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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in starch. Iodine forms complexes with both amylose and long chains within amylopectin. After the addition of iodine to a starch sample, the maximum absorption of amylose and amylopectin occurs at 620 and 550 nm, respectively. The amylose/amylopectin ratio can be estimated from the ratio of the 620 and 550 nm absorbance values and comparing them to a standard curve in which specific known concentrations are plotted against absorption values. This high throughput, inexpensive method is reliable and reproducible, allowing the evaluation of large populations of potato clones. 

Abstract

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in starch. Iodine forms complexes with both amylose and long chains within amylopectin. After the addition of iodine to a starch sample, the maximum absorption of amylose and amylopectin occurs at 620 and 550 nm, respectively. The amylose/amylopectin ratio can be estimated from the ratio of the 620 and 550 nm absorbance values and comparing them to a standard curve in which specific known concentrations are plotted against absorption values. This high throughput, inexpensive method is reliable and reproducible, allowing the evaluation of large populations of potato clones. 

Introduction

Approximately 80% of the fresh weight of a potato tuber is water; nearly all of the remaining dry matter is starch1. Most of the starch (70%) is composed of amylopectin, while the remainder is amylose. The ratio between amylose and amylopectin is the most important property influencing the physical properties of starch. Amylose is a linear alpha 1-4 glucose chain, while amylopectin is a linear alpha 1-4 chain with alpha 1-6 branches2. Methods such as iodine binding, differential scanning calorimetry (DSC), high performance size exclusion chromatography (HPSEC) and concanavalin A interactions have been developed for starch determination in differe....

Protocol

1. Amylose Determination from Potato Tuber Starch

  1. Peel and cut fresh tubers into small cubes.
  2. Place potato cubes in small brown bags and store overnight at -80 °C.
  3. Transfer potato cubes to nylon bags and for freeze drying.
  4. After freeze dried, grind the potato sample into powder using mortar and pestle or a Wiley Mill.
  5. In a 50 ml tube, add 20-30 mg of the freeze-dried, ground tuber sample.
  6. Prepare a solution of 45% (w/v) perchloric acid (mix 24.4 ml of 60% perchloric acid and 25.6 ml of ultra-pure water.
  7. Add 500 μl of 45% (w/v) perchloric acid and shake or swirl it to disperse starch gran....

Representative Results

The standard curve constructed from blanked data of different amylose/amylopectin concentration solutions shows approximate absorbance ratios ranging from 0.7 to 1.6 (Table 1). A linear regression trend line from these data is later used for inferring amylose content in freeze dried potato samples (Figure 1).

The plant production environment has some effect on the amylose content in the potato tuber7. Replicated amylose content determinations in two.......

Discussion

Amylose content in potato tubers typically ranges from 20-35%7,8. If most values are outside this range, consider these possible errors: 1) raw data were used instead of blanked data for the construction of the amylose/amylopectin standard curve; 2) raw data were used instead of blanked data for the determination of the amylose/amylopectin ratios from the potato samples; or 3) the water used for the analysis was not pure enough. We noticed that by using distilled water and not ultra-pure water in the analysis,.......

Disclosures

We have nothing to disclose.

Acknowledgements

Funding for this research was provided in part by the USDA-ARS Research Associate Program and the USDA Crop Germplasm Committee.

....

Materials

NameCompanyCatalog NumberComments
Reagents
Amylose Sigma859656
Amylopectin SigmaA8515
Perchloric Acid 60%Sigma311413Very hazardous to skin and eyes
IodineSigma23214TD
Potassium iodideSigma08625JE
Equipment
Plate ReaderBio TekELx800

References

  1. Composition Hoover, R. molecular structure, and physicochemical properties of tuber and root starches: a review. Carbohydr. 45 (3), 253-267 (2001).
  2. Bertoft, E., Blennow, A. Structure of Potato Starch. A....

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Amylose DeterminationFreeze Dried Potato TuberHigh ThroughputColorimetric MethodIodine starch ComplexAmyloseAmylopectinAbsorptionStandard CurvePotato Clones

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