method according to claim 1, characterized in that: In step (3), the acid solution is glacial acetic acid solution or phosphate buffer solution; the concentration of the glacial acetic acid solution is 0.3-0.6 mol / L; and the pH value of the phosphate buffer solution is 2.5-4.

0.

7. The preparation method according to claim 1, characterized in that: In step (4), the ultrafiltration is performed 8 to 10 times in total; wherein the replenishing liquid for the 1st to 7th ultrafiltrations is acid solution, and the replenishing liquid for the 8th to 10th ultrafiltrations is a mixture of acid solution, glycerol and 1-ethyl-3-methylimidazolium acetate ionic liquid.

8. The preparation method according to claim 7, characterized in that: The acid solution is a glacial acetic acid solution or a phosphate buffer solution; the concentration of the glacial acetic acid solution is 0.3-0.6 mol / L; the pH value of the phosphate buffer solution is 2.5-4.0; in the 8th to 10th replenishing solution of the ultrafiltration, the volume ratio of the acid solution, glycerol and 1-ethyl-3-methylimidazolium acetate ion liquid is 100:0.3-1.2:5-15.

9. The preparation method according to claim 1, characterized in that: In step (4), the protein content of the diluted collagen solution is controlled at 1.5-3.5 mg / g; and the 0.45 μm filter element is filtered 2-3 times.

10. A low-viscosity collagen solution prepared by the preparation method according to any one of claims 1 to 9.

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