Rice - Determination of amylose content - Part 1: Spectrophotometric method with a defatting procedure by methanol and with calibration solutions of potato amylose and waxy rice amylopectin (ISO 6647-1:2020)

This document specifies a reference method for the determination of the amylose content of milled rice,
non-parboiled. The method is applicable to rice with an amylose mass fraction higher than 5 %.
This document can also be used for husked rice, maize, millet and other cereals if the extension of this
scope has been validated by the user.
NOTE Amylose values determined with this document can be compared with PDO and PGI legislation.

Reis - Bestimmung des Amylosegehalts - Teil 1: Referenzmethode: Spektrophotometrische Methode mit Entfettungsverfahren durch Methanol und mit Kalibrierlösungen von Kartoffelamylose und wachsartigem Reisamylopektin (ISO 6647-1:2020)

Dieses Dokument legt ein Referenzverfahren für die Bestimmung des Amylosegehalts von gemahlenem Nicht-Parboiled-Reis fest. Das Verfahren ist anwendbar auf Reis mit einem Amylose-Massenanteil von mehr als 5 %.
Dieses Dokument kann auch für geschälten Reis, Mais, Hirse und andere Getreidearten angewendet werden, wenn die Erweiterung dieses Anwendungsbereichs von dem Anwender überprüft wurde.
ANMERKUNG Mit diesem Dokument bestimmte Amylose-Werte können mit den Rechtsvorschriften der geschützten Ursprungsbezeichnung (g. U., en: Protected Designation of Origin, PDO) und der geschützten geografischen Angabe (g. g. A., en: Protected Geographical Indication, PGI) verglichen werden.

Riz - Détermination de la teneur en amylose - Partie 1: Méthode spectrophotométrique avec un mode opératoire de dégraissage au méthanol et des solutions d'étalonnage d'amylose de pomme de terre et d'amylopectine de riz gluant (ISO 6647-1:2020)

Le présent document spécifie une méthode de référence pour la détermination de la teneur en amylose du riz usiné, non étuvé. La méthode est applicable à du riz avec une fraction massique d'amylose supérieure à 5 %.
Le présent document peut aussi être utilisé pour le riz décortiqué, le maïs, le millet et d'autres céréales si l'extension de son domaine d'application a été validée par l'utilisateur.
NOTE Les valeurs de teneur en amylose déterminées selon le présent document peuvent être comparées à la législation AOP et IGP.

Riž - Določevanje amiloze - 1. del: Spektrofotometrijska metoda s postopkom razmaščevanja z metanolom in s kalibracijskima raztopinama amiloza krompirja ter voskast amilopektin riža (ISO 6647-1:2020)

General Information

Status
Published
Public Enquiry End Date
19-Dec-2019
Publication Date
10-Sep-2020
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
25-Aug-2020
Due Date
30-Oct-2020
Completion Date
11-Sep-2020

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SLOVENSKI STANDARD
SIST EN ISO 6647-1:2020
01-oktober-2020
Riž - Določevanje amiloze - 1. del: Spektrofotometrijska metoda s postopkom
razmaščevanja z metanolom in s kalibracijskima raztopinama amiloza krompirja
ter voskast amilopektin riža (ISO 6647-1:2020)

Rice - Determination of amylose content - Part 1: Spectrophotometric method with a

defatting procedure by methanol and with calibration solutions of potato amylose and

waxy rice amylopectin (ISO 6647-1:2020)
Reis - Bestimmung des Amylosegehalts - Teil 1: Referenzmethode:
Spektrophotometrische Methode mit Entfettungsverfahren durch Methanol und mit

Kalibrierlösungen von Kartoffelamylose und wachsartigem Reisamylopektin (ISO 6647-

1:2020)

Riz - Détermination de la teneur en amylose - Partie 1: Méthode spectrophotométrique

avec un mode opératoire de dégraissage au méthanol et des solutions d'étalonnage
d'amylose de pomme de terre et d'amylopectine de riz gluant (ISO 6647-1:2020)
Ta slovenski standard je istoveten z: EN ISO 6647-1:2020
ICS:
67.060 Žita, stročnice in proizvodi iz Cereals, pulses and derived
njih products
SIST EN ISO 6647-1:2020 en

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
SIST EN ISO 6647-1:2020
---------------------- Page: 2 ----------------------
SIST EN ISO 6647-1:2020
EN ISO 6647-1
EUROPEAN STANDARD
NORME EUROPÉENNE
August 2020
EUROPÄISCHE NORM
ICS 67.060 Supersedes EN ISO 6647-1:2015
English Version
Rice - Determination of amylose content - Part 1:
Spectrophotometric method with a defatting procedure by
methanol and with calibration solutions of potato amylose
and waxy rice amylopectin (ISO 6647-1:2020)

Riz - Détermination de la teneur en amylose - Partie 1: Reis - Bestimmung des Amylosegehalts - Teil 1:

Méthode spectrophotométrique avec un mode Referenzmethode: Spektrophotometrische Methode

opératoire de dégraissage au méthanol et des solutions mit Entfettungsverfahren durch Methanol und mit

d'étalonnage d'amylose de pomme de terre et Kalibrierlösungen von Kartoffelamylose und

d'amylopectine de riz gluant (ISO 6647-1:2020) wachsartigem Reisamylopektin (ISO 6647-1:2020)

This European Standard was approved by CEN on 18 July 2020.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this

European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references

concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN

member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by

translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management

Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 6647-1:2020 E

worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
SIST EN ISO 6647-1:2020
EN ISO 6647-1:2020 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

---------------------- Page: 4 ----------------------
SIST EN ISO 6647-1:2020
EN ISO 6647-1:2020 (E)
European foreword

This document (EN ISO 6647-1:2020) has been prepared by Technical Committee ISO/TC 34 "Food

products" in collaboration with Technical Committee CEN/TC 338 “Cereal and cereal products” the

secretariat of which is held by AFNOR.

This European Standard shall be given the status of a national standard, either by publication of an

identical text or by endorsement, at the latest by February 2021, and conflicting national standards

shall be withdrawn at the latest by February 2021.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

This document supersedes EN ISO 6647-1:2015.

According to the CEN-CENELEC Internal Regulations, the national standards organizations of the

following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,

Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,

Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of

North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the

United Kingdom.
Endorsement notice

The text of ISO 6647-1:2020 has been approved by CEN as EN ISO 6647-1:2020 without any

modification.
---------------------- Page: 5 ----------------------
SIST EN ISO 6647-1:2020
---------------------- Page: 6 ----------------------
SIST EN ISO 6647-1:2020
INTERNATIONAL ISO
STANDARD 6647-1
Third edition
2020-07
Rice — Determination of amylose
content —
Part 1:
Spectrophotometric method with a
defatting procedure by methanol and
with calibration solutions of potato
amylose and waxy rice amylopectin
Riz — Détermination de la teneur en amylose —
Partie 1: Méthode spectrophotométrique avec un mode opératoire de
dégraissage au méthanol et des solutions d'étalonnage d'amylose de
pomme de terre et d'amylopectine de riz gluant
Reference number
ISO 6647-1:2020(E)
ISO 2020
---------------------- Page: 7 ----------------------
SIST EN ISO 6647-1:2020
ISO 6647-1:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
---------------------- Page: 8 ----------------------
SIST EN ISO 6647-1:2020
ISO 6647-1:2020(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

5 Reagents ........................................................................................................................................................................................................................ 2

6 Apparatus ..................................................................................................................................................................................................................... 3

7 Sampling ........................................................................................................................................................................................................................ 4

8 Procedure..................................................................................................................................................................................................................... 4

8.1 Preparation of test sample ............................................................................................................................................................ 4

8.2 Test portion and preparation of the test solution ..................................................................................................... 4

8.3 Preparation of the blank solution ........................................................................................................................................... 4

8.4 Preparation of the calibration graph .................................................................................................................................... 4

8.4.1 Preparation of the set of calibration solutions ...................................................................................... 4

8.4.2 Colour development and spectrophotometric measurements ................................................ 5

8.4.3 Plotting the calibration graph ............................................................................................................................... 5

8.5 Determination ......................................................................................................................................................................................... 5

9 Expression of results ........................................................................................................................................................................................ 5

10 Precision ....................................................................................................................................................................................................................... 5

10.1 Interlaboratory test............................................................................................................................................................................. 5

10.2 Repeatability ............................................................................................................................................................................................. 6

10.3 Reproducibility ....................................................................................................................................................................................... 6

11 Test report ................................................................................................................................................................................................................... 6

Annex A (informative) Determination of the quality of the potato amylose stock suspension ...............7

Annex B (informative) Results of an interlaboratory test .............................................................................................................. 9

Annex C (informative) Example of a flow injection analyser (FIA) for the determination of

amylose ........................................................................................................................................................................................................................10

Bibliography .............................................................................................................................................................................................................................11

© ISO 2020 – All rights reserved iii
---------------------- Page: 9 ----------------------
SIST EN ISO 6647-1:2020
ISO 6647-1:2020(E)
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.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).

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. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/ patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO’s adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/

iso/ foreword .html.

This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 4,

Cereals and pulses, in collaboration with the European Committee for Standardization (CEN) Technical

Committee CEN/TC 338, Cereal and cereal products, in accordance with the Agreement on technical

cooperation between ISO and CEN (Vienna Agreement).

This third edition cancels and replaces the second edition (ISO 6647-1:2015), which has been technically

revised. The main changes compared with the previous edition are as follows.

— A spectrophotometric method with a defatting procedure by methanol and with calibration solutions

of potato amylose and waxy rice amylopectin has replaced the size exclusion chromatography method.

A list of all parts in the ISO 6647 series can be found on the ISO website.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/ members .html.
iv © ISO 2020 – All rights reserved
---------------------- Page: 10 ----------------------
SIST EN ISO 6647-1:2020
INTERNATIONAL STANDARD ISO 6647-1:2020(E)
Rice — Determination of amylose content —
Part 1:
Spectrophotometric method with a defatting procedure by
methanol and with calibration solutions of potato amylose
and waxy rice amylopectin
1 Scope

This document specifies a reference method for the determination of the amylose content of milled rice,

non-parboiled. The method is applicable to rice with an amylose mass fraction higher than 5 %.

This document can also be used for husked rice, maize, millet and other cereals if the extension of this

scope has been validated by the user.

NOTE Amylose values determined with this document can be compared with PDO and PGI legislation.

2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements 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 712, Cereals and cereal products — Determination of moisture content — Reference method

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

ISO 15914, Animal feeding stuffs — Enzymatic determination of total starch content

3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
amylose
DEPRECATED: apparent amylose

polysaccharide constituent of starch, the macromolecules of which have glucose units linked in a

predominantly linear structure
3.2
amylopectin

polysaccharide constituent of starch, the macromolecules of which have from 6 to 100 linked glucose

units in a branched structure
© ISO 2020 – All rights reserved 1
---------------------- Page: 11 ----------------------
SIST EN ISO 6647-1:2020
ISO 6647-1:2020(E)
4 Principle

Rice is ground to a very fine flour to break up the endosperm structure in order to aid complete

dispersion and gelatinization; the flour is then defatted. A test portion is dispersed in a sodium

hydroxide solution. An aliquot portion is taken to which an iodine solution is added. The absorbance, at

720 nm, of the colour complex
...

SLOVENSKI STANDARD
oSIST prEN ISO 6647-1:2019
01-december-2019
Riž - Določevanje amiloze - 1. del: Referenčna metoda: spektrofotometrijska
metoda s postopkom razmaščevanja z metanolom in s kalibracijsko raztopino
amiloze krompirja ter voskastim riževim amilopektinom (ISO/DIS 6647-1:2019)

Rice - Determination of amylose content - Part 1: Reference method: Spectrophotometric

method with a defatting procedure by methanol and with calibration solutions of potato

amylose and waxy rice amylopectin (ISO/DIS 6647-1:2019)
Reis - Bestimmung des Amylosegehalts - Teil 1: Referenzmethode:
Spektrophotometrische Methode mit Entfettungsverfahren durch Methanol und mit
Kalibrierlösungen von Kartoffelamylose und wachsartigem Reisamylopektin (ISO/DIS
6647-1:2019)

Riz - Détermination de la teneur en amylose - Partie 1: Méthode de référence : méthode

spectrophotométrique avec un mode opératoire de dégraissage au méthanol et des

solutions d'étalonnage d'amylose de pomme de terre et d'amylopectine de riz gluant

(ISO/DIS 6647-1:2019)
Ta slovenski standard je istoveten z: prEN ISO 6647-1
ICS:
67.060 Žita, stročnice in proizvodi iz Cereals, pulses and derived
njih products
oSIST prEN ISO 6647-1:2019 en

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
oSIST prEN ISO 6647-1:2019
---------------------- Page: 2 ----------------------
oSIST prEN ISO 6647-1:2019
DRAFT INTERNATIONAL STANDARD
ISO/DIS 6647-1
ISO/TC 34/SC 4 Secretariat: SAC
Voting begins on: Voting terminates on:
2019-10-21 2020-01-13
Rice — Determination of amylose content —
Part 1:
Reference method: Spectrophotometric method with a
defatting procedure by methanol and with calibration
solutions of potato amylose and waxy rice amylopectin
ICS: 67.060
THIS DOCUMENT IS A DRAFT CIRCULATED
This document is circulated as received from the committee secretariat.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
ISO/CEN PARALLEL PROCESSING
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 6647-1:2019(E)
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. ISO 2019
---------------------- Page: 3 ----------------------
oSIST prEN ISO 6647-1:2019
ISO/DIS 6647-1:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
---------------------- Page: 4 ----------------------
oSIST prEN ISO 6647-1:2019
ISO/DIS 6647-1:2019(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

5 Reagents ........................................................................................................................................................................................................................ 2

6 Apparatus ..................................................................................................................................................................................................................... 3

7 Sampling ........................................................................................................................................................................................................................ 4

8 Procedure..................................................................................................................................................................................................................... 4

8.1 Preparation of test sample ............................................................................................................................................................ 4

8.2 Test portion and preparation of the test solution ..................................................................................................... 4

8.3 Preparation of the blank solution ........................................................................................................................................... 4

8.4 Preparation of the calibration graph .................................................................................................................................... 4

8.4.1 Preparation of the set of calibration solutions ...................................................................................... 4

8.4.2 Colour development and spectrophotometric measurements ................................................ 5

8.4.3 Plotting the calibration graph ............................................................................................................................... 5

8.5 Determination ......................................................................................................................................................................................... 5

9 Expression of results ........................................................................................................................................................................................ 5

10 Precision ....................................................................................................................................................................................................................... 5

10.1 Interlaboratory test............................................................................................................................................................................. 5

10.2 Repeatability ............................................................................................................................................................................................. 6

10.3 Reproducibility ....................................................................................................................................................................................... 6

11 Test report ................................................................................................................................................................................................................... 6

Annex A (informative) Determination of the quality of the potato amylose stock suspension ...............7

Annex B (informative) Results of an interlaboratory test .............................................................................................................. 9

Annex C (informative) Example of a flow injection analyzer (FIA) for the determination of

amylose ........................................................................................................................................................................................................................11

Bibliography .............................................................................................................................................................................................................................12

© ISO 2019 – All rights reserved iii
---------------------- Page: 5 ----------------------
oSIST prEN ISO 6647-1:2019
ISO/DIS 6647-1:2019(E)
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.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).

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. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso

.org/iso/foreword .html.

This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 4,

Cereals and pulses.

This third edition cancels and replaces the second edition (ISO 6647-1:2015), which has been technically

revised.
The main changes compared to the previous edition are as follows:

— Spectrophotometric method with a defatting procedure by methanol and with calibration

solutions of potato amylose and waxy rice amylopectin was used to replace the size exclusion

chromatography method.
A list of all parts in the ISO 6647 series can be found on the ISO website.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/members .html.
iv © ISO 2019 – All rights reserved
---------------------- Page: 6 ----------------------
oSIST prEN ISO 6647-1:2019
DRAFT INTERNATIONAL STANDARD ISO/DIS 6647-1:2019(E)
Rice — Determination of amylose content —
Part 1:
Reference method: Spectrophotometric method with a
defatting procedure by methanol and with calibration
solutions of potato amylose and waxy rice amylopectin
1 Scope

This part of ISO 6647 specifies a reference method for the determination of the amylose content of milled

rice, non-parboiled. The method is applicable to rice with an amylose mass fraction higher than 5 %.

This part of ISO 6647 can also be used for husked rice, maize, millet and other cereals if the extension of

this scope has been validated by the user.

Note Amylose values determined with this standard can be compared with PDO and PGI legislation.

2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements 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 712, Cereals and cereal products — Determination of moisture content — Reference method

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

ISO 15914, Animal feeding stuffs — Enzymatic determination of total starch content

3 Terms and definitions

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1
Amylose

text of the definition polysaccharide constituent of starch, the macromolecules of which have glucose

units linked in a predominantly linear structure

Note 1 to entry: The term "apparent amylose" is historically used as a synonym of the term "amylose".

3.2
amylopectin

polysaccharide constituent of starch, the macromolecules of which have from 6 to 100 linked glucose

units in a branched structureClause title autonumber
© ISO 2019 – All rights reserved 1
---------------------- Page: 7 ----------------------
oSIST prEN ISO 6647-1:2019
ISO/DIS 6647-1:2019(E)
4 Principle

Rice is ground to a very fine flour to break up the endosperm structure in order to aid complete

dispersion and gelatinization; the flour is then defatted. A test portion is dispersed in a sodium

hydroxide solution, to an aliquot portion of which an iodine solution is added. The absorbance, at 720

nm, of the colour complex formed is then determined using a spectrophotometer.

The amylose mass fraction of the sample is then read from a calibration graph, which is prepared using

mixtures of potato amylose and amylopectin to make allowance for the effect of amylopectin on the

colour of the amylose–iodine complex of the test solution.
5 Reagents

Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or

demineralized water or water of equivalent purity.
5.1 Methanol, a volume fraction of 85 %.
5.2 Ethanol, a volume fraction of 95 %.
5.3 Sodium hydroxide dispersal solutions.
5.3.1 Sodium hydroxide, 1 mol/l solution.
5.3.2 Sodium hydroxide, 0,09 mol/l solution.
5.4 Deproteination solutions
5.4.1 Detergent solution

Dissolve sodium dodecylbenzene sulfonate corresponding to a concentration of 20 g/l. Just before use,

add sodium sulfite to a final concentration of 2 g/l.
5.4.2 Sodium hydroxide, for protein removal, 3 g/l solution.
5.5 Acetic acid, 1 mol/l solution.
5.6 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 (6.6), make

up to volume with water and mix.
Prepare a fresh solution on each day of use and protect it from light.
5.7 Stock potato amylose suspension, free of amylopectin, 1 g/l.

5.7.1 Defat the potato amylose by refluxing with methanol (5.1) for 4 h to 6 h in an extractor at a rate of

5 to 6 droplets per second.

The potato amylose should be pure and should be tested by amperometric or potentiometric titration.

Some commercial preparations are impure and would give erroneously high results for the amylose

mass fraction of rice samples. Pure am
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