Method for rapidly determining amylose content of rice
The detection of amylose in rice was simplified by using a high-throughput ELISA plate colorimetric method. By mixing brown rice with ethanol and sodium hydroxide solution and calculating the absorbance ratio at a specific wavelength, the method solves the problem of cumbersome and time-consuming detection in existing technologies, and achieves efficient and accurate determination of amylose content.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- YAZHOUWAN NATIONAL LABORATORY
- Filing Date
- 2026-04-16
- Publication Date
- 2026-05-12
AI Technical Summary
Existing technologies for detecting amylose content in rice suffer from problems such as cumbersome operation, long time consumption, and low throughput. Furthermore, spectroscopic detection technologies are costly and require complex instrument maintenance, Raman spectroscopy is subject to fluorescence interference, and automated chemical analysis requires cumbersome sample pretreatment steps.
A high-throughput microplate colorimetric method was adopted. Brown rice was mixed with ethanol and sodium hydroxide solution, and after standing, the absorbance ratio was measured within a specific wavelength range. The amylose content was calculated by combining the mixture with a standard curve, simplifying the pretreatment steps. A 96-well microplate was used for detection.
It improves detection efficiency, shortens pretreatment time, reduces costs, and enhances detection accuracy, making it suitable for large-scale screening of rice mutants with altered amylose content.
Smart Images
Figure CN122016689A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of analytical testing technology, and specifically, to a method for rapidly determining the amylose content in rice. Background Technology
[0002] Amylose content (AC) is a key factor determining the quality of rice. Oryza sativa L. (rice) is one of the core indicators of rice's cooking and flavor quality. It directly affects the hardness, viscosity, gloss, retrogradation speed, and digestibility of rice, and is a key parameter for rice breeding improvement and food processing.
[0003] Common methods for detecting amylose content have the following problems: (1) The traditional national standard method (GB / T 15683-2025 Grain and Oil Inspection: Determination of Amylose Content in Rice) is the most accurate method for detecting the amylose content in rice, but it has disadvantages such as complicated operation, long time consumption, and low throughput, which makes it difficult to meet the needs of large-scale screening in rice breeding and rapid quality inspection in industrialization.
[0004] (2) Emerging spectroscopic detection technologies in recent years, such as near-infrared spectroscopy, Raman spectroscopy, and automated chemical analysis technologies, such as flow injection analysis and high-throughput 96-well microplate colorimetry, can be used for high-throughput detection and can avoid the problem of low throughput in traditional national standard methods. However, these spectroscopic detection technologies require expensive spectroscopic detection instruments, and the establishment and maintenance of detection models are costly. Moreover, Raman spectroscopy has problems with fluorescence interference and weak signals, which will affect the accuracy of detection. Automated chemical analysis technologies require sample pretreatment steps (rice polishing and grinding), which are cumbersome and time-consuming. In addition, the instrument tubing needs to be maintained regularly and contamination should be prevented. Summary of the Invention
[0005] This invention proposes a method for rapidly determining the amylose content of rice to solve or alleviate at least one of the above-mentioned problems.
[0006] The technical solution of the present invention is as follows: This invention proposes a method for rapidly determining the amylose content of rice, comprising the following steps: S100. Add ethanol and sodium hydroxide solution to brown rice and let it stand for 8-24 hours to obtain the sample to be tested. S200. The sample to be tested is detected using a high-throughput microplate colorimetric method. S300, Obtain the absorbance of the sample to be tested at the first detection wavelength and the second detection wavelength respectively; S400. Calculate the ratio of the absorbance of the sample to be tested at the first detection wavelength and the second detection wavelength; S500. Substitute the absorbance ratio into the standard curve to calculate the amylose content; The first detection wavelength is 620±10nm, and the second detection wavelength is 530±10nm.
[0007] Microplate readers are typically equipped with filters or grating systems with specific bandwidths. The actual selectable detection wavelengths are often in intervals of 5nm or 10nm, and there are inherent wavelength accuracy deviations between different models of equipment. Therefore, those skilled in the art can reasonably foresee that the present invention can achieve essentially the same high-throughput analysis effect as detection at 620nm and 530nm by setting the detection wavelength to any value in the range of 620±10nm and 530±10nm.
[0008] As a further technical solution, the vertical axis of the standard curve represents the amylose content of the standard, and the horizontal axis represents the ratio of the absorbance of the standard at the first detection wavelength and the second detection wavelength.
[0009] The inventors discovered that, due to improvements in the pretreatment steps of this invention, the absorbance ratio of amylose to amylopectin (i.e., A0) is used. 620 / A 530 The Quick AC analysis method (QAAM) is used as a rapid screening index for amylose content. It can effectively reduce errors and improve detection accuracy. QAAM shows a strong linear positive correlation with amylose content. .
[0010] As a further technical solution, in S100, the ratio of brown rice, ethanol, and sodium hydroxide solution is 80~120 mg:1 mL:9 mL.
[0011] As a further technical solution, the concentration of the sodium hydroxide solution is 0.9~1.1 mol / L.
[0012] This invention ensures that the amylose in brown rice is fully dissolved in the reaction system by limiting the amount and concentration of reagents in the pretreatment step. This avoids problems such as incomplete extraction and low detection accuracy due to insufficient reagents, and also avoids problems such as excessive ethanol or sodium hydroxide affecting the stability of subsequent colorimetric reactions and wasting reagents.
[0013] As a further technical solution, the high-throughput ELISA plate colorimetric method uses a 96-well plate.
[0014] As a further technical solution, S200 includes the following steps: mixing the sample to be tested with the colorimetric reaction solution and aspirating it into an ELISA plate for detection.
[0015] As a further technical solution, the colorimetric reaction solution includes glacial acetic acid and a mixture containing iodine.
[0016] As a further technical solution, the volume ratio of the glacial acetic acid to the iodine-containing mixture is 0.5~2:2.
[0017] As a further technical solution, the concentration of the glacial acetic acid is 0.5~2 mol / L.
[0018] As a further technical solution, the iodine-containing mixture is a mixed solution of 0.05 wt%~0.2 wt% I2 and 0.5 wt%~2 wt% KI.
[0019] As a further technical solution, the ratio of the amount of the sample to be tested to the amount of the colorimetric reaction solution is (4.9~5.1):(995.1~994.9).
[0020] As a further technical solution, the brown rice is dehulled rice.
[0021] As a further technical solution, the method for obtaining the standard curve includes the following steps: Provide standard samples with gradients in amylose content; The amylose content of the standard was determined; The absorbance ratio of the standard sample at the first detection wavelength and the second detection wavelength is obtained by using the steps S100~S400; A standard curve is established by using the amylose content of the standard as the ordinate and the ratio of the absorbance of the standard at the first detection wavelength and the second detection wavelength as the abscissa.
[0022] As a further technical solution, the linear range of the standard curve is 0~38.64%.
[0023] This invention also proposes an application of a rapid method for determining the amylose content of rice, applicable to any one of the following A~C: A. Identifying rice mutants with altered amylose content; B. Screening for rice mutants with altered amylose content; C. Select and breed rice varieties with low amylose content.
[0024] The beneficial effects of this invention are as follows: Compared to traditional methods for testing amylose content, this invention optimizes the pretreatment steps, eliminating the need for polished rice and the grinding process. Homogenization is achieved simply by soaking brown rice in a solvent, allowing for direct testing. Unlike the national standard method, which requires polished rice to a specific fineness, this method uses brown rice directly, achieving "chemical homogenization" through prolonged soaking in an alkaline solution, replacing "physical grinding and homogenization."
[0025] Meanwhile, the pretreatment steps of this invention avoid degradation or oxidation of amylose during the milling process due to mechanical friction or exposure to air, thus improving the accuracy of the test results. Furthermore, the soaking process can be performed overnight, significantly shortening the pretreatment time. Based on this, this invention uses fewer samples, is low-cost, simple to operate, and highly efficient. Approximately 160 samples can be measured per day with an 8-hour workday, and approximately 200 samples can be measured with extended work hours. Compared to traditional amylose content determination methods, this represents an efficiency improvement of nearly 10 times, making it suitable for large-scale screening of rice mutants with altered amylose content. Attached Figure Description
[0026] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.
[0027] Figure 1 The standard curve established for Embodiment 1 of the present invention; Figure 2 For Embodiment 2 of the present invention, R7954 and b10 QAAM was repeated 27 times. Detailed Implementation
[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0029] The samples used in the specific embodiments of the present invention include R7954. rs4 , b10， All of these are existing technologies, readily available to anyone skilled in the art. R7954 is a hybrid rice restorer line, belonging to the wild type of indica rice. Indica rice ( Oryza sativa L. spp. indica R7954 rs4 , b10 From collaborator Professor Wu Dianxing of Zhejiang University, the hybrid rice restorer line R7954 underwent sequential space mutagenesis and... 60Co irradiation breeding yielded a highly resistant starch mutant. rs4 (Shu et al., 2009), b10 Then from rs4 A lineage derived from backcrossing with R7954, b10 The resistant starch content was 5.8% (Zhou et al., 2016), R7954, rs4, b10 They share the same genetic background and are basically the same in other agronomic traits except for differences in resistant starch content.
[0030] References Shu, X., Jia, L., Ye, H., Li, C., and Wu, D. (2009). Slow digestionproperties of rice different in resistant starch. J. Agric. Food Chem. 57,7552-7559. Zhou, H., Wang, L., Liu, G., Meng, 12844-12849. This invention proposes a method for rapidly determining the amylose content of rice, comprising the following steps: S100. Add ethanol and sodium hydroxide solution to brown rice and let it stand for 8-24 hours to obtain the sample to be tested. Specifically, take 6 grains of brown rice (approximately 80-120 mg) in a test tube, add 1 mL of ethanol and 9 mL of sodium hydroxide solution, tighten the cap of the test tube and let it stand for 8-24 hours, or overnight; the concentration of sodium hydroxide solution is 0.9-1.1 mol / L, preferably 1 mol / L, and the volume concentration of ethanol is 95%. S200. The sample to be tested is detected using a high-throughput microplate colorimetric method. Preferably, a 96-well plate is used for the high-throughput microplate colorimetric method. Specifically, the following steps are included: S210. For each reaction, add 5-20 μL of 0.5-2 M glacial acetic acid, 20 μL of a mixed solution of 0.05 wt%-0.2 wt% I2 and 0.5 wt%-2 wt% KI, and purified water to prepare a 995 μL colorimetric reaction mixture. Preferably, for each reaction, add 10 μL of 1 M glacial acetic acid, 20 μL of a mixed solution of 0.1 wt% I2 and 1 wt% KI, and 965 μL of purified water to prepare the colorimetric reaction mixture. Use a pipette or mobile workstation to dispense 995 μL ± 0.1 μL of the colorimetric reaction mixture into a 96-well deep plate consisting of eight 1 mL tubes. S220: Pipette 4.9-5.1 μL of the sample to be tested into the colorimetric reaction mixture, tighten the silicone cap, vortex to mix, let stand for 5-10 min, and then use a pipette or mobile workstation to aspirate 200 μL into a 96-well microplate for detection. S300: Obtain the absorbance of the sample at specific wavelengths of 620±10 nm and 530±10 nm. S400. Calculate the ratio of absorbance of the sample at specific wavelengths of 620±10 nm and 530±10 nm, denoted as QAAM. S500, substitute QAAM into the standard curve to calculate the amylose content.
[0031] In a specific embodiment of the present invention, setting the detection wavelength to any value within the range of 620±10nm and 530±10nm can achieve the same high-throughput analysis effect as detection at 620nm and 530nm. The experimental results are similar when determining the amylose content of rice, and both methods can improve the detection efficiency and the accuracy of the detection results.
[0032] Example 1: Establishing a Standard Curve To establish a standard curve, mutant rice varieties with known gradient changes in amylose content were used as standards. Their amylose content (denoted as AAC) was accurately determined according to the method of NY / T 2639-2014. The results are shown in Table 1.
[0033] Table 1. AAC of Standard Products
[0034] Obtain the QAAM for the above standard products by following these steps: S100. Place 6 grains of brown rice in a test tube, add 1 mL of 95% ethanol and 9 mL of 1 mol / L sodium hydroxide solution, tighten the cap of the test tube and leave it overnight. S210. Prepare a colorimetric reaction mixture by adding 10 μL of 1 M glacial acetic acid, 20 μL of a mixed solution of 0.1 wt% I2 and 1 wt% KI, and 965 μL of purified water for each reaction. Use a pipette or mobile workstation to aliquot 995 μL of the colorimetric reaction mixture into a 96-well deep plate consisting of eight 1 mL tubes. S220. Pipette 5 μL of the sample to be tested into the colorimetric reaction mixture, tighten the silicone cap, vortex to mix, let stand for 10 min, and then use a pipette to aspirate 200 μL into a 96-well microplate for detection. S300: Obtain the absorbance of the sample at specific wavelengths of 620 nm and 530 nm. S400. Calculate the ratio of absorbance of the sample at specific wavelengths of 620 nm and 530 nm, denoted as QAAM.
[0035] A standard curve was constructed with AAC as the ordinate and QAAM as the abscissa. A simple linear regression model revealed a strong positive linear correlation between QAAM and AAC in the standard. RAAM 2 =0.95 (standard curve as shown) Figure 1 As shown in the figure, QAAM can be used to characterize AAC, thus enabling rapid analysis of changes in amylose content.
[0036] Example 2 R7954 and b10 The amylose content of the two rice samples was determined according to the following steps: S100. Take 6 grains of brown rice for each sample and put them into a test tube. Add 1 mL of 95% ethanol and 9 mL of 1 mol / L sodium hydroxide solution. After tightening the cap of the test tube, leave it overnight to obtain the sample to be tested. S210. Prepare a colorimetric reaction mixture by adding 10 μL of 1 M glacial acetic acid, 20 μL of a mixed solution of 0.1 wt% I2 and 1 wt% KI, and 965 μL of purified water for each reaction. Use a pipette or mobile workstation to aliquot 995 μL of the colorimetric reaction mixture into a 96-well deep plate consisting of eight 1 mL tubes. S220. Pipette 5 μL of the sample to be tested into the colorimetric reaction mixture, tighten the silicone cap, vortex to mix, let stand for 10 min, and then use a pipette to aspirate 200 μL into a 96-well microplate for detection. S300: Obtain the absorbance of the sample at specific wavelengths of 620 nm and 530 nm. S400. Calculate the ratio of absorbance of the sample at specific wavelengths of 620 nm and 530 nm, denoted as QAAM. S500. Substitute QAAM into the standard curve obtained in Example 1 to calculate the amylose content.
[0037] R7954 and b10 The experiment was repeated 27 times for each of the two rice samples, and the calculated QAAM was as follows: Figure 2 As shown, the average QAAM values for 27 repeated experiments were 1.4249 and 1.5825, with standard deviations of 0.0409 and 0.0280, respectively. Substituting the average QAAM value into the standard curve, the calculated amylose content in the R7954 rice sample was 24.25%. b10 The amylose content in the rice sample was 29.02%. R7954 content was determined using the national standard GB / T 15683-2025. b10 The amylose content in the two rice samples was 27.52% and 30.83%, respectively, with Δ values ranging from 1.81% to 3.27%.
[0038] Comparative Example 1 Brown rice samples from the same batch as in Example 2 were pretreated according to step S100 in Example 2, and then tested according to methods 9.2 and 9.3 in GB / T 15683-2025. The results showed that R7954... b10 The amylose content in the two rice samples was 33.42% and 41.08%, respectively. The measured... b10 The amylose content in the rice sample showed a Δ value exceeding 10% compared to the result determined by the national standard GB / T15683-2025 method. This indicates that the pretreatment steps of this invention are incompatible with traditional methods, resulting in low detection accuracy. The pretreatment steps of this invention need to be used in conjunction with subsequent testing methods to improve both detection efficiency and accuracy.
[0039] Comparative Example 2 Table 2 shows a comparison of the efficiency of detection using existing technology (NY / T 2639-2014 method) and the method of the present invention (hereinafter referred to as QAAM method).
[0040] Table 2 Comparison of detection efficiency between existing technologies and the present invention
[0041] Excluding the ridge process, the pretreatment and detection time for each sample using the NY / T 2639-2014 method is 23 minutes, while the QAAM method reduces the pretreatment steps, with a single sample detection time of only 3 minutes. Based on a daily working time of 8 hours, the NY / T 2639-2014 method can only process and detect about 21 samples, while the QAAM method can detect 160 samples, significantly improving detection efficiency and making it very suitable for high-throughput screening of materials.
[0042] Application Example 1 Mutagenesis was induced using EMS (ethyl methanesulfonate) according to traditional methods. rs4 Rice (corresponding to standard rice with serial number 18 in Example 1) was used to construct an M2 generation seed bank containing 24,000 mutants. Table 3 shows... rs4 Statistical results of screening the EMS mutagenesis library.
[0043] 7,043 M2 single-plant seeds were sampled and QAAM was determined according to the method described in Example 1. Due to... rs 4. The QAAM of rice is 1.68, and the QAAM fluctuates between 1.62 and 1.72. Therefore, two thresholds were set: QAAM above 1.72 or below 1.62. 332 candidate materials were initially screened out.
[0044] Accurate determination of resistant starch (RS) content (using the Megazyme Resistant Starch Detection Kit K-RSTAR, method according to AOAC 2002.02) and AAC content (NY / T2639-2014 method) was performed on 332 candidate materials. Finally, through statistical significance analysis, the RS content or AAC of 67 materials was found to be significantly lower than that of the parents. rs4 Mutants showing significant changes (Table 3). Among them, 15 mutants showed no change in RS content but a decrease in AAC, 29 mutants showed a decrease in both RS content and AAC, and 23 mutants showed an increase in both RS content and AAC.
[0045] Table 3 rs4 Screening and statistics of EMS mutagenesis library
[0046] The screening results revealed a positive correlation between QAAM and AAC; that is, mutants with high QAAM also had high AAC, and mutants with low QAAM also had low AAC. Table 4 shows some representative examples. rs4 The results of QAAM and AAC for the EMS mutant. 8170-1 is a mutant with decreased RS content and AAC, and its QAAM is much lower than that of the parent. rs48128-6 is also a mutant with decreased AAC, and its QAAM is less than 1. rs4 The QAAM of 8238-1 and 8238-9 is greater than... rs4 Its AAC is also higher than rs4 .
[0047] Table 4. QAAM and AAC of some rs4 EMS mutants
[0048] The results in Table 4 further confirm the reliability of using the method of the present invention to screen mutants with altered amylose content. This method is an efficient and reliable screening tool that can be used to quickly identify mutants with altered amylose content. It avoids the complex pretreatment process of traditional methods, shortens the detection time, and greatly improves the screening efficiency, providing a practical technology for the genetic improvement of rice starch quality.
[0049] The above are merely preferred embodiments of the present invention and are 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 within the protection scope of the present invention.
Claims
1. A method for rapidly determining the amylose content of rice, characterized in that, Includes the following steps: S100. Add ethanol and sodium hydroxide solution to brown rice and let it stand for 8-24 hours to obtain the sample to be tested. S200. The sample to be tested is detected using a high-throughput microplate colorimetric method. S300, Obtain the absorbance of the sample to be tested at the first detection wavelength and the second detection wavelength respectively; S400. Calculate the ratio of the absorbance of the sample to be tested at the first detection wavelength and the second detection wavelength; S500. Substitute the absorbance ratio into the standard curve to calculate the amylose content; The first detection wavelength is 620±10nm, and the second detection wavelength is 530±10nm; The vertical axis of the standard curve represents the amylose content of the standard, and the horizontal axis represents the ratio of the absorbance of the standard at the first detection wavelength and the second detection wavelength.
2. The method for rapidly determining the amylose content of rice according to claim 1, characterized in that, In the S100, the ratio of brown rice, ethanol, and sodium hydroxide solution is 80~120 mg:1 mL:9 mL.
3. The method for rapidly determining the amylose content of rice according to claim 2, characterized in that, The concentration of the sodium hydroxide solution is 0.9~1.1 mol / L.
4. The method for rapidly determining the amylose content of rice according to claim 1, characterized in that, The high-throughput ELISA plate colorimetric method uses a 96-well plate.
5. The method for rapidly determining the amylose content of rice according to claim 1, characterized in that, S200 includes the following steps: The sample to be tested is mixed with the colorimetric reaction solution and then aspirated into an ELISA plate for detection.
6. The method for rapidly determining the amylose content of rice according to claim 5, characterized in that, The colorimetric reaction solution includes glacial acetic acid and a mixture containing iodine.
7. The method for rapidly determining the amylose content of rice according to claim 6, characterized in that, The volume ratio of the glacial acetic acid to the iodine-containing mixture is 0.5~2:
2.
8. A method for rapidly determining the amylose content of rice according to claim 7, characterized in that, The concentration of the glacial acetic acid is 0.5~2 mol / L.
9. A method for rapidly determining the amylose content of rice according to claim 7, characterized in that, The iodine-containing mixture is a mixture of 0.05 wt%~0.2 wt% I2 and 0.5 wt%~2 wt% KI.
10. The method for rapidly determining the amylose content of rice according to claim 5, characterized in that, The volume ratio of the sample to be tested to the colorimetric reaction solution is (4.9~5.1):(995.1~994.9).
11. A method for rapidly determining the amylose content of rice according to any one of claims 1 to 10, characterized in that, The brown rice mentioned is dehulled rice.
12. A method for rapidly determining the amylose content of rice according to any one of claims 1 to 10, characterized in that, The method for obtaining the standard curve includes the following steps: Provide standard samples with gradients in amylose content; The amylose content of the standard was determined; The absorbance ratio of the standard sample at the first detection wavelength and the second detection wavelength is obtained by using the steps S100~S400; A standard curve is established by using the amylose content of the standard as the ordinate and the ratio of the absorbance of the standard at the first detection wavelength and the second detection wavelength as the abscissa.
13. The method for rapidly determining the amylose content of rice according to claim 12, characterized in that, The linear range of the standard curve is 0 to 38.64%.
14. The application of the method for rapid determination of amylose content in rice according to any one of claims 1 to 13, characterized in that, The application is for any one of the following A~C: A. Identifying rice mutants with altered amylose content; B. Screening for rice mutants with altered amylose content; C. Select and breed rice varieties with low amylose content.