Location: Sugarbeet and Potato Research
Title: Microplate assay for rapid determination of sucrose, glucose, fructose and raffinose Authors
|Martins, Daniel - UNIV VICOSA, BRAZIL|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: November 16, 2006
Publication Date: August 1, 2007
Citation: Klotz, K.L., Martins, D.N. 2007. Microplate assay for rapid determination of sucrose, glucose, fructose, and raffinose [abstract]. In: Proceedings from the 34th Biennial Meeting of the American Society of Sugar Beet Technologists, Operations, February 29-March 3, 2007. Salt Lake City, Utah. p. 125. Technical Abstract: Current methods for the quantification of carbohydrates in sugarbeet roots have limitations. Polarimetry and refractometry measure only sucrose content and are inaccurate with deteriorated roots. High performance liquid chromatography (HPLC) and gas chromatography (GC) quantify all simple carbohydrates regardless of root quality, but are time-consuming, costly (HPLC) or require the use of noxious reagents (GC). Research was conducted to develop a rapid, high-throughput microtiter plate assay for accurate determination of sucrose, glucose, fructose and raffinose in sugarbeet roots. The assay modifies a previously described enzyme-based microtiter plate assay for quantification of sucrose, glucose, and fructose (J. Sci. Food Agric. 82:80, 2001) to decrease sample preparation and analysis time, and increase the range of sugar concentrations that can be accurately quantified. In addition, the assay was expanded to quantify raffinose in addition to sucrose, glucose and fructose. Using 15 minute, enzyme-coupled reactions and commercially available reagents, sucrose, glucose, fructose and raffinose were quantified with high reproducibility. The assay was linear for samples containing 2 to 100 mM sucrose, 0.2 to 10 mM glucose, 0.2 to 10 mM fructose, and 0.1 to 20 mM raffinose. Aluminum sulfate concentrations typically used to clarify beet extracts did not affect the assay indicating that sample preparation methods currently used for polarimetry measurements can be used with this assay. The assay was used to quantify sucrose, glucose, fructose and raffinose concentrations in clarified extracts from stored roots exhibiting varying degrees of rot. Comparison of these results with those obtained by HPLC was used to validate the assay.