CN101726486B - Quick analyzing method for glutelin content of wheat - Google Patents

Abstract

The invention relates to a quick analyzing method for glutelin content of wheat. 5 to 30 grams of representative kernels is selected from a wheat sample to be tested, the kernels are milled into whole powder by a laboratory mill, the powder is screened by a screen of which aperture is 0.5 millimeter, then the screened powder is filled in a plastic self-sealing bag, the powder is stood for at least 2 to 4 hours at the room temperature to balance the moisture, then the moisture content is measured, and the powder is used as a tested sample. The method is characterized by comprising the following steps: accurately weighing 100 milligrams of the tested sample, putting the tested sample in a centrifugal tube, separately extracting monomer protein from the tested sample by a solvent, or extracting monomer protein and soluble glutelin polymer with lower molecular weight at the same time, and then removing supernate after centrifugation to keep the glutelin in the centrifugal tube; and adding 1 milliliter of biuret reagent into the centrifugal tube, treating the mixture by using an ultrasonic crusher, and measuring the content of the glutelin in the tested sample through the color development reaction of glutelin components, wherein the content of the glutelin in the tested sample refers to the total amount of the glutelin in the tested sample or the content of large glutelin polymer.

Description

Rapid Analysis Method of Glutenin Content in Wheat

technical field

The invention relates to a method for separating and quantifying wheat gluten, belonging to the field of grain chemistry.

Background technique

Protein accounts for about 8% to 20% of the weight of wheat grains, and is mainly composed of two types of storage proteins, glutenin and gliadin. Among them, glutenin is the main influencing factor of wheat gluten quality and food quality, and is the most important target trait in wheat quality improvement. It has been regarded as an important content of wheat grain chemistry research. In the quality evaluation of wheat gluten, protein content, sedimentation value and high molecular weight glutenin subunit marker selection were mostly used in early generations; protein content and dough rheological properties were mostly used in high generations, basically according to genes with high protein and high quality subunits Based on the general principle of good gluten quality, it played an important role in the early stage of wheat quality breeding in my country and greatly improved the processing quality of wheat products. However, with the improvement of the quality breeding level, it is more and more found that those genotypes with high protein and high quality subunits do not necessarily have good processing quality. Wheat gluten exists in the form of a polymer (Glutenin polymer), and the part with a high degree of aggregation that is not easily extracted is called glutenin macropolymer (GMP), which has a significant positive correlation with gluten strength . Therefore, the total amount of glutenin aggregates and the content of glutenin macromers are important contents of quality evaluation in wheat breeding generations.

The current methods for the determination of gluten content mainly include the following:

1. Biuret colorimetric method

R.J.，张立平.Glu-1和Glu-3等位变异对不溶性谷蛋白含量的影响.作物学报，2004，30：1086-1092)，主要有两个步骤，第一步利用70％乙醇或50％丙醇将面粉样本中的醇溶蛋白部分提取，经离心后谷蛋白部分保留在沉淀中，随后利用手工碾磨的方法在双缩脲试剂中将沉淀破碎，并对其中的谷蛋白组分进行显色反应，随后测定其含量。这种方法存在两个问题，一是只能分析谷蛋白聚合体的总量，而不能分离测定谷蛋白大聚体的含量；二是预提醇溶蛋白后，在双缩脲试剂中使用手工碾磨含有谷蛋白的沉淀，很难使沉淀充分破碎，造成平行样本间的重复性很差，重复分析使得工作量大幅增加，费时费力；同时使得测定值偏低，偏离客观值。The biuret colorimetric method is simple and fast, and is a common method for protein content determination. This method measures the protein content through the color reaction between the peptide bond and Cu ²⁺ under alkaline conditions. It is suitable for all kinds of protein content determination, and has the characteristics of simple operation, rapidity and low cost. Biuret colorimetry has been applied in the determination of gluten content (Liu Li, Zhou Yang, He Zhonghu,

RJ, Zhang Liping. The effect of Glu-1 and Glu-3 allelic variation on the content of insoluble gluten. Acta Crops Sinica, 2004, 30: 1086-1092), there are two main steps, the first step is to use 70% ethanol or 50% Propanol extracts the prolamin part of the flour sample, and the gluten part remains in the precipitate after centrifugation, and then the precipitate is broken in the biuret reagent by manual milling, and the gluten component in it is analyzed. Color reaction, followed by determination of its content. There are two problems with this method. One is that it can only analyze the total amount of glutenin polymers, but cannot separate and measure the content of glutenin macropolymers; Grinding the precipitate containing gluten makes it difficult to fully break the precipitate, resulting in poor repeatability between parallel samples. Repeated analysis will greatly increase the workload, time-consuming and laborious; at the same time, the measured value will be low and deviate from the objective value.

Two, n-propanol or propanol precipitation method

There are two main ways in which this law can be applied.

The first application mode is to directly use 50% n-propanol to extract the flour sample. After centrifugation, the protein in the flour is divided into two parts. The supernatant part contains albumin, globulin, gliadin, and a small part of Glutenin aggregates of lower molecular weight; the precipitated fraction contains most of the glutenin aggregates. The protein content of the pellet was determined after drying and was referred to as the content of "insoluble gluten aggregates". Although this method is simple, it still does not separate and quantify the total glutenin and glutenin macropolymers (Bean S R, Lyne R K, Tilley K A, Chung O K, Lookhart G L. A rapid method for quantitation of insoluble polymeric proteins in flour. Cereal Chem, 1998, 75: 374-379).

In the second mode of application, the first mode was first adopted, and then the concentration of n-propanol in the supernatant was increased to 70%, so that those small amounts of glutenin aggregates with lower molecular weight were precipitated. As a result, the flour protein is divided into three parts, that is, monomer protein (albumin, globulin, gliadin), a small part of glutenin aggregates with low molecular weight, and most of the glutenin in the precipitate. This method is not only complicated, but also does not separate and quantify the total amount of glutenin and the glutenin macropolymer (Fu B X, Sapirstein H D. Procedure for isolating monomeric proetins and polymeric glutenin of wheat flour. Cereal Chem, 1996, 73 : 143-152).

3. Densitometric scanning method of single subunit gel electrophoresis

Glutenin polymers are natural macromolecules with the largest molecular weight known in nature, and it is difficult to fully extract them using conventional non-reducing solutions and mechanical action. Proteins are reduced to monomeric subunits. The stained subunit bands were quantified by densitometric scanning after separation by gel electrophoresis. This method (Huang D Y, KhanK. Quantitative determination of high molecular weight glutenin subunits of hard red spring wheat by SDS-PAGE. II. Quantitative effects of individual subunits on breadmaking quality characteristics. Cereal Chem, 1997-74: 78) exists The following problems: (1) the determination is of a single subunit or the total amount of glutenin, and the content of glutenin macromers cannot be determined; (2) the subunit quantification of this method is usually done by gel electrophoresis, and the cost is relatively high , Time-consuming and labor-intensive and often use toxic chemicals, such as acrylamide, 4-vinylpyridine, etc.

4. SDS phosphate buffer extraction, multi-layer stacking gel separation + optical density scanning method

This method uses SDS phosphate buffer to divide flour protein into two parts. The first part includes monomeric proteins (albumin, globulin, gliadin) and soluble glutenin polymers with lower molecular weight; the second part includes insoluble glutenin aggregates. Higher molecular weight gluten macromers. Then the first part is separated by multi-layer stacking gel, stained and densitometrically scanned, combined with Kjeldahl nitrogen or near-infrared protein content determination of the first part and the second part, and the protein components with different molecular weights in the first part and the second part are calculated. Content of some glutenin macromers (Zhu J, Khan K. Effects of genotype and environment on glutenin polymers and bread making quality. Cereal Chem, 2001, 78: 125-130). The main problems of this method are: (1) the measurement process and technology are complicated, the cost is high, time-consuming and laborious; (2) the simultaneous use of different measurement methods and multiple calculations may cause the measured value to deviate from the objective value.

5. Ultrasonic crushing + gel chromatography

The method combined with SDS phosphate buffer and ultrasonic crushing can flexibly determine the content of different gluten components. Direct sonication in SDS phosphate buffer and gel chromatography can determine the total amount of glutenin aggregates; using only SDS phosphate buffer shock extraction and gel chromatography can determine the amount of glutenin aggregates with lower molecular weight Content, after the precipitation is treated by ultrasonic, the content of glutenin macromer can be determined by gel chromatography (Gupta R B, Khan K, MacRitchie F.Biochemical basis of flour properties in bread wheats.I.Effects of variation in the quantity and size distribution of polymeric protein. J Cereal Sci, 1993, 18: 23-41). This method is simple to extract the total amount of glutenin aggregates, glutenin aggregates with low molecular weight and large glutenin aggregates, but it needs to use chromatographic equipment in quantification. Not only the chromatographic equipment itself is expensive, but also the cost of determination consumables is high, and the sample pretreatment and instrument operation are relatively complicated, requiring full-time quality inspection personnel, which reduces work efficiency.

After the tested sample is crushed by ultrasonic waves in the biuret reagent, the color reaction of the biuret reagent is used to measure the gluten content after colorimetry, and there is no report at present.

Contents of the invention

The object of the present invention is: in view of the characteristics of the above-mentioned various measuring methods, aiming at the problems existing in their measuring process, provide a kind of wheat gluten content rapid analysis method which is different from the above-mentioned various measuring methods.

The purpose of the present invention is achieved in this way: a rapid analysis method for the glutenin content of wheat, the representative grain of 5g～30g of the wheat sample to be tested is cleaned, and the whole powder is made by using the experimental mill, and then packed after passing through a 0.5mm aperture sieve. Put it into a plastic ziplock bag, let it stand at room temperature for at least 24 hours to balance the moisture, and measure the water content. As the sample to be tested, it is characterized in that: accurately weigh 100 mg of the sample to be tested and place it in a centrifuge tube, and extract the monomer from the sample to be tested separately by solvent Protein, or after extracting monomeric protein and soluble glutenin aggregates with low molecular weight at the same time, discard the supernatant after centrifugation to keep the glutenin in the centrifuge tube; add 1mL biuret reagent to the centrifuge tube, use ultrasonic After the crushing instrument is processed, the gluten content in the tested sample is determined through the color reaction of the gluten component; the gluten content in the tested sample refers to the total amount of gluten or gluten in the tested sample macromer content.

In the present invention: the solvent for separately extracting monomeric protein from the tested sample is 70% (v/v) ethanol; the solvent for simultaneously extracting monomeric protein and soluble glutenin aggregates with a lower molecular weight from the tested sample It is SDS phosphate buffer solution with pH 6.90, and the monomeric protein includes white egg, globulin and gliadin.

In the present invention: the method for separately extracting monomeric protein from the tested sample is: accurately weigh 100 mg of the tested sample and place it in a 1.5 mL centrifuge tube, add 1 mL of 70% (v/v) ethanol, and continuously shake at room temperature for 30 min. Centrifuge at 12000rpm for 15min, discard the supernatant; add the same solvent again, shake continuously for 15min, and centrifuge at 12000rpm for 15min, so that the prolamin in the tested sample is fully dissolved in the solvent.

In the present invention: after extracting the monomeric protein, discard the supernatant by centrifugation, invert the centrifuge tube for 5 minutes to keep the gluten in the centrifuge tube, add 1 mL of biuret reagent to the centrifuge tube, and use an ultrasonic breaker to process for 15 seconds. Immediately transfer the suspension to a capped 10mL centrifuge tube filled with 4mL biuret reagent with a pipette gun, mix well and immediately put it in a dry heat oven at 40°C for 20min, and shake it upside down during 5min, 10min and 15min Once; take it out and centrifuge it at 4000rpm for 10min, compare the color of the supernatant at 560nm, refer to the background as the biuret reagent, substitute the absorbance value into the curve equation established with the standard protein, and calculate the total amount of gluten in the tested sample; In the curve equation, the correlation coefficient r≥0.9998.

In the present invention: the method for extracting monomeric protein and soluble glutenin polymers with lower molecular weight from the tested sample is: accurately weigh 100 mg of the tested sample and place it in a 1.5 mL centrifuge tube, add 1 mL of SDS with a pH of 6.90 Phosphate buffer solution, continuously shake at room temperature for 20 minutes, and centrifuge at 12,000 rpm for 15 minutes to fully dissolve the monomeric protein and soluble glutenin polymers with lower molecular weight in the test sample in the solvent.

In the present invention: after extracting monomeric protein and soluble glutenin aggregates with lower molecular weight, the supernatant is discarded by centrifugation, the centrifuge tube is inverted for 5 minutes, so that gluten is retained in the centrifuge tube, and 1 mL of double gluten is added to the centrifuge tube. For uret reagent, use an ultrasonic breaker to treat it for 15 seconds, then use a pipette gun to transfer the suspension to a capped 10mL centrifuge tube containing 4mL of biuret reagent, mix well and immediately put it in a dry heat oven at 40°C for 20min Take it out and centrifuge it at 4000rpm for 10min, compare the color of the supernatant at 560nm, refer to the background as the biuret reagent, substitute the absorbance value into the curve equation established with the standard protein, and calculate the glutenin macromer content in the tested sample , in the curve equation, the correlation coefficient r≥0.9998.

In the present invention: the probe diameter of the ultrasonic breaker is Φ3 mm, and the ultrasonic breaker processing means that the ultrasonic breaker performs processing under the output power state of 12W.

The invention has the advantages that: the ultrasonic breaker has the outstanding advantages of rapidity, high strength and uniform action, which can make the extraction rate and speed of wheat glutenin higher; 70% ethanol can extract monomeric protein including gliadin , SDS phosphate buffer at pH 6.90 can simultaneously extract monomeric protein and soluble glutenin aggregates with low molecular weight, so that glutenin remains in the centrifuge tube; use the color reaction of biuret reagent to determine the concentration of gluten in the tested sample The total amount of glutenin or the content of glutenin macromers is not only simple to operate, but also has the obvious advantages of rapidity, low cost and accuracy compared with various determination methods currently used.

Detailed ways

Example 1

Among the wheat samples to be tested, 20 g of representative grains after cleaning were selected, and whole wheat flour was ground by FOSSCyclotec 1093 experimental cyclone. Spoon fully stir evenly and seal the bag mouth, place at room temperature for more than 24 hours until the moisture content is uniform, according to GB 5497-85, that is, grain oil detection-moisture determination method to measure the moisture content of the whole wheat flour sample, as the sample to be tested.

Example 2

a. Biuret reagent preparation:

4% CuSO ₄ (w/v), 2.5% sodium potassium tartrate (w/v) and 5M NaOH solutions were prepared at room temperature according to the amount of sample required for determination. Add 30mL of CuSO4 solution and 100mL of potassium sodium tartrate solution to a _500mL volumetric flask, then slowly add 30mL of NaOH solution, and finally distill the volume to 500mL with distilled water to form a spare solution;

Before starting to measure the sample to be tested, take an appropriate amount of the stock solution and mix it with an equal volume of isopropanol to form a biuret reagent.

b. SDS phosphate buffer preparation:

Solution 1: 0.05mol/L Na ₂ HPO ₄ , 0.5% SDS (w/v);

Solution 2: 0.05mol/L NaH ₂ PO ₄ , 0.5% SDS (w/v);

Take an appropriate amount of solution 1 in a beaker, slowly add solution 2, and continue to stir until the pH of the mixed solution drops to 6.90, which is the SDS phosphate buffer solution.

Example 3

Standard curve drawing:

Dilute protein standard solution (7g/dL) with 0.1mol/L NaOH solution to 5mg/mL standard solution. Take protein standard solution (5mg/mL) 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0mL in 10mL test tubes, add 1.0, 0.9, 0.8, 0.7, 0.6, 0.5 , 0.4, 0.3, 0.2, 0.1, 0.0ml of distilled water, add 4.0mL biuret reagent (provided in a in Example 2, the same below) to each test tube, mix well, 40 ℃ water bath 20min, 560nm wavelength colorimetry, measure the absorbance value. Take the absorbance value as the abscissa, and take the protein content as the ordinate to make a standard curve, and obtain the following one-variable linear equation:

y=12.516x-0.0316

In the formula: x is the absorbance value, y is the percentage of protein, and in this embodiment, their correlation coefficient r=0.9999.

Example 4

Analysis of total gluten in the tested sample:

Accurately weigh 100 mg of the tested sample (provided in Example 1) and place it in a 1.5 mL centrifuge tube. There were 12 samples in each group, and each sample was repeated 2 times. Add 1 mL of 70% (v/v) ethanol. Shake continuously at room temperature for 30 minutes, centrifuge at 12,000 rpm for 15 minutes, discard the supernatant; add 1 mL of 70% (v/v) ethanol. Shake continuously at room temperature for 15 minutes, centrifuge at 12,000 rpm for 15 minutes, and invert the centrifuge tube for 5 minutes;

Add 1mL of biuret reagent to the 1.5mL centrifuge tube containing the precipitate, use an ultrasonic breaker VCX130PB, equipped with a Φ3mm probe, power 12W, and process for 15s until the precipitate is fully suspended.

Use a pipette gun to move the sonicated suspension into a capped 10mL centrifuge tube containing 4mL of biuret reagent, and repeat the blowing once to fully transfer the sample and make the suspension more uniform.

Cover the test tube tightly and immediately place it in a dry heat oven at 40°C for 20 minutes, and shake it upside down once at 5 minutes, 10 minutes and 15 minutes respectively. Take it out and centrifuge at 4000rpm for 10min, and compare the color of the supernatant at 560nm. The reference background is the biuret reagent.

The total amount of gluten in the sample can be calculated by substituting the absorbance value into the curve equation, and the dry basis content can be calculated according to the water content of the sample.

Example 5

Analysis of the content of glutenin macromers in the sample:

Accurately weigh 100mg sample and place it in a 1.5mL centrifuge tube. 12 samples per group, 2 replicates per sample. Add 1 mL of SDS phosphate buffer solution (provided in b in Example 2), shake continuously at room temperature for 20 min, centrifuge at 12000 rpm for 15 min, discard the supernatant, and invert the centrifuge tube for 5 min.

Add 1mL of biuret reagent to the 1.5mL centrifuge tube containing the precipitate, use an ultrasonic breaker VCX130PB, equipped with a Φ3mm probe, power 12W, and process for 15s until the precipitate is fully suspended.

Use a pipette gun to move the sonicated suspension into a capped 10mL centrifuge tube containing 4mL of biuret reagent, and repeat the blowing once to fully transfer the sample and make the suspension more uniform.

Cover the test tube tightly and immediately place it in a dry heat oven at 40°C for 20 minutes, and shake it upside down once at 5 minutes, 10 minutes and 15 minutes respectively. Take it out and centrifuge at 4000rpm for 10min, and compare the color of the supernatant at 560nm. The reference background is the biuret reagent.

By substituting the absorbance value into the curve equation, the glutenin macromer content in the sample can be calculated, and the dry basis content can be calculated according to the water content of the sample.

Example 6

According to the method of Examples 1-5, we analyzed the total glutenin content, glutenin macromer content and gluten strength of 85 varieties (lines) of wheat respectively, and the results are shown in Table 1. In Table 1, the determination of grain protein content uses the NIR method (Perten DA7200); the determination of water content uses the GB 5497-85 method; the sedimentation value is according to the AACC 44-15A method; National Mfg). "-" indicates that the data is missing; the percentage of glutenin macromers, namely: the percentage of glutenin macromers (%)=(glutenin macromers content/glutenin total amount)×100.

It can be seen from Table 1 that the peak time of the kneading instrument, which represents the strength of gluten, is significantly positively correlated with the total amount of gluten, the content of large aggregates, and the percentage of large aggregates obtained by ultrasonic extraction, and the correlation coefficients are 0.60, 0.77, and 0.87 (P<0.001). However, there was no significant correlation between grain protein content and sedimentation value and peak time, indicating that the content of gluten components measured by this method, especially the percentage of large aggregates, can predict gluten quality well.

It can also be told from Table 1 that the variation ranges of the total amount of gluten measured by the milling method and the ultrasonic method are 0.74% to 2.47% and 0.84% to 2.60%, respectively. The comparison between genotypes showed that the extraction rate of milling method was 79.6%-99.4% of that of ultrasonic method. It shows that this method has a higher extraction rate of gluten, more accurate measurement value, and is more conducive to the evaluation of gluten quality and the development of wheat quality breeding.

Table 185 varieties (lines) of quality traits and gluten content analysis

The above embodiments do not specifically limit the present invention.

Claims (5)

1. A rapid analysis method for the glutenin content of wheat. The wheat sample to be tested is cleaned to select 5g to 30g of representative grains, and the whole powder is ground by an experimental mill. After passing through a 0.5mm aperture sieve, put it into a plastic ziplock bag, and keep the room temperature at least Stand still for 24 hours to balance the moisture and determine the water content. As the sample to be tested, accurately weigh 100mg of the sample to be tested and place it in a centrifuge tube. Extract monomeric protein from the sample to be tested, or extract monomeric protein and soluble molecular weight at the same time After lowering the glutenin aggregates, discard the supernatant after centrifugation to keep the gluten in the centrifuge tube; add 1mL biuret reagent to the centrifuge tube, and use an ultrasonic breaker to process the glutenin component. color reaction to measure the gluten content in the tested sample; the gluten content in the tested sample refers to the gluten total amount or the gluten macropolymer content in the tested sample; It is characterized by: Centrifuge the extracted monomeric protein to discard the supernatant, then invert the centrifuge tube for 5 minutes to keep gluten in the centrifuge tube, add 1mL biuret reagent to the centrifuge tube, use ultrasonic breaker to treat for 15s, and then use a pipette gun Transfer the suspension to a capped 10mL centrifuge tube filled with 4mL of biuret reagent, mix well and immediately place it in a dry heat oven at 40°C for 20min, and shake it upside down once at 5min, 10min and 15min respectively; take it out at 4000rpm Centrifuge for 10 minutes, colorimetric the supernatant at 560nm, refer to the background as the biuret reagent, substitute the absorbance value into the curve equation established with the standard protein, and calculate the total amount of gluten in the tested sample; Centrifuge the simultaneously extracted monomeric protein and soluble glutenin polymers with low molecular weight to discard the supernatant, then invert the centrifuge tube for 5 minutes to keep the gluten in the centrifuge tube, add 1mL biuret reagent to the centrifuge tube , use an ultrasonic breaker to treat for 15 seconds, then use a pipette gun to transfer the suspension to a capped 10mL centrifuge tube containing 4mL biuret reagent, mix well and immediately put it in a dry heat oven at 40°C for 20min; take it out at Centrifuge at 4000rpm for 10min, compare the color of the supernatant at 560nm, refer to the background as the biuret reagent, substitute the absorbance value into the curve equation established with the standard protein, and calculate the glutenin macromer content in the tested sample; In the curve equation, the correlation coefficient r≥0.9998. 2. the glutenin content rapid analysis method of wheat according to claim 1 is characterized in that: the solvent that separately extracts monomeric protein from tested sample is 70% ethanol; The solvent for soluble glutenin aggregates with low molecular weight is SDS phosphate buffer at pH 6.90, and the monomeric proteins include albumin, globulin and gliadin. 3. The method for rapid analysis of gluten content of wheat according to claim 1 or 2, characterized in that: the method for separately extracting monomeric protein from the tested sample is: accurately weigh 100 mg of the tested sample and place it in a 1.5 mL centrifuge Add 1mL of 70% ethanol to the tube, shake continuously at room temperature for 30min, centrifuge at 12000rpm for 15min, discard the supernatant; add the same solvent again, shake continuously for 15min, and centrifuge at 12000rpm for 15min, so that the monomeric protein in the tested sample is fully dissolved in the solvent . 4. according to claim 1 and 2 described glutenin content rapid analysis methods of wheat, it is characterized in that: the method for extracting monomeric protein and soluble glutenin polymer with the lower molecular weight from tested sample is: accurately weigh Take 100mg of the tested sample and place it in a 1.5mL centrifuge tube, add 1mL of SDS phosphate buffer solution with pH 6.90, shake continuously at room temperature for 20min, and centrifuge at 12000rpm for 15min to polymerize monomeric protein in the tested sample with soluble glutenin with low molecular weight The body is fully soluble in the solvent. 5. The fast analysis method for glutenin content of wheat according to claim 1, characterized in that: the probe diameter of the ultrasonic breaker is Φ3mm, and the processing of the ultrasonic breaker refers to: the output power state of the ultrasonic breaker at 12W below for processing.