Rapid High Throughput Amylose Determination in Freeze Dried Potato Tuber Samples

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....

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,.......

Materials

Name	Company	Catalog Number	Comments
			Reagents
Amylose	Sigma	859656
Amylopectin	Sigma	A8515
Perchloric Acid 60%	Sigma	311413	Very hazardous to skin and eyes
Iodine	Sigma	23214TD
Potassium iodide	Sigma	08625JE
			Equipment
Plate Reader	Bio Tek	ELx800