CN1821421A

CN1821421A - Method for high efficiency extracting collagen by alkali swelling acid enzymolysis

Info

Publication number: CN1821421A
Application number: CN 200510057437
Authority: CN
Inventors: 伍津津
Original assignee: Third Affiliated Hospital of TMMU
Current assignee: Weili biological technology (Chengdu) Co., Ltd.
Priority date: 2005-12-13
Filing date: 2005-12-13
Publication date: 2006-08-23
Anticipated expiration: 2025-12-13
Also published as: CN1333082C

Abstract

The alkali swelling and acid enzymolysis process of extracting collagen effectively adopts animal tendon or animal skin tissue as material and includes the steps of pre-treatment, separating and refining, and drying and sterilization. The process features the alkali swelling step in 0.1~2 M NaOH solution and the acid enzymolysis step in 0.03~0.8 M acetic acid solution containing pepsase of 0.1~0.5 concentration after the pre-treatment and before the separating and refining. The process has raised collagen solubility, high efficiency and high yield, and the extracted collagen is un-denatured collagen, and has less multimer and high material utilizing rate. The product is used as rack material for tissue engineering.

Description

The method of a kind of alkali swelling-sour enzymolysis high efficiency extraction collagen

One, technical field

The invention belongs to the macromolecule product of what by protein derived, particularly a kind of by animal horn, hoof, tendon, skin or leather deutero-macromolecule product.

Two, background technology

Collagen is to be present in the animal body protein the most widely, and type i collagen mainly is present in skin, tendon and the ligament.In the last thirty years, continuous development along with tissue engineering technique, collagen protein becomes very important timbering material, is used widely in medical absorbable suture, biological dressing and tissue engineering product, is used for the treatment of burn, beauty and shaping, keratopathy and hard tissue repair etc.; In addition,, can strengthen skin elasticity effectively, reduce wrinkle, so also be used for the cosmetics of super quality because collagen has the good moisture preserving performance and promotes the effect that epidermal growth is bred.

Extract collagen at present and mainly contain two class methods, the one, to destroy in the tropocollagen molecule and the hydrogen bond and the chemical bond of intermolecular existence, thereby obtain the bigger collagen protein of molecular weight, this method is simpler relatively, and it is big to obtain the collagen protein amount; The 2nd, the completely destroy collagen structure is reformulated predetermined prod with it, this method complexity, expense height, yields poorly.Can be divided into three kinds of methods by the solvent acid-basicity, i.e. acid process, neutral method and basic process.What neutrality and acid process were extracted is soluble collagen, and the neutral salt solvent generally adopts the NaCl solution of 0.15M, 0.45M and 1M; Acid-soluble method can will not have crosslinked tropocollagen molecule dissolving, also solubilized contains the crosslinked collegen filament of aldehyde amines, when the collagen in the animal tissues that grows up with the acid-soluble method extraction, can only extract type i collagen, crosslinked stronger collagen then is difficult to dissolving and is difficult for extracting, and acid solvent adopts the acetic acid (Hac) of 0.05M～0.5M of pH3.5 or the citrate buffer of 0.15M more.

In the body generating process during constructing tissue, collagen since with the pathoklisis of protein-polysaccharide and glycoprotein, be insoluble and cause it, in addition, with advancing age, between tropocollagen molecule and and other composition between also can form bridge formation, these factors have all caused certain difficulty to the extraction of collagen.In order to increase the solubility of insoluble collagen as far as possible, generally adopt mutual extraction process or enzyme liberating method.Mutual extracting is promptly extracted alternately at acid and neutrallty condition, makes more collagen become solubility; The enzyme liberating method is carried out enzyme liberating with stomach en-, 537 aspartic proteases to substrates such as skin, tendons and is handled, or with wooden pawl proteolytic enzyme substrates such as skin, tendon are carried out enzyme liberating and handle under neutrallty condition normally under acidic conditions.In the prior art, there is a denomination of invention to be " preparation method who is used for the cell matrix protein biology timbering material of organizational project ", publication number is that the Chinese invention patent of CN1493366A applies on May 5th, 2004 openly, it adopts molten the saltouing with neutrality of acid to carry out repeatedly extractive mutual extraction process exactly, its defective is to need dialysis, operation steps is various, and collagen is lost and sex change in the leaching process, and insoluble collagen is difficult to effectively extract; Also having a denomination of invention is " preparation method of water-soluble not sex change natural collagen "; publication number is that the Chinese invention patent of CN1597742A applies on March 23rd, 2005 openly; what it adopted is the method for enzyme liberating and the neutral acidylate of saltouing again; its defective is to have increased operation steps; technology is complicated more, still insoluble collagen can not be extracted fully.

Three, summary of the invention

The objective of the invention is to defective at above-mentioned prior art existence, the method of a kind of alkali swelling-sour enzymolysis high efficiency extraction collagen is provided, so that can improve the utilization ratio of tendon, skin histology, fully dissolving is present in the collagen protein in tendon or the skin histology, the least possible its ground destroys the molecular structure in the collagen protein, improve the solubility of collagen, enable to be used for the timbering material of organization of production engineering skin.

Realize purpose of the present invention by the following technical solutions.

The method of a kind of alkali swelling-sour enzymolysis high efficiency extraction collagen, its tendon or animal skin tissue after with animal tendon or the amputation of people's surgery is starting material, is made up of pre-treatment, alkali swelling, sour enzymolysis, separation and purification and five step of dry sterilization process:

One) pre-treatment

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.

Two) alkali swelling

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, add 10 times in water, homogenate repeatedly once more obtains colloidal solution.

Three) sour enzymolysis

It is neutral with 1N HCl colloidal solution being neutralized to the pH value, 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.

Four) separation and purification

With ultrasonic wave the collegen filament coarse particles in the enzyme liberating crude product is pulverized, centrifugal 1 hour of 0～4 ℃ of low-temperature and high-speed, removed fully dissolved collagen and impurity then, 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.

Five) dry sterilization

With liquid collagen lyophilize, again through sterilization, obtain off-white powder shape collagen protein product, place 4 ℃ of refrigerators to preserve.

In aforesaid method, adopt the alkali swelling in conjunction with grinding, and after sour enzymolysis, adopt ultrasonic wave that the collegen filament coarse particles in the enzyme liberating crude product is pulverized, can effectively improve the solubility of collagen protein, the collagen that is extracted is denatured collagen not, fiber molecule length is relatively low, polymer is few, has improved the availability of raw material greatly.The collagen protein that is extracted still has the formation characteristic again and the excellent biological compatibility of fiber, and the rough collagen solution of extraction can be directly used in the timbering material of organizational project; Preparation method of the present invention does not contain Toxic matter simultaneously, can not influence the growing multiplication of cell, can mix with inoblast, endotheliocyte etc., is used for the dimensional culture of cell.The inventive method is extracted collagen protein, the characteristics that have rapidly and efficiently, output are high, for making casing, collagen gel provide very ideal method, the collagen protein that extracts also can be made into cast, sheet material, sponge net etc., artificial or natural materials is combined to form gel, sponge or film with other, is used for the timbering material of organizational projects such as skin, blood vessel, nerve.

The method of alkali swelling of the present invention-sour enzymolysis high efficiency extraction collagen compared with prior art has following advantages: the one, and the collagen solution concentration height that is extracted, more effective than simple sour enzyme dissolution method; The 2nd, no matter the tendon of adult animals or growing animal, skin extracts collagen and all can adopt this method; The 3rd, insoluble collagen is few, collagen extraction yield height, animal tissues's utilization ratio height, thereby the collagenous tissue residue that abandons is few, the 4th, when the neutralization of collagenic acid solution is solidified in the physiological pH scope.

Four, embodiment

Further specify the method for alkali swelling of the present invention-sour enzymolysis high efficiency extraction collagen below with an embodiment, but it never be confined to for embodiment.

The ox tendon is fully washed, and removes fat, fascia tissue, is cut into little of 10 * 3mm length.Be immersed in room temperature among the 0.9M NaOH, 15 days, tendon was expanded to sparkling and crystal-clear bright, the flowing water thorough washing, and distilled water immersion 2 days is neutralized to neutrality with 1N HCl again.Shred tendon tissue to pasty state with scissors, use homogenizer homogenate, add 10 times in water, homogenate repeatedly once more transfers to neutrality with 1N HCl with pH again, under 4 ℃, 10000 rev/mins conditions centrifugal 1 hour, abandon supernatant, get precipitation ,=1: 50 ratio joins in the 0.8M acetum that includes stomach en-0.1～0.5wt% by weight again, vibration digestion 3 days is thick to liquid in 4 ℃ of refrigerators, under 4 ℃, 10000 rev/mins conditions centrifugal 1 hour again.Remove undissolved collagen and impurity, supernatant is rough collagen solution; In rough collagen solution, add analytical pure NaCl, make the NaCl concentration of solution reach 2M, be settled out collagen, with distilled water centrifugal repeatedly three times, at last with collagenolysis in the 0.5M acetum, be refining liquid collagen, lyophilize again, sterilization obtains off-white powder shape collagen protein product, places 4 ℃ of refrigerators to preserve.

The tendon collagen that extracts adopts daily output 835-50 type automatic analyzer for amino acids to analyze aminoacids content result such as following table as stated above:

Amino acid	Content (mg/ml)	Amino acid	Content (mg/ml)
Amino acid	Content (mg/ml)	Amino acid	Content (mg/ml)	Aspartic acid ASP	4.70	Xie Ansuan VAL	0.76
Threonine THR	0.84	Isoleucine ILE	0.49	Aspartic acid ASP	4.70	Xie Ansuan VAL	0.76
Threonine THR	0.84	Isoleucine ILE	0.49	Serine SER	4.76	Leucine IEU	2.48
L-glutamic acid GLU	11.43	Tyrosine TYR	3.11	Serine SER	4.76	Leucine IEU	2.48
L-glutamic acid GLU	11.43	Tyrosine TYR	3.11	Proline(Pro) PRO	10.20	Phenylalanine PHE	2.14
Glycine GLY	33.78	Histidine HIS	0.98	Proline(Pro) PRO	10.20	Phenylalanine PHE	2.14
Glycine GLY	33.78	Histidine HIS	0.98	L-Ala ALA	5.28	Methionin LYS	2.88
Arginine AGR	5.92	Oxyproline HYPRO	6.98	L-Ala ALA	5.28	Methionin LYS	2.88

Claims

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, separation and purification and dry sterilization process, it is characterized in that: also have alkali swelling and sour enzymolysis process after pre-treatment and before the separation and purification, 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.