CN117534847A - Preparation method of collagen gel for removing sensitized telopeptide - Google Patents
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Abstract
The invention discloses a preparation method of collagen gel for removing sensitized telopeptide, which comprises the following steps: s100: cutting pigskin into pieces and cleaning; s200: immersing pigskin in 0.5M acetic acid solution for 12 to 36 hours; s300: pepsin is added into acetic acid solution for enzymolysis; s400: detecting the content of collagen in the solution after enzymolysis; s500: adjusting the pH value of the solution, separating out collagen precipitate, and adding the collagen precipitate into sodium chloride solution for salting out; s600: purifying the salted-out collagen; s700: preparing purified collagen into 35mg/ml aqueous suspension according to the content of the collagen, homogenizing uniformly, adding succinic anhydride powder into the aqueous suspension, adjusting the pH value of the solution by sodium hydroxide at the same time, maintaining the pH value at 8-10, and carrying out acylation reaction for 1-3 hours at room temperature until flocculent collagen solid disappears; s800: ultrafiltration treatment was performed using a 30kD ultrafiltration membrane. The collagen is made to be more hydroscopic by the way of acylation modification.
Description
Technical Field
The invention relates to a preparation method of collagen gel, in particular to a preparation method of collagen gel for removing sensitized telopeptide.
Background
Collagen is widely present in tissues such as skin, blood vessels, tendons, and bones of vertebrates, and is a major component of extracellular matrix. In human skin, the collagen accounts for more than 70%, has rich proline and lysine, forms a unique triple helix structure by intertwining three Gly-X-Y tripeptide repeats, has a molecular weight of 30 ten thousand, can be spontaneously assembled into collagen fibers with supermolecular high-grade structures, endows collagen with good biocompatibility, and has good promotion effects in cell behaviors such as cell adhesion, migration, proliferation and differentiation, so that collagen-based materials such as adsorption materials, plastic and cosmetic skin care materials can be widely applied to the possibility.
Before collagen can be used as a biomaterial or a functional food material, it is often necessary to modify collagen to impart more excellent properties. The collagen molecules contain a large number of hydroxyl, carboxyl, amino and other active groups, so that the collagen can be modified. Collagen is a typical amphiphilic polymer and has an isoelectric point of about 7.0, meaning that it has low solubility in physiological environments, whereas biological materials for clinical application, particularly implant materials, must ensure that the material is in physiological environment, so that unmodified natural collagen materials are poorly hydrophilic and almost insoluble in water. Researches show that the hydrophilic group carboxyl is introduced into the side chain of the collagen, so that the collagen has higher hydroscopicity, the charge on the side chain of the collagen molecule is changed, and the isoelectric point of the collagen is reduced, so that the solubility of the collagen under neutral conditions is greatly improved, and the collagen becomes soluble collagen gel, and has good bioactivity. In the fields of health food and cosmetology, the collagen gel can prevent water loss and has moisturizing effect, and can be used for preparing collagen gel Shui Guangji; on the adsorption material, the collagen gel can enhance the capability of collagen absorption and metal ion combination, and a slow-release drug release material is prepared; on biomedical materials, the negative charge amount of collagen molecules is greatly increased, so that the adhesion energy of modified collagen platelets and the formation energy of fibrin are relatively weak, and the modified collagen platelets have certain anti-inflammatory and anti-thrombus properties. The complete triple helix of collagen is a carrier with collagen function and is the basis for good biocompatibility and tissue induction, and if the modified triple helix structure is destroyed to form denatured product gelatin, the gelatin can not activate the growth of cells under the simulated physiological condition, and the biological activity is basically lost. The acylated modified collagen should therefore maintain the integrity of the triple helix structure.
Research shows that the non-helical terminal peptide domains at two ends of the three-strand helical structure of the collagen are the main reasons for sensitization of the collagen, so that the collagen gel from which the collagen terminal peptide is excised in the preparation process is more suitable for being used as a biological material for clinical application.
Disclosure of Invention
The invention aims to provide a preparation method of collagen gel for removing sensitized telopeptide, which is characterized in that on the premise of safety of SPF pigs not carrying pathogenic microorganisms, the telopeptide collagen without sensitized telopeptide is extracted from SPF pigskin by an enzyme cutting mode, and then the telopeptide collagen is subjected to succinic anhydride grafting modification under the condition of not changing a triple helix structure, so that the telopeptide collagen gel which can be dissolved under physiological conditions is prepared.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for preparing a collagen gel with the sensitized telopeptide removed, comprising the steps of:
s100: cutting pigskin into pieces and cleaning;
s200: immersing pigskin in 0.5M acetic acid solution for 12 to 36 hours;
s300: pepsin is added into acetic acid solution for enzymolysis;
s400: detecting the content of collagen in the solution after enzymolysis;
s500: adjusting the pH value of the solution, separating out collagen precipitate, and adding the collagen precipitate into sodium chloride solution for salting out;
s600: purifying the salted-out collagen;
s700: preparing purified collagen into 35mg/ml aqueous suspension according to the content of the collagen, homogenizing uniformly, adding succinic anhydride powder into the aqueous suspension, adjusting the pH value of the solution by sodium hydroxide at the same time, maintaining the pH value at 8-10, and carrying out acylation reaction for 1-3 hours at room temperature until flocculent collagen solid disappears;
s800: ultrafiltering with 30kD ultrafilter membrane, washing with water for 2 times, and removing excessive small molecule components to obtain the final product.
In the aforementioned preparation method of the collagen gel with the end peptide removed, the step S100 specifically includes the following steps: cutting pig skin into 5mm small pieces after shaving and fat-removing, degreasing with degreasing liquid, adding salt for washing, washing the pig skin small pieces after salt washing with water, wherein the degreasing liquid is 2% sodium carbonate solution, the solid-liquid ratio is 1:10, and the degreasing time is 2-6 hours; the salt washing liquid is 10% sodium chloride solution, the solid-liquid ratio is 1:10, and the salt washing time is 2 to 6 hours.
In the aforementioned preparation method of the collagen gel with the end peptide removed, the step S200 specifically includes the following steps: soaking the treated pigskin small pieces in 0.5M acetic acid solution for 12-36 hours, homogenizing and homogenizing the soaked pigskin small pieces, wherein the solid-liquid ratio is 1:50-100. The soaking is to break the homogenate more fully, otherwise the pigskin is difficult to break completely, and the collagen extraction rate of the pigskin is higher when the homogenate is more complete.
In the aforementioned preparation method of the collagen gel with the end peptide removed, the step S300 specifically includes the following steps: pepsin is added into acetic acid solution, the mass ratio of the pepsin to the pigskin is 1:100-200, the enzymolysis temperature is 10-20 ℃, the enzymolysis time is 12-24 hours, and the sensitized telopeptide of the collagen is excised.
In the aforementioned preparation method of the collagen gel with the end peptide removed, the step S400 specifically includes the following steps: after the enzymolysis is finished, the reaction solution is filtered by a filter membrane to remove impurities, so as to obtain clean enzymolysis solution, sampling is carried out, the content of collagen is detected by using a sirius red kit, the total amount of the collagen is calculated, and the pore diameter of the filter membrane is 1.2 mu m, 0.45 mu m and 0.22 mu m. The principle of detecting collagen concentration by using sirius red kit is as follows: sirius red is specifically combined with collagen, red compound is obtained after elution and centrifugation by sodium hydroxide solution, absorbance is measured under the condition of 540nm wavelength, and the collagen content in a sample is determined.
In the aforementioned preparation method of the collagen gel with the end peptide removed, the step S500 specifically includes the following steps: and regulating the pH value of the obtained enzymolysis liquid to 7-8 by using sodium hydroxide, separating out collagen precipitate, then adding 10% sodium chloride solution, and salting out for 12-24 hours.
In the aforementioned preparation method of the collagen gel with the end peptide removed, the step S600 specifically includes the following steps: collecting the collagen precipitate after salting out after centrifugation, and washing with water for 2 times to obtain the atelopeptide collagen.
Compared with the prior art, the invention extracts the atelopeptide collagen without the sensitized telopeptide from the SPF pigskin by an enzyme cutting method on the premise that the SPF pig does not carry pathogenic microorganisms, and then carries out succinic anhydride grafting modification on the atelopeptide collagen under the condition of not changing a triple helix structure to prepare the atelopeptide collagen gel which can be dissolved under physiological conditions.
The invention provides a complete preparation process of the atelopeptide collagen gel, which is suitable for industrial production and clinical application. SPF pig skin is selected, and pathogenic microorganisms of animal-derived materials are controlled from the source. The amino acid sequence of the collagen is detected by mass spectrometry to screen the optimal enzymolysis condition, so that the collagen with the terminal peptide removed is obtained, meanwhile, a membrane filtration step is added in the preparation process, impurities are removed, the collagen with higher purity is obtained, and the clinical application safety is improved.
Then on the premise of not damaging the three-strand spiral structure of the collagen, active groups in the collagen molecules are utilized, hydrophilic groups-COOH of succinic anhydride are introduced into the side chains of the collagen molecules, the acylation modification reaction mechanism is shown in figure 1, and the water solubility of the collagen is improved and improved, so that the collagen gel soluble under physiological conditions is prepared. The succinic anhydride powder is added in the reaction to replace the traditional method of dissolving succinic anhydride by using organic solvents such as acetone and the like and then carrying out the reaction, so that the reaction efficiency is not reduced, the organic solvent residue is not increased, the safety of clinical application is further improved, and the method is more suitable for production and clinical application.
Drawings
FIG. 1 is a schematic diagram of the reaction mechanism of succinic anhydride and collagen;
FIGS. 2a to 2f are mass spectra of collagen prepared in example 1;
FIG. 3 is a circular dichroism spectrum of the acylated modified collagen prepared in example 1;
FIG. 4 shows an infrared spectrum of the acylated modified collagen prepared in example 1.
The invention is further described below with reference to the drawings and the detailed description.
Detailed Description
Example 1 of the present invention: a method for preparing a collagen gel from which a sensitized telopeptide has been removed, comprising the steps of:
s100: cutting pig skin into 5mm small pieces after shaving and fat-shaving, degreasing with degreasing liquid, adding salt for washing, washing the pig skin small pieces after salt washing, wherein the degreasing liquid is 2% sodium carbonate solution, the solid-liquid ratio is 1:10, and the degreasing time is 4 hours; the salt washing liquid is 10% sodium chloride solution, the solid-liquid ratio is 1:10, and the salt washing time is 4 hours.
S200: soaking the treated pigskin small pieces in 0.5M acetic acid solution for 24 hours, homogenizing and homogenizing the soaked pigskin small pieces, wherein the solid-liquid ratio is 1:100.
S300: pepsin is added into acetic acid solution, the mass ratio of the pepsin to the pigskin is 1:100, the enzymolysis temperature is 15 ℃, the enzymolysis time is 16 hours, and the sensitized telopeptide of the collagen is excised.
S400: after the enzymolysis is finished, the reaction solution is filtered by a filter membrane to remove impurities, so as to obtain clean enzymolysis solution, sampling is carried out, the content of collagen is detected by using a sirius red kit, the total amount of the collagen is calculated, and the pore diameter of the filter membrane is 1.2 mu m, 0.45 mu m and 0.22 mu m.
S500: and regulating the pH value of the obtained enzymolysis liquid to 7-8 by using sodium hydroxide, separating out collagen precipitate, then adding 10% sodium chloride solution, and salting out for 12-24 hours.
S600: collecting the collagen precipitate after salting out after centrifugation, and washing with water for 2 times to obtain the atelopeptide collagen.
S700: preparing purified collagen into 35mg/ml aqueous suspension according to the content of the collagen, homogenizing uniformly, adding succinic anhydride powder into the aqueous suspension, adjusting the pH value of the solution by sodium hydroxide at the same time, maintaining the pH value at 8-10, and carrying out acylation reaction for 1-3 hours at room temperature until flocculent collagen solid disappears;
s800: ultrafiltering with 30kD ultrafilter membrane, washing with water for 2 times, and removing excessive small molecule components to obtain the final product.
Mass spectrometry detection was performed on the atelopeptide collagen obtained in example 1, and the porcine type I collagen a 1 chain amino acid sequence was compared, as shown in fig. 2, and the labeled sequence was the collagen amino acid sequence obtained in example 1, and the result showed that the N-terminal and C-terminal peptide sequences had been excised.
The structure of collagen gel after succinic anhydride acylation was measured as follows:
(1) The collagen is a protein with optical activity, and has a fixed characteristic peak position in a circular dichroism image, a positive peak at 220nm and a negative peak at 190nm, and is a characteristic peak of a three-helix structure of the collagen. Therefore, circular dichroism is an effective detection means for verifying whether collagen is denatured. Calculating the ratio R of positive peak intensity to negative peak intensity pn The value should be 0.09-0.15, the larger the value is, the more triple-flighted is indicatedThe higher the intermolecular assembly degree of collagen with a rotary structure, the CD spectrum of the acylated modified collagen gel prepared in example 1 is shown in a third graph, the maximum negative peak appears at 195-200nm, the maximum positive peak appears at 220-222nm, and the R of the spectrum is calculated pn The value is 0.15, which indicates that the acylated type I collagen has a complete triple helix structure.
(2) The configuration of the polypeptide chain was detected by infrared spectroscopy. In fig. 4, in the infrared spectrum, the acylated modified collagen gel prepared in example 1 has typical absorption peaks of collagen, as well as unmodified collagen. The characteristic absorption peaks include: amide A belt (3400-3440 cm) -1 N-H stretching vibration), amide B band (3080-3100 cm) -1 N-H stretching vibration), amide I band (1600-1660 cm) -1 C=O stretching vibration, is a marker region of alpha helical structure), amide II band (1500-1600 cm -1 N-H bending vibration and C-N stretching vibration) and an amide III band (1200-1300 cm -1 C-N and N-H flexural vibrations, glycine backbone and proline side chains-CH 2 Non-planar rocking vibration) of (a). Indicating that the acylation modification reaction does not change the triple helix structure of the collagen.
According to the technical scheme, collagen is enabled to be more hydroscopic through an acylation modification mode. Therefore, the application range of the collagen is enlarged to a certain extent, and the moisturizing effect of preventing water loss is achieved in the aspect of cosmetics; in the aspect of the adsorption material, the adsorption capacity of the collagen and the binding capacity of metal ions are enhanced; in the biomedical material, the negative charge amount of collagen increases after acylation, so that the platelet adhesion and the fibrin formation ability are weakened, and the thrombus resistance is presented.
Claims (7)
1. A method for preparing a collagen gel from which a sensitized telopeptide has been removed, comprising the steps of:
s100: cutting pigskin into pieces and cleaning;
s200: immersing pigskin in 0.5M acetic acid solution for 12 to 36 hours;
s300: pepsin is added into acetic acid solution for enzymolysis;
s400: detecting the content of collagen in the solution after enzymolysis;
s500: adjusting the pH value of the solution, separating out collagen precipitate, and adding the collagen precipitate into sodium chloride solution for salting out;
s600: purifying the salted-out collagen;
s700: preparing purified collagen into 35mg/ml aqueous suspension according to the content of the collagen, homogenizing uniformly, adding succinic anhydride powder into the aqueous suspension, adjusting the pH value of the solution by sodium hydroxide at the same time, maintaining the pH value at 8-10, and carrying out acylation reaction for 1-3 hours at room temperature until flocculent collagen solid disappears;
s800: ultrafiltering with 30kD ultrafilter membrane, washing with water for 2 times, and removing excessive small molecule components to obtain the final product.
2. The method for preparing a collagen gel with the end peptide removed as claimed in claim 1, wherein the step S100 specifically comprises the following steps: cutting pig skin into 5mm small pieces after shaving and fat-removing, degreasing with degreasing liquid, adding salt for washing, washing the pig skin small pieces after salt washing with water, wherein the degreasing liquid is 2% sodium carbonate solution, the solid-liquid ratio is 1:10, and the degreasing time is 2-6 hours; the salt washing liquid is 10% sodium chloride solution, the solid-liquid ratio is 1:10, and the salt washing time is 2 to 6 hours.
3. The method for preparing a collagen gel with the end peptide removed as claimed in claim 1, wherein the step S200 specifically comprises the following steps: soaking the treated pigskin small pieces in 0.5M acetic acid solution for 12-36 hours, homogenizing and homogenizing the soaked pigskin small pieces, wherein the solid-liquid ratio is 1:50-100.
4. The method for preparing a collagen gel with the end peptide removed as claimed in claim 1, wherein the step S300 specifically comprises the following steps: pepsin is added into acetic acid solution, the mass ratio of the pepsin to the pigskin is 1:100-200, the enzymolysis temperature is 10-20 ℃, the enzymolysis time is 12-24 hours, and the sensitized telopeptide of the collagen is excised.
5. The method for preparing a collagen gel with the end peptide removed as claimed in claim 1, wherein the step S400 specifically comprises the following steps: after the enzymolysis is finished, the reaction solution is filtered by a filter membrane to remove impurities, so as to obtain clean enzymolysis solution, sampling is carried out, the content of collagen is detected by using a sirius red kit, the total amount of the collagen is calculated, and the pore diameter of the filter membrane is 1.2 mu m, 0.45 mu m and 0.22 mu m.
6. The method for preparing a collagen gel with the end peptide removed as claimed in claim 1, wherein the step S500 specifically comprises the following steps: and regulating the pH value of the obtained enzymolysis liquid to 7-8 by using sodium hydroxide, separating out collagen precipitate, then adding 10% sodium chloride solution, and salting out for 12-24 hours.
7. The method for preparing a collagen gel with the end peptide removed as claimed in claim 1, wherein the step S600 specifically comprises the following steps: collecting the collagen precipitate after salting out after centrifugation, and washing with water for 2 times to obtain the atelopeptide collagen.
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