SIST EN ISO 30024:2010

Animal feeding stuffs - Determination of phytase activity (ISO 30024:2009)

This European Standard describes the determination of phytase activity in complete feedingstuff 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.

Futtermittel - Bestimmung der Phytaseaktivität (ISO 30024:2009)

Diese Internationale Norm legt die Bestimmung der Phytaseaktivität in Futtermittelproben fest.
Das Verfahren unterscheidet nicht, ob die Phytase als Futtermittelzusatz oder als endogene Phytase vorliegt, die bereits in den Futtermittel Ausgangsstoffen vorhanden war.
Das Verfahren kann nicht zur Bewertung oder zum Vergleich der in vivo Wirksamkeit von Phytase Produkten angewendet werden. Es dient auch nicht zur Vorhersage der in vivo Wirksamkeit von auf dem Markt angebotenen Phytasen, da sich diese in vivo Wirksamkeit in Abhängigkeit von der Einheit für die Aktivität unterschiedlich entwickeln kann.
Das Verfahren ist ausschließlich zur Bestimmung der Phytaseaktivität und nur für Alleinfuttermittel geeignet und validiert.
ANMERKUNG   Das harmonisierte Verfahren wurde für die gegenwärtig existierenden Phytase Produkte [E1600 (EC 3.1.3.8, 3-Phytase), E1614 (EC 3.1.3.26, 4-Phytase) und E1640 (EC 3.1.3.8, 3-Phytase)] entwickelt. Das Verfahren kann daher z. B. für zukünftig entwickelte Phytase Produkte ungeeignet sein. Dieses harmonisierte Verfahren ist somit lediglich ein Hilfsmittel zur Bewertung der gesamten Phytaseaktivität in Futtermittelproben.

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

L'ISO 30024:2009 spécifie la détermination de l'activité phytasique dans des échantillons d'aliments pour animaux.
Le méthode ne fait pas la distinction entre la phytase ajoutée comme additif aux aliments pour animaux et la phytase endogène déjà présente dans les matières premières entrant dans la composition des aliments.
La méthode ne peut pas être utilisée pour évaluer ou comparer l'efficacité in vivo des phytases. Il ne s'agit pas d'une méthode prédictive de l'efficacité in vivo des phytases disponibles sur le marché car celles-ci peuvent développer une efficacité in vivo différente par unité d'activité.
La méthode est adaptée et validée exclusivement pour la détermination de l'activité phytasique dans les aliments complets pour animaux.

Krma - Določevanje aktivnosti fitaz (ISO 30024:2009)

Ta mednarodni standard opredeljuje aktivnost fitaz v vzorcu krme. Metoda je primerna in potrjena samo za določevanje aktivnosti fitaz in samo za celovita krmila.

General Information

• Status: Published
• Publication Date: 19-Nov-2009
• ICS: 65.120 - Animal feeding stuffs
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 03-Nov-2009
• Due Date: 08-Jan-2010
• Completion Date: 20-Nov-2009
• Directive: 70/373/EC - Council Directive 70/373/EEC of 20 July 1970 on the introduction of Community methods of sampling and analysis for the official control of feeding-stuffs
• Mandate: M/382 - Mandate for standardisation addressed to CEN in the field of Methods of analysis for animal Feedingstuffs
• Ref Project: EN ISO 30024:2009 - Animal feeding stuffs - Determination of phytase activity (ISO 30024:2009)

Relations

• Revised: oSIST prEN ISO 30024:2023 - Animal feeding stuffs - Determination of phytase activity (ISO/DIS 30024:2023)
• Effective Date: 19-Jan-2023

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 30024:2010
01-januar-2010
.UPD'RORþHYDQMHDNWLYQRVWLILWD],62
Animal feeding stuffs - Determination of phytase activity (ISO 30024:2009)
Futtermittel - Bestimmung der Phytaseaktivität (ISO 30024:2009)
Aliments des animaux - Détermination de l'activité phytasique (ISO 30024:2009)
Ta slovenski standard je istoveten z: EN ISO 30024:2009
ICS:
65.120 Krmila Animal feeding stuffs
SIST EN ISO 30024:2010 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 30024:2010

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SIST EN ISO 30024:2010
EUROPEAN STANDARD
EN ISO 30024
NORME EUROPÉENNE
EUROPÄISCHE NORM
July 2009
ICS 65.120
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.

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SIST EN ISO 30024:2010
EN ISO 30024:2009 (E)
Contents Page
Foreword .3

2

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SIST EN ISO 30024:2010
EN ISO 30024:2009 (E)
Foreword
This document (EN ISO 30024:2009) has been prepared by Technical Committee CEN/TC 327 “Animal
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

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SIST EN ISO 30024:2010

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SIST EN ISO 30024:2010

INTERNATIONAL ISO
STANDARD 30024
First edition
2009-07-15


Animal feeding stuffs — Determination of
phytase activity
Aliments des animaux — Détermination de l'activité phytasique





Reference number
ISO 30024:2009(E)
©
ISO 2009

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SIST EN ISO 30024:2010
ISO 30024:2009(E)
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©  ISO 2009
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or
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Published in Switzerland

ii © ISO 2009 – All rights reserved

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SIST EN ISO 30024:2010
ISO 30024:2009(E)
Contents Page
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

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SIST EN ISO 30024:2010
ISO 30024:2009(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.
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

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SIST EN ISO 30024:2010
ISO 30024:2009(E)
Introduction
This International Standard has been developed to quantify phytase products in feed samples to enable the
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

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SIST EN ISO 30024:2010

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SIST EN ISO 30024:2010
INTERNATIONAL STANDARD ISO 30024:2009(E)

Animal feeding stuffs — Determination of phytase activity
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

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SIST EN ISO 30024:2010
ISO 30024:2009(E)
4 Reagents
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and distilled or
demineralized water or water of equivalent purity.
WARNING — This method requires the handling of hazardous substances. Observe local regulations
for potentially hazardous chemicals to minimize risks to organizational, technical, and personal safety.
4.1 Ammonia solution, 25 % mass fraction; NH .
3
4.2 Ammonium heptamolybdate tetrahydrate, (NH ) Mo O ⋅4H O.
4 6 7 24 2
4.3 Ammonium monovanadate, NH VO .
4 3
4.4 Hydrochloric acid, 25 % mass fraction; HCl.
4.5 Nitric acid, 65 % mass fraction; HNO .
3
4.6 Potassium dihydrogenphosphate, KH PO .
2 4
1)
4.7 Phytate, phytic acid, dodecasodium salt, C H Na O P⋅xH O, from rice, Sigma® P0109 .
6 6 12 24 6 2
4.8 Sodium acetate trihydrate, CH COONa⋅3H O.
3 2
2)
4.9 Polysorbate 20 .
4.10 Dilute nitric acid. Dilute 1 volume nitric acid 65 % mass fraction (4.5) with 2 volumes water. Store at
room temperature. The maximum storage time is indefinite.
4.11 Ammonium heptamolybdate reagent. Dissolve 100,0 g ammonium heptamolybdate tetrahydrate (4.2)
in approximately 800 ml water. Add 10 ml 25 % mass fraction ammonia solution (4.1) and make up with water
to 1 000 ml. Store at room temperature in the dark. The maximum storage time is 2 months.
4.12 Ammonium vanadate reagent. Dissolve completely 2,35 g of ammonium monovanadate (4.3) in
approximately 400 ml water (50 °C to 60 °C). Add 20 ml dilute nitric acid (4.10) and make up with water to
1 000 ml. Store at room temperature in the dark. The maximum storage time is 2 months.
4.13 Molybdate/vanadate STOP reagent. Mix 1 volume ammonium vanadate reagent (4.12) with 1 volume
ammonium heptamolybdate reagent (4.11) and add 2 volumes dilute nitric acid (4.10). Mix and store at room
temperature. The maximum storage time is 1 day.
4.14 Polysorbate 20, 10 % mass fraction. Dissolve 10,0 g of polysorbate 20 (4.9) with water and make up
to 100 ml. Store at room temperature. The maximum storage time is 6 months.
4.15 Acetate buffer, pH 5,5; 0,25 mol/l. Dissolve 34,0 g of sodium acetate trihydrate (4.8) in approximately
900 ml water. Adjust the pH with 25 % mass fraction hydrochloric acid (4.4) to 5,50 ± 0,02 and make up to
1 000 ml with water. Store at room temperature. The maximum storage time is 2 weeks.
4.16 Acetate buffer with 0,01 % mass fraction polysorbate 20, pH 5,5; 0,25 mol/l. Dissolve 34,0 g of
sodium acetate trihydrate (4.8) in approximately 900 ml water. Adjust the pH with 25 % mass fraction
hydrochloric acid (4.4) to 5,50 ± 0,02. Add 1 ml 10 % mass fraction polysorbate 20 (4.14) and make up to
1 000 ml with water. Store at room temperature. The maximum storage time is 2 weeks.

1) Example of a suitable product available commercially. This information is given for the convenience of users of this
International Standard and does not constitute an endorsement by ISO of this product.
2) Tween 20 is an example of a suitable product available commercially. This information is given for the convenience of
users of this International Standard and does not constitute an endorsement by ISO of this product.
2 © ISO 2009 – All rights reserved

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SIST EN ISO 30024:2010
ISO 30024:2009(E)
4.17 Acetate buffer with 0,01 % mass fraction polysorbate 20, pH 5,5; 0,50 mol/l. Dissolve 68,0 g of
sodium acetate trihydrate (4.8) in approximately 900 ml water. Adjust the pH with 25 % mass fraction
hydrochloric acid (4.4) to 5,50 ± 0,02. Add 1 ml 10 % mass fraction polysorbate 20 (4.14) and make up to
1 000 ml with water. Store at room temperature. The maximum storage time is 2 weeks.
4.18 Phytate substrate solution, 7,5 mmol/l (5 mmol/l end-concentration in the reaction). Dissolve 2,00 g of
dodecasodium phytate (4.7) whose inorganic phosphorus content is u 0,1 % mass fraction (see 9.3) in
approximately 200 ml acetate buffer (4.15). Adjust the pH with 25 % mass fraction hydrochloric acid (4.4) to
5,50 ± 0,02 and make up with acetate buffer (4.15) to 250 ml. The maximum storage time is 2 weeks at 4 °C.
4.19 Phosphate stock standard solution, 50 mmol/l. Dry approximately 10 g of potassium
dihydro
...