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ISO TC 34/SC 4 N XXX

Date: 2010-08-10 ISO/NP 6647-2 ISO TC 34/SC 4 Secretariat: SAC

Rice Determination of amylose content Part 2: Routine method

Rice Dtermination de la teneur en amylose Partie 2: Mthode de pratique

Warning This document is not an ISO International Standard. It is distributed for review and comment. It is subject to change without notice and may not be referred to as an International Standard. Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are aware and to provide supporting documentation.

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Copyright notice
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Contents
1 2 3 4 5 6 7 8 8.1 8.2 8.3 8.4 8.4.1 8.4.2 8.4.3 8.5 9 10 10.1 10.2 10.3 11

Page

Scope ......................................................................................................................................................1 Normative references ............................................................................................................................1 Terms and definitions ...........................................................................................................................1 Principle..................................................................................................................................................1 Reagents.................................................................................................................................................2 Apparatus ...............................................................................................................................................2 Sampling.................................................................................................................................................2 Procedure ...............................................................................................................................................3 Preparation of test sample ...................................................................................................................3 Test portion and preparation of the test solution ..............................................................................3 Blank test................................................................................................................................................3 Preparation of the calibration graph....................................................................................................3 Preparation of the set of calibration solutions...................................................................................3 Colour development and spectrometric measurements ...................................................................3 Plotting the calibration graph...............................................................................................................3 Determination.........................................................................................................................................4 Expression of results ............................................................................................................................4 Precision.................................................................................................................................................4 Interlaboratory test ................................................................................................................................4 Repeatability ..........................................................................................................................................4 Reproducibility.......................................................................................................................................4 Test report ..............................................................................................................................................4

Annex A (informative) Results of an interlaboratory test ...............................................................................1

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Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 6647-2 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 4, Cereals and pulses. The third edition replaces ISO 6647-2:2007, which has been technically revised. ISO 6647 consists of the following parts, under the general title Rice Determination of amylose content: Part 1: Reference method Part 2: Routine method

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Rice Determination of amylose content Part 2: Routine method

Scope

This part of ISO 6647 specifies a simplified routine method for the determination of the amylose content of milled, non parboiled rice in the range from 1 % - 30 %. (limits to be determined after statistical evaluation of the interlaboratory trial)

Normative references

The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 6647-1, Rice Determination of amylose content Part 1: Reference method ISO 1666, Starch Determination of moisture content Oven-drying method ISO 24333, Cereals and cereal products Sampling ISO 7301, Rice Specification ISO 8466-1, Water quality Calibration and evaluation of analytical methods and estimation of performance characteristics Part 1: Statistical evaluation of the linear calibration function

Terms and definitions

For the purposes of this document, the terms and definitions given in ISO 6647-1 and ISO 7301 apply.

Principle

Grinding of the rice to a very fine flour to break up the endosperm structure in order to aid complete dispersion and gelatinization. Dispersion of a test portion in sodium hydroxide solution, then addition of an iodine solution to an aliquot portion and spectrometric determination of the absorbance at 720 nm of the colour complex formed. Reading of the amylose content of the sample from a calibration graph, prepared using rice samples with known amylose content, determined using the reference method (part 1 of this standard).
NOTE Rice samples with certified amylose content according to ISO 6647-1 are used as standards.

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Reagents

All the reagents used shall be of recognized analytical quality and the water used shall be distilled or demineralised water or water of equivalent purity. 5.1 5.2 5.3 5.4 5.5 Ethanol, 95 % (v/v). Sodium hydroxide, 1 mol/l solution. Sodium hydroxide, 0,09 mol/l solution. Acetic acid, 1 mol/l solution. lodine solution.

Weigh, to the nearest 5 mg, 2,000 g of potassium iodide in a weighing bottle fitted with a stopper. Add sufficient water to form a saturated solution. Add 0,200 g of iodine, weighed to the nearest 1 mg. When all the iodine has dissolved, transfer the solution quantitatively to a 100 ml volumetric flask (6.4), make up to volume with water and mix. Prepare a fresh solution on each day of use and protect it from light.

Apparatus

Usual laboratory equipment, and in particular 6.1 Grinder, capable of reducing uncooked milled rice to a fine flour which will pass through a 150 m 180 m (80 - 100 Mesh) sieve. A cyclone mill with 0,5 mm screen is recommended. 6.2 Sieve, size 150 m 180 m (80 -100 Mesh).

6.3 Spectrophotometer, with matching cells, usually of 1 cm path length, capable of measuring absorbance at 720 nm. 6.4 6.5 Volumetric flasks, 100 ml. Boiling water bath.

6.6 Analytical balance, capable of weighing to the nearest 0.0001 g. 6.7 Test tubes, 20 ml 6.8 Pipettes, capacity of 0.2 ml, 1 ml, 5 ml and 10 ml. 6.9 Conical flask, 100 ml

Sampling

It is important that the laboratory receives a sample which is truly representative and has not been damaged or changed during transport or storage. Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 24333.

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Procedure

On a separate portion of the laboratory sample and the standard samples a moisture determination according to ISO 1666 has to be made.

8.1

Preparation of test sample

In the grinder (6.1) grind at least 10 g of milled rice to a very fine flour which will pass through the sieve (6.2).

8.2

Test portion and preparation of the test solution

Weigh 100 mg 0,5 mg of the test sample into a 100 ml conical flask (6.9). To this test portion carefully add 1 ml of ethanol (5.1) using a pipette, washing down any of the test portion adhering to the side of the flask. Shake slightly in order to wet all of the sample. Add 9,0 ml of sodium hydroxide solution (5.2) from a pipette and mix. Disperse starch completely by either heating the mixture on a boiling water bath (6.5) for 10 min or by standing overnight. Allow cooling to room temperature and transfer to 100 ml volumetric flask (6.4). Make up to volume with water and mix vigorously.
NOTE It is recommended to vortex before adding the water and after making up to volume.

8.3

Blank test

Carry out a blank test in parallel with the determination, by the same procedure, using the same quantities of all the reagents as in the determination, but using 5,0 ml of sodium hydroxide solution (5.3) instead of the test solution.

8.4
8.4.1

Preparation of the calibration graph

Preparation of the set of calibration solutions

Use 5 calibrated rice samples with a distribution of the amylose content from 0% to 30% and for which the amylose content had been certified by the reference method in Part 1 of this InternationalStandard. Alternatively, create a set of standards from different rice varieties calibrated against a standard curve made from the calibrated standards. Prepare the calibration solutions as in 8.1 and 8.2. 8.4.2 Colour development and spectrometric measurements

Pipette a 0.5 ml aliquot of each calibration solution into a series of five test tubes (6.7). Add 5.00 ml of water, 0.1 ml of acetic acid (5.4) and 0.2 ml of iodine solution (5.5). Place an additional 4.20 ml of water into the tube to make the volume of the reaction mixture up to 10.00 ml. Cover the tube with parafilm, and mix well on a vortex mixer or by inverting several times. Measure the absorbance at either 620 or 720 nm against the blank (8.3), immediately after mixing using the spectrophotometer (6.3). 8.4.3 Plotting the calibration graph

Prepare a calibration graph by plotting absorbance against the amylose content, expressed as a percentage by mass, in the milled rice on the dry basis.
NOTE Instead of manual spectrometric measurements an automatic analyser, e.g. a flow injection analyser, may be used (see example given in Annex C of part 1).

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8.5

Determination

Pipette a 0.5 ml aliquot of the test solution (8.2) into a test tube (6.7). Add 5.00 ml of water, 0.1 ml of acetic acid (5.4) and 0.2 ml of iodine solution (5.5). Put an additional 4.20 ml of water into the tube to make the volume of the reaction mixture up to 10.00 ml. Cover the tube with parafilm, and mix well on a vortex mixer or by inverting several times..Measure the absorbance at either 620 or 720 nm against the blank (8.3), immediately after mixing using the spectrometer (6.3). Ensure that the standards and test solutions are measured at the same wavelength, either 620 or 720 nm.
NOTE Instead of manual spectrometric measurements an automatic analyser, e.g. a flow injection analyser, may be used (see example given in Annex C of part 1).

Carry out two determinations on separate test portions taken from the same test sample.
NOTE: If double determinations are made, based on two independent preparations of the sample (8.1), this should be noted in the test report.

Expression of results

The amylose content, expressed as a percentage by mass on dry basis is obtained by referring the absorbance (8.5) to the calibration graph (8.4.3) according to ISO 8466-1. Take as the result the arithmetic mean of the two determinations.

10 Precision
10.1 Interlaboratory test
Details of an international interlaboratory test on the precision of the method are summarized in Annex A. The values derived from this test may not be applicable to concentration ranges and matrices other than those given.

10.2 Repeatability
The absolute difference between two independent single test results, obtained using the same method on identical test material in the same laboratory by the same operator using the same equipment within a short interval of time will in not more that 5 % of cases exceed the repeatability limit r (r will be deduced from the results of the interlaboratory test)

10.3 Reproducibility
The absolute difference between two single test results, obtained using the same method on identical test material in different laboratories by different operators using different equipment will in not more than 5 % of cases be greater than the reproducibility limit R (R will be deduced from the results of the interlaboratory test)

11 Test report
The test report shall specify: a) all information necessary for the complete identification of the sample; b) the sampling method used, if known; c) the test method used, with reference to this part of ISO 6647;

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d) all operating details not specified in this part of ISO 6647, or regarded as optional, together with details of any incidents which may have influenced the test result(s); e) the test result(s) obtained.

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Annex A Results of an interlaboratory test

An international inter-laboratory test involving 33 laboratories in 24 countries was carried out on 18 samples The test was organized by the INQR/IRRI and the results obtained were subjected to statistical analysis in accordance with ISO 5725-1 [1] and ISO 5725-2 [2] to give the precision data shown in Table A.1. Table A.1 Samples
E 28 10.43 0.56 5.35 1.56 1.08 1.55 1.55 2.16 1.44 1.15 2.13 2.74 2.10 2.97 -45.31 3.31 3.43 5.31 1.99 2.08 0.51 0.41 0.76 0.98 0.71 17.31 -0.91 22.92 28.51 13.38 6.63 0.58 8.79 1.63 0.98 30 30 28 28 31 29 29 6.57 0.56 8.49 1.56 1.02 F G H I J K L M 31 16.67 1.05 6.30 2.94 1.98 N 31 20.38 1.02 5.02 2.86 1.79 O 32 25.58 0.89 3.50 2.50 2.50 P 31 -0.81 0.36 -44.21 1.00 1.14 Q 27 -0.97 0.30 -30.72 2.30 1.23 R 30 28.66 1.06 2.97 3.70 2.87

Parameter Number of laboratories after elimination of outliers

29

27

28

27

3.69

14.05

27.46

26.49

Mean amylose content, % m/m Repeatability standard deviation (sr), %

0.51

0.74

0.91

0.89

13.94

5.27

3.33

3.38

Repeatability relative standard deviation, % Repeatability limit r (r = 2,77x sr),%

1.44

2.07

2.56

2.50

Reproducibility standard deviation (sR), % 10.32 3.02 4.34 8.95 -170.23 4.33 9.44 6.06

1.09

1.40

2.39

2.02

Reproducibility relative standard deviation, %

29.58

9.93

8.72

7.63

7.36 5.88

15.55 5.82

14.75 2.74

15.46 2.84

11.89 5.55

8.77 5.00

9.79 7.01

-141.97 3.20

-126.64 3.46

10.02 8.04

Reproducibility limit R (R = 2,77 sR), %

3.05

3.91

6.71

5.66

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Bibliography

[1] ISO 5725-1, Accuracy (trueness and precision) of measurement methods and results Part 1: General principles and definitions [2] ISO 5725-2, Accuracy (trueness and precision) of measurement methods and results Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method [3] [4] B.O. Juliano: A simplified assay for milled rice amylose, Cereal Sci. Today, 16, pp 334-340, 360, (1971).

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