CN104006998A - Method for separating different components of starch of rice and determining contents thereof - Google Patents

Abstract

A method for the separation and content determination of different components of rice starch, comprising randomly taking more than 10 rice grains, and performing the separation and content determination of different starch components on each rice grain: adding 10ml of pure water to the rice grains and gelatinizing in a water bath at 80°C Obtain a starch suspension; use a relative centrifugal force of 20,000g, centrifuge at 4°C for 30 minutes to obtain the supernatant and precipitate, take out the supernatant as the sample to be tested, add 10ml of 1.5mg/ml KOH solution to the precipitate, and then centrifuge to remove the supernatant The solution is another sample to be tested; the concentration of the KOH solution added to the precipitate is increased by a gradient of 1.5 mg/ml each time, and the precipitate is centrifuged. Repeat this until the obtained precipitate cannot be dissolved, and a series of samples to be tested are obtained; then press The national standard GB/T15683-2008 measures the apparent amylose starch content of the samples to be tested respectively. The method of the invention can measure the apparent amylose content of different starch components in the rice breeding material rice, and provides a basis for the breeding work of new rice varieties with excellent taste and quality.

Description

A method for separation and content determination of different components of rice starch

technical field

The invention belongs to the technical field of rice quality measuring methods, and in particular relates to a method for separating starch components with different rice polymerization degrees and measuring their content.

Background technique

According to the taste and texture of rice, rice varieties can be basically divided into four categories: ordinary indica rice varieties, ordinary japonica rice varieties, soft rice varieties, and glutinous rice varieties. There are great differences in the taste and texture of rice in these four types of rice varieties. In order to improve the quality of rice varieties and meet people's different needs for rice taste and texture, breeders need to study the factors that affect the taste and texture of rice. It has long been found that there are large differences in rice texture between varieties with similar amylose content. Japanese scholars and the International Rice Institute also found that the long chain of amylopectin plays an important role in determining the texture of rice. It is now clear that the real reason for the difference in rice texture is not the starch content (amylose content, AC), but the number of long chains on the amylopectin and the length of the chain (degree of polymerization).

In 1958, Williams et al. found that there was a correlation between "iodine staining value" and starch content (amylose content, AC), and proposed a colorimetric method for the determination of rice amylose. Later, the improved method became an international standard. However, there is a certain difference between the so-called "AC" calculated from the "iodine staining value" and the real AC of rice. Because part of the long-chain amylopectin can also react with iodine. Therefore, Takeda et al. in 1987 called the AC value measured by the iodine colorimetric method the apparent amylose content. In terms of composition, the apparent amylose actually consists of two parts, that is, the long chain of real amylose and part of amylopectin. That is to say, the long chains of real amylose and partial amylopectin can react with iodine reagent (color developer) to develop color, and any starch that can combine with iodine reagent (color developer) can be visually observed To the color, therefore, the absorbance of the solution that develops the color can be measured by a spectrophotometer, which is called apparent amylose.

Both amylose and amylopectin are glucosides formed by the polymerization of multiple glucose molecules through hydroxyl dehydration. Rice starch is composed of glucoside chains with different degrees of polymerization and contains a large number of hydroxyl groups. Because the hydroxyl groups in starch exist in the state of hydrogen bonds, there is a huge aggregation force between molecules, which prevents the penetration and solvation of starch granules by water molecules; at the same time, there are amylose and amylopectin with different branching degrees, which are entangled together The rice starch is almost insoluble in water. Therefore, in order to improve the solubility of starch, it is necessary to destroy the retrogradation phenomenon caused by hydrogen bonds.

Common methods for breaking hydrogen bonds between starch glucose chains include heating starch liquid at high temperature and adding reagents such as sodium hydroxide or potassium hydroxide.

The main steps of the method for determining the starch content of rice in the prior art are to grind the rice into powder, add a certain concentration of dilute lye to dissolve it, and then detect the absorbance value of the color-developed part, and only the partial content of apparent amylose can be calculated. This method cannot show the apparent amylose content in different components with different degrees of polymerization and starch branching in starch, so it cannot better reflect the apparent amylose content that affects the mouthfeel. The deficiencies and deficiencies of the prior art cannot meet the needs of determining the content of different components of rice starch.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the existence of the prior art, which can only calculate the partial content of apparent amylose in general, and cannot reflect the content of apparent amylose and its distribution of different components with different degrees of polymerization. As a result, the starch components of rice varieties with different mouthfeel textures cannot be better reflected, which affects the selection of rice breeding materials for improving mouthfeel quality.

The purpose of the present invention is to directly measure the content of apparent amylose in different components with different degrees of polymerization in rice that affect the taste quality, so as to provide a basis for screening breeding materials for improving the taste quality of rice. Therefore, the present invention provides a method for the separation and content determination of different components of rice starch.

The method of the invention is to separate the starches with different degrees of polymerization and branching degrees that are intertwined and mixed together, so as to separate and measure the apparent amylose content of different components with different degrees of polymerization in the starch. the

A kind of technical proposal of the method for the separation and content determination of different components of rice starch provided by the present invention comprises the following steps:

(1) Randomly take more than 10 complete rice grains from which chaff, seed coat and embryo have been removed, and operate as follows for each rice grain:

A. Weigh a grain of rice, add 10ml of pure water, gelatinize in a water bath at 80°C for 1 hour, and cool to room temperature to obtain a starch suspension;

B. Centrifuge the starch suspension according to the centrifugal conditions to obtain the supernatant and precipitate, the supernatant is the sample to be tested, marked 0; add 10ml of 1.5mg/ml KOH solution to the precipitate, shake well and save it for the next time centrifugation; the centrifugation conditions are: relative centrifugal force 20000g, centrifugation at 4°C for 30min;

C. Centrifuge the precipitate added with KOH solution in step (1) B. The centrifugation conditions are: relative centrifugal force 20,000g, centrifuge at 4°C for 30min to obtain supernatant and precipitate, and use the obtained supernatant as the sample to be tested, press The order is marked as 1, and the resulting precipitate is then added to KOH solution and centrifuged again, and the centrifugation operation is repeated until the obtained precipitate cannot be dissolved. The KOH solution added to the obtained precipitate is 10ml, but the concentration of the added KOH solution increases with a gradient of 1.5mg/ml, and the supernatant obtained from each centrifugation is the sample to be tested and marked in order, that is, a series of samples to be tested are obtained. Sample;

(2) According to the national standard GB/T 15683-2008 Determination of Amylose Content in Rice, the amylose content of a series of samples to be tested obtained in step (1) C was respectively measured.

On the basis of comparative experiments on different centrifugal forces (10000g, 20000g, 40000g) and centrifugation time (10min, 20min, 30min), the present invention draws: insufficient relative centrifugal force and insufficient time may lead to incomplete separation of sediment and supernatant . After repeated tests, the relative centrifugal force is 20000g, the centrifugation time is 30min, and the temperature is 4 degrees, the effect is the best.

Compared with prior art, the beneficial effect of the present invention is:

The present invention centrifuges the starch several times until the precipitate can no longer be dissolved, and appropriate parameters (centrifugation conditions and the concentration gradient of the concentration of the KOH solution) achieve different components with different degrees of polymerization in the starch from low degrees of polymerization to high degrees of polymerization The effect of continuous separation can effectively measure the apparent amylose content and distribution of the different components of starch in rice of different rice varieties with different mouthfeel qualities (larger differences in mouthfeel quality). Therefore, the use of the present invention The method can measure the apparent amylose content and distribution of different starch components in rice breeding materials (distribution visual map), and provide a direct basis for the improvement of rice taste quality and breeding of new varieties.

Description of drawings

Figure 1 is a direct view of the distribution of relative apparent amylose content in different components of rice starch in indica rice variety 93-11. The abscissa in the figure is the KOH solution concentration of 1.5 mg/ml, 3 mg/ml, ... 13.5 mg/ml, and the ordinate is the relative apparent amylose content.

Figure 2 is a direct view of the distribution of relative apparent amylose content in different components of rice starch of soft rice variety Yunhui 290. The abscissa in the figure is the KOH solution concentration of 1.5 mg/ml, 3 mg/ml, ... 13.5 mg/ml, and the ordinate is the relative apparent amylose content.

Figure 3 is a direct view of the distribution of relative apparent amylose content in different components of japonica rice Nipponbare rice starch. The abscissa in the figure is the KOH solution concentration of 1.5 mg/ml, 3 mg/ml, ... 13.5 mg/ml, and the ordinate is the relative apparent amylose content.

Figure 4 is a direct view of the distribution of relative apparent amylose content in different components of rice starch of the glutinous rice variety Wennuo 1. The abscissa in the figure is the KOH solution concentration of 1.5 mg/ml, 3 mg/ml, ... 13.5 mg/ml, and the ordinate is the relative apparent amylose content.

Detailed ways

The present invention will be further described below with four implementation examples.

In the examples, four recognized rice varieties with significant differences in taste and quality were used: indica rice variety 93-11, soft rice variety Yunhui 290, japonica rice Nipponbare, and glutinous rice variety Wennuo 1. These four rice varieties and their rice are commercially available.

Example 1 is to measure the amylose content of the rice grains of the indica rice variety 93-11, that is, the determination of the apparent amylose content.

Example 2 is to measure the amylose content of the rice grains of the soft rice variety Yunhui 290 (Yunnan high-quality soft rice), that is, the determination of the apparent amylose content.

Example 3 is the determination of the amylose content of the rice grains of the japonica rice Nipponbare, that is, the determination of the apparent amylose content.

Example 4 is the determination of the amylose content of the rice grains of the glutinous rice variety Wennuo 1, that is, the determination of the apparent amylose content.

The methods for the separation of different components of rice starch of the above four embodiments and the content determination are all as follows:

(1) Randomly take more than 10 complete rice grains from which chaff, seed coat and embryo have been removed, and operate as follows for each rice grain:

A. Weigh a grain of rice, add 10ml of pure water, gelatinize in a water bath at 80°C for 1 hour, and cool to room temperature to obtain a starch suspension;

B. Centrifuge the starch suspension according to the centrifugal conditions to obtain the supernatant and precipitate, the supernatant is the sample to be tested, marked 0; add 10ml of 1.5mg/ml KOH solution to the precipitate, shake well and save it for the next time centrifugation; the centrifugation conditions are: relative centrifugal force 20000g, centrifugation at 4°C for 30min;

C. In step (1) B, add 1.5mg/ml KOH solution to the precipitate and centrifuge, use the obtained supernatant as the sample to be tested, mark it as 1 in order, add the obtained precipitate to the KOH solution and centrifuge, so Repeat the centrifugation operation (that is, add KOH solution to the precipitate obtained by each centrifugation and then centrifuge) until the obtained precipitate cannot be dissolved. The KOH solution added to the obtained precipitate is 10ml, but the concentration of the added KOH solution increases with a gradient of 1.5 mg/ml, such as 1.5 mg/ml, 3 mg/ml, ... 1.35 mg/ml. The supernatant obtained from each centrifugation is the sample to be tested and labeled in sequence, that is, a series of samples to be tested are obtained;

(2) According to the People's Republic of China national standard GB/T 15683-2008 The mensuration of the amylose content of rice carries out the mensuration of the amylose content respectively to a series of samples to be tested obtained in the step (1) C (the amylose content Determination is the determination of apparent amylose content).

The rice starch of rice varieties with different eating quality is separated by KOH solution with a concentration gradient of 1.5mg/ml. Starch solutions with different degrees of polymerization and branching degrees are reacted with iodine colorimetrically, and the absorbance value A is measured with a spectrophotometer. According to the standard curve Calculate the apparent amylose content of the different fractions of starch. And use the following formula to calculate the relative apparent amylose content of different components of starch, the results are shown in Table 1.

Calculation formula:

Apparent amylose content = apparent amylose mass of a single component/total mass of a single rice grain

Relative apparent amylose content = apparent amylose content of a single component / total apparent amylose content of a single rice grain

According to the data in Table 1, take the KOH concentration as the abscissa, and the relative apparent amylose content as the ordinate, see accompanying drawings 1, 2, 3 and 4.

the

The results showed that rice varieties with different taste qualities could be clearly distinguished according to the peak shape. Indica rice varieties with ordinary rice quality (such as 93-11) are bimodal, soft rice varieties (such as Yunhui 290) also have bimodal, but the rear peak is wider and shorter; japonica rice varieties with normal quality (such as Nipponbare) are 3 Peak type, glutinous rice varieties (such as Wennuo 1) are unimodal.

The ordinate of each sample in the map is the relative apparent amylose content percentage, which represents the percentage of the apparent amylose content of a single component to the total apparent amylose content of a single rice grain. The difference in peak value can also reflect the difference among varieties, and the method of separation and content determination can more intuitively and accurately show the characteristics of starch components among different varieties.

This method can be applied to other rice varieties, and can also expand the number of rice grains sampled to increase the overall representativeness of individual samples. Using this processing method, the characteristics of the comprehensive starch components of a certain variety or a certain batch of samples can be summarized.

It can be seen from the examples that the method can distinguish the differences among different rice varieties, and obtain a comprehensive index through a statistical method. It provides a direct basis for the breeding of new rice varieties with good taste and quality.

Claims (1)

1. a method for the separation of rice starch different component and assay, is characterized in that comprising the following steps:

(1) get at random 10 above complete grain of rices of removing clever shell, planting skin and embryo, each grain of rice is operation as follows all:

A. weigh and add the gelatinization 1 hour in 80 ℃ of water-baths of 10ml pure water after a grain of rice, be cooled to room temperature and obtain starch suspension;

B. starch suspension is obtained to supernatant and precipitation by centrifugal condition centrifuging, supernatant is for treating test sample, mark 0; In this precipitation, add 1.5mg/ml KOH solution 10ml, after shaking up, give over to centrifuging next time; Described centrifugal condition is: relative centrifugal force(RCF) 20000g, 4 ℃ of centrifugal 30min;

C. the precipitation and centrifugal separation of KOH solution will have been added in step (1) B, centrifugal condition is: relative centrifugal force(RCF) 20000g, 4 ℃ of centrifugal 30min, obtain supernatant and precipitation, using the supernatant of gained as treating test sample, be labeled as in order 1, the precipitation of gained is added to the centrifuging again of KOH solution again, and so repeated centrifugation lock out operation, can not dissolve until gained precipitates, each centrifugal condition is: relative centrifugal force(RCF) 20000g, 4 ℃ of centrifugal 30min; The KOH solution at every turn gained precipitation being added is 10ml, but the concentration of the KOH solution adding increases progressively with 1.5mg/ml gradient, and the supernatant of each centrifuging gained is treats test sample mark in order, obtains a series of test samples for the treatment of;

(2) by the mensuration of State Standard of the People's Republic of China GB/T 15683-2008 Rice Amylose content, to what obtain in step (1) C, a series ofly treat that test sample carries out respectively the mensuration of amylose content.