Validation of a simple, colorimetric, microplate assay using amplex red for the determination of glucose and sucrose in potato tubers and other vegetables

Authors: Bethke, Paul; Busse, James

Submitted to: American Journal of Potato Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/7/2008
Publication Date: 7/12/2008
Citation: Bethke, P.C., Busse, J.S. 2008. Validation of a simple, colorimetric, microplate assay using amplex red for the determination of glucose and sucrose in potato tubers and other vegetables. American Journal of Potato Research. 85(6):414-421.

Interpretive Summary: Accurate quantification of potato tuber glucose and sucrose content is important for scientific as well as commercial purposes. Precise quantification of free sugars in potato tubers is highly beneficial to potato growers and processors at several times in the potato production cycle. For example, tuber sucrose concentration is as an indicator of tuber maturity and sucrose measurements are used to make decisions about when to harvest. Potatoes destined for high temperature frying, such as those that will be used for french fries or potato chips, produce unacceptably dark and bitter products if glucose concentrations exceed empirically determined thresholds. To guard against this, storage managers routinely monitor tuber glucose and base processing decisions on trends in the data. Senescent sweetening occurs after long-duration storage and is characterized by an increase in tuber sucrose content that is followed by an increase in tuber reducing sugars. Senescent sweetening indicates to the storage manager that the affected potatoes need to be processed or sold quickly. Individuals breeding potatoes for processing need to know how susceptible advanced breeding lines are to the accumulation of reducing sugars. We have developed a reliable, cost-effective 96 well microplate assay that allows us to determine quickly sugar concentrations in tuber samples. Sample size can be as little as 1g of fresh material. This colorimetric assay utilizes enzyme-coupled reactions to measure glucose concentrations as low as 10 µM. and sucrose. Data from this improved enzymatic assay compare favorably with HPLC data, with the added benefits of lower capital outlay and faster sample turn around times.

Technical Abstract: Accurate quantification of tuber glucose and sucrose content is important for scientific as well as commercial purposes. High pressure liquid chromatography (HPLC) and enzyme coupled assays are accepted methods for determining sugar concentrations. HPLC assays require expensive equipment and lengthy sample preparation times. Because of this, HPLC is rarely used for routine monitoring of potato tuber sugars. Sample preparation times for enzyme-based assays are much shorter than for traditional HPLC methods, and this makes them attractive management tools for routine sugar monitoring. We have developed a reliable, cost-effective 96 well microplate enzymatic assay that allows us to determine quickly sugar concentrations in tuber samples. Sample size can be as little as 1g of fresh material. This colorometric assay utilizes coupled reactions of glucose oxidase and peroxidase to accurately measure glucose concentrations as low as 10 µM. Sucrose content can be determined at the same time by adding invertase, an additional enzyme mediated step. Data from this improved enzymatic assay compare favorably with HPLC data, with the added benefits of lower capital outlay and faster sample turn around times.