Infant formula and adult nutritionals - Determination of fructans - High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) after enzymatic treatment (ISO 22579:2020)

This International Standard specifies a method for the determination of inulin-type fructans (including oligofructose, and fructooligosaccharides) in infant formula and adult nutritionals containing 0,03 g/100 g to 5,0 g/100 g of fructan in the product as prepared ready for consumption.
A high performance anion exchange chromatographic method in combination with pulsed amperometric detection (HPAEC-PAD) is applied.

Säuglingsnahrung und Nahrungsergänzungsmittel für Erwachsene - Bestimmung von Fructanen - Hochleistungs-Anionenaustausch-Chromatographieverfahren mit gepulster amperometrischer Detektion (HPAEC-PAD) nach enzymatischer Behandlung (ISO 22579:2020)

WARNUNG — Das in diesem Dokument beschriebene Verfahren verwendet ätzende (Natrium¬hydroxid, Essigsäure) und giftige (Natriumazid) Chemikalien. Es sind die Sicherheitsdatenblätter zu beachten und entsprechende zusätzliche Sicherheitsvorkehrungen für die Handhabung und Ent¬sorgung zu treffen.
Dieses Dokument legt ein Verfahren zur Bestimmung von Fructanen des Inulintyps (einschließlich Oligo-fruktose, Fruktooligosaccharide) in Säuglingsnahrung und Nahrungsergänzungsmitteln für Erwachsene (sowohl Pulver als auch Flüssigkeit) fest, die 0,03 g/100 g bis 5,0 g/100 g Fructane in dem verzehrfertigen Enderzeugnis enthalten.
Das Verfahren wurde in einer Multi-Laborstudie [1] mit rekonstituiertem Standardreferenzmaterial (SRM) für Säuglingsnahrung/Nahrungsergänzungsmittel für Erwachsene mit einem Gehalt von 0,204 g/100 g, Nahrungsergänzungsmittel für Erwachsene als Fertignahrung (RTF, en: ready-to-feed) mit Gehalten von 1,28 g/100 g und 2,67 g/100 g, Säuglingsnahrung als Fertignahrung mit einem Gehalt von 0,300 g/100 g, rekonstituierte Folgenahrung im Bereich von 0,209 g/100 g bis 0,275 g/100 g, rekonstituierte Säuglingsnahrung im Bereich von 0,030 8 g/100 g bis 0,264 g/100 g validiert. Während der Validierungs-studie im Einzellabor [2] wurden Versuche zur Wiederfindungsrate bei Aufstockung von bis zu 5 g/100 g in rekonstituierten Säuglingsnahrungspulvern (auf Milchbasis, auf teilweise hydrolysierter Milchbasis und Sojabasis), Nahrungsergänzungsmitteln für Erwachsene als Fertignahrung und rekonstituierten pulver-förmigen Nahrungsergänzungsmitteln für Erwachsene durchgeführt.

Formules infantiles et produits nutritionnels pour adultes - Détermination de la teneur en fructanes - Chromatographie d’échange d’anions à haute performance couplée à la détection par ampérométrie pulsée « CEAHP-DAP » après traitement enzymatique (ISO 22579:2020)

Le présent document spécifie une méthode de dosage des fructanes de type inuline (tels que l'oligofructose, les fructo-oligosaccharides) dans les formules pour nourrissons et les produits nutritionnels pour adultes (en poudre ou liquides) contenant 0,03 g/100 g à 5,0 g/100 g de fructanes dans le produit préparé sous une forme prête à la consommation.
La méthode a été validée lors d'une étude interlaboratoires[1] avec un matériau de référence standard (SRM) reconstitué de type préparation nutritionnelle pour nourrissons/adultes à une concentration de 0,204 g/100 g, produits nutritionnels prêts à l'emploi (RTF) pour adultes à des concentrations de 1,28 g/100 g et 2,67 g/100 g, préparation RTF pour nourrissons à une concentration de 0,300 g/100 g, préparation de suite reconstituée à des concentrations de 0,209 g/100 g à 0,275 g/100 g, préparation reconstituée pour nourrissons à des concentrations de 8 g/100 g à 0,264 g/100 g. Pendant l'étude de validation intralaboratoire[2], des essais de recouvrement ont été menés jusqu'à 5 g/100 g dans les préparations en poudre reconstituées pour nourrissons (à base de lait, de lait partiellement hydrolysé et de soja), le produit nutritionnel RTF et les produits nutritionnels reconstitués pour adultes.

Hrana za dojenčke in prehranska dopolnila za odrasle - Določevanje fruktanov - Anionska izmenjevalna kromatografija z visoko ločljivostjo s pulzno amperometrično detekcijo (HPAEC-PAD) po encimski obdelavi (ISO 22579:2020)

General Information

Status
Published
Public Enquiry End Date
19-Feb-2020
Publication Date
16-Mar-2021
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
02-Mar-2021
Due Date
07-May-2021
Completion Date
17-Mar-2021

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SLOVENSKI STANDARD
SIST EN ISO 22579:2021
01-april-2021
Hrana za dojenčke in prehranska dopolnila za odrasle - Določevanje fruktanov -
Anionska izmenjevalna kromatografija z visoko ločljivostjo s pulzno
amperometrično detekcijo (HPAEC-PAD) po encimski obdelavi (ISO 22579:2020)

Infant formula and adult nutritionals - Determination of fructans - High performance anion

exchange chromatography with pulsed amperometric detection (HPAEC-PAD) after
enzymatic treatment (ISO 22579:2020)
Säuglingsnahrung und Nahrungsergänzungsmittel für Erwachsene - Bestimmung von

Fructanen - Hochleistungs-Anionenaustausch-Chromatographieverfahren mit gepulster

amperometrischer Detektion (HPAEC-PAD) nach enzymatischer Behandlung (ISO
22579:2020)

Formules infantiles et produits nutritionnels pour adultes - Détermination de la teneur en

fructanes - Chromatographie déchange danions à haute performance couplée à la

détection par ampérométrie pulsée « CEAHP-DAP » après traitement enzymatique (ISO

22579:2020)
Ta slovenski standard je istoveten z: EN ISO 22579:2021
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
SIST EN ISO 22579:2021 en

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

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SIST EN ISO 22579:2021
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SIST EN ISO 22579:2021
EN ISO 22579
EUROPEAN STANDARD
NORME EUROPÉENNE
February 2021
EUROPÄISCHE NORM
ICS 67.050
English Version
Infant formula and adult nutritionals - Determination of
fructans - High performance anion exchange
chromatography with pulsed amperometric detection
(HPAEC-PAD) after enzymatic treatment (ISO
22579:2020)

Préparations pour nourrissons et produits Säuglingsnahrung und Nahrungsergänzungsmittel für

nutritionnels pour adultes - Dosage des fructanes - Erwachsene - Bestimmung von Fructanen -

Chromatographie échangeuse d'anions haute Hochleistungs-Anionenaustausch-

performance couplée à la détection par ampérométrie Chromatographieverfahren mit gepulster

pulsée (CEAHP-DAP) après traitement enzymatique amperometrischer Detektion (HPAEC-PAD) nach

(ISO 22579:2020) enzymatischer Behandlung (ISO 22579:2020)
This European Standard was approved by CEN on 22 September 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

© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 22579:2021 E

worldwide for CEN national Members.
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SIST EN ISO 22579:2021
EN ISO 22579:2021 (E)
Contents Page

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

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SIST EN ISO 22579:2021
EN ISO 22579:2021 (E)
European foreword

This document (EN ISO 22579:2021) has been prepared by Technical Committee ISO/TC 34 "Food

products" in collaboration with Technical Committee CEN/TC 302 “Milk and milk products - Methods of

sampling and analysis” the secretariat of which is held by NEN.

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 August 2021, and conflicting national standards shall

be withdrawn at the latest by August 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.

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 22579:2020 has been approved by CEN as EN ISO 22579:2021 without any modification.

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SIST EN ISO 22579:2021
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SIST EN ISO 22579:2021
INTERNATIONAL ISO
STANDARD 22579
IDF 241
First edition
2020-09
Infant formula and adult
nutritionals — Determination of
fructans — High performance anion
exchange chromatography with pulsed
amperometric detection (HPAEC-PAD)
after enzymatic treatment
Préparations pour nourrissons et produits nutritionnels pour
adultes — Dosage des fructanes — Chromatographie échangeuse
d'anions haute performance couplée à la détection par ampérométrie
pulsée (CEAHP-DAP) après traitement enzymatique
Reference numbers
ISO 22579:2020(E)
IDF 241:2020(E)
ISO and IDF 2020
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SIST EN ISO 22579:2021
ISO 22579:2020(E)
IDF 241:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO and IDF 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 International Dairy Federation
CP 401 • Ch. de Blandonnet 8 Silver Building • Bd Auguste Reyers 70/B
CH-1214 Vernier, Geneva B-1030 Brussels
Phone: +41 22 749 01 11 Phone: +32 2 325 67 40
Fax: +32 2 325 67 41
Email: copyright@iso.org Email: info@fil-idf.org
Website: www.iso.org Website: www.fil-idf.org
Published in Switzerland
ii © ISO and IDF 2020 – All rights reserved
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SIST EN ISO 22579:2021
ISO 22579:2020(E)
IDF 241:2020(E)
Contents Page

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

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

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

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

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

5 Chemicals and reagents ................................................................................................................................................................................ 2

5.1 List of chemicals and reagents .................................................................................................................................................. 2

5.2 Preparation of reagents ................................................................................................................................................................... 3

5.3 Preparation of mobile phases using column A (6.13.1) or equivalent.................................................... 5

5.4 Preparation of mobile phases using columns B (6.13.2) or equivalent ................................................. 5

5.5 Preparation of standard solutions ......................................................................................................................................... 6

6 Apparatus ..................................................................................................................................................................................................................... 6

7 Procedure..................................................................................................................................................................................................................... 8

7.1 Sample preparation ............................................................................................................................................................................ 8

7.1.1 Powdered or concentrated products on a ready-to feed (RTF) basis and

powder products inhomogeneous at the subgram level ............................................................... 8

7.1.2 Reconstituted products as prepared in 7.1.1 or products sold as RTF ............................. 8

7.1.3 Homogeneous powdered products without prior reconstitution ........................................ 8

7.1.4 Dilution..................................................................................................................................................................................... 8

7.1.5 Hydrolysis of sucrose and α-glucans .............................................................................................................. 8

7.1.6 Carrez clarification (optional, use in case of difficulties passing sample

through SPE) ...................................................................................................................................................................... 8

7.1.7 Removal of monosaccharides ............................................................................................................................... 9

7.1.8 Hydrolysis of fructans .................. ......................................................................................................................... ....... 9

7.2 Chromatographic conditions using column A (6.13.1) ........................................................................................ 9

7.3 Chromatographic conditions using columns B (6.13.2) ...................................................................................10

7.4 System suitability test ....................................................................................................................................................................11

7.5 Calibration ...............................................................................................................................................................................................11

8 Calculation ...............................................................................................................................................................................................................11

9 Reporting ...................................................................................................................................................................................................................13

10 Precision data .......................................................................................................................................................................................................13

10.1 General ........................................................................................................................................................................................................13

10.2 Repeatability ..........................................................................................................................................................................................13

10.3 Reproducibility ....................................................................................................................................................................................13

11 Test report ................................................................................................................................................................................................................14

Annex A (informative) Example chromatograms and calibration curves ...................................................................15

Annex B (informative) Precision data ..............................................................................................................................................................18

Annex C (informative) Check test enzyme mixture of sucrase, β-amylase, pullulanase and

maltase.........................................................................................................................................................................................................................19

Bibliography .............................................................................................................................................................................................................................22

© ISO and IDF 2020 – All rights reserved iii
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SIST EN ISO 22579:2021
ISO 22579:2020(E)
IDF 241:2020(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 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with

AOAC INTERNATIONAL, and in collaboration with the European Committee for Standardization

(CEN) Technical Committee CEN/TC 302, Milk and milk products — Methods of sampling and analysis, in

accordance with the Agreement on technical cooperation between ISO and CEN (Vienna Agreement).

It is being published jointly by ISO and IDF and separately by AOAC INTERNATIONAL. The method

described in this document is equivalent to the AOAC Official Method 2016.14: Fructans in Infant

Formula and Adult Nutrition.

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 and IDF 2020 – All rights reserved
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SIST EN ISO 22579:2021
ISO 22579:2020(E)
IDF 241:2020(E)

IDF (the International Dairy Federation) is a non-profit private sector organization representing the

interests of various stakeholders in dairying at the global level. IDF members are organized in National

Committees, which are national associations composed of representatives of dairy-related national

interest groups including dairy farmers, dairy processing industry, dairy suppliers, academics and

governments/food control authorities.

ISO and IDF collaborate closely on all matters of standardization relating to methods of analysis

and sampling for milk and milk products. Since 2001, ISO and IDF jointly publish their International

Standards using the logos and reference numbers of both organizations.

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

patent rights. IDF 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.

This document was prepared by the IDF Standing Committee on Analytical Methods for Composition and

ISO Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is

being published jointly by ISO and IDF.

The work was carried out by the IDF-ISO Action Team on C41 of the Standing Committee on Analytical

Methods for Composition under the aegis of its project leader Mr S. Austin (CH).
© ISO and IDF 2020 – All rights reserved v
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SIST EN ISO 22579:2021
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SIST EN ISO 22579:2021
ISO 22579:2020(E)
INTERNATIONAL STANDARD
IDF 241:2020(E)
Infant formula and adult nutritionals — Determination
of fructans — High performance anion exchange
chromatography with pulsed amperometric detection
(HPAEC-PAD) after enzymatic treatment

WARNING — The method described in this document employs corrosive (sodium hydroxide,

acetic acid) and toxic (sodium azide) chemicals. Refer to the materials safety data sheets and

take appropriate additional safety precautions for handling and waste disposal.
1 Scope

This document specifies a method for the determination of inulin-type fructans (including oligofructose,

fructooligosaccharides) in infant formula and adult nutritionals (both powder and liquid) containing

0,03 g/100 g to 5,0 g/100 g of fructans in the product as prepared ready for consumption.

[1]

The method has been validated in a multi laboratory study with reconstituted standard reference

material (SRM), infant/adult nutritional formula at a level of 0,204 g/100 g, adult nutritionals ready-

to-feed (RTF) at levels of 1,28 g/100 g and 2,67 g/100 g, infant formula RTF at a level of 0,300 g/100 g,

reconstituted follow-up formula at levels of 0,209 g/100 g to 0,275 g/100 g, reconstituted infant formula

[2]

at levels from 0,030 8 g/100 g to 0,264 g/100 g. During the single laboratory validation study , spike-

recovery experiments were performed up to 5 g/100 g in reconstituted infant formula powders (milk-

based, partially hydrolysed milk-based and soy-based), adult nutritional RTF and reconstituted adult

nutritional powders.
2 Normative references
There are no normative references in this document.
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
adult nutritional

nutritionally complete, specially formulated food, consumed in liquid form, which may constitute the

sole source of nourishment, made from any combination of milk, soy, rice, whey, hydrolysed protein,

starch and amino acids, with or without intact protein
3.2
infant formula

breast-milk substitute specially manufactured to satisfy, by itself, the nutritional requirements of

infants during the first months of life up to the introduction of appropriate complementary feeding

[SOURCE: Codex Standard 72-1981]
© ISO and IDF 2020 – All rights reserved 1
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SIST EN ISO 22579:2021
ISO 22579:2020(E)
IDF 241:2020(E)
3.3
follow-up formula

food intended for use as a liquid part of the weaning diet for an infant from the age of six months, and

for young children up to the age of three years
Note 1 to entry: Follow-up formula includes child formula and toddler formula.
[SOURCE: Codex Standard 156-1987]
4 Principle

Samples are reconstituted in water (if required) and further diluted until the concentration of fructans

in solution is such that after hydrolysis the fructose and glucose concentration will be within the

range covered by the standard curve. The diluted sample is treated with a mixture of a highly specific

sucrase and α-glucanases to hydrolyse sucrose and α-glucooligosaccharides to their constituent

monosaccharides. The sample is passed through a solid phase extraction (SPE) column packed with

graphitized carbon. Salts and monosaccharides pass through and are washed away, while the fructans

are retained. Fructans are released from the column using an acetonitrile solution. The released

fructans are hydrolysed with a fructanase mixture, and the released glucose and fructose are analysed

by high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-

PAD). The fructan content is calculated by summing the glucose and 0,9 times the fructose contents

measured. In some matrices, a blank correction may be necessary and can be applied.

5 Chemicals and reagents
5.1 List of chemicals and reagents

Use only reagents of recognized analytical grade, unless otherwise specified. Solvents shall be of quality

for HPLC analysis, unless otherwise specified.
5.1.1 Deionized water, purified with resistivity ≥ 18 MΩ.
5.1.2 Maleic acid, purity ≥ 99,0 %.
5.1.3 Acetonitrile.
5.1.4 Acetic acid, glacial 100 %, anhydrous.
5.1.5 Potassium hexacyanoferrate(II)trihydrate, optional.
5.1.6 Zinc acetate, optional.
5.1.7 Trifluoroacetic acid (TFA).
5.1.8 Hydrochloric acid, substance concentration c = 1 mol/l.

5.1.9 Sodium acetate anhydrous, purity ≥ 99,0 %, only when using columns B (6.13.2) for HPAEC-PAD.

5.1.10 Sodium hydroxide solution, a mass fraction of 50 %.
5.1.11 Sodium hydroxide pellets.
5.1.12 Sodium chloride.
2 © ISO and IDF 2020 – All rights reserved
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SIST EN ISO 22579:2021
ISO 22579:2020(E)
IDF 241:2020(E)
5.1.13 Sodium azide, optional.
5.1.14 D-(-)-fructose, purity ≥ 99,0 % (dry weight basis).
5.1.15 D-(+)-glucose, purity ≥ 99,5 % (dry weight basis).
5.1.16 N,N′-diacetylchitobiose, purity > 90 %.

5.1.17 Mixture of highly purified sucrase, β-amylase, pullulanase and maltase, from Fructan Assay

Kit K-FRUC (Megazyme International Ireland Ltd or equivalent). 200 µl of enzyme mixture working

solution (5.2.10) should be able to completely hydrolyse 2 mg of sucrose under the conditions described

in the method (90 min at 40 °C) without hydrolysing any fructans.

5.1.18 Mixture of highly purified recombinant exo- and endo-inulinases and recombinant endo-

levanase, from Fructan Assay Kit K-FRUC (Megazyme International Ireland Ltd or equivalent). 100 µl

of enzyme mixture working solution (5.2.11) should be able to hydrolyse 70 µg of fructans under the

conditions described in the method (40 min at 40 °C).
5.2 Preparation of reagents
5.2.1 Sodium hydroxide solution, c = 2 mol/l.

Dissolve 40 g ± 1 g of sodium hydroxide pellets in 250 ml of deionized water in a 500 ml volumetric

flask. After cooling down to room temperature, make up to the mark with deionized water and mix

well. This solution is stable for six months at room temperature.
5.2.2 Sodium maleate buffer solution, c = 0,100 mol/l, pH = 6,5.

Into a large beaker (>500 ml), weigh 5,8 g of maleic acid and dissolve with 450 ml of deionized water

using a magnetic stirrer. Adjust to pH = 6,5 with sodium hydroxide solution (5.2.1). Transfer the solution

to a 500 ml volumetric flask and make up to the mark with deionized water. This solution is stable for

three months at 4 °C.
5.2.3 Sodium acetate buffer solution, c = 0,100 mol/l, pH = 4,5.

Into a large beaker (>500 ml) containing 450 ml of deionized water, pipette 2,9 ml of glacial acetic acid.

Adjust to pH = 4,5 with sodium hydroxide solution (5.2.1). Transfer the solution to a 500 ml volumetric

flask and make up to the mark with deionized water. This solution is stable for three months at 4 °C.

5.2.4 N,N′diacetylchitobiose internal standard solution, mass concentration ρ = 600 µg/ml.

Into a 25 ml volumetric flask, weigh 15 mg of N,N′diacetylchitobiose and make up to the mark with

deionized water. This solution is stable for one year at −20 °C.
5.2.5 Glucose stock solution, ρ = 5 mg/ml.

Into a 25 ml volumetric flask, weigh 125 mg of glucose and make up to the mark with deionized water.

This solution is stable for one year at −20 °C.

1) This is an example of a suitable product available commercially. This information is given for the convenience

of users of this document and does not constitute an endorsement by ISO or IDF of the product named. Equivalent

products may be used if they can be shown to lead to the same results.
© ISO and IDF 2020 – All rights reserved 3
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SIST EN ISO 22579:2021
ISO 22579:2020(E)
IDF 241:2020(E)
5.2.6 Fructose stock solution, ρ = 10 mg/ml.

Into a 25 ml volumetric flask, weigh 250 mg of fructose and make up to the mark with deionized water.

This solution is stable for one year at −20 °C.
5.2.7 Carrez I solution

Dissolve 10,6 g of potassium hexacyanoferrate(II)trihydrate in 100 ml of deionized water and store in a

brown bottle. This solution is stable for six months at room temperature (optional reagent).

5.2.8 Carrez II solution

In a 100 ml volumetric flask, dissolve 22,0 g of zinc acetate in 90 ml of deionized water and add 2,9 ml

of glacial acetic acid. Make up to the mark with deionized water and homogenize. This solution is stable

for six months at room temperature (optional reagent).
5.2.9 Sodium azide solution, ρ = 5 g/l.
Dissolve 1 g of sodium azide in 200 ml of deionized water (optional reagent).
5.2.10 Mixture of sucrase, β-amylase, pullulanase and maltase solution.

Dissolve the contents of the bottle containing freeze-dried powdered sucrase, β-amylase, pullulanase

and maltase in 22,0 ml of sodium maleate buffer solution (5.2.2). Mix well and divide into aliquots of

2 ml each and store frozen at −20 °C in polypropylene tubes until use. This solution is stable for five

years at −20 °C.

IMPORTANT — For the development and validation of this method, a commercially available

pre-prepared enzyme mixture from Megazyme (5.1.17) was used. When enzymes from

another source are used, the buffer composition (type, concentration and pH) may need to be

adapted to the recommendations of the supplier. It is also imperative to ensure the enzyme

mixture employed will completely hydrolyse any sucrose in the product without hydrolysing

the fructan. This can be checked by performing an analysis with sucrose as an analyte and

with a pure fructan as an analyte. No fructan should be measured when sucrose is analysed,

and more than 90 % recovery should be achieved when a fructan of known purity is analysed

(recommended to check using both long-chain inulin and short-chain fructooligosaccharides).

An alternative test for checking the suitability and performance of the enzyme mixture is

described in Annex C.
5.2.11 Fructanase solution (exo-/endo-inulinases and endo-levanase).

Dissolve the contents of the bottle containing freeze-dried powdered exo- and endo-inulinases and

endo-levanase in 22,0 ml of sodium acetate buffer solution (5.2.3). Mix well a
...

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