Method for determining protein content in nucleotide-containing collagen implant

Abstract

The invention discloses a method for determining the protein content in a nucleotide-containing collagen implant, and relates to the technical field of protein content detection. The collagen implant contains collagen and nucleotide. The determination method comprises the following steps: determining the nitrogen content N0 in a nucleotide raw material; measuring the nucleotide content Y in the collagen implant, and calculating the total nitrogen amount N2 of the nucleotide in the sampled collagen implant according to the Y, the N0 and the sampling amount m2 of the collagen implant; measuring the total nitrogen amount N1 in the sampled collagen implant, calculating the total nitrogen amount N3 of collagen in the sampled collagen implant according to the N1 and the N2, and calculating the protein content X in the collagen implant according to the N3, the conversion factor B and the total mass m2 of the sampled collagen implant. According to the method, the protein content in the nucleotide-containing collagen implant can be accurately measured.

Description

Technical Field

[0001] This application relates to the field of protein content detection technology, and more specifically, to a method for determining the protein content in a nucleotide-containing collagen implant. Background Technology

[0002] When manufacturing implant products, it is usually necessary to test and verify the content of their components. However, when testing and verifying the protein content in implants containing both nucleotides and collagen, nucleotides do not contain protein, while collagen does. Since nucleotides and collagen in implants cannot be separated, the protein content cannot be detected by isolating nucleotides. Therefore, accurately detecting the protein content in implants has become a challenge.

[0003] Based on this, this application provides a method for determining the protein content in a nucleotide-containing collagen implant. Summary of the Invention

[0004] This application provides a method for determining the protein content in a nucleotide-containing collagen implant, which can accurately determine the protein content in the nucleotide-containing collagen implant.

[0005] This application is implemented as follows:

[0006] This application provides a method for determining the protein content in a nucleotide-containing collagen implant. The collagen implant contains collagen and nucleotides, and the determination method includes the following steps:

[0007] The nitrogen content (N0) in the nucleotide raw materials was determined.

[0008] The nucleotide content Y in the collagen implant was determined, and the total nitrogen content N2 of the nucleotides in the sampled collagen implant was calculated based on Y, N0 and the sampling amount m2.

[0009] The total nitrogen content N1 in the sampled collagen implant was determined. The total nitrogen content N3 of collagen in the sampled collagen implant was calculated based on N1 and N2. The protein content X in the collagen implant was calculated based on N3, conversion factor B, and the total mass m2 of the sampled collagen implant.

[0010] This application has at least the following beneficial effects:

[0011] The method for determining the protein content in nucleotide-containing collagen implants disclosed in this application involves measuring the nitrogen content in the nucleotide raw material, the nucleotide content in the sampled collagen implant, the total nitrogen content in the sampled collagen implant, and the total mass of the sampled collagen implant. The protein content in the collagen implant can then be calculated through specific methods. Verification has shown that the method for determining the protein content in nucleotide-containing collagen implants disclosed in this application can accurately determine the protein content in nucleotide-containing collagen implants. Detailed Implementation

[0012] The embodiments of this application will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this application. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0013] The following is a detailed description of the method for determining the protein content in the nucleotide-containing collagen implant according to embodiments of this application:

[0014] This application provides a method for determining the protein content in a nucleotide-containing collagen implant. The collagen implant contains collagen and nucleotides, wherein the nucleotides include at least one selected from polydeoxynucleotides (PDRN) and polynucleotides (PN). The determination method includes the following steps:

[0015] (1) Determine the nitrogen content N0 in the nucleotide raw material; wherein, the nitrogen content N0 in the nucleotide raw material is calculated by determining the total nitrogen content in the nucleotide raw material by the total mass m1 of the nucleotide raw material and the Kjeldahl nitrogen determination method.

[0016] Specifically, the nitrogen content in nucleotide raw materials Where C is C 1 / 2 Concentration of sulfuric acid standard titrant (mol / L); V1 is the volume of sulfuric acid titrant consumed by the nucleotide raw material (mL); V0 is the volume of sulfuric acid titrant consumed by the blank group (mL); total mass of nucleotide raw material m1 (g). It should be noted that the Kjeldahl method is an existing method for determining nitrogen content. Specific steps can be found in GB5009.5-2016 "National Food Safety Standard - Determination of Protein in Food".

[0017] (2) The nucleotide content Y in the sampled collagen implant is determined. Based on Y, N0, and the sampling amount m2 of the collagen implant, the total nitrogen content N2 of the nucleotides in the sampled collagen implant is calculated. Specifically, the total nitrogen content N2 of the nucleotides in the sampled collagen implant is N0*Y*m2 / 1000, where the nucleotide content Y is in mg / g and the total mass m2 of the collagen implant is in g.

[0018] The step of determining the nucleotide content Y in the sampled collagen implant can be referenced from the steps of the applicant's previous patent application 202411968275.4. Specifically, the sampled collagen implant is diluted 100 times with phosphate buffer to obtain a colorless and transparent solution. The absorbance of the diluted colorless and transparent solution at 260 nm and 280 nm under ultraviolet light is measured and recorded as A1 and A2, respectively. The absorbance of the implant at 260 nm under ultraviolet light is A260 = A1 * 100, and the absorbance of the implant at 280 nm under ultraviolet light is A280 = A2 * 100. The nucleotide content Y in the collagen implant is calculated using the formula: Y = 14.68A260 - 18.1A280.

[0019] (3) The total nitrogen content N1 in the sampled collagen implant was determined, and the total nitrogen content N3 of the collagen in the sampled collagen implant was calculated based on N1 and N2.

[0020] The total nitrogen content N1 in the sampled collagen implant was determined by the Kjeldahl method. The total nitrogen content N1 in the sampled collagen implant was calculated as: N1 = C * 2 * 0.0140 * (V2 - V0), where C is C 1 / 2 The concentration of the standard sulfuric acid titrant is (mol / L), V2 is the volume of sulfuric acid titrant consumed by the collagen implant (mL), and V0 is the volume of sulfuric acid titrant consumed by the blank group (mL).

[0021] The total nitrogen content of collagen in the sampled collagen implant is N3 = N1 - N2.

[0022] (4) Calculate the protein content X in the collagen implant based on N3, conversion factor B, and the sampling amount m2 of the collagen implant. For example, the conversion factor B is 5.79. Here, the unit of X is mg / g.

[0023] The following describes in further detail the method for determining the protein content in the nucleotide-containing collagen implant of this application, with reference to the embodiments.

[0024] Example 1

[0025] This embodiment provides a method for determining the protein content in a nucleotide-containing collagen implant, wherein the collagen implant is composed of polynucleotides (PN) and collagen, the polynucleotide content in the collagen implant is 5 mg / g, and the protein content in the collagen implant is 34 mg / g.

[0026] The determination method includes the following steps:

[0027] (1) The nitrogen content N0 in the polynucleotide raw material was determined; wherein, the nitrogen content N0 in the polynucleotide raw material was calculated by determining the total nitrogen content in the polynucleotide raw material by the total mass m1 of the polynucleotide raw material and the Kjeldahl nitrogen determination method.

[0028] Specifically, the nitrogen content in polynucleotide raw materials Where C is C 1 / 2 Concentration of sulfuric acid standard titrant (mol / L); V1 is the volume of sulfuric acid titrant consumed by the polynucleotide raw material (mL); V0 is the volume of sulfuric acid titrant consumed by the blank group (mL); total mass of polynucleotide raw material m1 (g).

[0029] (2) The polynucleotide content Y in the sampled collagen implant was determined, and the total nitrogen content N2 of the nucleotides in the sampled collagen implant was calculated based on Y, N0, and the sampling amount m2 of the collagen implant. Specifically, the total nitrogen content N2 of the nucleotides in the sampled collagen implant is N2 = N0 * Y * m2 / 1000, where the nucleotide content Y is in mg / g and the sampling amount m2 of the collagen implant is in g.

[0030] The step of determining the nucleotide content Y in the sampled collagen implant includes: diluting the collagen implant 100 times with phosphate buffer to obtain a colorless and transparent solution; measuring the absorbance of the diluted colorless and transparent solution at 260 nm and 280 nm under ultraviolet light, denoted as A1 and A2, respectively; wherein the absorbance of the implant at 260 nm under ultraviolet light A260 = A1 * 100, and the absorbance of the implant at 280 nm under ultraviolet light A280 = A2 * 100. The nucleotide content Y in the collagen implant is calculated using the formula: Y = 14.68A260 - 18.1A280.

[0031] (3) The total nitrogen content N1 in the sampled collagen implant is determined, and the total nitrogen content N3 of collagen in the sampled collagen implant is calculated based on N1 and N2. Wherein, the total nitrogen content N3 of collagen in the sampled collagen implant is N1-N2.

[0032] The total nitrogen content N1 in the sampled collagen implant was determined by the Kjeldahl method. The total nitrogen content N1 in the sampled collagen implant was calculated as: N1 = C * 2 * 0.0140 * (V2 - V0), where C is C 1 / 2 The concentration of the standard sulfuric acid titrant is (mol / L), V2 is the volume of sulfuric acid titrant consumed by the collagen implant (mL), and V0 is the volume of sulfuric acid titrant consumed by the blank group (mL).

[0033] (4) Calculate the protein content X in the collagen implant based on N3, conversion factor B, and the sampling amount m2 of the collagen implant. The conversion factor B is 5.79.

[0034] Example 2

[0035] This embodiment provides a method for determining the protein content in a nucleotide-containing collagen implant, wherein the collagen implant is composed of polynucleotides (PN) and collagen. The only difference from Example 1 is that the polynucleotide content in the collagen implant is 10 mg / g and the protein content in the collagen implant is 34 mg / g.

[0036] The specific steps of the determination method are the same as those in Example 1.

[0037] Example 3

[0038] This embodiment provides a method for determining the protein content in a nucleotide-containing collagen implant, wherein the collagen implant is composed of polynucleotides (PN) and collagen. The only difference from Example 1 is that the polynucleotide content in the collagen implant is 8 mg / g and the protein content in the collagen implant is 40 mg / g.

[0039] The specific steps of the determination method are the same as those in Example 1.

[0040] Example 4

[0041] This embodiment provides a method for determining the protein content in a nucleotide-containing collagen implant, wherein the collagen implant is composed of polynucleotides (PN) and collagen. The only difference from Example 1 is that the polynucleotide content in the collagen implant is 15 mg / g and the protein content in the collagen implant is 34 mg / g.

[0042] The specific steps of the determination method are the same as those in Example 1.

[0043] The measurement and calculation results of each parameter in Examples 1 to 4 are shown in Tables 1 and 2.

[0044] Table 1. Measurement and calculation results of various parameters of the nucleotide raw materials used in Examples 1 to 4

[0045]

[0046] Table 2. Measurement and calculation results of parameters in Examples 1 to 4

[0047]

[0048] The experimental results above show that the method for determining the protein content in the nucleotide-containing collagen implant of this application can accurately determine the protein content in the collagen implant. The measurement results of Examples 1, 2 and 4 have an error of 0.5% to 2.5%, while the measurement result of Example 3 has an error of 8.7%.

[0049] The above description is merely a specific embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for determining the protein content in a nucleotide-containing collagen implant, characterized in that, The collagen implant contains collagen and nucleotides, and the assay method includes the following steps: The nitrogen content (N0) in the nucleotide raw materials was determined. The nucleotide content Y in the collagen implant is determined, and the total nitrogen content N2 of the nucleotides in the sampled collagen implant is calculated based on Y, N0 and the sampling amount m2 of the collagen implant. The total nitrogen content N1 in the sampled collagen implant is determined. The total nitrogen content N3 of the collagen in the sampled collagen implant is calculated based on N1 and N2. The protein content X in the collagen implant is calculated based on N3, conversion factor B, and the total mass m2 of the sampled collagen implant.

2. The method for determining the protein content in a nucleotide-containing collagen implant according to claim 1, characterized in that, The total nitrogen content N1 in the sampled collagen implant was measured by the Kjeldahl method.

3. The method for determining the protein content in a nucleotide-containing collagen implant according to claim 2, characterized in that, The total nitrogen content N1 in the sampled collagen implant is calculated as: N1 = C * 2 * 0.0140 * (V2 - V0), where C is C 1 / 2 The concentration of the standard sulfuric acid titrant, V2 is the volume of sulfuric acid titrant consumed by the collagen implant, and V0 is the volume of sulfuric acid titrant consumed by the blank group.

4. The method for determining the protein content in a nucleotide-containing collagen implant according to claim 1, characterized in that, The nitrogen content N0 in the nucleotide raw material is calculated by determining the total nitrogen content in the nucleotide raw material using the Kjeldahl method based on the total mass m1 of the nucleotide raw material.

5. The method for determining the protein content in a nucleotide-containing collagen implant according to claim 4, characterized in that, Nitrogen content in the nucleotide raw material Where C is C 1 / 2 The concentration of the sulfuric acid standard titrant, V1 is the volume of sulfuric acid titrant consumed by the nucleotide raw material, and V0 is the volume of sulfuric acid titrant consumed by the blank group.

6. The method for determining the protein content in a nucleotide-containing collagen implant according to claim 5, characterized in that, The total nitrogen content of the nucleotides in the sampled collagen implant is N2 = N0 * Y * m2 / 1000.

7. The method for determining the protein content in a nucleotide-containing collagen implant according to claim 6, characterized in that, The total nitrogen content of the collagen in the sampled collagen implant is N3 = N1 - N2.

8. The method for determining the protein content in a nucleotide-containing collagen implant according to claim 7, characterized in that, The protein content of the collagen implant 9. The method for determining the protein content in a nucleotide-containing collagen implant according to any one of claims 1 to 8, characterized in that, The conversion factor B is 5.

79.

10. The method for determining the protein content in a nucleotide-containing collagen implant according to any one of claims 1 to 8, characterized in that, The nucleotides include at least one of polydeoxynucleotides and polynucleotides.

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