CN115124613A - High-fluidity high-concentration collagen solution and preparation method thereof - Google Patents
High-fluidity high-concentration collagen solution and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a high-fluidity high-concentration collagen solution and a preparation method thereof. The preparation method comprises the steps of carrying out irradiation treatment on the collagen raw material after degreasing treatment, and then carrying out enzymolysis. The preparation method of some embodiments of the invention is simple to operate, does not change the subsequent production process, and can directly prepare the collagen solution with the concentration of 10-20 mg/mL. The viscosity of the prepared collagen solution at 20 ℃ is 100-4500 cP, and the prepared collagen solution has good fluidity; the obtained collagen molecule has the structural characteristics of typical type I collagen, no obvious miscellaneous band appears under 97.2 kDa, the molecular structure in the collagen solution has higher integrity, the molecular weight is about 300 kDa, and good biological activity is kept; the viscosity stability of the collagen solution is good, the requirements of long-term storage, transportation, further processing and filling can be met, and the defect of short processing time caused by easy rise of the viscosity of the collagen solution subjected to the homogenizing viscosity reduction treatment is overcome.
Description
Technical Field
The invention belongs to the field of biology, and particularly relates to a high-fluidity high-concentration collagen solution and a preparation method thereof.
Background
Collagen (Collagen) is a structural protein having a triple helix structure and a molecular weight of about 300 kDa, is one of the major components of extracellular matrix (ECM), and is widely present in tendons, skin, blood vessels, and cornea. In vivo, collagen and polysaccharide form a precise and ordered intercellular lattice structure, which plays an important role in proliferation, migration and differentiation of body cells, and influences body tissue differentiation and function exertion. The characteristics of good cell compatibility, low immunogenicity and the like cause the related research of collagen to attract wide attention, and the collagen is widely applied to the fields of cosmetics, functional foods, biological materials and the like.
The collagen solution is generally prepared by pulverizing the raw materials, defatting, performing enzymolysis, and purifying. The collagen solution prepared by the process has the collagen content of no more than 7 mg/mL generally, and the lower concentration limits the processing and transportation of the collagen solution. In order to further increase the concentration of the collagen solution, salting out or directly increasing the feeding ratio is generally adopted for treatment, and the salting out method can achieve a good concentration effect, but the treatment time is long, pollution is easily caused, and the amount of generated wastewater is also large. Meanwhile, in the enzymolysis process of the collagen, the viscosity of the enzymolysis liquid is obviously increased along with the release of the collagen in the enzymolysis process, and the subsequent enzymolysis process is seriously affected by the high-viscosity enzymolysis liquid, so that the collagen solution with higher concentration is difficult to obtain by directly increasing the reaction ratio for enzymolysis.
Collagen is a biological macromolecule whose solution viscosity increases with increasing concentration, and highly concentrated collagen solutions generally have a high viscosity (low fluidity). The collagen solution with low viscosity has better processability. If the viscosity is low, the collagen solution has high fluidity, and better filling precision can be obtained when the machine is used for filling; the high concentration collagen solution has low fluidity and is difficult to handle during filling and processing steps. If the viscosity is more than 8000 cP, on one hand, the flowability is low, and the machine filling property is poor, and on the other hand, the phenomenon of solution retention in the filling process is obviously increased, so that the actual filling quantity is unstable, and the uniformity and the stability of the final product are possibly influenced.
In order to improve the fluidity of collagen solutions, the prior art generally achieves this by terminal homogenization and/or adjustment of the pH. For example, Schrochenshun (Schrochenshun. extraction of collagen from pigskin and study of viscosity Properties [ J ]]Chinese Adhesives 2007, 16(3):4.) and the like, using pigskin as a raw material, extracting collagen under the synergistic action of citric acid and pepsin. The concentration, pH, shear rate, NaCl and CaCl were investigated using a rotational rheometer system 2 Ethanol and glycerol on collagen viscosity. The results show that the viscosity of the collagen solution increases exponentially with the concentration; viscosity is maximal at pH =3, viscosity decreases at pH =4 and pH =6-9, and viscosity increases abruptly again at pH = 10; the viscosity shows a logarithmic decrease trend along with the increase of the shear rate; NaCl and CaCl 2 The addition of (2) can reduce the viscosity of the collagen solution; when the amount of the added ethanol or glycerin is less than 10%, the concentration of the added ethanol or glycerin is in direct proportion to the viscosity. These methods require adjustment of pH while adding other components, and their methods of use are limited.
Homogenization, which does not introduce foreign components, is a more common method. However, homogenization has the following disadvantages: 1) the homogenization can generate a large amount of heat, and the existence of high-speed shearing easily causes the denaturation of temperature-sensitive collagen molecules, so that the activity of the collagen is reduced; 2) a large amount of bubbles are generated during homogenization, and degassing treatment needs to be carried out synchronously; 3) the homogenizer has higher cost and larger occupied area; 4) the viscosity of the homogenized sample is reduced in a limited way, and the viscosity of the homogenized sample can be gradually recovered along with the prolonging of the storage time, so that the collagen solution with the viscosity reduced by shearing needs to be processed and used as soon as possible, and the problem that the viscosity rises after the homogenized sample is stored for a long time to cause difficulty in filling is avoided. These deficiencies of homogeneity result in a limited range of applications.
Irradiation is a safe and efficient sterilization method. Irradiation destroys the structure of the molecule and, under certain conditions, can also initiate crosslinking. Natural macromolecular collagen can be broken into small molecules by irradiation, the irradiation is also commonly used for preparing small molecule collagen, such as CN112341537A, CN107022592A and the like, after enzymolysis, the enzymolysis liquid is treated by irradiation, and finally the small molecule collagen which is easy to absorb is obtained. CN105063154A discloses washing, acid treatment, alkali treatment, grinding, cooking, cooling of fish scales as raw materials; carrying out irradiation treatment; enzymolysis, primary enzymolysis and secondary enzymolysis; adsorbing by activated carbon; filtering; concentrating; sterilizing; and (5) spray drying. In the whole production process, a large amount of chemical raw materials such as acid, alkali and the like are not needed, the production period is short, and finally the micromolecular collagen is obtained. In the field of preparation of active collagen (type i collagen), irradiation is generally not used to treat collagen raw materials, but other relatively milder sterilization methods are used because irradiation destroys the molecular structure, resulting in the destruction of the triple helix structure of active collagen. Additional studies have shown that irradiation initiates free radical polymerization when processing biomaterials with high water content, resulting in increased viscosity. While the water content of the defatted connective tissue powder is generally not less than 20%, irradiation treatment may increase the viscosity of the enzymatic hydrolysate. In the prior art, no irradiation treatment of raw materials is known during the production of active collagen.
The development of a high-fluidity high-concentration collagen solution and a preparation method thereof have very important significance for improving the processability of the collagen solution.
Disclosure of Invention
The present invention is directed to overcoming at least one of the disadvantages of the prior art and providing a high fluidity and high concentration collagen solution and a method for preparing the same.
The technical scheme adopted by the invention is as follows:
in the research process, the inventor finds that the collagen raw material (connective tissue) powder after degreasing is subjected to irradiation treatment by using a specific dose, and then is subjected to enzymolysis according to a conventional process, so that the biological activity of collagen is not obviously reduced, a high-fluidity collagen solution can be obtained, the processability of the collagen solution is greatly improved, and the high-fluidity high-concentration collagen solution can be further directly prepared by increasing the feeding proportion due to the reduction of the viscosity, and the method has unexpected effects. Based on this finding, the inventors have developed a high fluidity and high concentration collagen solution and a method for preparing the same.
In a first aspect of the present invention, there is provided:
a preparation method of a collagen solution comprises the following steps:
s1) taking fresh animal connective tissue, cleaning, crushing, and degreasing to obtain degreased connective tissue powder;
s2) carrying out irradiation treatment on the degreased connective tissue powder, wherein the irradiation dose is 10-20 kGy;
s3) performing enzymolysis and purification on the irradiated connective tissue powder to obtain collagen solution.
In some embodiments, the connective tissue is maintained at a temperature of-20 to 0 ℃ during the irradiation treatment.
In some examples of the preparation method, the irradiation source of the irradiation is gamma rays or electron beams.
In some embodiments, the defatted connective tissue powder is placed in a sealed bag, and the bag is sealed with a coolant and irradiated after sealing.
In some examples of the manufacturing method, the cold storage agent is at least one of an ice bag, ice cubes, dry ice, and a polyvinyl alcohol-borax high-efficiency cold storage agent.
In some examples of the method of making, the connective tissue is selected from animal tendon, skin, cartilage, or scales.
In some examples of the method of making, the animal is selected from at least one of a pig, a cow, a mouse, a sheep, and a horse.
In some examples of the preparation method, a mixing ratio of the cold storage agent and the defatted connective tissue powder is (0.5-20): 1.
in some examples of the preparation method, the collagen solution has a concentration of 10 to 20 mg/mL, a viscosity of 100 to 4500 cP at 20 ℃, a molecular weight of about 300 kDa, and a complete triple helix structure.
In some examples of the preparation method, the mixing ratio of the connective tissue powder to the enzyme solution is 1: (40-80).
In some examples of preparation methods, the collagen solution has a positive and negative absorption peak near 220 nm and 197 nm, respectively, as determined by circular dichroism scanning, and the complete triple-helix structure is confirmed to be retained.
In a second aspect of the present invention, there is provided:
a collagen solution prepared by the method of the first aspect of the present invention.
In some examples of the collagen solution, the collagen has a concentration of 10 to 20 mg/mL, a viscosity of 100 to 4500 cP at 20 ℃, a molecular weight of about 300 kDa, and a complete triple helix structure.
In some examples of the collagen solution, when the circular dichroism scanning measurement is carried out, the collagen solution has a positive absorption peak and a negative absorption peak near 220 nm and 197 nm respectively, which proves that the collagen solution retains a complete triple-helix structure, the collagen triple-helix structure is the key for exerting the complete efficacy, and the collagen molecule with the complete triple-helix structure has better bioactivity than that of the small molecule collagen.
The invention has the beneficial effects that:
the preparation method of some embodiments of the invention is simple to operate, and compared with the traditional collagen solution production process, the preparation method only needs to additionally use a specific dose of irradiation treatment on the degreased collagen raw material (connective tissue) powder, does not need to change the subsequent production process, and can directly prepare the collagen solution with the concentration of 10-20 mg/mL.
1) According to the preparation method of some embodiments of the invention, the mass mixing ratio of the connective tissue powder and the enzyme liquid can be increased to 1: (40-80), the difficulties that the traditional preparation of high-concentration collagen liquid needs salting out and concentration and other complicated steps, such as long time consumption, large loss and more water consumption are overcome;
2) the viscosity of the prepared collagen solution at 20 ℃ is 100-4500 cP, and the prepared collagen solution has good fluidity;
3) collagen molecules in the collagen solution have typical type I collagen structural characteristics, no obvious miscellaneous band appears under 97.2 kDa, the molecular structure in the collagen solution is high in integrity, the molecular weight is about 300 kDa, and the collagen solution retains good biological activity.
According to the preparation method of some embodiments of the invention, the prepared collagen solution has good viscosity stability, can meet the requirement of long-term storage, can meet the requirements of further processing, storage and transportation, and overcomes the defect of short processing time caused by easy viscosity rise of the collagen solution subjected to homogenizing viscosity reduction treatment.
Drawings
FIG. 1: the viscosity change graphs of examples 1 to 4 of the present invention and comparative examples 1 to 4.
FIG. 2: circular dichroism graphs of examples 1-4 of the present invention and comparative examples 1-4.
FIG. 3: polyacrylamide gel electrophoresis patterns of collagen molecules of examples 1-4 and comparative examples 1-4 of the present invention.
Detailed Description
In a first aspect of the present invention, there is provided:
a method for preparing a collagen solution, comprising the steps of:
s1) taking fresh animal connective tissue, crushing and degreasing after cleaning to obtain degreased connective tissue powder;
s2) carrying out irradiation treatment on the degreased connective tissue powder, wherein the irradiation dose is 10-20 kGy;
s3) performing enzymolysis and purification on the irradiated connective tissue powder to obtain a collagen solution.
Fresh animal connective tissue can be processed in accordance with known methods to produce a defatted connective tissue powder. The enzymatic hydrolysis can also be carried out according to known methods. Generally speaking, the temperature of enzymolysis is controlled to be 25-35 ℃, and the enzymolysis time is controlled to be 24-72 h.
In order to better maintain the activity of collagen, in some examples of the preparation method, the temperature of the connective tissue is maintained at-20 to 0 ℃ during the irradiation treatment. Within this temperature range, it is easy to implement, while avoiding the additional costs of maintaining a lower temperature.
In some examples of the preparation method, the irradiation source of the irradiation is gamma rays or electron beams. The irradiation sources have better penetrating power and can better ensure the irradiation treatment effect.
In some embodiments of the method, the defatted connective tissue powder is placed in a sealed bag, cold storage agent is added, and the bag is sealed and then irradiated. This allows for better maintenance of the connective tissue powder temperature during the irradiation treatment.
Connective tissue is rich in type i collagen and, in some examples of methods of preparation, is selected from the group consisting of animal tendon, skin, cartilage, or scales. The collagen content of the tissues is high, and the type of the collagen is more single, so that high-quality collagen solution can be obtained more favorably.
The species of the animal is not particularly limited. For reasons of cost, stability of production, in some examples of manufacturing methods, the animal includes, but is not limited to, at least one of mammals such as swine, cattle, mice, sheep, horses, and the like. The source of the mammal is more stable and the treatment difficulty is relatively low.
The cold accumulating agent absorbs the heat generated by irradiation and has no special requirement on its kind. In order to avoid adverse effects of the cold storage agent on the collagen, in some examples of the preparation method, the cold storage agent includes, but is not limited to, at least one of an ice bag, ice cubes, dry ice, and a polyvinyl alcohol-borax high-efficiency cold storage agent. The ice bag and the ice block have large specific heat capacity and low cost, and cannot cause adverse effect on collagen even if being melted; the dry ice absorbs heat and volatilizes, and does not have adverse effect on the collagen.
The dosage of the cold storage agent can be adjusted according to the heat generated in irradiation, and in some preparation method examples, the mixing ratio of the cold storage agent and the degreased connective tissue powder is (0.5-20): 1. this ratio of mixing provides good assurance that the connective tissue powder remains below 0 c during irradiation treatment.
In some examples of the preparation method, the collagen solution has a concentration of 10 to 20 mg/mL, a viscosity of 100 to 4500 cP at 20 ℃, a molecular weight of about 300 kDa, and a complete triple helix structure.
In some examples of preparation methods, the collagen solution has a positive and negative absorption peak near 220 nm and 197 nm, respectively, as determined by circular dichroism scanning, and the complete triple-helix structure is confirmed to be retained.
In a second aspect of the present invention, there is provided:
a collagen solution prepared by the method of the first aspect of the present invention.
In some examples of the collagen solution, the concentration is 10-20 mg/mL, the viscosity at 20 ℃ is 100-4500 cP, the molecular weight is about 300 kDa, and the collagen solution has a complete triple helix structure.
In some examples of the collagen solution, when the circular dichroism scanning measurement is carried out, the collagen solution has a positive absorption peak and a negative absorption peak near 220 nm and 197 nm respectively, which proves that the collagen solution retains a complete triple-helix structure, the collagen triple-helix structure is the key for exerting the complete efficacy, and the collagen molecule with the complete triple-helix structure has better bioactivity than that of the small molecule collagen.
The technical scheme of the invention is further explained by combining the embodiment.
In the following examples, the connective tissue is washed, pulverized, defatted, and enzymatically hydrolyzed according to conventional methods. The coolant is used in an amount to maintain the temperature of the defatted connective tissue at no more than 0 ℃ during the irradiation treatment.
Example 1:
the embodiment provides a preparation method of a high-fluidity high-concentration collagen solution, which specifically comprises the following steps:
s1) taking 1 kg of fresh bovine achilles tendon, and washing bloodiness and attachments on the surface by using 5 kg of solution; then placing the mixture into a mould, freezing the mixture for 10 hours at the temperature of minus 20 ℃, and then finishing crushing and degreasing treatment.
S2) sealing the degreased achilles tendon by a sealing bag, adding 15 kg of coolant, and carrying out irradiation treatment under the irradiation dose of 10 kGy, wherein the irradiation source is gamma rays;
s3), adding the mixed solution of the acid enzyme, reacting for 48 hours at the temperature of 28-33 ℃, and then purifying to obtain the high-concentration collagen solution.
The final concentration of the collagen solution at 20 ℃ was measured to be 19.36 mg/mL and the viscosity was measured to be 4056 cP, and after leaving for 45min, the viscosity was measured to be 4073 cP.
Example 2:
the embodiment provides a preparation method of a high-fluidity high-concentration collagen solution, which specifically comprises the following steps:
s1) taking 2.5 kg of fresh pig achilles tendon, and washing blood stain and attachments on the surface by using 7.5 kg of solution; then placing the mixture into a mould, and freezing the mixture for 28 hours at the temperature of minus 20 ℃; and then the crushing and degreasing treatment are finished.
S2) sealing the degreased pig achilles tendon by a sealing bag, adding 12 kg of coolant, and carrying out irradiation treatment under the irradiation dose of 13 kGy, wherein the irradiation source is an electron beam;
s3), adding the mixed solution of the enzyme, reacting for 72 hours at the temperature of 28-33 ℃, and then purifying to obtain the high-concentration collagen solution.
The final concentration of the collagen solution at 20 ℃ was determined to be 16.53 mg/mL, the viscosity was determined to be 3098 cP, and the viscosity was determined to be 3103 cP after standing for 45 min.
Example 3
The embodiment provides a preparation method of a high-concentration high-fluidity collagen solution, which specifically comprises the following steps:
s1) taking 0.5 kg of fresh rat achilles tendon, and washing blood stain and attachments on the surface by using 2 kg of solution; placing the cleaned Achilles tendrils into a mould, and freezing for 48 h at-80 ℃; and then the crushing and degreasing treatment are finished.
S2) sealing the degreased achilles tendon by a sealing bag, adding 17 kg of coolant, and performing irradiation treatment under the irradiation dose of 15 kGy, wherein the irradiation source is gamma rays;
s3), adding the mixed solution of the acid enzyme, reacting for 48 hours at the temperature of 28-33 ℃, and then purifying to obtain the high-concentration collagen solution.
The final concentration of the collagen solution at 20 ℃ was measured to be 13.06 mg/mL and the viscosity to be 2182 cP, and after leaving for 45min the viscosity to be measured was 2163 cP.
Example 4:
the embodiment provides a preparation method of a high-concentration high-fluidity collagen solution, which specifically comprises the following steps:
s1) taking 3.5 kg of fresh pigskin, and washing bloodiness and attachments on the surface by using 10 kg of solution; placing the cleaned pigskin in a mould, and freezing for 36 h at-20 ℃; and then the crushing and degreasing treatment are finished.
S2) sealing the degreased pigskin by a sealing bag, adding 12 kg of coolant, and carrying out irradiation treatment under the irradiation dose of 20 kGy, wherein the irradiation source is gamma rays;
s3), adding the mixed solution of the enzyme, reacting for 72 hours at the temperature of 28-33 ℃, and then purifying to obtain the high-concentration collagen solution.
The final concentration of the collagen solution at 20 ℃ was determined to be 10.01 mg/mL, the viscosity to be 856 cP, and the viscosity to be 871 cP after leaving for 45 min.
Comparative example 1:
the comparative example provides a preparation method of a high-concentration collagen solution, which specifically comprises the following steps:
taking fresh bovine achilles tendon, completing cleaning, slicing, crushing and degreasing treatment according to a conventional method, then carrying out no irradiation treatment, adjusting the feeding proportion to be half of that of irradiation treatment in consideration of higher viscosity when carrying out non-irradiation enzymolysis, and preparing collagen solution after carrying out enzymolysis treatment and purification.
The final concentration of the collagen solution at 20 ℃ was measured as 11.59 mg/mL, the viscosity was measured as 50256 cP, and the viscosity was measured as 50250 cP after standing for 45 min.
Comparative example 2:
the comparative example provides a preparation method of a high-concentration high-fluidity collagen solution, which specifically comprises the following steps:
homogenizing the collagen solution obtained in the comparative example 1 at 20-28 ℃ at a homogenizing speed of 3200 rpm, a stirring speed of 20 rpm and a vacuum degree of-0.08 MPa for 10 min to obtain a homogeneous collagen solution.
The final concentration of the collagen solution was measured at 11.59 mg/mL and the viscosity was measured at 3086 cP at 20 ℃ and 12056 cP after standing for 45 min.
Comparative example 3:
the comparative example provides a preparation method of a high-concentration collagen solution, which specifically comprises the following steps:
homogenizing the collagen solution of the comparative example 1 at 20-28 ℃ at a homogenizing speed of 2300 rpm, a stirring speed of 25 rpm and a vacuum degree of-0.08 MPa for 8 min to obtain the homogeneous collagen solution.
The final concentration of the collagen solution was measured at 11.59 mg/mL and the viscosity was measured at 5351 cP at 20 ℃ and after standing for 45min the viscosity was measured to 18632 cP.
Comparative example 4:
the comparative example provides a method for preparing a collagen solution, specifically comprising the steps of:
s1) taking fresh achilles tendon, and finishing the cleaning, slicing, crushing and degreasing treatment according to the conventional method.
S2) sealing the degreased pig achilles tendon by a sealing bag, adding 18 kg of coolant, and carrying out irradiation treatment under the irradiation dose of 25 kGy, wherein the irradiation source is gamma rays;
s3) directly carrying out enzymolysis treatment according to the same feeding proportion as in the embodiment, and purifying to obtain the collagen solution.
The final concentration of the collagen solution at 20 ℃ was measured to be 8.46 mg/mL and the viscosity was measured to be 263 cP, and after standing for 45min, the viscosity was measured to be 242 cP.
Test example 1: change in collagen solution irradiation viscosity
The viscosity change of the collagen solution extracted in examples 1 to 4 and comparative examples 1 to 4 was measured, and a rotational viscometer was used for the measurement (specifically, the measurement method was referred to "Chinese pharmacopoeia" 2020 edition 0633 third method of viscosity measurement). The results of the experiment are shown in FIG. 1.
FIG. 1 shows the viscosity change of collagen solution under different pretreatment conditions and homogenization conditions for raw materials in examples 1 to 4 and comparative examples 1 to 4, wherein:
the viscosity of the collagen solution obtained in the comparative example 1 (without irradiation) is 50256 cP, while the viscosities of the collagen solutions obtained in the examples 1-4 and the comparative example 4 after raw material irradiation treatment are 4056 cP, 3098 cP, 2182 cP, 856 cP and 263 cP respectively, which shows that the raw material is extracted after irradiation treatment, the viscosity of the extraction solution can be greatly reduced, and the viscosity is in a linear descending trend along with the increase of irradiation dose; after the examples 1-4 and the comparative example 4 are left for 45min, the viscosities of the solutions are 4073 cP, 3103 cP, 2163 cP, 871 cP and 242 cP respectively, which shows that the viscosity of the collagen solution extracted by the irradiation post-treatment is stable and no re-rise occurs.
Comparative examples 2 to 3 are viscosity measurement results of the solutions obtained in comparative example 1 immediately after the homogenization treatment and after the solutions are left for 45min, and the results show that the viscosities of the solutions obtained in comparative examples 2 and 3 immediately after the homogenization treatment are respectively reduced to 3086 cP and 5351 cP, and that the homogenization has a certain shear thinning effect on the high-viscosity solutions, but after the solutions are left for 45min, the viscosities of the solutions are obviously increased to 12056 cP and 18632 cP, which indicates that the viscosity retention period of the solutions after the homogenization treatment is short. The collagen solution obtained by the treatment of the embodiments 1 to 4 can be determined to have better machine filling performance.
TABLE 1 viscosity change Table for collagen solution of different treatments
Test example 2: yield of collagen solution
The yields of the solutions obtained in examples 1 to 4 and comparative examples 1 to 4 were measured by a biuret method (the test method is referred to in "Chinese pharmacopoeia" 2020 edition, and the results are shown in Table 2.
Table 2 shows the collagen yields obtained under different treatment conditions of examples 1 to 4 and comparative examples 1 to 4, wherein comparative example 1 shows that the extraction yield of the raw material without irradiation treatment is about 34.7%, and comparative examples 2 and 3 show that the extraction yield of the raw material does not change after homogenization treatment; examples 1 to 4 and comparative example 4 are extraction results of different irradiation dose treatments, and the results show that the solution yield decreases as the irradiation dose increases, wherein the solution yield is higher under the irradiation dose not exceeding 20 kGy. While the result of the extraction yield of the solution treated with the irradiation dose of 30 kGy in comparative example 4 shows that the yield of the solution treated with the irradiation dose is only 9.69%, which suggests that a collagen solution having a yield comparable to that of the conventional extraction and homogenization treatment can be obtained by controlling the irradiation dose appropriately.
TABLE 2 collagen solution yield table
Test example 3: results of circular dichroism test of collagen solution
The results of the circular dichroism scan measurement of the collagen liquids (after standing for 45 min) obtained in examples 1 to 4 and comparative examples 1 to 4 are shown in fig. 2.
In general, the CD spectrum of a collagen molecule with an intact triple helix structure shows a positive peak at a wavelength of 220 nm and a negative peak at a wavelength of 197 nm. The denatured collagen was destroyed due to the triple helix structure, resulting in disappearance of its positive peak at 220 nm and weakening of the negative peak at 197 nm. Examples 1-4 and comparative example 1 show that the collagen solution extracted without irradiation treatment and with appropriate dose of irradiation treatment has a positive and negative absorption peak near 220 nm and 197 nm, respectively, which indicates that the raw material is less affected by the irradiation treatment with appropriate dose, and collagen molecules in the extracted solution still maintain a complete triple-helical structure. Compared with comparative example 1 and comparative examples 2 and 3, the CD result of the solution after homogenization treatment shows that the collagen solution has no obvious change before and after homogenization treatment under the parameter; while comparative example 4 shows that the positive and negative peaks of the collagen solution irradiated at a dose of 30 kGy are significantly reduced, indicating that the collagen molecules have been partially denatured by irradiation at this dose.
Test example 4: molecular structure change of collagen solution
The collagen solutions obtained in examples 1 to 4 and comparative examples 1 to 4 were subjected to polyacrylamide gel electrophoresis (after leaving for 45 min). The results of the assay according to SDS-polyacrylamide gel electrophoresis are shown in FIG. 3.
Examples 1 to 4 and comparative examples 1 to 3 each have alpha 1 Chain and alpha 2 Chain and a 1 Chain belt ratio of about alpha 2 Twice the chain, which is a structural feature of typical type I collagen, has a molecular weight of about 300 kDa (type I collagen molecular weight =2 x α) 1 Chain molecular weight + α 2 Chain molecular weight). Meanwhile, the samples of examples 1 to 4 and comparative examples 1 to 3 have no obvious impurity band under 97.2 kDa, which indicates that neither irradiation treatment nor solution homogenization treatment damages the extraction solution and the molecular structure in the collagen solution has high integrity. The results show that the raw material irradiation treatment can obtain a high-concentration and high-fluidity glue solution without destroying the biological activity of the glue solution. In comparative example 4, it is possible to see that the electrophoretic band becomes obviously shallow after the treatment with too high irradiation dose, which indicates that the collagen is partially denatured under the high irradiation dose, and the result is consistent with the CD result.
The above results show that:
1) according to the method, the high-concentration high-fluidity collagen solution can be obtained only by optimizing the irradiation dose without changing other processes, and the method has small influence on the yield of the collagen;
2) the collagen molecules in the collagen solution prepared by the method have typical type I collagen structural characteristics and a complete triple helix structure, the molecular weight is about 300 kDa, and the collagen solution has good biological activity;
3) the collagen solution prepared by the method has good viscosity stability, keeps stable viscosity after long-term storage, basically has no rise, and has high processability.
Possible reasons for the reduction in viscosity of the final collagen solution due to the irradiation treatment
The mechanism of the viscosity reduction of collagen solution after enzymolysis caused by irradiation treatment of defatted connective tissue powder is not clear, and based on the detection results, the collagen extraction yield of the method is slightly lower than that of collagen solution without irradiation treatment, the viscosity is mainly the macroscopic expression of intermolecular hydrogen bond and ionic bond, and the inventor conjectures the possible reason to be that: firstly, the irradiation treatment has higher energy, so that part of collagen molecules are damaged, and the number of small molecules in the solution is increased; secondly, small molecules compete with collagen molecules through hydrogen bonds and ionic bonds, and the interaction between the collagen molecules is reduced due to the increase of the number of the small molecules, so that the entanglement and aggregation of the collagen molecules in the purification process are reduced. Finally, the viscosity of the whole body is remarkably reduced, and the collagen molecule still has the molecular weight of about 300 kDa and also has a triple-helical structure.
The foregoing is a more detailed description of the invention and is not to be taken in a limiting sense. It will be apparent to those skilled in the art that various modifications, additions and substitutions can be made without departing from the spirit and scope of the invention.
Claims (10)
1. A preparation method of a collagen solution comprises the following steps:
s1) taking fresh animal connective tissue, crushing and degreasing after cleaning to obtain degreased connective tissue powder;
s2) carrying out irradiation treatment on the degreased connective tissue powder, wherein the irradiation dose is 10-20 kGy;
s3) performing enzymolysis and purification on the irradiated connective tissue powder to obtain a collagen solution.
2. The production method according to claim 1, characterized in that: maintaining the connective tissue at a temperature of
-20～0 ℃。
3. The method of claim 1, wherein: the irradiation source of the irradiation is gamma rays or electron beams.
4. The method of claim 1, wherein: placing the defatted connective tissue powder in a sealed bag, adding cold storage agent, sealing, and performing irradiation treatment.
5. The method of claim 4, wherein: the cold storage agent is at least one of an ice bag, an ice block, dry ice and a polyvinyl alcohol-borax high-efficiency cold storage agent.
6. The production method according to any one of claims 1 to 4, characterized in that: the connective tissue is selected from tendon, skin, cartilage or scales of animals.
7. The production method according to any one of claims 1 to 4, characterized in that: the animal is selected from at least one of pig, cattle, mouse, sheep, and horse.
8. The production method according to claim 1 or 4, characterized in that: the mixing ratio of the cold accumulation agent to the degreased connective tissue powder is (0.5-20): 1; and/or the mass mixing ratio of the connective tissue powder to the enzyme liquid is 1: (40-80).
9. A collagen solution obtained by the method according to any one of claims 1 to 8.
10. The collagen solution according to claim 9, wherein: the concentration of the nano-particles is 10-20 mg/mL, the viscosity at 20 ℃ is 100-4500 cP, the molecular weight is about 300 kDa, and the nano-particles have a complete triple helix structure.
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