CN108659117B - Method for quantitatively detecting content of collagen triple helix structure - Google Patents
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Abstract
The invention discloses a method for quantitatively detecting the content of a collagen triple helix structure, and belongs to the technical field of biological detection. The invention utilizes the principle that collagen with a complete triple helical structure can not be enzymolyzed by trypsin, and collagen damaged by the triple helical structure can be enzymolyzed by trypsin, and calculates the content of the complete triple helical structure of the collagen by measuring the content change of the characteristic amino acid-hydroxyproline of the collagen in a collagen solution system before and after the enzymolysis of the trypsin. The method can quantitatively judge the damage degree of the triple helix structure of the collagen under various process conditions by quantitatively detecting the triple helix of the collagen, makes up for the deficiency of qualitative detection, is suitable for complex biological systems containing the collagen, has strong specificity, good result reproducibility and simple operation, and can be used as an application means for detecting the development process of collagen products.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a method for quantitatively detecting the content of a collagen triple helix structure.
Background
Collagen is an important component of the extracellular matrix of vertebrates, is the most abundant protein in mammals, and accounts for about 20-30% of the total protein content. Because the collagen has the advantages of obvious fiber forming structure, good biodegradability, excellent biocompatibility, lower antigenicity and the like, the collagen is widely applied to the fields of beauty industry, pharmaceutical industry, food and biomedicine. The triple helix structure of collagen allows cells to adsorb, migrate on the collagen matrix and even regulate and promote cellular differentiation, and as an important extracellular matrix, collagen helps to maintain the stability of tissues or organs and maintain their functional integrity. Therefore, the triple helix structure is an important mark of the bioactivity of the collagen and is a precondition for the functional performance of the collagen. However, the triple helical structure of native collagen is very susceptible to the processing environment, and physical heat, mechanical stirring, shearing, chemical reagents and the like are inevitably introduced in the production and processing of collagen, which can cause partial or even complete destruction of the triple helical structure of collagen, resulting in the loss of the native bioactive function of collagen.
At present, the qualitative detection method of the collagen triple helix structure mainly comprises a circular dichroism method and an infrared spectrum method, and for example, the circular dichroism characteristics of the collagen are as follows: a strong negative absorption at about 198nm and a weaker positive absorption at about 220nm, the intensity of the absorption being reflected in the degree of retention of the triple helical structure of collagen; as another example, infrared spectroscopy reflects changes in the secondary structure of a protein by observing the absorption of specific bond energies. However, the above methods can only qualitatively judge the destruction degree of triple helix structure in collagen by comparing with a reference substance, and cannot quantitatively detect the content of triple helix structure.
In addition, patent No. CN 106383232, "a method for detecting a collagen triple helix structure using a disulfide nanomaterial", discloses a method for detecting a collagen triple helix structure, but the method has the following drawbacks: 1) the operation is complex when the fluorescent probe is prepared; 2) the method comprises the steps of forming a collagen triple helix structure by probe polypeptide and polypeptide to be detected, and further detecting the triple helix structure, wherein the triple helix structure is not a collagen triple helix structure originally existing in a detection system; 3) it is known that high viscosity is a characteristic of collagen solution, but the method is difficult to measure the content of triple helix structure in high viscosity collagen system.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for quantitatively detecting the content of the triple helical structure of collagen.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for quantitatively detecting the content of a collagen triple helix structure comprises the steps of adding trypsin into a collagen sample solution for enzymolysis reaction, separating out collagen with a complete triple helix structure by using a trichloroacetic acid precipitation method, measuring the content of hydroxyproline in the collagen before and after enzymolysis, and calculating the ratio of the content of hydroxyproline in the collagen with the complete triple helix structure separated out after the enzymolysis to the content of hydroxyproline in the collagen before the enzymolysis, so that the content of the collagen with the triple helix structure in the sample is obtained.
The invention utilizes the principle that collagen with a complete triple helix structure can not be enzymolyzed by trypsin, and collagen with a complete triple helix structure or a part of the triple helix structure damaged can be enzymolyzed by the trypsin, and calculates the content of the complete triple helix structure in the collagen by detecting the content change of the characteristic amino acid-hydroxyproline of the collagen in a collagen solution system before and after the enzymolysis of the trypsin. The detection method provided by the invention overcomes the defect that the prior art can only qualitatively detect the triple helical structure of the collagen, is simple to operate, and is not influenced by the high viscosity of a collagen system.
As a preferred embodiment of the method for quantitatively detecting the content of the triple helical structure of the collagen, the method comprises the following steps:
(1) adjusting the pH value of the sample liquid to 3.6-3.7 for later use;
(2) adding the trypsin liquid into the sample liquid, and uniformly mixing to obtain a system liquid to be subjected to enzymolysis;
(3) weighing part of the solution to be subjected to enzymolysis, adding a hydrochloric acid solution, hydrolyzing, and determining the content of hydroxyproline in the hydrolysate to obtain the content of hydroxyproline in the solution to be subjected to enzymolysis before enzymolysis;
(4) placing the sampled solution to be subjected to enzymolysis under the constant-temperature oscillation condition for enzymolysis reaction;
(5) weighing the solution after enzymolysis, treating and separating out collagen precipitate and supernatant with a complete triple helical structure by a trichloroacetic acid precipitation method, hydrolyzing the precipitate or supernatant, determining the content of hydroxyproline in hydrolysate, and calculating the ratio of the content of hydroxyproline in the collagen with the complete triple helical structure to the content of hydroxyproline in the collagen before enzymolysis.
According to the invention, the content of hydroxyproline in the system liquid to be subjected to enzymolysis before enzymolysis is the content of hydroxyproline in collagen before enzymolysis, after enzymolysis reaction, collagen with a complete triple helix structure cannot be subjected to enzymolysis by trypsin, collagen with a triple helix structure damaged/partially damaged can be subjected to enzymolysis by trypsin to form polypeptide, the molecular weight of the polypeptide is much smaller than that of the collagen, the collagen is easily dissolved by trichloroacetic acid solution, and the collagen without enzymolysis is difficultly dissolved in trichloroacetic acid solution and precipitated at the bottom through centrifugation, and the polypeptide is dissolved in supernatant, so that the content of the collagen with the complete triple helix structure can be calculated by measuring the content of hydroxyproline in hydrolysate of protein precipitate.
As a preferred embodiment of the method for quantitatively detecting the content of the triple helical structure of collagen according to the present invention, in the step (2), the preparation method of the trypsin solution comprises: weighing trypsin, adding 0.05M CaCl2In 0.001MHCl solution.
In a preferred embodiment of the method for quantitatively detecting the content of the triple helical structure of collagen according to the present invention, in the step (2), the mass ratio of trypsin to total protein in the sample solution is 1:20 to 1: 100.
As a preferred embodiment of the method for quantitatively detecting the content of the triple helical structure of the collagen, in the step (4), the enzymolysis temperature is 15-30 ℃, the oscillation speed is 50-120 rmp, and the enzymolysis time is 1-24 h.
In the technical scheme, through a plurality of tests, the inventor optimizes the mass ratio of the trypsin to the total protein of the sample solution, the enzymolysis temperature, the oscillation speed, the enzymolysis time and other parameters, so that the collagen with the damaged/partially damaged triple helical structure in the collagen can be fully enzymolyzed into the polypeptide by the trypsin, and the accuracy of the detection result is improved.
As a preferred embodiment of the method for quantitatively determining the content of the collagen triple helix structure according to the present invention, in the step (5), the step of processing and separating the collagen precipitate and the supernatant having the complete triple helix structure comprises: adding trichloroacetic acid solution with the mass concentration of 10-30% at 4 ℃ into the solution after enzymolysis, wherein the mass ratio of the solution after enzymolysis to the trichloroacetic acid solution is 1: 0.5-2, shaking for 10s, uniformly mixing, and centrifuging for 10min at 4 ℃ at 5000 rmp.
As a preferred embodiment of the method for quantitatively determining the content of the triple helical structure of collagen according to the present invention, in the step (5), a precipitation treatment is selected: discarding the supernatant, cleaning the surface of the precipitate with trichloroacetic acid solution with the mass concentration of 10% at 4 ℃ for 3 times, adding hydrochloric acid solution, hydrolyzing, and determining the content of hydroxyproline in the hydrolysate, wherein the content of the collagen triple helix structure in the sample is calculated according to the formula (1):
in the formula:
NC is the content of the collagen triple helix structure in the sample, and the unit is;
X1content of hydroxyproline in the solution of the system to be subjected to enzymolysis before enzymolysis, X2And (4) after enzymolysis, the content of hydroxyproline in the enzymolysis system liquid is mg/g.
As a preferred embodiment of the method for quantitatively detecting the content of the triple helical structure of collagen according to the present invention, in the step (5), a supernatant treatment is selected: weighing the supernatant, adding a hydrochloric acid solution, hydrolyzing, and measuring the content of the hydroxyproline in the hydrolysate, wherein the content of the collagen triple helix structure in the sample is calculated according to the formula (2):
in the formula:
NC is the content of the collagen triple helix structure in the sample, and the unit is;
X1content of hydroxyproline in the solution of the system to be subjected to enzymolysis before enzymolysis, X2And (4) after enzymolysis, the content of hydroxyproline in the enzymolysis system liquid is mg/g.
As a preferred embodiment of the method for quantitatively detecting the content of the triple helix structure of the collagen, after the hydrolysis is finished, the content of hydroxyproline in the hydrolysate is determined by a waters amino acid derivation method, an ultraviolet spectrophotometry method or an amino acid analyzer method.
Compared with the prior art, the invention has the advantages that:
1) the invention makes up the defects of the existing method for qualitatively detecting the triple helical structure of the collagen, and provides a method for quantitatively detecting the content of the triple helical structure of the collagen by utilizing the enzymolysis of trypsin;
2) the method quantitatively judges the damage degree of the collagen triple-helical structure under various process conditions by quantitatively detecting the collagen triple-helical structure;
3) the invention is suitable for complex biological systems containing collagen, has strong specificity and good result reproducibility, and can be used as an application means for detecting the development process of collagen products.
Drawings
FIG. 1 is a schematic diagram of the principle of detecting the triple helix structure of collagen by trypsin enzymolysis according to the present invention.
FIG. 2 is a gel electrophoresis diagram of collagen liquid and gelatin liquid at 25 deg.C, wherein C in C0h 25 at 25 deg.C represents collagen liquid, 0h represents enzymolysis for 0h, 25 deg.C represents enzymolysis at 25 deg.C, and so on; in addition, G represents the enzymatic hydrolysis of gelatin solution by trypsin, and S is a collagen standard.
FIG. 3 is a gel electrophoresis of collagen at 30 and 37 deg.C, wherein C in C0h 30 deg.C represents collagen solution, 0h represents enzymolysis for 0h, 30 deg.C represents enzymolysis at 30 deg.C, and so on; further, G represents the enzymatic hydrolysis of gelatin solution with trypsin.
Figure 4 circular dichroism chromatogram of collagen sample.
FIG. 5 is an infrared absorption spectrum of a collagen sample.
Figure 6 enthalpy diagrams of collagen samples.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. It will be understood by those skilled in the art that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The test methods used in the following examples are all conventional methods unless otherwise specified; materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
FIG. 1 is a schematic diagram of the principle of detecting the triple helix structure of collagen by trypsin enzymolysis according to the present invention. According to the invention, after trypsin is added into a collagen sample liquid for enzymolysis reaction, collagen with a damaged triple helical structure is converted into polypeptide through trypsin enzymolysis, collagen with a complete triple helical structure is separated by a trichloroacetic acid precipitation method, and the ratio of hydroxyproline in the collagen with the complete triple helical structure separated after enzymolysis to hydroxyproline in the collagen sample liquid before enzymolysis is calculated by measuring the content of hydroxyproline in the collagen before and after enzymolysis, so that the content of the collagen with the triple helical structure in the sample is obtained.
The method for quantitatively detecting the content of the collagen triple helix structure comprises the following steps:
(1) putting 50mL of sample liquid into a 150mL conical flask, and adjusting the pH of the sample liquid to 3.6-3.7 by using 0.5M acetic acid solution or NaOH solution;
(2) preparation of trypsin solution: weighing trypsin, adding 0.05M CaCl2To obtain a trypsin solution in 0.001M HCl solution;
(3) adding 1mL of trypsin solution into the sample solution, wherein the mass ratio of trypsin to total protein of the sample solution is 1: 20-1: 100, and uniformly stirring to obtain a system solution to be subjected to enzymolysis;
(4) treatment of the sample before enzymolysis: accurately weighing the solution to be subjected to enzymolysis into a hydrolysis tube, adding a hydrochloric acid solution, hydrolyzing, measuring the content of hydroxyproline in the hydrolysate, and calculating the content of hydroxyproline in the solution to be subjected to enzymolysis before enzymolysis;
(5) placing the sampled solution to be subjected to enzymolysis under the constant-temperature oscillation condition for enzymolysis reaction;
(6) treating a sample after enzymolysis: weighing the liquid after enzymolysis, adding trichloroacetic acid solution with the mass concentration of 10-30% at 4 ℃, oscillating for 10s, uniformly mixing, and centrifuging for 10min at 4 ℃ at 5000rmp to separate precipitate and supernatant; selecting supernatant or sediment for processing, and performing the following operations after selection:
the selection treatment is a precipitation operation: discarding the supernatant, cleaning the surface of the precipitate with trichloroacetic acid solution with the mass concentration of 10% at 4 ℃ for 3 times, adding hydrochloric acid solution, hydrolyzing, and determining the content of hydroxyproline in the hydrolysate, wherein the content of the collagen triple helix structure in the sample is calculated according to the formula (1):
in the formula:
NC is the content of the collagen triple helix structure in the sample, and the unit is;
X1before enzymolysis, the content of hydroxyproline in the system liquid to be subjected to enzymolysis, namely the total content of collagen in the system liquid to be subjected to enzymolysis, wherein the unit is mg/g;
X2and (3) the hydroxyproline content in the system liquid to be subjected to enzymolysis, namely the collagen content with a complete triple-helix structure in the system liquid to be subjected to enzymolysis, wherein the unit is mg/g.
Selecting and processing a supernatant fluid operation: weighing the supernatant, adding a hydrochloric acid solution, hydrolyzing, and measuring the content of the hydroxyproline in the hydrolysate, wherein the content of the collagen triple helix structure in the sample is calculated according to the formula (2):
in the formula:
NC is the content of the collagen triple helix structure in the sample, and the unit is;
X1the hydroxyproline content in the system liquid to be subjected to enzymolysis before enzymolysis, namely the total content of collagen in the system liquid to be subjected to enzymolysis, wherein the unit is mg/g;
X2after enzymolysis, the content of hydroxyproline in the system liquid to be subjected to enzymolysis, namely the content of collagen damaged by a triple-helix structure in the system liquid to be subjected to enzymolysis is mg/g;
in the above formulae (1) and (2),
C1the content of hydroxyproline in the hydrolysate before enzymolysis is mg/g;
C3the content of hydroxyproline in the hydrolysate after enzymolysis is mg/g;
M1-mass of hydrolysate in g before enzymolysis;
M2the sampling amount of the solution of the system to be enzymolyzed before enzymolysis is g;
M3the mass of the hydrolysate after enzymolysis is g;
M4the sample volume of the liquid after the enzymatic hydrolysis, in g.
The invention adopts a waters amino acid derivation method, an ultraviolet spectrophotometry method or an amino acid analyzer method to determine the content of hydroxyproline in hydrolysate.
The invention can adjust the hydrolysis temperature and hydrolysis time according to the existing collagen hydrolysis method, and fully convert the collagen into the amino acid.
Example 1 determination of the temperature of the enzymatic digestion of Trypsin
Respectively taking 2 parts of 50mL collagen solution with the concentration of 4mg/mL, putting one part of the collagen solution in a water bath kettle at the temperature of 60 ℃, heating for 10min to destroy the three-strand helical structure of the collagen, and then cooling to 25 ℃ to prepare gelatin solution; the other is ready for use. Adjusting pH of the two solutions to 3.7 with 0.5M acetic acid solution, adding trypsin solution with a mass ratio of 1:20 of trypsin to total protein of the sample solution, oscillating at 25 deg.C with rotation speed of 95 + -2 rmp, and performing enzymolysis for 24 h. The samples were taken at intervals and analyzed in gel electrophoresis with 8% separation gel, and the results are shown in fig. 2, the beta chain and the alpha chain of the collagen liquid are not degraded after 24h enzymolysis at 25 ℃, while the alpha chain and the bands below the alpha chain are gradually reduced to disappear with the extension of the enzymolysis time under the action of trypsin. It can be seen that the temperature of the trypsin digestion can be 25 ℃.
FIG. 3 is a gel electrophoresis of collagen enzymatic hydrolysis at 30 and 37 ℃. The inventor finds that the beta chain and the alpha chain of the collagen liquid are not degraded after enzymolysis for 24 hours at 25 ℃ and 30 ℃ respectively; under the action of trypsin, the gelatin solution gradually becomes smaller and smaller with the extension of enzymolysis time until the alpha chain and bands below the alpha chain disappear. At 37 ℃, the main characteristic bands (gamma, beta and alpha) of the collagen become lighter with the prolongation of enzymolysis time, which indicates that the whole bands of the collagen in the solution are gradually degraded by enzyme. According to the thermal denaturation of collagen, the triple-helical structure of collagen is partially or completely destroyed at a temperature higher than the denaturation temperature of collagen; when the enzymolysis temperature is too low, the enzyme activity is reduced, and the enzymolysis time is prolonged. Therefore, the enzymolysis temperature is lower than the denaturation temperature of the collagen.
In conclusion, the enzymolysis temperature of the trypsin in the invention is preferably 15-30 ℃; the enzymolysis time is preferably 1-24 h.
Example 2 summary of results calculated using pellets or supernatants after tryptic digestion
(1) Diluting collagen protein solution with concentration of 4.0mg/mL to 1.0mg/mL with distilled water at 4 deg.C; 50mL of collagen with a concentration of 4.0mg/mL and 1.0mg/mL respectively was taken, and the pH of the solution was adjusted to 3.7. + -. 0.05 with 0.5M acetic acid solution. Adding trypsin liquid respectively, wherein the mass ratio of trypsin to total protein of the sample liquid is 1:40, uniformly stirring to obtain a system liquid to be subjected to enzymolysis, and treating;
(2) respectively putting 2.00 +/-0.005 g of system liquid to be enzymolyzed into a hydrolysis tube, adding 5mL of hydrochloric acid solution with the concentration of 8.4M, placing at 110 ℃ for hydrolysis, and determining the content of hydroxyproline in the hydrolysate, namely the total content of collagen in the system liquid to be enzymolyzed;
(3) oscillating the sampled collagen liquid at 25 ℃, and carrying out enzymolysis for 4h at the rotating speed of 95 +/-2 rmp;
(4) treating a sample after enzymolysis: accurately weighing 2.00 + -0.005 g of the liquid after enzymolysis, adding 1.00 + -0.005 g of trichloroacetic acid solution with the mass concentration of 30% at 4 ℃, vibrating for 10s, uniformly mixing, and centrifuging for 10min at 4 ℃ and 5000rmp to separate precipitate and supernatant. Selecting supernatant or sediment for processing, and performing the following operations after selection:
the selection treatment is a precipitation operation: the supernatant was discarded, the surface of the precipitate was washed with 10% trichloroacetic acid solution at 4 ℃ for 3 times, and 5mL of 6M hydrochloric acid solution was added and hydrolyzed at 110 ℃ for 24 hours.
Selecting and processing a supernatant fluid operation: 2.00. + -. 0.005g of the supernatant was weighed out accurately, and 5mL of 8.4M hydrochloric acid solution was added and hydrolyzed at 110 ℃ for 24 hours.
(5) After hydrolysis is finished, determining the content of hydroxyproline in the hydrolysate by using a waters amino acid derivation method, calculating the content of hydroxyproline in a system liquid to be subjected to enzymolysis before and after the enzymolysis, calculating the content of collagen with a triple helix structure in a sample liquid by using precipitation according to a formula (1), and calculating the content of collagen with a triple helix structure in the sample liquid by using a supernatant according to a formula (2).
As shown in table 1, under this method, there was no significant difference in the content of triple helical structure in collagen, whether calculated using pellet or supernatant; in addition, the same collagen sample is diluted to different concentrations, and the content of the triple helix structure in the collagen obtained by the method has no significant difference.
In step (3) of this embodiment, the oscillation rotation speed is adjusted to 50rmp and 120rmp respectively to ensure sufficient enzymolysis reaction of collagen.
TABLE 1 content of triple helix in collagen calculated using pellet and supernatant of samples after enzymatic hydrolysis
Example 3 content of collagen triple helix Structure in Complex System
(1) Heating collagen solution in 60 deg.C water bath for 10min to destroy three-strand spiral structure of collagen, cooling to 25 deg.C, and preparing gelatin solution; mixing the prepared gelatin solution and collagen solution to prepare a mixed solution of gelatin and collagen solution, wherein the mixing ratio is shown in table 2;
(2) respectively taking 50mL of mixed solution, adjusting the pH value of the solution to 3.7 +/-0.05 by using 0.5M acetic acid solution, respectively adding trypsin solution, wherein the mass ratio of trypsin to total protein of the sample solution is 1:100, uniformly stirring to obtain a to-be-enzymolyzed system solution for treatment;
(3) respectively putting 2.00 +/-0.005 g of system liquid to be enzymolyzed into a hydrolysis tube, adding 5mL of hydrochloric acid solution with the concentration of 8.4M, placing at 110 ℃ for hydrolysis, and determining the content of hydroxyproline in the hydrolysis liquid;
(4) carrying out enzymolysis on the sampled system liquid to be subjected to enzymolysis at 30 ℃ for 1 h; after the enzymolysis is finished, respectively weighing 2.00 +/-0.005 g of liquid after enzymolysis, adding 1.00 +/-0.005 g of trichloroacetic acid solution with the mass concentration of 30% at 4 ℃, oscillating for 10s, uniformly mixing, and centrifuging for 10min at 4 ℃ and 5000rmp to separate precipitates and supernatant. Discarding supernatant, washing the surface of the precipitate with 10% trichloroacetic acid solution at 4 deg.C for 3 times, adding hydrochloric acid solution, and hydrolyzing at 110 deg.C.
(5) After the hydrolysis is finished, respectively measuring the content of hydroxyproline in the hydrolysate by using a liquid chromatography pre-column derivatization method, calculating the content of hydroxyproline in the system liquid to be subjected to enzymolysis before and after the enzymolysis, and further calculating according to the formula (1) to obtain the content of the collagen with the triple helical structure in the sample liquid.
As shown in Table 2, in the mixed solution system of gelatin and collagen, the method can accurately measure the content of collagen triple helix in a complex system.
TABLE 2 content of triple helix in gelatin collagen mixture of different percentages
Example 4 content of collagen triple helix Structure by different processing
(1) Respectively taking collagen solution which is dialyzed by filter membranes with the diameter of 0.1 mu M (ultrafiltration treatment 1) and the diameter of 0.2 mu M (ultrafiltration treatment 2), adjusting the pH of the solution to be 3.7 +/-0.05 by using 0.5M acetic acid solution, respectively adding trypsin liquid, wherein the mass ratio of the trypsin to the total protein of the sample liquid is 1:80, uniformly stirring to obtain a system liquid to be subjected to enzymolysis, and treating;
(2) respectively weighing the solution to be subjected to enzymolysis into hydrolysis tubes, adding a hydrochloric acid solution, placing at 110 ℃ for hydrolysis, and determining the content of hydroxyproline in the hydrolysate;
(3) placing the sampled system liquid to be subjected to enzymolysis at 15 ℃, and oscillating and performing enzymolysis for 24 hours at the rotating speed of 50 rmp;
(4) after enzymolysis, respectively weighing the liquid after enzymolysis, adding trichloroacetic acid solution with the mass concentration of 10% at 4 ℃, wherein the mass ratio of the liquid after enzymolysis to the trichloroacetic acid solution is 1:2, oscillating for 10s, uniformly mixing, and centrifuging for 10min at the temperature of 4 ℃ at 5000rmp to separate precipitate and supernatant. Discarding the supernatant, washing the surface of the precipitate with 10% trichloroacetic acid solution at 4 deg.C for 3 times, adding hydrochloric acid solution, and hydrolyzing at 110 deg.C.
(5) After the hydrolysis is finished, respectively measuring the content of hydroxyproline in the hydrolysate by using an ultraviolet spectrophotometry, calculating the content of hydroxyproline in the system liquid to be subjected to enzymolysis before and after the enzymolysis, and further calculating according to the formula (1) to obtain the content of the collagen with the triple helical structure in the sample liquid.
As shown in Table 3, the triple helix structure of collagen was destroyed at 0.1 μm of the ultrafiltration membrane.
TABLE 3 collagen triple helix structure content after different process treatments
Example 5 content of highly viscous collagen triple helix structures of different extraction batches
(1) Three separate batches (20171120, 20171121 and 20171217) of viscous collagen solution (viscosity about 4000 mm) extracted from Achilles tendon of cattle2And/s), adjusting the pH value of the solution to 3.7 +/-0.05 by using 0.5M acetic acid solution, respectively adding trypsin liquid, wherein the mass ratio of the trypsin to the total protein of the sample liquid is 1:50, uniformly stirring to obtain a system liquid to be subjected to enzymolysis, and treating;
(2) respectively putting the solution to be enzymolyzed into a hydrolysis tube, adding a hydrochloric acid solution, hydrolyzing at 110 ℃, and determining the content of hydroxyproline in the hydrolysate;
(3) placing the sampled system liquid to be enzymolyzed at 25 ℃, and oscillating and enzymolyzing at the rotating speed of 120rmp for 4 h;
(4) after the enzymolysis is finished, respectively weighing the liquid after the enzymolysis, adding trichloroacetic acid solution with the mass concentration of 20% at 4 ℃, wherein the mass ratio of the liquid after the enzymolysis to the trichloroacetic acid solution is 1:1, oscillating for 10s, uniformly mixing, and centrifuging for 10min at 4 ℃ and 5000rmp for separating precipitate and supernatant. Discarding the supernatant, washing the surface of the precipitate with 10% trichloroacetic acid solution at 4 deg.C for 3 times, adding hydrochloric acid solution, and hydrolyzing at 110 deg.C.
(5) After the hydrolysis is finished, the content of hydroxyproline in the hydrolysate is respectively determined by using an amino acid analyzer method, the content of hydroxyproline in the system liquid to be subjected to enzymolysis before and after the enzymolysis is respectively calculated according to the sampling dosage of the system liquid to be subjected to enzymolysis and the liquid after the enzymolysis, and then the content of collagen with a triple helix structure in the sample liquid is calculated according to the formula (1).
Using 20171120 batches of extracted collagen liquid as a sample, and respectively measuring the secondary structure, the characteristic absorption peak and the thermal denaturation temperature of the sample by using a Circular Dichroism (CD), an infrared spectrum (FTIR) and a Differential Scanning Calorimeter (DSC), and comparing with the method disclosed by the patent.
As shown in Table 4, the method can detect the triple helical structure in the extracted viscous collagen liquid, and solves the problem that the content of the triple helical structure in a high-viscosity collagen system is difficult to measure by the conventional method. Furthermore, in comparison to existing CD, FTIR and DSC, it was found that: the collagen liquid in FIG. 4 has a negative at 198nmAbsorption, wherein a positive absorption exists at a 222nm position, which accords with the typical absorption wavelength of collagen, and shows that the three-strand helical structure of the batch of collagen liquid is relatively complete; while in the FTIR spectrum (FIG. 5) the amide II bond of the sample (1638 cm)-1) And amide III bond (1239 cm)-1) The absorption of the collagen accords with the characteristic absorption of the complete secondary structure of the collagen, which shows that the triple helical structure of the collagen is relatively complete; figure 6 shows that the thermal denaturation temperature of the collagen batch is 41.90 ℃, the enthalpy is 1.843J/g, and the thermal denaturation temperature conforms to the thermal denaturation temperature of the collagen with the complete triple helix structure. However, these methods can only qualitatively determine the presence or absence of the triple helix structure of collagen, and cannot quantitatively determine the content of the triple helix structure. The method overcomes the defects of the existing qualitative detection method.
Table 4 content of triple helical structure in different extraction batches of high viscosity collagen liquid
In conclusion, the invention utilizes the principle that collagen with a complete triple helical structure cannot be enzymolyzed by trypsin, and collagen with a complete triple helical structure or a partially destroyed triple helical structure can be enzymolyzed by trypsin, and calculates the content of the complete triple helical structure of the collagen through the content change of the characteristic amino acid-hydroxyproline of the collagen in a collagen solution system before and after the enzymolysis of the trypsin, thereby making up the defect of the existing detection method for qualitatively detecting the triple helical structure of the collagen. The invention can quantitatively judge the damage degree of the collagen under various process conditions by quantitatively detecting the collagen triple helix. The invention is suitable for complex biological systems containing collagen, has strong specificity, good result reproducibility and simple operation, and can be used as an application means for detecting the development process of collagen products.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (8)
1. A method for quantitatively detecting the content of a collagen triple helix structure is characterized in that trypsin is added into a collagen sample solution for enzymolysis reaction, collagen with a complete triple helix structure is separated by using a trichloroacetic acid precipitation method, the content of hydroxyproline in the collagen before and after enzymolysis is measured, and the ratio of the content of hydroxyproline in the collagen with the complete triple helix structure separated after enzymolysis to the content of hydroxyproline in the collagen before enzymolysis is calculated, so that the content of the collagen with the triple helix structure in the sample is obtained; the content of hydroxyproline in the collagen before enzymolysis is the content of hydroxyproline in a system to be subjected to enzymolysis before enzymolysis, and the content of hydroxyproline in the collagen before enzymolysis comprises the sum of collagen with a complete triple-helix structure and the hydroxyproline in the collagen with a damaged triple-helix structure; the content of hydroxyproline in the collagen after enzymolysis is the content of hydroxyproline in the collagen with a complete triple-helix structure after the solution after enzymolysis is separated by trichloroacetic acid.
2. The method for quantitatively determining the content of the triple helical structure of collagen according to claim 1, comprising the steps of:
(1) adjusting the pH value of the sample liquid to 3.6-3.7 for later use;
(2) adding the trypsin liquid into the sample liquid, and uniformly mixing to obtain a system liquid to be subjected to enzymolysis;
(3) weighing part of the system liquid to be subjected to enzymolysis, adding a hydrochloric acid solution, hydrolyzing, and determining the content of hydroxyproline in the hydrolysate to obtain the content of hydroxyproline in the system liquid to be subjected to enzymolysis before enzymolysis;
(4) placing the sampled solution to be subjected to enzymolysis under the constant-temperature oscillation condition for enzymolysis reaction;
(5) weighing an enzymolysis solution with the same volume as the liquid phase of the part of the system to be subjected to enzymolysis in the step (3), treating and separating collagen precipitate and supernatant with a complete triple helical structure by using a trichloroacetic acid precipitation method, hydrolyzing the precipitate or the supernatant, determining the content of hydroxyproline in hydrolysate, and calculating the ratio of the content of hydroxyproline in collagen with a complete triple helical structure to the content of hydroxyproline in collagen before enzymolysis.
3. The method for quantitatively determining the content of the triple helical structure of collagen according to claim 2, wherein in the step (2), the trypsin solution is prepared by: weighing trypsin, adding 0.05M CaCl2In 0.001M HCl solution.
4. The method for quantitatively detecting the content of the triple helical structure of collagen according to claim 2, wherein in the step (2), the mass ratio of trypsin to total protein in the sample solution is 1:20 to 1: 100.
5. The method for quantitatively detecting the content of the triple helical structure of collagen according to claim 2, wherein in the step (4), the enzymolysis temperature is 15-30 ℃, the oscillation speed is 50-120 rmp, and the enzymolysis time is 1-24 h.
6. The method for quantitatively determining the content of the triple helical structure of collagen according to claim 2, wherein in the step (5), the step of processing and separating the collagen precipitate and the supernatant having the complete triple helical structure comprises: adding trichloroacetic acid solution with the mass concentration of 10-30% at 4 ℃ into the solution after enzymolysis, wherein the mass ratio of the solution after enzymolysis to the trichloroacetic acid solution is 1: 0.5-2, shaking for 10s, uniformly mixing, and centrifuging for 10min at 4 ℃ at 5000 rmp.
7. The method for quantitatively determining the content of a triple helical structure of collagen according to claim 6, wherein in the step (5), a precipitation treatment is selected: discarding supernatant, cleaning the surface of the precipitate with 10% trichloroacetic acid solution at 4 deg.C for 3 times, adding hydrochloric acid solution, hydrolyzing, and determining hydroxyproline content in the hydrolysate.
8. The method for quantitatively determining the content of triple helical structure of collagen according to claim 6, wherein in the step (5), the supernatant is selected for treatment: and weighing the supernatant, adding a hydrochloric acid solution, hydrolyzing, and determining the content of hydroxyproline in the hydrolysate.
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| Title |
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| 《Quantitation of Collagen Fragments and Gelatin by Deconvolution of Polarimetry Denaturation Curves》;RICHARD A.CONDELL;《Collagen Rel.Res.》;19880930;第8卷(第5期);第407-418页 * |
| 《胶原与明胶的结构研究：方法、结构与分析》;朱欣星;《皮革科学与工程》;20121031;第22卷(第5期);第9-14页 * |
| 《胶原蛋白的高效制备及与明胶的鉴别》;汤俊;《食品科学》;20100401;第31卷(第7期);第54-60页 * |
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