Dansk standard DS/EN ISO 30024

1. udgave

2009-09-03
COPYRIGHT Danish Standards. NOT FOR COMMERCIAL USE OR REPRODUCTION. DS/EN ISO 30024:2009

Foderstoffer – Bestemmelse af
fytaseaktivitet
Animal feeding stuffs – Determination of
phytase activity
 DS/EN ISO 30024
København
DS projekt: M229748
ICS: 65.120

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COPYRIGHT Danish Standards. NOT FOR COMMERCIAL USE OR REPRODUCTION. DS/EN ISO 30024:2009

IDT med: ISO 30024:2009.

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 EUROPEAN STANDARD EN ISO 30024
NORME EUROPÉENNE
EUROPÄISCHE NORM July 2009

ICS 65.120
COPYRIGHT Danish Standards. NOT FOR COMMERCIAL USE OR REPRODUCTION. DS/EN ISO 30024:2009

English Version

Animal feeding stuffs - Determination of phytase activity (ISO

30024:2009)

Aliments des animaux - Détermination de l'activité Futtermittel - Bestimmung der Phytaseaktivität (ISO
phytasique (ISO 30024:2009) 30024:2009)

This European Standard was approved by CEN on 17 June 2009.

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 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 Management Centre has the same status as the
official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.

EUROPEAN COMMITTEE FOR STANDARDIZATION

COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels

© 2009 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 30024:2009: E
worldwide for CEN national Members.
 EN ISO 30024:2009 (E)

Contents Page

Foreword ..............................................................................................................................................................3
COPYRIGHT Danish Standards. NOT FOR COMMERCIAL USE OR REPRODUCTION. DS/EN ISO 30024:2009

2
 EN ISO 30024:2009 (E)

Foreword
This document (EN ISO 30024:2009) has been prepared by Technical Committee CEN/TC 327 “Animal
COPYRIGHT Danish Standards. NOT FOR COMMERCIAL USE OR REPRODUCTION. DS/EN ISO 30024:2009

feeding stuffs - Methods of sampling and analysis”, the secretariat of which is held by NEN, in collaboration
with Technical Committee ISO/TC 34 "Food products".

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 January 2010, and conflicting national standards shall be withdrawn at
the latest by January 2010.

Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] 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, Cyprus, Czech
Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland and the United Kingdom.

3
COPYRIGHT Danish Standards. NOT FOR COMMERCIAL USE OR REPRODUCTION. DS/EN ISO 30024:2009
 INTERNATIONAL ISO
STANDARD 30024

First edition
2009-07-15
COPYRIGHT Danish Standards. NOT FOR COMMERCIAL USE OR REPRODUCTION. DS/EN ISO 30024:2009

Animal feeding stuffs — Determination of

phytase activity
Aliments des animaux — Détermination de l'activité phytasique

Reference number
ISO 30024:2009(E)

© ISO 2009
 ISO 30024:2009(E)

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ii © ISO 2009 – All rights reserved

 ISO 30024:2009(E)

Contents Page
COPYRIGHT Danish Standards. NOT FOR COMMERCIAL USE OR REPRODUCTION. DS/EN ISO 30024:2009

Foreword............................................................................................................................................................ iv
Introduction ........................................................................................................................................................ v
1 Scope ..................................................................................................................................................... 1
2 Terms and definitions........................................................................................................................... 1
3 Principle ................................................................................................................................................. 1
4 Reagents ................................................................................................................................................ 2
5 Apparatus .............................................................................................................................................. 3
6 Sampling ................................................................................................................................................ 4
7 Sample preparation .............................................................................................................................. 4
8 Procedure .............................................................................................................................................. 4
8.1 Blank solutions ..................................................................................................................................... 4
8.2 Standards .............................................................................................................................................. 4
8.3 Standard curve...................................................................................................................................... 5
8.4 Phytase level control ............................................................................................................................ 5
8.5 Feed test portions................................................................................................................................. 6
9 Calculations........................................................................................................................................... 7
9.1 Formulation standard curve ................................................................................................................ 7
9.2 Calculation phytase activity ................................................................................................................ 8
9.3 Correction for phytic acid purity and water content ......................................................................... 9
9.4 Interference with blank values ............................................................................................................ 9
10 Precision ................................................................................................................................................ 9
10.1 Limit of detection and limit of quantification ..................................................................................... 9
10.2 Interlaboratory test ............................................................................................................................. 10
10.3 Repeatability........................................................................................................................................ 10
10.4 Reproducibility.................................................................................................................................... 10
11 Test report ........................................................................................................................................... 10
Annex A (informative) Interlaboratory study results .................................................................................... 11
Bibliography ..................................................................................................................................................... 12

© ISO 2009 – All rights reserved iii

 ISO 30024:2009(E)

Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
COPYRIGHT Danish Standards. NOT FOR COMMERCIAL USE OR REPRODUCTION. DS/EN ISO 30024:2009

(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 30024 was prepared by the European Committee for Standardization (CEN) Technical Committee
CEN/TC 327, Animal feeding stuffs — Methods of sampling and analysis, in collaboration with Technical
Committee ISO/TC 34, Food products, Subcommittee SC 10, Animal feeding stuffs, in accordance with the
Agreement on technical cooperation between ISO and CEN (Vienna Agreement).

iv © ISO 2009 – All rights reserved

 ISO 30024:2009(E)

Introduction
This International Standard has been developed to quantify phytase products in feed samples to enable the
COPYRIGHT Danish Standards. NOT FOR COMMERCIAL USE OR REPRODUCTION. DS/EN ISO 30024:2009

European Commission to control the phytase content of animal feed products. However, the method cannot
be used to evaluate the in vivo efficacy of the phytase products.

© ISO 2009 – All rights reserved v

COPYRIGHT Danish Standards. NOT FOR COMMERCIAL USE OR REPRODUCTION. DS/EN ISO 30024:2009
 INTERNATIONAL STANDARD ISO 30024:2009(E)

Animal feeding stuffs — Determination of phytase activity

COPYRIGHT Danish Standards. NOT FOR COMMERCIAL USE OR REPRODUCTION. DS/EN ISO 30024:2009

1 Scope
This International Standard specifies the determination of phytase activity in feed samples.

The method does not distinguish between phytase added as a feed additive and endogenous phytase already
present in the feed materials.

The method cannot be used to evaluate or compare the in vivo efficacy of the phytase product. It is not a
predictive method of the in vivo efficacy of phytases present on the market as they can develop different
in vivo efficacy per unit of activity.

The method is suitable and validated exclusively for the determination of phytase activity and exclusively in
complete feeds.

NOTE The harmonized method was developed on the basis of the presently existing phytase products
[E1600 (EC 3.1.3.8, 3-phytase), E1614 (EC 3.1.3.26, 4-phytase), and E1640 (EC 3.1.3.26, 4-phytase)]. Therefore, it might
not necessarily be suitable as such for phytase products that are developed in the future. The harmonized method is thus
a tool which is useful only to evaluate the total phytase activity in feed samples.

2 Terms and definitions

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

2.1
phytase unit
U
amount of enzyme that releases 1 µmol of inorganic phosphate from phytate per minute under the reaction
conditions specified in this International Standard

3 Principle
Phytase releases phosphate from the substrate myo-inositol hexakisphosphate (phytate). The released
inorganic phosphate is determined by forming a yellow complex with an acidic molybdate/vanadate reagent.
The optical density (OD) of the yellow complex is measured at a wavelength of 415 nm and the inorganic
phosphate released is quantified from a phosphate standard calibration curve.

© ISO 2009 – All rights reserved 1