CN112410392A - Extraction method and application of type I collagen - Google Patents
Extraction method and application of type I collagen Download PDFInfo
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- CN112410392A CN112410392A CN202011253634.XA CN202011253634A CN112410392A CN 112410392 A CN112410392 A CN 112410392A CN 202011253634 A CN202011253634 A CN 202011253634A CN 112410392 A CN112410392 A CN 112410392A
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
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Abstract
The invention belongs to the technical field of cartilage, and particularly relates to an extraction method and application of type I collagen, wherein the extraction method of the type I collagen comprises the following steps: degreasing and decalcifying animal bones, and performing protease enzymolysis to obtain an enzymolysis liquid; after extracting the enzymolysis liquid, adding a protein denaturation solution to obtain collagen precipitation; dissolving the collagen precipitate, and salting out the dissolved collagen to obtain a crude collagen product; and purifying the crude collagen product to obtain the type I collagen. Type I collagen is used in practice. The invention can extract type I collagen from animal hard bones, and greatly improves the comprehensive utilization value of edible bones. The extraction method of the type I collagen provided by the invention has the advantages of proper crosslinking degree, good biological activity, stable property and difficult degradation, and has the potential application of generating fibrocartilage by using a novel cartilage substitute.
Description
Technical Field
The invention belongs to the technical field of cartilage, and particularly relates to an extraction method and application of type I collagen.
Background
Type I Collagen (COL), heterotrimer composed of two alpha 1 chains and one alpha 2 chain, each peptide chain contains multiple (Gly-Xaa-Yaa) n repeated sequences, wherein Gly is glycine, and X and Y are proline and hydroxyproline respectively, which are helpful to form stable triple helix chain structure, and then become protocollagen and cut off amino terminal and carboxyl terminal to become type I collagen. Type I collagen is one of the most abundant various collagens contained in human body, is present in muscle, skin, artery wall, fibrocartilage, can be extracted from animal tissue, has excellent biological properties such as low antigenicity, blood coagulation, and the ability to regulate cell adhesion, proliferation, differentiation, etc., is the best substitute material in terms of biocompatibility, and is advantageous for cell ingrowth and soft tissue healing.
The human body has a large amount of cartilage, when the cartilage has problems, corresponding cartilage substitutes are often needed for corresponding treatment, and type I collagen which is the best substitute material in the aspect of biocompatibility is particularly important.
In order to solve the problems, the invention provides an extraction method of type I collagen and application thereof.
Disclosure of Invention
The invention aims to provide an extraction method of type I collagen, which can extract the type I collagen from animal hard bones and greatly improve the comprehensive utilization value of edible bones.
The invention also aims to provide the application of the method for extracting the type I collagen, which has the advantages of proper crosslinking degree, good biological activity, stable property, difficult degradation and potential application of a novel cartilage substitute in the generation of fibrocartilage.
To achieve these objects and other advantages in accordance with the purpose of the invention, there is provided a method for extracting type I collagen, comprising the steps of:
degreasing and decalcifying animal bones, and performing protease enzymolysis to obtain an enzymolysis liquid;
after extracting the enzymolysis liquid, adding a protein denaturation solution to obtain collagen precipitation;
dissolving the collagen precipitate, and salting out the dissolved collagen to obtain a crude collagen product;
and purifying the crude collagen product to obtain the type I collagen.
Preferably, the animal bone is degreased and decalcified, and then is subjected to protease enzymolysis to obtain an enzymolysis solution, wherein the enzymolysis solution comprises:
crushing animal bones, washing with water, and degreasing with 0.5-0.8mol/L sodium hydroxide or 70-75% ethanol solution;
decalcifying the degreased animal bone with hydrochloric acid with the concentration of 0.01-0.02 mol/L;
adding protease into the decalcified material, and performing enzymolysis to obtain an enzymolysis solution.
Preferably, the performing protease enzymolysis to obtain an enzymolysis solution comprises:
adding a first protease into the decalcified material, and performing enzymolysis at 50-55 deg.C and pH of 5-7 for 20-40min to obtain a first enzymolysis solution;
adding a second protease into the first enzymolysis liquid, and carrying out enzymolysis at 50-55 ℃ and pH 5-7 to obtain an enzymolysis liquid;
the mass fraction of the first protease is 0.2-0.5 per mill, the sum of the mass fractions of the first protease and the second protease is 3-5 per mill, and the sum of the two enzymolysis times is 5-7 hours.
Preferably, after the extracting the enzymatic hydrolysate, adding a protein denaturation solution to obtain a collagen precipitate comprises:
extracting an enzymolysis solution, slowly stirring and centrifuging the enzymolysis solution to obtain a supernatant;
and adding a sodium hydroxide solution into the supernatant, stirring, and adjusting the pH value to obtain the collagen precipitate without the protease.
Preferably, after dissolving the collagen precipitate, salting out the dissolved collagen to obtain a crude collagen product comprising:
adding a disodium hydrogen phosphate solution containing peroxyacetic acid into the collagen precipitate, standing for salting out, filtering and cleaning after the collagen precipitate is dissolved, thereby obtaining a crude collagen product.
Preferably, said purifying said crude collagen product to obtain type I collagen comprises:
adding acetic acid solution containing peroxyacetic acid into the collagen crude product to obtain 1g/L collagen solution;
performing ultrafiltration purification column chromatography separation on the collagen solution, and entering a double-effect falling film vacuum concentrator through a nanofiltration membrane concentration system to obtain unsterilized type I collagen;
irradiating the type I collagen by using cobalt 60-gamma rays to obtain the type I collagen.
Preferably, the animal bone is bovine achilles tendon or bovine bone.
The application of the type I collagen extracted by the extraction method in the production of cartilage substitutes and the production of fibrocartilage according to the cartilage substitutes.
Preferably, collagen molecules of the type I collagen are covalently cross-linked by lysine oxidase to form lysine-hydroxylysine, dense collagen fibers are formed, cartilage substitutes are generated, and fibrocartilage is generated according to the cartilage substitutes.
The invention has the advantages of
1. The extraction method of the type I collagen provided by the invention can extract the type I collagen from animal hard bones, so that the comprehensive utilization value of the edible bones is greatly improved;
2. the extraction method of the type I collagen provided by the invention has the advantages of simple preparation method, high production efficiency and outstanding economic benefit;
3. according to the extraction method of the type I collagen provided by the invention, the membrane method and the vacuum falling film gradient concentration are adopted in the process to hydrolyze the collagen, so that the energy-saving effect is obvious;
4. the extraction process of the type I collagen provided by the invention contains peracetic acid, so that the aseptic operation environment in the production process can be ensured, and meanwhile, the whole extraction process can be sterilized and aseptic by combining the irradiation of cobalt 60-gamma rays in the subsequent steps and the matching of the front step and the rear step.
5. The application of the type I collagen provided by the invention adopts the type I collagen, has proper crosslinking degree, good biological activity, stable property and difficult degradation, and has potential application of a novel cartilage substitute in generating fibrocartilage.
Drawings
FIG. 1 is a schematic flow chart of a method for extracting type I collagen according to an embodiment;
FIG. 2 is a schematic illustration of toluidine staining of type I collagen white plate chondrocytes, according to an embodiment;
FIG. 3 is a microscope image of type I collagen fibers provided in an embodiment.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It should be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other combinations.
The invention provides a method for extracting type I collagen, which comprises the following steps:
step 1, degreasing and decalcifying animal bones, and performing protease enzymolysis to obtain an enzymolysis solution;
specifically, animal bones are crushed and washed with water, and then degreased by using sodium hydroxide with the concentration of 0.5-0.8mol/L or ethanol solution with the volume fraction of 70-75%;
specifically, hydrochloric acid with the concentration of 0.01-0.02mol/L is used for decalcifying the degreased animal bones;
specifically, adding a first protease into the decalcified material, and carrying out enzymolysis for 20-40min at 50-55 ℃ and pH 5-7 to obtain a first enzymolysis liquid;
specifically, adding a second protease into the first enzymolysis liquid, and carrying out enzymolysis at 50-55 ℃ and pH 5-7 to obtain an enzymolysis liquid;
wherein the mass fraction of the first protease is 0.2-0.5 per mill, the total mass fraction of the first protease and the second protease is 3-5 per mill, and the total time of the two times of enzymolysis is 5-7 h.
Step 2, adding a protein denaturation solution after extracting the enzymolysis liquid to obtain collagen precipitation;
specifically, extracting an enzymolysis solution, slowly stirring and centrifuging the enzymolysis solution, and collecting to obtain a supernatant;
specifically, a sodium hydroxide solution with a certain concentration is added into the supernatant, and after slowly stirring and adjusting the pH value, the collagen precipitate with the protease removed is obtained.
Step 3, dissolving the collagen precipitate, and salting out the dissolved collagen to obtain a crude collagen product;
specifically, collagen is dissolved in disodium hydrogen phosphate (0.02mol/L) containing low-concentration peroxyacetic acid, and the collagen is salted out, kept stand for a period of time, filtered, and quickly washed with distilled water for several times to obtain a crude collagen product.
And 4, purifying the crude collagen product to obtain the type I collagen.
Specifically, the crude collagen product is dissolved in 0.5mol/L acetic acid (containing peroxyacetic acid) to obtain 1g/L collagen solution;
performing ultrafiltration purification column chromatography separation on the collagen solution (purified water is continuously added until the pH of the purified collagen solution is close to 5, and high-purity collagen is obtained), then entering a nanofiltration membrane concentration system, and then entering a double-effect falling film vacuum concentrator to obtain unsterilized type I collagen;
irradiating the type I collagen by using cobalt 60-gamma rays to obtain the type I collagen.
Wherein, the animal bone is bovine achilles tendon or bovine bone.
The application of the type I collagen extracted by the extraction method in the production of cartilage substitutes and the production of fibrocartilage according to the cartilage substitutes.
Specifically, lysine-hydroxylysine covalent cross-linking is formed among collagen molecules of the type I collagen through lysine oxidase to form compact collagen fibers, so that a cartilage substitute is generated, and fibrocartilage is generated according to the cartilage substitute.
The following examples are provided to illustrate the method and application of the present invention.
Example 1
A method for extracting type I collagen comprises the following steps:
freezing and slicing 500g of bovine achilles tendon, soaking in 75% ethanol solution by volume fraction for 6h for degreasing and disinfection, washing with water, pulverizing, dispersing in 0.5mol/L acetic acid-pepsin solution (m (bovine achilles tendon):m (pepsin) ═ 1: 0.03) according to the solid-to-liquid ratio of 1: 60(g/mL), performing enzymolysis extraction at 10 ℃ or below by using a cold circulation device, and slowly stirring during extraction. After 7h, the extract is centrifuged for 5min at about 8000r/min at 4 ℃, and the supernatant is collected and enters the purification stage. Firstly, pepsin is removed by isoelectric precipitation: and slowly adding 0.5mol/L sodium hydroxide solution into the supernatant, slowly stirring, adjusting the pH of the solution to 5.8-7.0, and observing that collagen is precipitated. Dissolving the precipitate in disodium hydrogen phosphate (0.02mol/L) containing low concentration peroxyacetic acid, salting out collagen, standing for 2 min, filtering, and rapidly cleaning the collagen precipitate with pure water for 3-4 times to obtain type I collagen crude extract. Dissolving the crude extract in 0.5mol/L acetic acid (containing peroxyacetic acid) to obtain 1g/L collagen solution, and performing ultrafiltration purification column chromatography separation under certain pressure, wherein the relative molecular mass intercepted by the purification column is 100kDa, and the pressure is kept below 0.8MPa during ultrafiltration purification. In order to ensure the stability of the collagen, a cold circulating device with the temperature kept at 4 ℃ is used as much as possible. And in the purification process, continuously adding purified water until the pH value of the purified collagen liquid is approximately equal to 5, thus obtaining the high-purity collagen liquid.
The detection proves that the extraction yield of the type I collagen in the example 1 of the application reaches 78.1 percent. But the product percent of pass is reduced without disinfection and sterilization by a cobalt 60-gamma ray irradiation method.
According to the above description, the method also includes the residual detection of pepsin in the purification process, specifically, the protein belongs to an amphoteric substance, when the pH value is at the isoelectric point, the net charge on the surface of the molecule is zero, the electrostatic repulsion between protein molecules is weakened, the attraction force is increased, and the protein molecules are aggregated and precipitated. The isoelectric precipitation method is adopted for testing, the recovery rate of the protein is high, and neutral fat can be effectively removed.
The pepsin can cut off the allergic telopeptide in the non-helical structural region of the collagen without damaging the triple-helical structure. If the collagen with the pepsin left is made into a product which is easy to have the phenomenon of yellow color and the like and is accompanied with adverse reactions such as stimulation and the like, the pepsin in the collagen is removed as much as possible. Multiple preliminary experiments show that the collagen is flocculated and precipitated when the pH of the solution is adjusted to be above 5.8 and is close to the isoelectric point of the collagen.
As the pepsin has stronger hydrophilicity and larger solubility in water, the pepsin can be removed by multiple quick water washes according to the difference of the solubilities. On the basis of the white precipitate formed by the combination of pepsin and tannic acid, whether pepsin remains can be judged. Adjusting pH to the isoelectric point of collagen by using 0.5mol/L sodium hydroxide solution to separate out collagen, adding 50g/L tannic acid solution into the filtrate to obtain a large amount of white turbidity, which indicates that the filtrate after collagen precipitation contains pepsin with higher concentration; after the filtrate obtained after washing the collagen for the 1 st time and the 2 nd time is added with 50g/L of tannic acid solution, white precipitate is gradually reduced, after the collagen is washed for the 3 rd time, flocculent precipitate can not be observed after the reaction of the washing filtrate and the tannic acid, and the pure water is clear if judged by naked eyes. Therefore, pepsin was substantially removed by 3 washes.
In this example, type I collagen is insoluble in water, but soluble in dilute acetic acid solution, so the extraction method using the enzyme-linked enzyme is suitable. Since collagen is easily denatured at 38 ℃ or higher, the extraction operation is performed at as low a temperature as possible. The pepsin protein has a relative molecular mass of 35-55kDa, can stimulate eyes, respiratory system and skin, is an allergen, and needs to be removed in the process of purifying collagen. The long-term action of pepsin with collagen results in the cleavage of collagen into collagen peptides and even amino acids of small molecular mass. To eliminate the effect of pepsin, pepsin is usually inactivated by salting out or heating. However, chloride ions are introduced during salting out, which increases the difficulty in later-stage collagen purification, and the heating operation step increases the risk of collagen denaturation, thereby destroying the structure and efficacy of collagen.
The type I collagen has a relative molecular mass of about 300kDa and can be retained by a semipermeable membrane, and some small-molecule impurities can pass through the semipermeable membrane so as to achieve the separation and purification effects. However, the purification process is long in period, needs about 7 days, is easy to breed microorganisms, and is not beneficial to industrial production. The method adopts an isoelectric precipitation method, and can quickly and effectively remove protease in the collagen solution; dissolving collagen in disodium hydrogen phosphate, salting out the collagen, standing, filtering, and cleaning a crude collagen product; the high-purity type I collagen can be obtained by using an ultrafiltration purification technology.
Example 2
Extracting type I collagen from bovine bones, comprising the following steps:
selecting 10 kilograms of frozen bone raw materials at the temperature of-18 ℃, washing impurities by using clear water, crushing by using a hard bone crusher, and controlling the crushing granularity to be 10 mm;
washing the crushed bone with clear water, cleaning to remove impurities and remove attachments;
adopting NaOH solution with the concentration of 0.5mol/L and the soaking time of 6h to degrease the cleaned broken bones, wherein the stirring speed is 12rpm in the soaking process;
carrying out decalcification treatment by adopting HCl solution with the concentration of 0.01mol/L and the soaking time of 10h, wherein the stirring speed is 12rpm in the soaking process;
cooking the material treated in the previous step at 90 ℃ for 2h, wherein the stirring speed in the cooking process is 12 rpm;
the used enzyme is pepsin, the addition amount is 0.3 per mill, the enzymolysis temperature is 50 ℃, the enzymolysis time is 5 hours, the pH is kept at 5, and the stirring speed is 12rpm in the enzymolysis process;
performing solid-liquid separation on the materials subjected to enzymolysis, and performing solid-liquid separation in a filtering mode; liquid-oil separation adopts a disc centrifuge to process the liquid-oil separation, and heavy phase (enzymolysis liquid) and light phase (bone oil) are separated by high-speed centrifugation;
slowly adding 0.5mol/L sodium hydroxide solution into the enzymolysis solution, slowly stirring, adjusting pH to obtain collagen precipitate, and removing protease;
dissolving collagen in disodium hydrogen phosphate (0.02mol/L) containing low-concentration peroxyacetic acid, salting out collagen, standing for 2 min, filtering, and washing crude collagen product with distilled water for 5 times;
dissolving the crude extract in 0.5mol/L acetic acid (containing peroxyacetic acid) to prepare 1g/L collagen solution, and performing ultrafiltration purification column chromatography separation under certain pressure, wherein the intercepted relative molecular mass of the purification column is 100kDa, and the pressure is kept below 0.8MPa in the ultrafiltration purification process. In order to ensure the stability of the collagen, a cold circulating device with the temperature kept at 4 ℃ is used as much as possible. And in the purification process, continuously adding purified water until the pH value of the purified collagen liquid is approximately equal to 5, thus obtaining the high-purity collagen liquid.
Allowing the collagen liquid to enter a nanofiltration membrane concentration system, wherein the aperture of the nanofiltration membrane is 1nm, and the collagen liquid is concentrated to the concentration of Brix10 degrees; then the mixture enters a double-effect falling film vacuum concentrator to be concentrated to the concentration of Brix40 degrees, and the volume is reduced to 31.4 percent of the original volume; the high-purity collagen concentrated solution is placed in a freeze dryer for freeze drying for 48 hours at the temperature of minus 50 ℃, and the energy consumption is saved by about 69 percent. Taking out, vacuum packaging, and sterilizing with cobalt 60-gamma ray irradiation.
Through detection, the extraction yield of the type I collagen in example 2 of the present application reaches 81.7%, and the calculated type I collagen yield (%) -the mass of the crude lyophilized type I collagen/the mass of the raw material bone for extraction × 100%, and the yield thereof is 3.53%.
Example 3
Extracting type I collagen from bovine achilles tendon comprising:
cleaning fresh bovine Achilles tendon with ultrapure water, removing fascia, broken bones, lean meat and fat with a scalpel blade, freezing the bovine Achilles tendon in a refrigerator at-20 ℃ until the bovine Achilles tendon is hardened, taking out the bovine Achilles tendon, and cutting the bovine Achilles tendon into Achilles tendon slices with the thickness of 1mm along the direction of tissue fibers.
100g Achilles tendon tablets were weighed in 75% ethanol: soaking the mixed solution of n-hexane (3: 1) for 12h at the temperature of 15-25 ℃, mechanically stirring, filtering the solution by using a stainless steel wire net, taking out, and washing with ultrapure water for 5 times.
Soaking in 0.05mol/L sodium carbonate solution at 15-25 deg.C for 24h, mechanically stirring, replacing sodium carbonate solution once at intervals of 8h, filtering with stainless steel wire net, taking out, and washing with ultrapure water for several times until the surface of Achilles tendon piece is neutral (pH value is 7).
Mincing the achilles tendon slice with the protein removed in a meat mincer, putting the minced achilles tendon tissue into 1000mL of 75% (volume fraction) acetic acid solution, homogenizing and crushing the solution for 5 times by a homogenizer at the temperature of 15-25 ℃, and homogenizing for 5min each time.
Placing the homogenate into a large-capacity stainless steel container, adding 1000mL of 75% (volume fraction) acetic acid solution into the homogenate, adding pepsin into the solution, wherein the concentration of the pepsin is 100mg/L, stirring and digesting the solution at 15-25 ℃ for 24h, and centrifuging the solution for 15-30 min. Taking the supernatant for later use.
Slowly adding 0.5mol/L sodium hydroxide solution into the supernatant, slowly stirring, adjusting pH to obtain collagen precipitate, and removing protease;
dissolving collagen in disodium hydrogen phosphate (0.02mol/L) containing low-concentration peroxyacetic acid, salting out collagen, standing for 2 min, filtering, and washing crude collagen product with distilled water for 4 times;
dissolving the crude extract in 0.5mol/L acetic acid (containing peroxyacetic acid) to prepare 1g/L collagen solution, and performing ultrafiltration purification column chromatography separation under certain pressure, wherein the intercepted relative molecular mass of the purification column is 100kDa, and the pressure is kept below 0.8MPa in the ultrafiltration purification process. In order to ensure the stability of the collagen, a cold circulating device with the temperature kept at 4 ℃ is used as much as possible. And in the purification process, continuously adding purified water until the pH value of the purified collagen liquid is approximately equal to 5, thus obtaining the high-purity collagen liquid.
And (3) placing the high-purity collagen stock solution into a freeze dryer, freeze-drying for 48h at-50 ℃, taking out, and carrying out vacuum packaging. The product is sterilized by cobalt 60-gamma ray irradiation.
The detection proves that the extraction yield of the type I collagen in the example 3 of the application reaches 78.6 percent.
Example 4
Extracting type I collagen from bovine achilles tendon comprising:
cleaning fresh bovine Achilles tendon with ultrapure water, removing fascia, broken bones, lean meat and fat with a scalpel blade, freezing the bovine Achilles tendon in a refrigerator at-20 ℃ until the bovine Achilles tendon is hardened, taking out the bovine Achilles tendon, and cutting the bovine Achilles tendon into Achilles tendon slices with the thickness of 1mm along the direction of tissue fibers.
100g Achilles tendon tablets were weighed in 70% ethanol: soaking the mixed solution of n-hexane (3: 1) for 12h at the temperature of 15-25 ℃, mechanically stirring, filtering the solution by using a stainless steel wire net, taking out, and washing with ultrapure water for 5 times.
Soaking in 0.04mol/L sodium carbonate solution at 15-25 deg.C for 24 hr, mechanically stirring, replacing sodium carbonate solution at intervals of 8 hr, filtering with stainless steel wire net, taking out, and washing with ultrapure water for several times until the surface of Achilles tendon piece is neutral (pH value of 7).
Mincing the achilles tendon slice with the protein removed in a meat mincer, putting the minced achilles tendon tissue into 1000mL of 72% (volume fraction) acetic acid solution, homogenizing and crushing the solution for 5 times by a homogenizer at the temperature of 15-25 ℃, and homogenizing for 5min each time.
Placing the homogenate into a large-capacity stainless steel container, adding 1000mL of 72% (volume fraction) acetic acid solution into the homogenate, adding pepsin with pepsin concentration of 80mg/L, stirring and digesting at 15-25 deg.C for 24h, and centrifuging for 15-30 min. Taking the supernatant for later use.
Neutralizing the supernatant with 0.6mol/L sodium hydroxide solution until the solution is neutral, adding sodium chloride until the concentration of sodium chloride is 2mol/L, stirring and salting out for 12h, and centrifuging for 15-30 min. Dissolving the precipitate in disodium hydrogen phosphate (0.02mol/L) containing low concentration peroxyacetic acid, salting out collagen, standing for 5min, filtering, and washing the crude collagen product with distilled water for 3 times;
dissolving the crude extract in 0.5mol/L acetic acid (containing peroxyacetic acid) to prepare 1g/L collagen solution, and performing ultrafiltration purification column chromatography separation under certain pressure, wherein the intercepted relative molecular mass of the purification column is 100kDa, and the pressure is kept below 0.8MPa in the ultrafiltration purification process. In order to ensure the stability of the collagen, a cold circulating device with the temperature kept at 4 ℃ is used as much as possible. And in the purification process, continuously adding purified water until the pH value of the purified collagen liquid is approximately equal to 5, thus obtaining the high-purity collagen liquid. And (3) placing the high-purity collagen stock solution into a freeze dryer, freeze-drying for 48h at-50 ℃, taking out, and carrying out vacuum packaging. The product is sterilized by cobalt 60-gamma ray irradiation.
The detection shows that the extraction yield of the type I collagen in the example 4 reaches 79.1 percent.
Example 5
In another embodiment of the present application, a method for extracting type I collagen from bovine achilles tendon comprises:
cleaning fresh bovine Achilles tendon with ultrapure water, removing fascia, broken bones, lean meat and fat with a scalpel blade, freezing the bovine Achilles tendon in a refrigerator at-20 ℃ until the bovine Achilles tendon is hardened, taking out the bovine Achilles tendon, and cutting the bovine Achilles tendon into Achilles tendon slices with the thickness of 1mm along the direction of tissue fibers.
100g Achilles tendon tablets were weighed into 73% ethanol: soaking the mixed solution of n-hexane (3: 1) for 12h at the temperature of 15-25 ℃, mechanically stirring, filtering the solution by using a stainless steel wire net, taking out, and washing with ultrapure water for 5 times.
Soaking in 0.06mol/L sodium carbonate solution at 15-25 deg.C for 24h, mechanically stirring, replacing sodium carbonate solution once at intervals of 8h, filtering with stainless steel wire net, taking out, and cleaning with ultrapure water for several times until the surface of Achilles tendon piece is neutral (pH value is 7).
Mincing the achilles tendon slice with the protein removed in a meat mincer, putting the minced achilles tendon tissue into 1000mL of 70% (volume fraction) acetic acid solution, homogenizing and crushing the solution for 5 times by a homogenizer at the temperature of 15-25 ℃, and homogenizing for 5min each time.
Placing the homogenate into a large-capacity stainless steel container, adding 1000mL of 70% (volume fraction) acetic acid solution into the homogenate, adding pepsin with pepsin concentration of 80mg/L, stirring and digesting at 15-25 deg.C for 24h, and centrifuging for 15-30 min. Taking the supernatant for later use.
Neutralizing the supernatant with 0.6mol/L sodium hydroxide solution until the solution is neutral, adding sodium chloride until the concentration of sodium chloride is 2mol/L, stirring and salting out for 12h, and centrifuging for 15-30 min. Dissolving the precipitate in disodium hydrogen phosphate (0.02mol/L) containing low concentration peroxyacetic acid, salting out collagen, standing for 5min, filtering, and washing the crude collagen product with distilled water for 3 times;
dissolving the crude extract in 0.5mol/L acetic acid (containing peroxyacetic acid) to prepare 1g/L collagen solution, firstly passing through a filtering and decolorizing column filled with activated carbon at an external water volume of 5 times, and then entering a resin column filled with anion and cation resin at an external water volume flow rate of 8 times in a serial connection mode;
centrifuging the liquid subjected to the decolorization and desalination treatment, and then feeding the liquid into a nanofiltration membrane concentration system, wherein the aperture of the nanofiltration membrane is 1nm, and the liquid is concentrated to the concentration of Brix10 degrees; then the mixture enters a double-effect falling film vacuum concentrator to be concentrated to the concentration of Brix40 degrees, and the volume is reduced to 30.9 percent of the original volume; the high-purity collagen concentrated solution is placed in a freeze dryer for freeze drying for 48 hours at the temperature of minus 50 ℃, and the energy consumption is saved by about 69 percent. Taking out, vacuum packaging, and sterilizing with cobalt 60-gamma ray irradiation.
Through detection, the extraction yield of the type I collagen in example 6 of the present application reaches 63.7%, and the calculated type I collagen yield (%) -the mass of the crude lyophilized type I collagen product/the mass of the raw material bone for extraction × 100%, and the yield thereof is 2.08%. Through analysis of enzyme soluble type I collagen (PSC), the protein has a complete 3-strand helical structure, a better network structure is maintained, and the protein structure is not damaged.
Example 6
Extracting type I collagen from bovine bone, comprising:
selecting 20 kg of frozen bone raw materials at the temperature of-18 ℃, washing impurities by using clear water, crushing by using a hard bone crusher, and controlling the crushing granularity to be 10 mm;
washing the crushed bone with clear water, cleaning to remove impurities and remove attachments;
adopting NaOH solution with the concentration of 0.8mol/L and the soaking time of 6h to degrease the cleaned broken bones, wherein the stirring speed is 12rpm in the soaking process;
carrying out decalcification treatment by adopting HCl solution with the concentration of 0.02mol/L and the soaking time of 10h, wherein the stirring speed is 12rpm in the soaking process;
cooking the material treated in the previous step at 90 ℃ for 2h, wherein the stirring speed in the cooking process is 12 rpm;
adding protease twice, adding protease 0.5 ‰, and maintaining pH at 5 at 50 deg.C for 20 min; adding protease for the second time, wherein the enzymolysis temperature is 50 ℃, the pH is maintained at 5, the adding amount of the protease added for the two times is 4 per mill, the enzymolysis time for the two times is 5h in total, and the stirring rotating speed in the enzymolysis process is 12 rpm;
performing solid-liquid separation on the materials subjected to enzymolysis, and performing solid-liquid separation in a filtering mode; liquid-oil separation adopts a disc centrifuge to process the liquid-oil separation, and heavy phase (enzymolysis liquid) and light phase (bone oil) are separated by high-speed centrifugation;
slowly adding 0.5mol/L sodium hydroxide solution into the enzymolysis solution, slowly stirring, adjusting pH to obtain collagen precipitate, and removing protease;
dissolving collagen in disodium hydrogen phosphate (0.02mol/L) containing low-concentration peroxyacetic acid, salting out collagen, standing for 2 min, filtering, and washing crude collagen product with distilled water for 5 times;
dissolving the crude extract in 0.5mol/L acetic acid (containing peroxyacetic acid) to prepare 1g/L collagen solution, and performing ultrafiltration purification column chromatography separation under certain pressure, wherein the intercepted relative molecular mass of the purification column is 100kDa, and the pressure is kept below 0.8MPa in the ultrafiltration purification process. In order to ensure the stability of the collagen, a cold circulating device with the temperature kept at 4 ℃ is used as much as possible. And in the purification process, continuously adding purified water until the pH value of the purified collagen liquid is approximately equal to 5, thus obtaining the high-purity collagen liquid.
Allowing the collagen liquid to enter a nanofiltration membrane concentration system, wherein the aperture of the nanofiltration membrane is 1nm, and the collagen liquid is concentrated to the concentration of Brix10 degrees; then the mixture enters a double-effect falling film vacuum concentrator to be concentrated to the concentration of Brix40 degrees, and the volume is reduced to 30.9 percent of the original volume; the high-purity collagen concentrated solution is placed in a freeze dryer for freeze drying for 48 hours at the temperature of minus 50 ℃, and the energy consumption is saved by about 70 percent. Taking out, vacuum packaging, and sterilizing with cobalt 60-gamma ray irradiation.
Through detection, the extraction yield of the type I collagen in example 6 of the present application reaches 82.6%, and the calculated type I collagen yield (%) -the mass of the crude lyophilized type I collagen product/the mass of the raw material bone for extraction × 100%, and the yield thereof is 3.69%. Through analysis of enzyme soluble type I collagen (PSC), the protein has a complete 3-strand helical structure, a better network structure is maintained, and the protein structure is not damaged.
Example 7
In another embodiment of the present application, a method for extracting type I collagen from bovine bone comprises:
crushing frozen ox bone at-18 deg.C with a crusher to obtain powder with particle size of 20 mm;
adopting NaOH solution with the concentration of 0.6mol/L and the soaking time of 7h to degrease the cleaned broken bones at the stirring speed of 24rpm in the soaking process;
carrying out decalcification treatment by adopting HCl solution with the concentration of 0.015mol/L and the soaking time of 12h, wherein the stirring speed is 24rpm in the soaking process;
cooking the material treated in the previous step at 95 ℃ for 3h, wherein the stirring speed in the cooking process is 24 rpm;
adding protease twice, adding protease 0.5 ‰, performing enzymolysis at 55 deg.C and pH7 for 40 min; adding protease for the second time, wherein the enzymolysis temperature is 55 ℃, the pH is maintained at 7, the adding amount of the protease added for the two times is 5 per mill, the enzymolysis time for the two times is 7h in total, and the stirring speed is 24rpm in the enzymolysis process;
performing solid-liquid separation on the materials subjected to enzymolysis, and performing solid-liquid separation on the ox bones by adopting a horizontal spiral centrifugal machine; liquid-oil separation adopts a disc centrifuge to process the liquid-oil separation, and heavy phase (enzymolysis liquid) and light phase (bone oil) are separated by high-speed centrifugation;
slowly adding 0.4mol/L sodium hydroxide solution into the enzymolysis solution, slowly stirring, adjusting pH to obtain collagen precipitate, and removing protease;
dissolving collagen in disodium hydrogen phosphate (0.02mol/L) containing low-concentration peroxyacetic acid, salting out collagen, standing for 2 min, filtering, and washing crude collagen product with distilled water for 5 times;
dissolving the crude extract in 0.5mol/L acetic acid (containing peroxyacetic acid) to prepare 1g/L collagen solution, and performing ultrafiltration purification column chromatography separation under certain pressure, wherein the intercepted relative molecular mass of the purification column is 100kDa, and the pressure is kept below 0.8MPa in the ultrafiltration purification process. In order to ensure the stability of the collagen, a cold circulating device with the temperature kept at 4 ℃ is used as much as possible. And in the purification process, continuously adding purified water until the pH value of the purified collagen liquid is approximately equal to 5, thus obtaining the high-purity collagen liquid.
Allowing the collagen liquid to enter a nanofiltration membrane concentration system, wherein the aperture of the nanofiltration membrane is 1nm, and the collagen liquid is concentrated to the concentration of Brix10 degrees; then the mixture enters a double-effect falling film vacuum concentrator to be concentrated to the concentration of Brix40 degrees, and the volume is reduced to 31.1 percent of the original volume; the high-purity collagen concentrated solution is placed in a freeze dryer for freeze drying for 48 hours at the temperature of minus 50 ℃, and the energy consumption is saved by about 69 percent. Taking out, vacuum packaging, and sterilizing with cobalt 60-gamma ray irradiation.
Through detection, the extraction yield of the type I collagen in example 7 of the present application reaches 82.3%, and the calculated type I collagen yield (%) -the mass of the crude lyophilized type I collagen/the mass of the raw material bone for extraction × 100%, which is 3.74%. Through analysis of enzyme soluble type I collagen (PSC), the protein has a complete 3-strand helical structure, a better network structure is maintained, and the protein structure is not damaged.
Since a comparative test is incorporated in the examples of the present invention, the following will be explained: example 1 is that only collagen is produced and subjected to structural analysis, no subsequent sterilization and production process is performed, the yield is only 78.1%, although the extraction process contains peracetic acid, the aseptic operation environment of the production process can be ensured, but the synergistic cooperation of the subsequent step of irradiation with cobalt 60-gamma rays is avoided, the sterilization and the sterility of the whole extraction process are ensured, so the product yield is influenced, and the yield is reduced;
the two steps of the embodiment 2, the embodiment 6 and the embodiment 7 are added with a membrane separation concentration method and a product sterilizing and killing process, so that the yield can be improved, the extraction yield is 81.7 percent, 82.6 percent and 82.3 percent respectively, the next step of drying is facilitated, and the energy-saving beneficial effect is achieved;
examples 3 and 4 were operable but without concentration, resulting in extraction yields of 78.6% and 79.1%, respectively, so the concentration step was also very important, essential to improve the extraction yield;
in example 5, the ultrafiltration purification, which is a very important step, was omitted, so the yield was reduced by a large amount of 69.7%, indicating that the ultrafiltration purification step is an important step for increasing the extraction yield in this example and is indispensable.
In conclusion, the method for extracting the type I collagen provided by the invention improves the extraction yield of the type I collagen, and the content of the foreign proteins in the collagen is less. The type I collagen extracted by the invention has good biocompatibility and bioactivity, can promote the hyperplasia of epithelial cells and the generation of collagenase, and meets the requirements of the clinical biomedical engineering field and the cosmetic industry. The preparation process is simple, can realize large-scale production, and is an economic and effective extraction method. The cartilage substitute is formed by the I-type collagen, can provide a basic structure place for bone mineralization by the I-type collagen, ensures the toughness of bones, plays an important role in the aspects of development, differentiation, activity regulation and the like of bone cells, and changes of the structure and the quantity of the collagen can cause the abnormity of the development, the differentiation and the function of the bone cells so as to change the structure and the performance of the bones.
Collagen is a huge family, has various varieties and highly complex structures, and has remarkable diversity from molecular structures, supermolecular structures to tissue distribution and functions. Nevertheless, the basic composition of collagen is largely homologous and contains (Gly-X-Y) n repeats in the amino acid sequence, which are the structural basis for the formation of trimers, where X is usually Pro and Y is usually Hy-Pm or Hy-Lys. Type I procollagen, the precursor of type I collagen, has 2 more terminal propeptide structures (C-propeptide and N-propeptide) than type I collagen. The type I procollagen protein has the typical characteristics of all procollagen proteins in a collagen protein family, and comprises a section of trimer structure with (Gly-X-Y) N repetitive sequences, wherein 2 propeptides at the N end and the C end form a non-helical structural domain (NC structural domain) which is positioned at two sides of the (Gly-X-Y) N repetitive sequences, and the NC structural domain further forms a spherical structure. The folded I-type procollagen protein is in a heterotrimeric structure, namely, the folded I-type procollagen protein is formed by winding 2 alpha 1 chains and 1 alpha 2 chain in an alternating way, and the collagen trimer has high tensile strength. After the type I procollagen protein is secreted into the extracellular matrix, the C-propeptide and the N-propeptide are removed by the cleavage of protease, and the mature collagen protein is formed. Mature collagen is capable of self-assembling into highly ordered structures, i.e., collagen fibers.
The invention also provides a type I collagen prepared by the method for preparing the type I collagen, which comprises the following steps: generating a novel cartilage substitute using the type I collagen, and generating fibrocartilage from the cartilage substitute; wherein, the collagen molecules of the type I collagen form lysine-hydroxylysine covalent cross-linking through lysine oxidase to form compact collagen fibers with regular arrangement. Glycosaminoglycan GAG secreted by chondrocytes is determined by toluidine blue staining, the phenomenon of chondrocyte heterosis in the type I plate is obvious, as shown in figure 2, the glycosaminoglycan GAG secreted by the chondrocytes of the third generation is obviously reduced, and the type I collagen can promote the glycosaminoglycan GAG to be secreted by the chondrocytes. The fibrocartilage is generated by the novel cartilage substitute, so that a basic structure place can be provided for bone mineralization, the toughness of bones is guaranteed, important effects on the aspects of development, differentiation, activity regulation and the like of osteocytes are played, and the abnormality of development, differentiation and function of the osteocytes, which can be caused, can be changed, and further the structure and performance of the bones are changed.
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are applicable to all kinds of fields suitable for the invention, and further modifications can be easily made by those skilled in the art, so that the invention is not limited to the specific details and the examples shown herein without departing from the general concept defined by the claims and the equivalent scope.
Claims (9)
1. A method for extracting type I collagen, which is characterized by comprising the following steps:
degreasing and decalcifying animal bones, and performing protease enzymolysis to obtain an enzymolysis liquid;
after extracting the enzymolysis liquid, adding a protein denaturation solution to obtain collagen precipitation;
dissolving the collagen precipitate, and salting out the dissolved collagen to obtain a crude collagen product;
and purifying the crude collagen product to obtain the type I collagen.
2. The method of extracting type I collagen according to claim 1, wherein the step of subjecting the animal bone to the enzymatic hydrolysis with a protease after the animal bone is defatted and decalcified comprises:
crushing animal bones, washing with water, and degreasing with 0.5-0.8mol/L sodium hydroxide or 70-75% ethanol solution;
decalcifying the degreased animal bone with hydrochloric acid with the concentration of 0.01-0.02 mol/L;
adding protease into the decalcified material, and performing enzymolysis to obtain an enzymolysis solution.
3. The method of extracting type I collagen according to claim 1, wherein said subjecting to protease hydrolysis to obtain an enzymatic hydrolysate comprises:
adding a first protease into the decalcified material, and carrying out enzymolysis for 20-40min at the temperature of 50-55 ℃ and under the condition that the pH =5-7 to obtain a first enzymolysis liquid;
adding a second protease into the first enzymolysis liquid, and carrying out enzymolysis at the temperature of 50-55 ℃ and under the condition that the pH =5-7 to obtain an enzymolysis liquid;
the mass fraction of the first protease is 0.2-0.5 per mill, the sum of the mass fractions of the first protease and the second protease is 3-5 per mill, and the sum of the two enzymolysis times is 5-7 hours.
4. The method of extracting type I collagen according to claim 1, wherein said extracting said enzymatic hydrolysate and then adding a protein denaturing solution to obtain a collagen precipitate comprises:
extracting an enzymolysis solution, slowly stirring and centrifuging the enzymolysis solution to obtain a supernatant;
and adding a sodium hydroxide solution into the supernatant, stirring, and adjusting the pH value to obtain the collagen precipitate without the protease.
5. The method of extracting type I collagen according to claim 1, wherein said dissolving said collagen precipitate and salting out said dissolved collagen to obtain a crude collagen product comprises:
adding a disodium hydrogen phosphate solution containing peroxyacetic acid into the collagen precipitate, standing for salting out, filtering and cleaning after the collagen precipitate is dissolved, thereby obtaining a crude collagen product.
6. The method of claim 1 wherein the purifying the collagen crude product to obtain type I collagen comprises:
adding acetic acid solution containing peroxyacetic acid into the collagen crude product to obtain 1g/L collagen solution;
performing ultrafiltration purification column chromatography separation on the collagen solution, and entering a double-effect falling film vacuum concentrator through a nanofiltration membrane concentration system to obtain unsterilized type I collagen;
irradiating the type I collagen by using cobalt 60-gamma rays to obtain the type I collagen.
7. The method of extracting type I collagen according to claim 1, wherein said animal bone is bovine achilles tendon or bovine bone.
8. Use of type I collagen extracted by the extraction process according to any one of claims 1 to 7 for the production of cartilage substitutes and for the production of fibrocartilage from said cartilage substitutes.
9. The use according to claim 8, wherein the collagen molecules of the type I collagen are covalently cross-linked by lysine oxidase to form lysine-hydroxylysine, to form compact collagen fibers, to form cartilage substitutes, and to form fibrocartilage from the cartilage substitutes.
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