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ARS Home » Plains Area » Manhattan, Kansas » Center for Grain and Animal Health Research » Grain Quality and Structure Research » Research » Publications at this Location » Publication #304905
Title: Development of a 96-well plate iodine binding assay for amylose content determination

Author
item Kaufman, Rhett
item Wilson, Jeff
item Bean, Scott
item Herald, Thomas
item SHI, Y - Kansas State University

Submitted to: Carbohydrate Polymers
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/1/2014
Publication Date: 9/19/2014
Publication URL: http://handle.nal.usda.gov/10113/59733
Citation: Kaufman, R.C., Wilson, J.D., Bean, S., Herald, T.J., Shi, Y.C. 2014. Development of a 96-well plate iodine binding assay for amylose content determination. Carbohydrate Polymers. 115:444-447.

Interpretive Summary: Starch is synthesized and deposited in the endosperm of cereal grains to function as an energy reserve. In wild-type grains starch consists of two distinct polymers, amylose (AM) a mostly linear polymer and amylopectin (AP) a large highly branched polymer. In wild-type starches amylose content is usually in the 20-30% range, however mutants exist for several cereals that contain very high (>40%) and very low (0-15%) levels of amylose. The ratio of amylose to amylopectin is important to both the functionality and the nutritional properties of starch and starch based products. Conventional methods for the determination of AM/AP of cereal starches are very time consuming and labor intensive making it very difficult to screen large sample sets. Studying large data sets is necessary for evaluating breeding samples and investigating the impact the environment has on cereal starch development. The objective of this study was to adapt and optimize the iodine binding assay (colorimetric) in a 96 well plate format for both single and dual wavelength (gamma 620nm and gamma 510nm respectively) assays. This method was tested on maize, wheat and sorghum starch providing excellent repeatability and consistent with larger scale iodine binding assays.

Technical Abstract: Cereal starch amylose/amylopectin (AM/AP) ratios are critical in functional properties for food and industrial applications. Conventional methods for the determination of AM/AP of cereal starches are very time consuming and labor intensive making it very difficult to screen large sample sets. Studying large data sets is necessary for evaluating breeding samples and investigating the impact the environment has on cereal starch development. The objective of this study was to adapt and optimize the iodine binding assay (colorimetric) in a 96 well plate format for both single and dual wavelength (gamma 620nm and gamma 510nm respectively) assays. The standard curve for amylose content was scaled down to work in a 96-well plate format as demonstrated by regression equations with R squared values of 0.999 and 0.993 for single and dual wavelength, respectively. The plate methods were applicable over large ranges of amylose contents: high amylose maize starch at 61.7±2.3%, normal wheat starch at 29.0±0.74%, and a waxy maize starch at 1.2±0.9%. The method exhibited slightly greater amylose content values than the Concanavalin A method for normal type starches; but is consistent with cuvette scale iodine binding assays. This method was tested on maize, wheat and sorghum starch providing excellent repeatability.