CN109781825B

CN109781825B - Extraction method and electrophoresis detection method of rice albumin

Info

Publication number: CN109781825B
Application number: CN201910071937.0A
Authority: CN
Inventors: 兰静; 张瑞英; 贾雯靖; 关海涛; 孙向东; 赵琳; 金海涛; 王冰
Original assignee: SAFETY AND QUALITY INSTITUTE OF AGRICULTURAL PRODUCTS HEILONGJIANG ACADEMY OF AGRICULTURAL SCIENCES
Current assignee: SAFETY AND QUALITY INSTITUTE OF AGRICULTURAL PRODUCTS HEILONGJIANG ACADEMY OF AGRICULTURAL SCIENCES
Priority date: 2019-01-25
Filing date: 2019-01-25
Publication date: 2021-06-15
Anticipated expiration: 2039-01-25
Also published as: CN109781825A

Abstract

The invention provides a method for extracting rice albumin and an electrophoresis detection method. Naturally drying the rice, grinding the rice into rice flour, and adding the rice flour into the rice flour according to a solid-liquid ratio of 1 g: adding 1mL of water, stirring uniformly, leaching for 1h, centrifuging, and taking supernatant, namely the rice albumin extracting solution. By utilizing the method for extracting the albumin from the rice, the albumin is extracted more fully, the albumin extraction rate is improved, and the obtained electrophoresis pattern is clearer. The invention applies the rice albumin electrophoresis technology to the rice taste quality evaluation for the first time, and evaluates the rice taste quality from the aspect of protein component structure, thus being more scientific. The method for extracting the albumin from the rice is suitable for identifying rice varieties from different sources, and has important significance for guiding rice quality breeding and the like.

Description

Extraction method and electrophoresis detection method of rice albumin

Technical Field

The invention relates to the technical field of biology, in particular to an extraction method and an electrophoresis detection method of rice albumin.

Background

The quality traits of rice varieties have become important targets for breeding work, and the practicability and rationality of early quality selection methods are important concerns for breeders. The taste quality of rice is the ultimate goal of rice quality breeding, and is always a blind area in the screening of early generation materials of rice, and no better evaluation technology exists. Protein is a main character of rice quality, and is an important aspect of rice quality improvement. Regarding the relationship between protein and rice quality, the existing reports at home and abroad mainly focus on the relationship change between the apparent protein content and the taste quality. Generally, the higher the protein content of rice, the lower the taste value, and for rice with the approximate protein content, the difference of taste values is larger, which needs to be explained from the aspects of protein components and structures.

The storage protein in the rice protein is the second most nutrient component of rice, and the content of the storage protein accounts for 5.5-12%. Proteins in rice are classified into albumin, globulin, prolamin and gluten according to their solubility in different solvents. The storage proteins are distributed unevenly in rice, and the albumin is mainly distributed in an aleurone layer and a secondary aleurone layer. Research shows that albumin accounts for about 2-5% of total storage protein of rice and is related to taste quality. Few studies on albumin are reported at present. Only one report on the albumin extraction method is found (influence of nitrogen on the content of protein components of rice and the quality of cooked food flavor, Yangjing, agricultural college of northeast university of agriculture, 2006), but the obtained albumin content is low, and the extraction is insufficient. The albumin electrophoresis technology has not been reported yet. Therefore, the development of the rice taste quality protein electrophoresis evaluation technology is of great significance for shortening the breeding period, accelerating the rice breeding process and improving the rice breeding efficiency by applying the technology to the screening of early generation materials of rice.

Disclosure of Invention

The invention aims to provide a method for extracting rice albumin with high speed and high extraction rate aiming at the problems in the prior method for extracting rice albumin.

The invention also aims to provide an electrophoresis method of the rice albumin, which is obviously related to the evaluation of the taste quality of rice, aiming at the current situation that the prior rice albumin electrophoresis technology is blank.

In order to achieve the object of the present invention, in a first aspect, the present invention provides a method for extracting albumin from rice, comprising the steps of naturally air-drying rice, grinding the rice into rice flour, and adding 1g of albumin: adding 1mL of water, stirring until uniform leaching is achieved for 1h, centrifuging, and taking supernatant, namely the rice albumin extracting solution.

In the above method, after leaching, centrifugation is preferably carried out at 15000r/min at room temperature for 5 min.

In a second aspect, the invention provides a method for electrophoretic detection of rice albumin, comprising the steps of mixing the obtained rice albumin extract with a protein extract, standing for a period of time, centrifuging, and taking the supernatant for SDS-PAGE electrophoretic detection.

Wherein the protein extracting solution is as follows: 0.125mol/L Tris-HCl pH6.8, 4% SDS, 20% glycerol, 4mol/L urea, 5% beta-mercaptoethanol.

The electrophoresis detection method comprises the following steps:

1) putting 100 μ L of the rice albumin extract in a centrifugal tube, adding 100 μ L of the protein extract, shaking, mixing, and standing at 4 deg.C for 30 min; centrifuging at 10000r/min for 5min, and performing SDS-PAGE electrophoresis on the supernatant;

2) after electrophoresis, taking out the gel, and fixing the gel in trichloroacetic acid solution;

3) dyeing and decoloring: the fixed gel was transferred to Coomassie Brilliant blue R-250 solution, stained with a shaker, and then destained.

Further, the gel prepared by performing SDS-PAGE electrophoresis in step 1) is: 15% of separation gel and 4% of concentrated gel; in the separation gel, the voltage is 100V, when the gel is concentrated, the voltage is reduced to 80V, and the total electrophoresis time is 2.5 h.

Step 2) the gel was fixed in 10% trichloroacetic acid solution for 2 h.

In the step 3), the rotating speed of a shaking table is 70r/min, and dyeing is carried out for 2 h.

Preferably, SDS-PAGE is run at a rate of 10. mu.L/well.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

by utilizing the method for extracting the albumin from the rice, the albumin is extracted more fully, the albumin extraction rate is improved, and the obtained electrophoresis pattern is clearer.

The invention firstly establishes an electrophoresis method of the rice albumin, and fills the blank of the research fields of the rice albumin electrophoresis detection technology and the taste quality evaluation.

And thirdly, the rice albumin is closely related to the taste quality, the invention combines the electrophoresis technology, provides a theoretical basis for identifying the taste quality of the rice, and simultaneously makes up the current situation of the blank of the rice albumin electrophoresis technology.

The invention firstly applies the rice albumin electrophoresis technology to the rice taste quality evaluation, and evaluates the rice taste quality from the aspect of protein component structure, thus being more scientific.

The method for extracting the albumin from the rice is suitable for identifying rice varieties from different sources, and has important significance for guiding rice breeding and the like.

Drawings

FIG. 1 shows the SDS-PAGE electrophoresis detection results of two methods for extracting albumin from rice in example 1; wherein A corresponds to method one and B corresponds to method two.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

The percent in the present invention means mass percent unless otherwise specified; but the percent of the solution, unless otherwise specified, refers to the grams of solute contained in 100mL of the solution.

Example 1 extraction and electrophoretic detection of Rice Albumin

1. Preparation of rice sample: after the rice was naturally air-dried, 100g of rice was weighed per rice sample. Grinding the brown rice into brown rice by a brown rice grinding machine, and grinding the brown rice into polished rice by a rice polishing machine. Milling the polished rice into powder by a milling machine, and storing in a refrigerator at-20 deg.C for use.

2. And (3) measuring the rice protein content: the measurement was carried out by using a Kjeldahl apparatus. The selected samples Longjing Xiang No. 1 and the sorghum rice crude protein content are shown in Table 1.

3. And (3) measuring the taste score of the rice: measured using a taste meter in combination with sensory scores. The taste scoring results are shown in table 1.

TABLE 1 crude protein content and taste score

Name of breed	Crude protein content	Taste scoring
			Longjing Xiang No. 1	7.91％	71
Sorghum rice	7.70％	90

4. Albumin extraction:

the method comprises the following steps: this method has been reported. Weighing 0.1g of polished rice flour in a 2mL centrifugal tube, adding 1mL of distilled water, shaking and extracting for 2h on a shaking table, and centrifuging at room temperature at 10000r/min for 10 min. Transferring the supernatant into a 10mL graduated test tube, repeatedly extracting for 3 times, and mixing the extractive solutions to obtain albumin solution, and storing at-20 deg.C for use.

The second method comprises the following steps: the method of the invention. Weighing 1g of fine rice powder in a 2mL centrifugal tube, adding 1mL of distilled water, stirring to be uniform, and leaching for 1 h. Centrifuge at 15000r/min for 5min at room temperature. The supernatant (albumin solution) was taken in a 2mL centrifuge tube and stored at-20 ℃ until use.

5. Preparing gel: SDS-PAGE electrophoresis was performed on 15% separation gel and 4% concentration gel, respectively.

6. Preparing a protein extracting solution: 0.125mol/L Tris-HCl pH6.8, 4% SDS, 20% glycerol, 4mol/L urea, 5% beta-mercaptoethanol.

7. And (3) electrophoresis detection: mu.L of albumin solution was taken in a 2mL centrifuge tube, and 100. mu.L of protein extract was added. Vortex and shake for 1min, mix well. Standing at 4 deg.C for 30 min. Then, the mixture is centrifuged at 10000r/min for 5 min. The supernatant was subjected to an electrophoresis test in an amount of 10. mu.L/well. In the separation gel, the voltage is 100V, and when the gel is concentrated, the voltage is reduced to 80V. The electrophoresis time is 2.5 h.

8. Protein fixation: and taking out the gel after the electrophoresis is finished. And (4) placing the mixture in 10% trichloroacetic acid solution for fixation for 2 h.

9. Dyeing and decoloring: the gel was placed in 40mL Coomassie Brilliant blue R-250 solution and stained on a shaker for 2 h. And then decolorized.

The results of the albumin electrophoresis are shown in FIG. 1, and it can be seen from FIG. 1 that the albumin electrophoresis pattern obtained in the first method is almost free from the band (A); the albumin spectrum obtained by the second method has clear and visible spectral bands (B), and the extraction effects of the second method in two rice varieties are basically the same, which shows that the method has wide applicability and good stability.

Different operators can carry out the extraction of the albumin of the rice and the electrophoresis detection according to the method, and the results are basically consistent.

In FIG. 1B, two rice varieties with similar crude protein content but different taste scores are shown. As can be seen from the figure, Longjing Xiang No. 1 has a band deletion at 70-105KDa, while the sorghum rice band is normal. It was preliminarily concluded that the absence of the 105kDa albumin band could be correlated with the taste score of rice.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. The electrophoresis detection method of the rice albumin is characterized in that the rice is naturally dried and ground into rice flour, and the rice flour is added with the following components in a solid-to-liquid ratio of 1 g: adding 1mL of water, stirring uniformly, leaching for 1h, centrifuging at 15000r/min for 5min, and taking supernatant, namely the rice albumin extracting solution;

mixing the rice albumin extract and the protein extract, standing for a period of time, centrifuging, and taking supernatant to perform SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis detection;

wherein the protein extracting solution is as follows: 0.125mol/L Tris-HCl pH6.8, 4% SDS, 20% glycerol, 4mol/L urea, 5% beta-mercaptoethanol;

the rice is from the rice variety Longjing Xiang No. 1 and sorghum rice;

the electrophoresis detection comprises the following steps:

3) dyeing and decoloring: transferring the fixed gel into a Coomassie brilliant blue R-250 solution, dyeing by a shaking table, and then decoloring;

the gel prepared by performing SDS-PAGE electrophoresis in the step 1) is as follows: 15% of separation gel and 4% of concentrated gel; in the separation gel, the voltage is 100V, when the gel is concentrated, the voltage is reduced to 80V, and the total electrophoresis time is 2.5 h.

2. The method of claim 1, wherein step 2) immobilizes the gel in 10% trichloroacetic acid solution for 2 hours.

3. The method as claimed in claim 1, wherein in step 3), the shaking table is rotated at 70r/min and the dyeing is carried out for 2 h.

4. The method according to any one of claims 1 to 3, wherein the amount of SDS-PAGE electrophoresis of step 1) is 10. mu.L/well.