Plant grain protein component 15N isotope abundance determination method based on sequential extraction

Abstract

The invention provides a plant grain protein component 15N isotope abundance determination method based on sequential extraction, and belongs to the technical field of isotope tracing. The method comprises the steps of protein sequential extraction, precipitation treatment, freeze drying, isotope mass spectrometry and the like. Key protein components such as albumin, globulin, prolamin and glutelin in the plant seeds are effectively separated through a sequential extraction method; carrying out precipitation treatment and freeze drying to obtain dried protein precipitate; and finally, measuring the abundance of the < 15 > N isotope in the protein component sample by using an isotope mass spectrometer. The method for determining the abundance of the 15N isotope of the plant grain protein component based on sequential extraction has the advantages of simplicity and convenience in operation, high accuracy, high sensitivity and the like, and provides powerful support for deeply researching the physiological mechanism of plant nitrogen nutrition.

Description

Technical Field

[0001] The present invention relates to the field of isotope tracing technology, in particular to plant seed protein components based on sequential extraction 15 N isotope abundance determination method. Background Art

[0002] 15 As an important tracer method, N stable isotope technology has been widely used in soil science and plant nutrition research. 15 The abundance changes of N isotopes can provide insights into the absorption, transport, distribution and metabolism of nitrogen. 15 The methods for determining the isotope abundance of N isotopes at the soil and plant organ levels are relatively mature, but there is still a large technical gap in the determination of isotopes in plant seed protein components.

[0003] Currently, only a few literatures have mentioned 15 However, these studies have not elaborated on the specific determination means and methods, so it is difficult to directly apply them to practical research. In addition, due to the complexity and diversity of plant seed protein components, traditional isotope determination methods are often difficult to accurately and quickly determine the content of protein components. 15 N isotopic abundance, thus limiting 15 Application of N tracing technology at the protein component level.

[0004] Therefore, it is necessary to develop a method suitable for plant seed protein components. 15 The N isotope abundance determination method not only helps to fill the gaps in existing technologies, but also provides more accurate and reliable data support for the study of the chemical and biological processes of nitrogen in plants. Summary of the invention

[0005] The object of the present invention is to provide a plant seed protein component based on sequential extraction 15 The N isotope abundance determination method has the advantages of simple operation, high accuracy and high sensitivity, and provides strong support for in-depth research on the physiological mechanism of plant nitrogen nutrition.

[0006] To achieve the above object, the present invention provides a plant seed protein component based on sequential extraction 15 The method for determining N isotope abundance comprises the following steps: S1. Weigh the whole wheat flour sample and place it in a centrifuge tube. Then add distilled water to the centrifuge tube, shake it on a constant temperature mixer, centrifuge it, extract the supernatant twice, and obtain an albumin solution. Continue to add 10% NaCl to the centrifuge tube containing whole wheat flour, stir and disperse the precipitate evenly, shake it on a constant temperature mixer, centrifuge it again, extract the supernatant twice, and obtain the globulin solution; Continue to add 75% ethanol to the centrifuge tube containing whole wheat flour, stir and disperse the precipitate evenly, shake on a constant temperature mixer, centrifuge again, extract the supernatant twice, and obtain the alcohol-soluble protein solution; Continue to add 0.2% NaOH solution to the centrifuge tube containing whole wheat flour, stir and disperse the precipitate evenly, shake it on a constant temperature mixer, and then use a centrifuge to extract the supernatant twice to obtain a gluten solution; S2, transferring the supernatant of each protein component extracted in step S1 into a new centrifuge tube respectively, and adding anhydrous ethanol to the centrifuge tube containing the albumin solution for precipitation treatment; Add 20% trichloroacetic acid solution containing 0.14% mercaptoethanol to the centrifuge tubes containing globulin, alcohol-soluble protein and glutenin solutions for precipitation treatment; S3. Collect the precipitate by centrifugation, and then wash the precipitate 2 to 3 times with precooled acetone solution containing 0.07% mercaptoethanol; S4, freeze-drying the protein precipitate in step S3 to obtain a dry protein precipitate; S5, grinding the dried protein precipitate in step S4 into powder, and using an isotope mass spectrometer to determine the protein component sample 15 N isotope abundance.

[0007] Preferably, in step S1, the ratio of whole wheat flour to distilled water, 10% NaCl, 75% ethanol and 0.2% NaOH solution is 2g:40ml.

[0008] Preferably, in step S1, when shaking on the constant temperature mixer, the shaking time is 30 min, the temperature is 20° C., and the rotation speed is 1800 r / min.

[0009] Preferably, in steps S1 and S3, during centrifugation, the centrifugation time is 15 min, the temperature is 20° C., and the rotation speed is 5000 r / min.

[0010] Preferably, in step S2, the ratio of albumin solution to anhydrous ethanol is 1:3.

[0011] Preferably, in step S2, the precipitation treatment is performed at -20°C for 60 minutes.

[0012] Preferably, in step S2, the ratio of the globulin, alcohol-soluble protein, glutenin solution to the 20% trichloroacetic acid solution containing 0.14% mercaptoethanol is 1:1.

[0013] Preferably, in step S4, during freeze drying, the temperature is -80°C and the vacuum pressure is less than 1 mbar, and then the temperature is increased to -20°C for secondary drying.

[0014] Therefore, the present invention adopts the above-mentioned plant grain protein component based on sequential extraction 15 The N isotope abundance determination method has the following beneficial technical effects: The present invention is directed to the protein components in plant seeds 15 The technical gap in the field of N isotope abundance determination is filled by providing a complete set of determination methods with significant technical innovation and practical application value.

[0015] First, the present invention fills the technical gap in this field. 15 N stable isotope technology has been widely used in the field of natural sciences, but it is not widely used in plant seed protein components. 15 There has been a lack of mature research or clear determination methods for N abundance determination. The few existing relevant literatures have also failed to elaborate on the specific operations and technical paths, so it is difficult to directly apply them to practical research. The present invention provides a clear and feasible determination method for this field for the first time through a complete technical solution based on protein sequential extraction, precipitation treatment, freeze drying and isotope mass spectrometry, providing important technical support for future related research.

[0016] Secondly, the present invention clarifies the precipitation treatment method and freeze-drying treatment technology applicable to grain protein components. In view of the solubility and precipitation characteristics of different protein components, the present invention adopts a specific precipitation treatment method to ensure the integrity of the protein and the accuracy of the measurement. At the same time, by precisely controlling the drying rate and environmental conditions, the present invention ensures the stability of the protein and provides a more stable and reliable sample for subsequent isotope mass spectrometry analysis. This technical treatment not only improves the repeatability and accuracy of isotope determination results, but also provides new ideas and methods for research in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The plant seed protein component based on sequential extraction of the present invention 15 Flowchart of the N isotope abundance determination method. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0019] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0020] Embodiment 1 like Figure 1 As shown, the plant seed protein components based on sequential extraction of the present invention are 15 The flowchart of the method for determining N isotope abundance includes the following steps: S1. Weigh 2 g of whole wheat flour sample and place it in a centrifuge tube. Then add 40 mL of distilled water to the centrifuge tube, shake it on a constant temperature mixer for 30 min (20°C, 1800 r / min), and then centrifuge it for 15 min (20°C, 5000 r / min). Extract the supernatant twice to obtain the albumin solution. Add 40 mL of 10% NaCl to the centrifuge tube containing whole wheat flour, stir and disperse the precipitate evenly, shake on a constant temperature mixer for 30 min (20°C, 1800 r / min), centrifuge for 15 min (20°C, 5000 r / min), extract the supernatant twice, and obtain the globulin solution; Add 40 mL of 75% ethanol to the centrifuge tube containing whole wheat flour, stir and disperse the precipitate evenly, shake on a constant temperature mixer for 30 min (20°C, 1800 r / min), centrifuge for 15 min (20°C, 5000 r / min), extract the supernatant twice, and obtain the alcohol-soluble protein solution; Add 40 mL of 0.2% NaOH solution to the centrifuge tube containing whole wheat flour, stir and disperse the precipitate evenly, shake on a constant temperature mixer for 30 minutes (20°C, 1800 r / min), and then centrifuge for 15 minutes (20°C, 5000 r / min), extract the supernatant twice, and obtain the gluten solution.

[0021] S2, take 10mL of albumin solution and add it into a 50mL centrifuge tube, then add 30mL of anhydrous ethanol and treat at -20℃ for 60min; Take 10 mL of globulin, alcohol-soluble protein and glutenin solution respectively and add them into 50 mL centrifuge tubes, then add 10 mL of 20% trichloroacetic acid (TCA) solution of 0.14% mercaptoethanol and treat at -20℃ for 60 min; S3. Collect the precipitate by centrifugation for 15 min (20°C, 5000 r / min), and then wash the precipitate 2 to 3 times with precooled acetone solution containing 0.07% mercaptoethanol; S4, freeze-drying the protein precipitate in step S3 to obtain a dry protein precipitate; During freeze drying, the protein precipitate is placed in a freeze drying bottle, which is quickly placed in -80°C for pre-freezing to ensure that the precipitate is completely frozen. The temperature in the freeze dryer is set to -60°C, the vacuum pressure is set to 0.8 mbar, and the drying time is determined according to the sample volume. The temperature is then raised to -20°C for secondary drying.

[0022] S5, grinding the dried protein precipitate in step S4 into powder, and using an isotope mass spectrometer to determine the protein component sample 15 N isotope abundance.

[0023] Whole wheat flour samples with different nitrogen supply levels (T1: N 0.6 g; T2: N 0.8 g) were selected for the experiment, and each treatment was repeated 3 times. The samples were processed according to the established protein extraction and precipitation methods, and the solids of different protein components obtained were freeze-dried and ground into powder. The samples were oxidized and calcined at 1120°C in an oxidation tube filled with WO3 using a vario PYRO cube elemental analyzer and an ISOPRIME-100 stable isotope mass spectrometer. The gas formed after combustion was reduced to N2 (850°C) in a tube filled with reducing copper. The gas was flowed through a water trap (phosphorus pentoxide) by a helium carrier gas. N2, CO2, and SO2 were separated by adsorption and desorption columns, and then entered the isotope mass spectrometer (IRMS) for isotope analysis to obtain the different protein components. 15 N abundance data are shown in Table 1.

[0024] Table 1 Protein composition of wheat grain protein 15 N abundance / % ;

[0025] The present invention also conducted the following related experiments for determining the protein precipitant.

[0026] Protein precipitant 1: anhydrous ethanol; Protein precipitant 2: 10% trichloroacetic acid solution; Protein precipitant 3: 10% trichloroacetic acid solution containing 0.07% mercaptoethanol; The protein component liquids were mixed with different precipitants in the ratio of 1:1, 1:2, and 1:3. The specific operations were as follows: 10 ml of albumin, globulin, alcohol-soluble protein, and glutenin solutions were respectively taken into 50 ml centrifuge tubes, and 10 ml, 20 ml, and 30 ml of protein precipitant 1, protein precipitant 2, and protein precipitant 3 were added respectively, and treated at -20°C for 60 min.

[0027] Table 2 Precipitation results of protein precipitant 1 ;

[0028] Table 3 Precipitation results of protein precipitant 2 ;

[0029] Table 4 Precipitation results of protein precipitant 3 ;

[0030] It is worth noting that the contents not elaborated in detail in the present invention are all prior art and are well known to those skilled in the art.

[0031] Therefore, the present invention adopts the above-mentioned plant grain protein component based on sequential extraction 15 The N isotope abundance determination method has the advantages of simple operation, high accuracy and high sensitivity, and provides strong support for in-depth research on the physiological mechanism of plant nitrogen nutrition.

[0032] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. Plant seed protein components based on sequential extraction 15 The method for determining N isotope abundance is characterized in that: The following steps are involved: S1. Weigh the whole wheat flour sample and place it in a centrifuge tube. Then add distilled water to the centrifuge tube, shake it on a constant temperature mixer, centrifuge it, extract the supernatant twice, and obtain an albumin solution. Continue to add 10% NaCl to the centrifuge tube containing whole wheat flour, stir and disperse the precipitate evenly, shake it on a constant temperature mixer, centrifuge it again, extract the supernatant twice, and obtain the globulin solution; Continue to add 75% ethanol to the centrifuge tube containing whole wheat flour, stir and disperse the precipitate evenly, shake on a constant temperature mixer, centrifuge again, extract the supernatant twice, and obtain the alcohol-soluble protein solution; Continue to add 0.2% NaOH solution to the centrifuge tube containing whole wheat flour, stir and disperse the precipitate evenly, shake it on a constant temperature mixer, and then use a centrifuge to extract the supernatant twice to obtain a gluten solution; S2, transferring the supernatant of each protein component extracted in step S1 into a new centrifuge tube respectively, and adding anhydrous ethanol to the centrifuge tube containing the albumin solution for precipitation treatment; Add 20% trichloroacetic acid solution containing 0.14% mercaptoethanol to the centrifuge tubes containing globulin, alcohol-soluble protein and glutenin solutions for precipitation treatment; S3. Collect the precipitate by centrifugation, and then wash the precipitate 2 to 3 times with precooled acetone solution containing 0.07% mercaptoethanol; S4, freeze-drying the protein precipitate in step S3 to obtain a dry protein precipitate; S5, grinding the dried protein precipitate in step S4 into powder, and using an isotope mass spectrometer to determine the protein component sample 15 N isotope abundance.

2. The plant seed protein component based on sequential extraction according to claim 1 15 The method for determining N isotope abundance is characterized in that: In step S1, the ratio of whole wheat flour to distilled water, 10% NaCl, 75% ethanol and 0.2% NaOH solution is 2g:40ml.

3. The plant seed protein component based on sequential extraction according to claim 1 15 The method for determining N isotope abundance is characterized in that: In step S1, when shaking on the constant temperature mixer, the shaking time is 30 minutes, the temperature is 20° C., and the rotation speed is 1800 r / min.

4. The plant seed protein component based on sequential extraction according to claim 1 15 The method for determining N isotope abundance is characterized in that: In steps S1 and S3, during centrifugation, the centrifugation time was 15 min, the temperature was 20° C., and the rotation speed was 5000 r / min.

5. The plant seed protein component based on sequential extraction according to claim 1 15 The method for determining N isotope abundance is characterized in that: In step S2, the ratio of albumin solution to anhydrous ethanol is 1:

3.

6. The plant seed protein component based on sequential extraction according to claim 1 15 The method for determining N isotope abundance is characterized in that: In step S2, the precipitation treatment is performed at -20°C for 60 minutes.

7. The plant seed protein component based on sequential extraction according to claim 1 15 The method for determining N isotope abundance is characterized in that: In step S2, the ratio of the globulin, alcohol-soluble protein, glutenin solution to the 20% trichloroacetic acid solution containing 0.14% mercaptoethanol is 1:

1.

8. The plant seed protein component based on sequential extraction according to claim 1 15 The method for determining N isotope abundance is characterized in that: In step S4, during freeze drying, the temperature is -80°C and the vacuum pressure is less than 1 mbar.

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