CN1333082C - Method for high efficiency extracting collagen by alkali swelling acid enzymolysis - Google Patents

Abstract

The present invention relates to a high efficiency collagen extracting method by alkali swelling and acid enzymolysis, which uses animal tendons or human tendons obtained after surgical amputations or animal skin tissues as raw materials. The present invention comprises processes of pretreatment, separation, refining, drying and sterilization. The present invention is characterized in that after pretreatment and before separation and refining, alkali swelling is carried out in 0.1M to 2M NaOH solution, and acid enzymolysis is carried out in 0.03 M to 0.8 M acetic acid solution which comprises pepsin of 0.1 to 0.5 wt%. The method of the present invention has the advantages that the solubility of collagen protein is effectively increased, extracted collagen is collagen which is not denatured, multimers are in a small quantity, and the raw materials are used at a high degree, and the method of the present invention also has the characteristics of quickness, high efficiency and high yield; thereby, an ideal method is offered to the manufacture of casings and collagen gels. Extracted collagen protein can be made into casts, sheets, sponge webs, etc. and can be combined with other artificial or natural materials to form gels or sponge or film which is used as support materials of tissue engineering.

Description

A method for efficiently extracting collagen by alkali swelling-acid enzymatic hydrolysis

1. Technical field

The present invention belongs to the polymer product derived from protein, in particular to a polymer product derived from animal horn, hoof, tendon, skin or leather.

2. Background technology

Collagen is the most widespread protein in animals, and type I collagen is mainly found in skin, tendons and ligaments. In the past 30 years, with the continuous development of tissue engineering technology, collagen has become a very important scaffold material, which is widely used in medical absorbable sutures, biological dressings and tissue engineering products for the treatment of burns, cosmetic surgery, Corneal disease and hard tissue repair, etc. In addition, because collagen has good moisturizing properties and promotes the growth and reproduction of epidermal cells, it can effectively enhance skin elasticity and reduce wrinkles, so it is also used in high-end cosmetics.

At present, there are two main methods for extracting collagen. One is to destroy the hydrogen bonds and chemical bonds existing in collagen molecules and between molecules, so as to obtain collagen with a larger molecular weight. This method is relatively simple and the amount of collagen obtained is large; the other is to completely destroy Collagen structure, reconstitute it into the intended product, this method is complex, expensive and low yield. According to the acidity and alkalinity of the solvent, it can be divided into three methods, namely acidic method, neutral method and alkaline method. The neutral and acidic methods extract soluble collagen. The neutral salt solvent generally uses 0.15M, 0.45M and 1M NaCl solutions; When extracting collagen fibers from adult animal tissues by acid dissolution, only type I collagen can be extracted. The cross-linked collagen is difficult to dissolve and not easy to extract. The acidic solvent is usually 0.05 of pH 3.5. M～0.5M acetic acid (Hac) or 0.15M citrate buffer.

When constructing tissues in the process of body development, collagen is insoluble due to its specific affinity with proteoglycans and glycoproteins. In addition, with age, the collagen molecules and other components will also These factors have caused certain difficulties in the extraction of collagen. In order to increase the solubility of insoluble collagen as much as possible, alternate extraction method or enzymatic degradation method is generally used. Alternative extraction refers to alternate extraction under acidic and neutral conditions to make more collagen soluble; enzymatic degradation method usually uses pepsin and 537 acid protease to degrade substrates such as skin and tendon under acidic conditions , or use wood paw protease to degrade substrates such as skin and tendon under neutral conditions. In the prior art, there is an invention titled "Preparation Method of Cell Matrix Protein Biological Scaffold Material for Tissue Engineering", and the Chinese invention patent application with the publication number CN1493366A was published on May 5, 2004. It uses acid The disadvantage is that dialysis is required, the operation steps are numerous, the collagen is lost and denatured during the extraction process, and the insoluble collagen is difficult to effectively extract; there is another invention titled "Water-soluble Collagen The preparation method of undenatured natural collagen", the Chinese invention patent application with the publication number CN1597742A was published on March 23, 2005. What it used was the method of enzymatic degradation and neutral salting out and acylation, and its defect was that it increased the operation Steps, the process is more complicated, still can not completely extract the insoluble collagen.

3. Contents of the invention

The object of the present invention is to aim at the defects in the above-mentioned prior art, to provide a method for efficiently extracting collagen by alkali swelling-acid enzymatic hydrolysis, so as to improve the utilization rate of tendon and skin tissue, and fully dissolve the collagen existing in tendon or skin tissue. Collagen, destroy the molecular structure of collagen as little as possible, improve the solubility of collagen, so that it can be used to produce scaffold materials for tissue engineering skin.

Adopt following technical scheme to realize the object of the present invention.

A method for efficiently extracting collagen by alkali swelling-acid enzymatic hydrolysis, which uses animal tendon or tendon after human surgical amputation, or animal skin tissue as raw materials, and consists of pretreatment, alkali swelling, acid enzymatic hydrolysis, separation and purification, and drying and sterilization The five-step process consists of:

1) Pretreatment

Remove fat tissue from tendon or skin tissue, then degrease and remove hair with chloroform/ethanol solution with a weight ratio of 2:1, rinse with distilled water, cut into thin slices, strips or small pieces, and finally centrifuge three times repeatedly with distilled water.

2) Alkali swelling

Immerse the pretreated raw materials in 0.1M-2M NaOH solution for at least 10 days at a weight ratio of 1:5-40 until the pH value reaches 12.2 to make the substrate crystal clear, then fully wash with water, and then rinse with distilled water Soak for at least 1 day, use a homogenizer to grind the swollen substrate until it becomes jelly in an ice bath, add 10 times of water, and homogenize repeatedly to obtain a colloidal solution.

3) Acid enzymatic hydrolysis

Use 1N HCl to neutralize the colloidal solution until the pH value is neutral, put the solution into a centrifuge and centrifuge at a low temperature and high speed for 1 hour at 0-4°C, take the precipitate, and then add it in a ratio of 1:20-100 by weight A 0.03M-0.8M acetic acid solution containing 0.1-0.5wt% pepsin was shaken and digested in a refrigerator at 4°C for at least 2 days until the liquid became viscous to obtain a crude product of enzymatic degradation.

Four) separation and refining

Use ultrasonic waves to crush the coarse collagen fiber particles in the enzyme-degraded crude product, and then centrifuge at a low temperature and high speed for 1 hour at 0-4°C to remove incompletely dissolved collagen and impurities. The supernatant after sedimentation is a crude collagen solution; Gradually add NaCl to the collagen solution to a concentration of 2M to precipitate the collagen, centrifuge it several times with distilled water, then dissolve it in 0.03M-0.8M acetic acid to obtain refined liquid collagen.

5) Drying and sterilization

The liquid collagen is freeze-dried, and then sterilized to obtain a milky white powdery collagen product, which is stored in a refrigerator at 4°C.

In the above method, alkali swelling is combined with grinding, and after acid enzymolysis, ultrasonic waves are used to crush the collagen fiber coarse particles in the enzymatic degradation crude product, which can effectively improve the solubility of collagen. The extracted collagen is undenatured collagen, fiber molecules The length is relatively low and there are few multimers, which greatly improves the utilization of raw materials. The extracted collagen still has fiber re-forming properties and good biocompatibility, and the extracted crude collagen solution can be directly used as a scaffold material for tissue engineering; meanwhile, the preparation method of the present invention does not contain toxic substances and will not affect The growth and proliferation of cells can be mixed with fibroblasts, endothelial cells, etc. for three-dimensional culture of cells. The method of the present invention extracts collagen, which has the characteristics of rapid efficiency and high yield, and provides a very ideal method for making sausage casings and collagen gels. The extracted collagen can also be made into tubes, sheets, sponge nets, etc., and other Artificial or natural materials are combined to form gels, sponges or membranes, which are used as scaffold materials for tissue engineering such as skin, blood vessels, and nerves.

Compared with the prior art, the method for efficiently extracting collagen by alkali swelling-acid enzymatic hydrolysis of the present invention has the following advantages: the first is that the extracted collagen solution has a high concentration, which is better than the simple acid enzyme dissolution method; This method can be used to extract collagen from the tendon and skin of young animals; the third is that there is less insoluble collagen, the extraction rate of collagen is high, and the utilization rate of animal tissue is high, so there are few residues of collagen tissue discarded; the fourth is that the collagen acid solution is neutralized and coagulated in the physiological pH range.

4. Specific implementation

An example is used below to further illustrate the method for efficiently extracting collagen by alkali swelling-acid enzymatic hydrolysis of the present invention, but it is by no means limited to the example given.

Wash the bovine tendon with water, remove fat and fascia tissue, and cut into 10×3mm long strips. Soaked in 0.9M NaOH at room temperature for 15 days, the tendon swelled to crystal clear, washed thoroughly in running water, soaked in distilled water for 2 days, and then neutralized to neutral with 1N HCl. Cut the tendon tissue with scissors until it becomes a paste, homogenize it with a homogenizer, add water 10 times, homogenize it again and again, adjust the pH to neutral with 1N HCl, and centrifuge at 4°C and 10,000 rpm for 1 hour , discard the supernatant, take the precipitate, and then add it to the 0.8M acetic acid solution containing pepsin 0.1-0.5wt% according to the ratio of weight ratio = 1:50, shake and digest in the refrigerator at 4°C for 3 days until the liquid is viscous , and then centrifuged at 4°C and 10,000 rpm for 1 hour. Remove the undissolved collagen and impurities, and the supernatant is the crude collagen solution; add analytically pure NaCl to the crude collagen solution to make the NaCl concentration of the solution reach 2M, precipitate the collagen, centrifuge three times with distilled water, and finally dissolve the collagen Dissolved in 0.5M acetic acid solution to obtain refined liquid collagen, then freeze-dried, sterilized and sterilized to obtain a milky white powdered collagen product, which was stored in a refrigerator at 4°C.

The tendon collagen extracted by the above method was analyzed by a Nissan 835-50 amino acid automatic analyzer, and the amino acid content results were as follows:

amino acid Content (mg/ml) amino acid Content (mg/ml) aspartic acid ASP 4.70 Valine VAL 0.76 Threonine THR 0.84 ILE 0.49 Serine SER 4.76 Leucine IEU 2.48 Glutamate GLU 11.43 Tyrosine TYR 3.11 Proline PRO 10.20 Phenylalanine PHE 2.14 GlycineGLY 33.78 HistidineHIS 0.98 Alanine ALA 5.28 Lysine LYS 2.88 Arginine AGR 5.92 Hydroxyproline HYPRO 6.98

Claims (3)

1, the method of a kind of alkali swelling-sour enzymolysis high efficiency extraction collagen, its tendon after with animal tendon or the amputation of people's surgery, perhaps animal skin tissue is starting material, comprise pre-treatment, the acid enzymolysis, separation and purification and dry sterilization process, it is characterized in that: also have the alkali swelling process after pre-treatment and before the sour enzymolysis, wherein the alkali swelling is to be immersed in the NaOH solution of 0.1M～2M at least 10 days through the ratio of pretreated raw material by weight=1: 5～40, reach 12.2 to the pH value, make substrate be the fully washing of sparkling and crystal-clear bright back, again with distilled water immersion at least 1 day, under condition of ice bath, be ground to the substrate after the swelling gelatin with homogenizer, 10 times of adding distil waters, homogenate once more obtains colloidal solution; Acid enzymolysis be with 1N HCl with colloidal solution be neutralized to the pH value be neutrality, solution is dropped in the whizzer centrifugal 1 hour of 0～4 ℃ of low-temperature and high-speed, taking precipitate, include 0.03M～0.8M acetum of stomach en-0.1～0.5wt% again by weight=1: 20～100 ratio adding, vibration digestion at least 2 days is thick to liquid in 4 ℃ of refrigerators, obtains the enzyme liberating crude product.

2, according to the method for the described alkali swelling of claim 1-sour enzymolysis high efficiency extraction collagen, it is characterized in that: said separation and purification is with ultrasonic wave the collegen filament coarse particles in the enzyme liberating crude product to be pulverized, then centrifugal 1 hour of 0～4 ℃ of low-temperature and high-speed, remove inabundant dissolved collagen and impurity, the supernatant after the sedimentation is rough collagen solution; In rough collagen solution, add NaCl to 2M concentration gradually, be settled out collagen, behind centrifugal repeatedly several times of distilled water, be dissolved in again in 0.03M～0.8M acetic acid, obtain the liquid collagen of purified.

3, according to the method for the described alkali swelling of claim 1-sour enzymolysis high efficiency extraction collagen, it is characterized in that: said pre-treatment is that tendon or skin histology are removed fatty tissue, use the degreasing of chloroform/ethanol solution, the unhairing of weight ratio=2: 1 again, thinly slice after clean with distilled water flushing, little or fritter, centrifugal repeatedly three times at last with distilled water.