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CN-122016689-A - Method for rapidly determining content of amylose in rice

CN122016689ACN 122016689 ACN122016689 ACN 122016689ACN-122016689-A

Abstract

The invention relates to the technical field of analysis and detection, and provides a method for rapidly determining the content of amylose in rice, which comprises the following steps of S100, adding ethanol and sodium hydroxide solution into brown rice, standing for 8-24 hours to obtain a sample to be detected, S200, detecting the sample to be detected by using a high-flux ELISA plate colorimetric method, S300, obtaining absorbance of the sample to be detected at a first detection wavelength and a second detection wavelength, S400, calculating the absorbance ratio of the sample to be detected at the first detection wavelength and the second detection wavelength, S500, substituting the absorbance ratio into a standard curve, and calculating to obtain the content of amylose, wherein the first detection wavelength is 620+/-10 nm, and the second detection wavelength is 530+/-10 nm. Through the technical scheme, the problems of complex operation and long time consumption of the detection method in the related technology are solved.

Inventors

  • LI JIAYANG
  • WANG ANQI
  • CHENG QIAO
  • JING YANHUI
  • YU HONG
  • ZHANG YUXIN

Assignees

  • 崖州湾国家实验室
  • 中国科学院遗传与发育生物学研究所

Dates

Publication Date
20260512
Application Date
20260416

Claims (14)

  1. 1. A method for rapidly determining the amylose content of rice, comprising the following steps: S100, adding ethanol and sodium hydroxide solution into brown rice, and standing for 8-24 hours to obtain a sample to be detected; S200, detecting the sample to be detected by using a high-flux ELISA plate colorimetric method; S300, respectively obtaining the absorbance of the sample to be detected at a first detection wavelength and a second detection wavelength; S400, calculating the absorbance ratio of the sample to be detected at the first detection wavelength and the second detection wavelength; s500, substituting the absorbance ratio into a standard curve, and calculating to obtain the amylose content; the first detection wavelength is 620+/-10 nm, and the second detection wavelength is 530+/-10 nm; And the ordinate of the standard curve is the amylose content of the standard substance, and the abscissa is the absorbance ratio of the standard substance at the first detection wavelength and the second detection wavelength.
  2. 2. The method for rapidly determining amylose content of rice according to claim 1, wherein the ratio of the amount of brown rice, ethanol and sodium hydroxide solution in S100 is 80~120 mg:1 mL:9 mL.
  3. 3. The method for rapidly determining the amylose content of rice according to claim 2, wherein the concentration of the sodium hydroxide solution is 0.9 to 1.1 mol/L.
  4. 4. The method for rapidly determining amylose content of rice according to claim 1, wherein the high-throughput elisa method employs 96-well plate.
  5. 5. The method for rapidly determining amylose content of rice according to claim 1, wherein S200 comprises the steps of: and mixing the sample to be detected with the chromogenic reaction solution, and sucking the mixture into an ELISA plate for detection.
  6. 6. The method for rapidly determining amylose content of rice as defined in claim 5, wherein the chromogenic reaction solution comprises glacial acetic acid and iodine-containing mixed solution.
  7. 7. The method for rapidly determining the amylose content of rice according to claim 6, wherein the volume ratio of the glacial acetic acid to the iodine-containing mixed solution is 0.5-2:2.
  8. 8. The method for rapidly determining the amylose content of rice according to claim 7, wherein the concentration of glacial acetic acid is 0.5-2 mol/L.
  9. 9. The method for rapidly determining amylose content of rice according to claim 7, wherein the iodine-containing mixed solution is a mixed solution of 0.05-wt% to 0.2-wt% I 2 and 0.5-wt-2-wt% KI.
  10. 10. The method for rapidly determining the amylose content of rice according to claim 5, wherein the volume ratio of the sample to be measured to the chromogenic reaction solution is (4.9-5.1): (995.1-994.9).
  11. 11. The method for rapidly determining amylose content of rice according to any one of claims 1 to 10, wherein the brown rice is dehulled rice.
  12. 12. The method for rapidly determining the amylose content of rice according to any one of claims 1 to 10, wherein the method for obtaining the standard curve comprises the steps of: Providing a standard substance with the amylose content changing in a gradient manner; Determining the amylose content of the standard; the step of S100-S400 is adopted to obtain the absorbance ratio of the standard substance at the first detection wavelength and the second detection wavelength; And establishing a standard curve by taking the amylose content of the standard substance as an ordinate and the absorbance ratio of the standard substance at the first detection wavelength and the second detection wavelength as an abscissa.
  13. 13. The method for rapidly determining amylose content of rice according to claim 12, wherein the linear range of the standard curve is 0-38.64%.
  14. 14. The use of a method for rapidly determining the amylose content of rice according to any one of claims 1 to 13, characterized in that it is used for any one of the following a to C: A. Identifying rice mutants having altered amylose content; B. screening rice mutants with altered amylose content; C. And breeding rice varieties with low amylose content.

Description

Method for rapidly determining content of amylose in rice Technical Field The invention relates to the technical field of analysis and detection, in particular to a method for rapidly determining the content of amylose in rice. Background Amylose content (Amylose Content, AC) is one of the core indicators determining the quality of rice (Oryza sativa l.) cooking taste. It directly affects hardness, viscosity, luster, retrogradation speed and digestion characteristics of rice, and is a key parameter for rice breeding improvement and food processing. The usual method for detecting amylose content has the following problems: (1) The traditional national standard method (GB/T15683-2025 determination of rice amylose content by grain and oil inspection) is the most accurate method for detecting rice amylose content, but has the defects of complicated operation, long time consumption, low flux and the like, and is difficult to meet the requirements of large-scale screening and industrialized rapid quality inspection of rice breeding. (2) In recent years, the emerging spectroscopy detection technologies such as near infrared spectroscopy, raman spectroscopy and automated chemical analysis technologies such as flow injection analysis, high-flux 96-well microplate colorimetry and the like can be used for high-flux detection, and can avoid the problem of low flux of the traditional national standard method, but the spectroscopy detection technologies need to be provided with expensive spectroscopy detection instruments, and the detection model is high in building and maintenance cost. The automatic chemical analysis technology needs to perform the steps of sample pretreatment (polished rice and grinding), has complex operation and long time consumption, and the instrument pipeline needs to be maintained regularly and prevent pollution. Disclosure of Invention The invention provides a method for rapidly determining the amylose content of rice, which aims to solve or alleviate at least one of the problems. The technical scheme of the invention is as follows: The invention provides a method for rapidly determining the content of amylose in rice, which comprises the following steps: S100, adding ethanol and sodium hydroxide solution into brown rice, and standing for 8-24 hours to obtain a sample to be detected; S200, detecting the sample to be detected by using a high-flux ELISA plate colorimetric method; S300, respectively obtaining the absorbance of the sample to be detected at a first detection wavelength and a second detection wavelength; S400, calculating the absorbance ratio of the sample to be detected at the first detection wavelength and the second detection wavelength; s500, substituting the absorbance ratio into a standard curve, and calculating to obtain the amylose content; The first detection wavelength is 620+/-10 nm, and the second detection wavelength is 530+/-10 nm. The microplate reader is usually equipped with a specific bandwidth optical filter or grating system, the actual optional detection wavelength of the microplate reader is usually 5nm or 10nm apart, and the inherent wavelength precision deviation exists between different types of equipment, so that those skilled in the art can reasonably foresee that the detection wavelength is set to any value in the range of 620+/-10 nm and 530+/-10 nm, and the detection at 620nm and 530nm can achieve the high-throughput analysis effect basically the same as that of the detection at 530 nm. As a further technical scheme, the ordinate of the standard curve is the amylose content of the standard substance, and the abscissa is the absorbance ratio of the standard substance at the first detection wavelength and the second detection wavelength. The inventor finds that, due to the improvement of the pretreatment step of the invention, the ratio of the absorbance of the amylose to the amylopectin (namely A 620/A530, which is marked as QAAM and Quick AC ANALYSIS method) is adopted as a rapid amylose content screening index, the error can be effectively reduced, the detection accuracy is improved, the QAAM and the amylose content show strong linear positive correlation,。 As a further technical scheme, in S100, the dosage ratio of brown rice, ethanol and sodium hydroxide solution is 80~120 mg:1 mL:9 mL. As a further technical scheme, the concentration of the sodium hydroxide solution is 0.9-1.1 mol/L. The method ensures that the amylose in the brown rice is fully dissolved in a reaction system by limiting the dosage and the concentration of the reagent in the pretreatment step, so that the problems of incomplete extraction and low detection accuracy caused by insufficient reagent can be avoided, and the problems of influence on the stability of the subsequent color development reaction and the waste of the reagent caused by excessive ethanol or sodium hydroxide can be avoided. As a further technical scheme, the high-throughput ELISA plate colorimetric method adopts a