Protein determination method

Abstract

The invention provides a protein determination method, and belongs to the technical field of detection methods. Comprising the following steps: S1, adding a sample to be detected into a digestion tube, adding concentrated sulfuric acid and a catalyst, and digesting by using a digestion instrument; s2, after digestion is completed, cooling the digestion tube in a fuming cupboard to room temperature, and detecting by using a Kjeldahl apparatus. According to the detection method, the digestion link of determining the protein content through the Kjeldahl method is improved, the detection efficiency is improved by changing the type and proportion of the catalyst, and the detection method for determining the nitrogen or the protein is more accurate, high in efficiency, low in cost and small in environmental pollution.

Description

Technical Field

[0001] The present invention relates to the technical field of detection methods, and in particular to a protein determination method. Background Art

[0002] Nitrogen is an essential nutrient element in the growth and development of plants. It is one of the main components of nucleic acids, chlorophyll, proteins, phospholipids and some growth hormones. Proteins, nucleic acids, phospholipids, etc. are the most basic substances that make up the cytoplasm and cell nucleus. Therefore, nitrogen is also called the element of life. Nitrogen generally accounts for 0.3%-0.5% of the dry weight in plant tissues, and nitrogen contributes 40%-50% to the final yield of crops. The nitrogen content affects the growth and development process of plants and also affects the quality of crops. Studies have shown that when the nitrogen content is within a reasonable range, the photosynthetic efficiency of plants will increase with the increase of nitrogen in the plant body. However, when the nitrogen content exceeds a certain critical value, the photosynthetic rate of plants begins to decrease with the increase of nitrogen content. [2] In short, nitrogen has a very direct impact on plant growth and development and the quality and yield of crop products.

[0003] At present, the main methods for measuring nitrogen are Dumas combustion method, Kjeldahl nitrogen determination method and infrared spectroscopy technology. Kjeldahl nitrogen determination method is the most important method for detecting nitrogen or protein content in grain and soil. It is one of the methods with high accuracy and simple operation among the existing methods for determining protein content in samples. It is used as a standard test method both at home and abroad. The Kjeldahl nitrogen determination method was proposed by Kjeldahl in 1883 when he was studying protein. The principle is that the protein in the sample is digested with concentrated sulfuric acid under the action of a catalyst to convert organic nitrogen into ammonia. Ammonia combines with sulfuric acid to form ammonium nitrogen. The ammonia is distilled out with sodium hydroxide solution, and then the distilled ammonia is absorbed with a boric acid solution with a specified pH value. Finally, it is titrated with a standard solution of hydrochloric acid. The nitrogen content is calculated by the formula and the consumption of hydrochloric acid of known concentration, and then multiplied by the conversion coefficient to convert it into protein content, which is the protein content in the sample.

[0004] my country's currently effective national standards such as LY / T 1228-2015, GB5009-2016, and NY / T 2017-2011 all choose the Kjeldahl method as the preferred method for nitrogen determination. Different standard methods are suitable for different samples. In GB50095-2016, the entire test process uses a lot of sodium hydroxide, sulfuric acid, etc., the detection cost is high, and a lot of chemical waste liquid is discharged. During the digestion process, concentrated sulfuric acid reacts to produce a large amount of sulfur dioxide, which will inevitably cause a certain degree of pollution to the test personnel and the environment. In the process of high-temperature distillation of ammonia with sodium hydroxide, the concentration of sodium hydroxide is 400 g / L. Due to the large amount of sulfuric acid added, in order to completely distill the ammonia, an excess of sodium hydroxide is added. A large amount of sodium hydroxide is consumed, the test cost is increased, and the damage to the environment is also increased. Therefore, this study improved and optimized the Kjeldahl method for determining the nitrogen content in samples through experiments, trying to reduce the amount of rice weighed, reduce the amount of sulfuric acid added, change the catalyst addition ratio, shorten the digestion time, reduce environmental pollution, and try to find more accurate, effective, and less polluting steps to detect the nitrogen content in samples. Summary of the invention

[0005] The purpose of the present invention is to propose a protein determination method, improve the digestion link of determining protein content by Kjeldahl method, improve the detection efficiency by changing the type and proportion of catalysts, and provide a detection method for determining nitrogen or protein with more accuracy, high efficiency, low cost and less environmental pollution.

[0006] The technical solution of the present invention is achieved in this way: The present invention provides a protein determination method, comprising the following steps: S1. Add the sample to be tested into a digestion tube, add concentrated sulfuric acid and a catalyst, and digest it using a digestion instrument; S2. After digestion is completed, cool the digestion tube to room temperature in a fume hood and then test it using a Kjeldahl nitrogen analyzer.

[0007] As a further improvement of the present invention, the mass ratio of the sample to be tested, concentrated sulfuric acid and catalyst is 0.2-1:20-25:0.1-0.2.

[0008] As a further improvement of the present invention, the catalyst is a mixture of potassium sulfate and copper sulfate.

[0009] As a further improvement of the present invention, the mass ratio of potassium sulfate to copper sulfate is 6:0.05-0.8.

[0010] As a further improvement of the present invention, the preparation method of the catalyst is as follows: T1. Dissolve potassium sulfate and copper sulfate in water to obtain a solution; T2. Add bentonite, polydopamine and Tris-HCl solution to the solution, heat and stir to react, centrifuge, wash and dry to obtain a catalyst.

[0011] As a further improvement of the present invention, the mass ratio of the potassium sulfate, copper sulfate, bentonite, polydopamine and Tris-HCl solution is 6:0.05-0.8:15-20:3-5:0.1-0.2.

[0012] As a further improvement of the present invention, the temperature of the heating and stirring reaction is 40-50°C.

[0013] As a further improvement of the present invention, the heating and stirring reaction time is 1-2h.

[0014] As a further improvement of the present invention, the pH value of the Tris-HCl solution is 8.5-9.5.

[0015] The present invention has the following beneficial effects: The present invention improves the digestion link of determining protein content by the Kjeldahl nitrogen method, improves the detection efficiency by changing the type and proportion of the catalyst, and has a more accurate, efficient, low-cost and less environmentally polluting detection method for determining nitrogen or protein. DETAILED DESCRIPTION

[0016] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0017] Preparation Example 1 The preparation method of the catalyst is as follows: T1. Dissolve 6 g of potassium sulfate and 0.05 g of copper sulfate in 200 mL of water to obtain a solution; T2. Add 15 g of bentonite, 3 g of polydopamine and 0.1 g of Tris-HCl solution with a pH value of 8.5 to the solution, heat to 40°C, stir to react for 1 hour, centrifuge, wash, and dry to obtain a catalyst.

[0018] Preparation Example 2 The preparation method of the catalyst is as follows: T1. Dissolve 6 g of potassium sulfate and 0.8 g of copper sulfate in 200 mL of water to obtain a solution; T2. Add 20 g of bentonite, 5 g of polydopamine and 0.2 g of Tris-HCl solution with a pH value of 9.5 to the solution, heat to 50°C, stir and react for 2 h, centrifuge, wash and dry to obtain a catalyst.

[0019] Preparation Example 3 The preparation method of the catalyst is as follows: T1. Dissolve 6 g of potassium sulfate and 0.4 g of copper sulfate in 200 mL of water to obtain a solution; T2. Add 17 g of bentonite, 4 g of polydopamine and 0.15 g of Tris-HCl solution with a pH value of 9 to the solution, heat to 45°C, stir and react for 1.5 h, centrifuge, wash and dry to obtain a catalyst.

[0020] Example 1 This embodiment provides a protein determination method, characterized in that it includes the following steps: S1. Add 0.2 g of the rice sample to be tested into a digestion tube, add 20 g of concentrated sulfuric acid and 0.1 g of the catalyst prepared in Preparation Example 1, and digest at 420 ° C for 1 h using a digestion instrument; S2. After digestion is completed, cool the digestion tube to room temperature in a fume hood and then test it using a Kjeldahl nitrogen analyzer.

[0021] Example 2 This embodiment provides a protein determination method, characterized in that it includes the following steps: S1. Add 1 g of the rice sample to be tested into a digestion tube, add 25 g of concentrated sulfuric acid, 0.2 g of the catalyst prepared in Preparation Example 2, and digest at 420 ° C for 1 h using a digestion instrument; S2. After digestion is completed, cool the digestion tube to room temperature in a fume hood and then test it using a Kjeldahl nitrogen analyzer.

[0022] Example 3 This embodiment provides a protein determination method, characterized in that it includes the following steps: S1. Add 0.5 g of the rice sample to be tested into a digestion tube, add 22 g of concentrated sulfuric acid, 0.15 g of the catalyst prepared in Preparation Example 3, and digest at 420 ° C for 1 h using a digestion instrument; S2. After digestion is completed, cool the digestion tube to room temperature in a fume hood and then test it using a Kjeldahl nitrogen analyzer.

[0023] Test Example 1 The N content results detected in Examples 1-3 are shown in Table 1 below.

[0024] Table 1

[0025] Test Example 2 Recovery rate determination is an important way to test the accuracy of instrument results. Taking advantage of the fact that the nitrogen content of ammonium sulfate is known and no digestion is required, recovery rate determination is more convenient. Determine the ammonium sulfate with a known nitrogen content and calculate the recovery rate to determine whether the accuracy and stability meet the requirements.

[0026] Method for determining the recovery rate: dry ammonium sulfate with a purity of ≥99.5% at 102 °C for 1 h, accurately weigh 2.5 g (accurate to 0.0001 g), and prepare a 5 g / L ammonium sulfate solution. Use a pipette to take 10 mL of ammonium sulfate solution and put it into a digestion tube, input a sample mass of 0.05 g, and perform the test according to the method of Example 3. Use a fully automatic Kjeldahl nitrogen analyzer to determine the nitrogen content of ammonium sulfate, repeat 6 times, use an empty digestion tube as a blank control, and repeat 3 times. The comparison of the nitrogen content data obtained from the 6 parallel comparison tests is shown in Table 2.

[0027] Table 2 Calibration of 5 g / L ammonium sulfate solution Test No. Weight / g Blank volume / mL Titration volume / mL Nitrogen content / % 1 0.5 0.0974 14.0725 20.9718 2 0.5 0.0974 14.25 20.9277 3 0.5 0.0974 14.275 20.9628 4 0.5 0.0974 14.2 20.9577 5 0.5 0.0974 14.2325 20.9132 6 0.5 0.0974 14.235 20.9167 The nitrogen content of ammonium sulfate is known to be 21.09%. After 6 repeated experiments, the recovery rates were calculated to be 99.44%, 99.23%, 99.40%, 99.37%, 99.16%, and 99.18%, with an average value of 99.297%. This shows that the recovery rate of the instrument meets the quantitative requirements and the stability of the Kjeldahl nitrogen analyzer meets the requirements.

[0028] Test Example 3 The measurement repeatability of the instrument is a standard for testing the accuracy and stability of the measured data. Calibration method for measurement repeatability: dry ammonium sulfate with a purity of ≥99.5% at 102 °C for 2 hours, weigh 2.5 g, and prepare a 5 g / L ammonium sulfate solution. Take 10 mL of ammonium sulfate solution with a concentration of 5 g / L, test it according to the method of Example 3, and then perform distillation and titration processes to calculate the nitrogen content data obtained in the test. The measurement repeatability of the instrument was tested, and its relative standard deviation RSD (%) was 0.12%. RSD≤0.3%, indicating that the instrument has good repeatability and high reliability.

[0029] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A protein determination method, characterized in that: The following steps are involved: S1. Add the sample to be tested into a digestion tube, add concentrated sulfuric acid and a catalyst, and digest it using a digestion instrument; S2. After digestion is completed, cool the digestion tube to room temperature in a fume hood and then test it using a Kjeldahl nitrogen analyzer.

2. The protein determination method according to claim 1, characterized in that The mass ratio of the sample to be tested, concentrated sulfuric acid and catalyst is 0.2-1:20-25:0.1-0.

2.

3. The protein determination method according to claim 1, characterized in that: The catalyst is a mixture of potassium sulfate and copper sulfate.

4. The protein determination method according to claim 3, characterized in that: The mass ratio of potassium sulfate to copper sulfate is 6:0.05-0.

8.

5. The protein determination method according to claim 3, characterized in that: The preparation method of the catalyst is as follows: T1. Dissolve potassium sulfate and copper sulfate in water to obtain a solution; T2. Add bentonite, polydopamine and Tris-HCl solution to the solution, heat and stir to react, centrifuge, wash and dry to obtain a catalyst.

6. The protein determination method according to claim 5, characterized in that: The mass ratio of the potassium sulfate, copper sulfate, bentonite, polydopamine and Tris-HCl solution is 6:0.05-0.8:15-20:3-5:0.1-0.

2.

7. The protein determination method according to claim 5, characterized in that: The temperature of the heating and stirring reaction is 40-50°C.

8. The protein determination method according to claim 5, characterized in that: The heating and stirring reaction time is 1-2h.

9. The protein determination method according to claim 5, characterized in that: The pH value of the Tris-HCl solution is 8.5-9.5.

Images