4 Estimation of amylose content in rice grains
 

 

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The formation of a helical complex between amylose and iodine gives rise to the typical deep blue color of starch dispersions stained with iodine and forms the basis for quantitative determination of amylose content. In I2/KI solutions, polyiodide ions such as I3− and/or I5− interact with amylose forming single left-handed V-type helices. The formation of these complexes is determined by colorimetry.

 

Materials and Reagents

 

95% Ethanol

1N NaOH

1N Acetic acid

Iodine - Potassium iodide solution

Standard amylose

Distilled water

100 ml volumetric flask

Water bath

Spectrophotometer

Quartz cuvette

 

Composition of reaction mixtures

 

A. 1N NaOH solution

Dissolve 40g of NaOH in 1000ml distilled water

 

B. 1N Acetic acid solution

Dilute 57.5 ml glacial acetic acid to 1000ml using distilled water

 

C. Iodine - Potassium iodide solution

Dissolve 0.26 g of Iodine in 10 ml of Potassium iodide solution containing 2.6 g of KI

 

D. Standard Amylose Solution:

Take 40mg of pure potato starch (amylose) in a 100 ml volumetric flask and add 1 ml of 95% ethanol and 9.0 ml of 1N NaOH. Shake well and boil over water bath for 10 minutes and make up the solution to 100 ml using distilled water.

 

Procedure

  1. Weigh 100 mg well powdered milled rice into 100 ml volumetric flask.

  2. Add 1 ml 95% ethanol and 9 ml 1 N NaOH.

  3. Heat the sample for 10 minutes in boiling water bath, cool it and make up the volume to 100 ml.

  4. Pipette 5 ml from the 100 ml into another 100 ml volumetric flask.

  5. Add 1 ml I N acetic acid and then 2 ml iodide solution and make up the volume to 100 ml.

  6. Shake, stand for 20 minutes and determine the per cent Transmittance at 620 nm using a colorimeter.

  7. Prepare a series of standard starch solution containing 0, 20, 40, 60, 80 and 100% amylose as in the steps 1 to 5

  8. Read the transmittance of the standards at 620nm and plot a standard graph.

  9. Amylose content of the sample is determined in reference to the standard curve and expressed on percent basis.

  10. Making of amylose standards:

  1. Pipette out 1, 2, 3, 4 and 5 ml of the standard amylose into 100 ml volumetric flasks in three replications.

  2. Keep one flask as blank without adding anything.

  3. Add 1.0 ml 1N acetic acid and 2.0 ml I-KI solution to all the flasks including blank.

  4. Make up all the flasks to 100ml using distilled water and cover all the flasks with a black cloth or aluminum foil to prevent direct light exposure. I-KI disintegrates on exposure to light.

  5. Keep for 20 minutes and take reading at 620nm in a spectrophotometer.

  6. The standards including balnk, correspond to 0%, 4%, 8%, 12%, 16% and 20% of amylose.

  7. Draw a standard curve using the absorbance reading.

 

Based on the amylose percentage varieties can be grouped as follows

 

Category

Amylose Content (%)

Waxy

1-2

Very low amylose

2-9

Low

10-20

Intermediate

20-25

High

25-30

 

Juliano, B.O. (1971). A simplified assay for milled rice amylose. Cereal Science Today, 16 : 334-338.