CN101307093B - Method for separating and purifying collagen for biopharmaceutical use from sharkskin - Google Patents

Abstract

A method for separating and purifying collagen for biomedical use from shark skin. The operation is as follows: take the shark skin and soak it in disodium hydrogen phosphate buffer solution with a concentration of 10mM and a pH value of 7.4-7.6 to remove scales and residual meat, cut it into small pieces, soak and rinse it with deionized water for 3 to 5 times; Clean fish skin is soaked in disodium hydrogen phosphate buffer solution with a concentration five times the amount of the raw material of 100mM and a pH value of 7.4-7.6 for 70-75 hours, homogenized for 15 minutes, and centrifuged at a temperature of 4°C and a speed of 10,000 rpm for 15 minutes. Take the supernatant; precipitate and repeat the previous step, combine the supernatant, add ammonium sulfate to a saturation of 80%, centrifuge at a temperature of 4°C and a speed of 8000rpm for 15min, and retain the precipitate; dissolve the precipitate in a concentration of 200mM, pH 7.0 in phosphate buffer solution; use G-75, G-100, G-150 dextran to chromatograph the above solution, collect the protein solution at a temperature of 4°C, rotate at 10,000 rpm, centrifuge for 15 minutes, and concentrate the supernatant. Freeze-dried collagen for biomedical use. The invention is used for the method of separating and purifying collagen for biomedicine from shark skin.

Description

Method for separating and purifying collagen for biomedicine from shark skin

Technical field:

The invention relates to a method for separating and purifying collagen for biomedicine, in particular to a method for separating and purifying collagen for biomedicine from shark skin.

Background technique:

Collagen or collagen is a biological polymer synthesized by animal cells. It is widely found in animal bones, feet, cartilage, skin and other connective tissues. It accounts for about 1/3 of the total protein of mammals. It is an important Collagen, a component of the extracellular matrix, has good biological properties and can be used as a support for tissues. It has a major impact on the normal function of cells, tissues and even organs, and has a significant impact on trauma repair. The utilization of collagen at home and abroad is mainly divided into two categories: one is to focus on the unique macroscopic physical properties of collagen, that is, fiber properties, which are used in photographic paper negatives, textiles, papermaking, etc.; the other is to focus on its intrinsic properties, namely The biological functions of collagen are used in food health care, feed, beauty cosmetics, medical materials, etc. Collagen has important biological properties, high mechanical properties, promotion of cell growth, hemostasis, biocompatibility and biodegradability, and can be used as skin The repair, treatment and care of burns and scalds can reduce the degree of burns and scalds and prevent infection. It plays a very important role in the treatment of burns and scalds. It can be processed into "artificial skin" and directly applied to burns and scalds On the surface of the skin, especially for patients with large areas and depths above grade III, human-like collagen can be used as artificial blood vessels, practical blood vessel grafting and repair of damaged human tissues, such as gastric bleeding, perforation, collagen Protein can be combined with antibiotic cords, cell growth-promoting factors, etc. to repair damaged tissues. Collagen can be combined with bone meal and bone protein for limb extension. Collagen can be used to make artificial organs, cardiac pacemakers, and filling of human internal organs. Collagen is also a new type of biological fermentation medium, which contains a complete range of amino acids and has a unique effect on microbial culture. It is one of the best raw materials for bacteriological culture and biological fermentation medium. In addition, the material made of collagen can diffuse into the deep layer of the skin and has a good nutritional effect on human skin. At the same time, it has a very good moisturizing effect due to the large number of hydroxyl groups in its molecules. According to the survey, collagen was the most popular among the best-selling skin care products in 2003. Collagen has been popular abroad for many years. Many cosmetic R&D centers in Europe and the United States have launched skin care products containing collagen ingredients. The current global collagen production value exceeds 4 billion US dollars. Regarding the extraction and purification of collagen, although the methods are different, most of them are extracted from the skins of small fish such as squid and cod and livestock. Small fish themselves contain less collagen and the raw materials are limited. The extracted protein has low solubility, and the use of cattle as raw materials is not easily accepted by many trade organizations in the world. The current steps of extracting collagen include extraction, separation and purification. Extraction is divided into acid method, alkali method, neutral salt method and Enzyme method, etc., the process is loaded down with trivial details, also useful genetic engineering method is prepared protein, but increased cost again at the same time, because these defects that above-mentioned these separation techniques exist, make the collagen production rate, yield, quality that make is not high, so that The high price of collagen for biomedical use has greatly affected the wide application of collagen. Therefore, reducing the cost of collagen purification and improving protein quality is the key.

Invention content:

The purpose of the present invention is to provide a method for separating and purifying collagen for biomedicine from shark skin, which not only can effectively extract high-quality collagen from shark skin, but also has simple process, low cost and high yield.

In order to achieve the above-mentioned purpose, the present invention is achieved in that the raw material used in the method for separating and purifying biomedical collagen from shark skin of the present invention is shark skin, and the method for separating and purifying biomedical collagen therefrom operates as follows:

(1) Soak the shark skin in a low-concentration phosphate buffer solution with a pH value of 7.4-7.6 to remove scales and residual meat, cut it into small pieces, and then soak and rinse it with deionized water for 3 to 5 times;

(2) After cleaning the fish skin and soaking it in a medium-concentration phosphate buffer solution with a pH value of 7.4-7.6 five times the amount of raw materials for 70-75 hours, homogenate for 15 minutes at a temperature of 4°C and a rotation speed of 10,000 rpm. Centrifuge for 15min, take the supernatant;

(3), repeat the process of (2) once again for reprecipitation, combine the supernatants, add ammonium sulfate to a saturation of 80%, at a temperature of 4 ° C, a rotation speed of 8000 rpm, centrifuge for 15 minutes, and retain the precipitate;

(4), the precipitate is dissolved in a medium-concentration phosphate buffer solution with a pH value of 7.0;

(5), use G-75, 100, 150 dextran to chromatograph the above solution, collect the protein solution at a temperature of 4°C, and a rotation speed of 10,000 rpm, centrifuge for 15 minutes, concentrate the supernatant, and freeze-dry to obtain a biomedical product. Collagen.

The low-concentration phosphate buffer is a disodium hydrogen phosphate buffer with a concentration of 8-12mM and a pH value of 7.4-7.6, and the medium-concentration phosphate buffer is a concentration of 80-120mM and a pH value of 7.4 - 7.6 disodium hydrogen phosphate buffer solution, the concentration of the medium-concentration phosphate buffer solution is 180-220mM, the pH value is 7.0, and its consumption is 1.5g/ml.

Because the present invention uses shark skin as a material, extracts and purifies collagen by methods such as alkali washing and dialysis, it has the following advantages:

1. Shark skin is rich in raw materials and low in price. It is generally used as leftovers and used as raw material for protein extraction, which greatly reduces the cost. It is reported that the current market price of shark skin and scalp is 6,000-8,000 yuan/ton, and the total annual output About 1000 tons;

2. The obtained collagen has been tested to be electrophoretic pure and of high quality;

3. The obtained shark skin collagen can be dissolved in neutral and acidic solutions, and has greater solubility than collagen from other sources, and can be used in more fields;

4. The denaturation temperature of the obtained shark skin collagen is significantly higher than that of other sources of protein, which has unique application value as a biomedical material;

5. The cost is low. When the process is enlarged, it is less limited by instruments and materials, and it only needs to increase the labor cost.

Detailed ways:

The method for separating and purifying collagen for biomedicine from shark skin of the present invention will be further described in detail below.

The material used in this embodiment is horse gum shark skin.

Specific steps are as follows:

1. Soak 30g of frozen horse gum shark skin in disodium hydrogen phosphate buffer solution with a concentration of 10mM and a pH value of 7.4-7.6 to remove scales and residual meat, cut into small pieces of 0.5cm×0.5cm, and then use Soak in ionized water and rinse 3 to 5 times;

2. Soak the cleaned fish skin in 250ml of disodium hydrogen phosphate buffer solution with a concentration of 100mM and a pH value of 7.4-7.6 for 72 hours, homogenate for 15 minutes, centrifuge at a temperature of 4°C and a speed of 10,000rpm for 15 minutes, and take Serum;

3. Repeat the process of 2 once for the precipitation, combine the supernatant, add ammonium sulfate to a saturation of 80%, centrifuge for 15 minutes at a speed of 8000rpm and a temperature of 4°C, and retain the precipitate;

4. Dissolve the precipitate in about 20ml of phosphate buffer solution with a concentration of 200mM and a pH value of 7.0;

5. Use G-75, 100, 150 dextran to chromatograph the above solution, collect the protein solution at a temperature of 4°C and a rotation speed of 10,000 rpm, centrifuge for 15 minutes, concentrate the supernatant, and obtain biomedical collagen after freeze-drying .

The above-mentioned separation and purification of horse gum sharkskin collagen showed three clear bands on the SDS-PAGE of 4-5% concentrated gel and 7.5-8% separating gel. The low molecular weight peptide chain content is relatively high, and the molecular masses are respectively 134kDa and 118kDa; high molecular weight peptide chain content is less, its molecular mass is about 205kDa. This high molecular weight peptide chain has a higher molecular weight than β-peptide chains obtained from terrestrial and other marine fishes.

The denaturation temperature of collagen isolated from horse gum shark skin is 40.31°C, which is consistent with the existence of β-sheets and random coils in the spatial structure of collagen. This denaturation temperature is significantly different from that of marine fish collagen determined by viscosity method The denaturation temperature of protein is estimated from the fact that the thermal denaturation temperature of Japanese perch collagen is 30°C, the denaturation temperature of tuna is 29.7°C, the thermal denaturation temperature of freshwater fish scale is 32.3°C, and the denaturation temperature of cowhide collagen is 38°C. The collagen isolated from shark scalp has unique spatial structure characteristics and amino acid composition characteristics, which has unique application value as biomedical materials.

Claims (1)

1. A method for separating and purifying biomedical collagen from shark skin, characterized in that choosing raw material is shark skin, and the preparation method is as follows: (1) Soak the shark skin in disodium hydrogen phosphate buffer solution with a concentration of 10mM and a pH value of 7.4-7.6 to remove the scales and residual meat, cut it into small pieces, then soak and rinse it with deionized water for 3 to 5 times ; (2), the cleaned fish skin is soaked in disodium hydrogen phosphate buffer solution with a concentration five times the amount of raw materials of 100mM and a pH value of 7.4-7.6 for 70-75 hours, homogenized for 15 minutes, and at a temperature of 4°C. Rotate at 10,000 rpm, centrifuge for 15 minutes, and take the supernatant; (3), repeat the process of (2) once for reprecipitation, combine the supernatant, add ammonium sulfate to a saturation of 80%, at a temperature of 4°C, a rotation speed of 8000rpm, centrifuge for 15min, and retain the precipitate; (4), the precipitate is dissolved in a concentration of 200mM, a pH value of 7.0 in phosphate buffer; (5) Use G-75, G-100, G-150 dextran to chromatograph the above dissolved solution, collect the protein dissolved solution at a temperature of 4°C, rotate at 10,000 rpm, centrifuge for 15 minutes, concentrate the supernatant, and freeze-dry Get collagen for biomedical use.