EN ISO 30024:2024

SLOVENSKI STANDARD
01-julij-2024
Krma - Določanje aktivnosti fitaze (ISO 30024:2024)
Animal feeding stuffs - Determination of phytase activity (ISO 30024:2024)
Futtermittel - Bestimmung der Phytaseaktivität (ISO 30024:2024)
Alimentation animale - Détermination de l'activité phytasique (ISO 30024:2024)
Ta slovenski standard je istoveten z: EN ISO 30024:2024
ICS:
65.120 Krmila Animal feeding stuffs
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 30024
EUROPEAN STANDARD
NORME EUROPÉENNE
May 2024
EUROPÄISCHE NORM
ICS 65.120 Supersedes EN ISO 30024:2009
English Version
Animal feeding stuffs - Determination of phytase activity
(ISO 30024:2024)
Alimentation animale - Détermination de l'activité Futtermittel - Bestimmung der Phytaseaktivität (ISO
phytasique (ISO 30024:2024) 30024:2024)
This European Standard was approved by CEN on 21 April 2024.

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, Türkiye 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
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 30024:2024 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 30024:2024) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with Technical Committee CEN/TC 327 “Animal feeding stuffs - 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 November 2024, and conflicting national standards
shall be withdrawn at the latest by November 2024.
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 30024:2009.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN website.
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, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 30024:2024 has been approved by CEN as EN ISO 30024:2024 without any modification.

International
Standard
ISO 30024
Second edition
Animal feeding stuffs —
2024-04
Determination of phytase activity
Alimentation animale — Détermination de l'activité phytasique
Reference number
ISO 30024:2024(en) © ISO 2024
ISO 30024:2024(en)
© ISO 2024
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 30024:2024(en)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Reagents . 3
6 Apparatus . 5
7 Sampling and sample preparation . 5
8 Sample extractions . 6
8.1 For compound feeds (excluding mineral feeds) .6
8.2 For mineral feeds and premixtures .6
8.3 For feed additives .6
9 Procedure . 7
9.1 General .7
9.2 Blank solution .7
9.3 Standards .7
9.3.1 Phosphate standard solution.7
9.3.2 Phytase level control.7
9.4 Standard curve .7
9.5 Phytase level control .8
9.6 For compound feed (excluding mineral feeds) .9
9.7 For mineral feeds and premixtures .10
9.8 For feed additives .10
10 Calculations .11
10.1 Formulation of standard curve .11
10.2 Calculation of phytase activity. 12
10.3 Correction for phytic acid purity and water content . 13
10.4 Interference with blank values .14
11 Precision . 14
11.1 Limit of detection and limit of quantification .14
11.2 Interlaboratory tests .14
11.3 Repeatability .14
11.4 Reproducibility . 15
12 Test report .15
Annex A (informative) Interlaboratory study results for compound feeds (excluding mineral
feeds) . 16
Annex B (informative) Interlaboratory study results for mineral feeds and premixtures . 19
Annex C (informative) Interlaboratory study results for feed additives .20
Bibliography .22

iii
ISO 30024:2024(en)
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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
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 10,
Animal feeding stuffs, in collaboration with the European Committee for Standardization (CEN) Technical
Committee CEN/TC 327, Animal feeding stuffs - Methods of sampling and analysis, in accordance with the
Agreement on technical cooperation between ISO and CEN (Vienna Agreement).
This second edition cancels and replaces the first edition (ISO 30024:2009), which has been technically
revised.
The main changes are as follows:
— the scope has been extended to include complementary compound feeds, mineral feeds, premixtures and
feed additives;
— phytic acid (phytate substrate) specifications have been added.
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 30024:2024(en)
Introduction
This document quantifies phytase products in feeding stuff samples 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.

v
International Standard ISO 30024:2024(en)
Animal feeding stuffs — Determination of phytase activity
1 Scope
This document specifies the determination of phytase activity in feeding stuff samples, including feed raw
materials from plant origin, compound feeds (complete, complementary, mineral feeds), premixtures and
feed additives.
The method is applicable to, and is collaboratively validated for, the determination of phytase activity in
complete feed, complementary feed including mineral feed, premixtures and feed additives.
The method does not distinguish between phytase added as a feed additive and endogenous phytase already
present in the feed materials. Therefore, the method is also applicable for feed materials from plant origin.
The method does not apply to evaluating or comparing 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.
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 terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
phytase unit
U
amount of enzyme that releases 1 µmol of inorganic phosphate from phytate per minute in acetate buffer at
pH 5,5 and 37 °C
Note 1 to entry: This is determined by the reaction conditions specified in this document.
3.2
premixture
premix
uniform mixture of one or more micro-ingredients/feed additives (3.4) with a diluent and/or carrier, and
that is not intended for direct feeding to animals
Note 1 to entry: Premixtures are used to facilitate the uniform dispersion of the micro-ingredients/additives in a
larger mix.
[SOURCE: ISO 20588:2019, 3.2.39]

ISO 30024:2024(en)
3.3
mineral feed
mineral mix
mineral supplement
feed that mainly consists of mineral elements, which is as an entire mix free-flowing
Note 1 to entry: In European legislation, a mineral feed contains at least 40 % of crude ash.
Note 2 to entry: to entry : Mineral feed is a form of compound feed (3.5) and of complementary feed (see Note 1 to entry
of 3.5).
[SOURCE: ISO 20588:2019, 3.2.37, modified — "mineral feed" replaced "mineral mix" as the preferred term.
Notes to entry added.]
3.4
feed additive
substance intentionally added to feed and/or water, not consumed as feed by itself, whether or not it has a
nutritional value, that affects the characteristics of feed including organoleptic properties, animal products,
animal production or performance or welfare, or the environment
Note 1 to entry: Microorganisms, enzymes, acidity regulators, trace elements, vitamins and other products fall within
the scope of this definition, depending on the purpose of use and the method of administration.
Note 2 to entry: Coccidiostats and histomonostats are a category of feed additives.
Note 3 to entry: Feed additive does not include feed materials (3.6) and premixtures (3.2).
[SOURCE: ISO 20588:2019, 3.2.18]
3.5
compound feed
formula feed
feed mixture
mixture of at least two feed materials (3.6), whether or not containing feed additives (3.4), for oral animal
feeding in the form of a complementary feed or a complete feed
Note 1 to entry: Complementary feed is a form of compound feed as defined in ISO 20588:2019, 3.2.9.
Note 2 to entry: Complete feed is a form of compound feed as defined in ISO 20588:2019, 3.2.10.
[SOURCE: ISO 20588:2019, 3.2.11]
3.6
feed materials
products of vegetable or animal origin, whether or not containing feed additives (3.4), that are intended for
use in oral animal feeding to meet animals’ nutritional needs
Note 1 to entry: Feed materials can be in their natural state, fresh or preserved, or products derived from industrial
processing, either organic or inorganic substances.
Note 2 to entry: Feed materials may be fed to animals either directly as such, or after processing, or in the preparation
of compound feed (3.5), or as carrier of premixtures (3.2).
[SOURCE: ISO 20588:2019, 3.2.23, modified — "and products derived from industrial processing, either
organic or inorganic substances" moved from the definition to Note 1 to entry.]
4 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 30024:2024(en)
5 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. It is the responsibility of
the user of this document to establish appropriate safety and health practices and determine the
applicability of regulatory limitations prior to use.
5.1 Ammonia solution, 25 % mass fraction; NH .
5.2 Ammonium heptamolybdate tetrahydrate, (NH ) Mo O ·4H O.
4 6 7 24 2
5.3 Ammonium monovanadate, NH VO .
4 3
5.4 Hydrochloric acid, 25 % mass fraction; HCl.
5.5 Nitric acid, 65 % mass fraction; HNO .
5.6 Potassium dihydrogenphosphate, KH PO .
2 4
5.7 Phytate (anionic form of phytic acid), phytic acid:
— all forms of phytate or phytic acid are may be used, e.g. phytic acid (PA), phytic acid dodecasodium salt
(Na-PA), phytic acid dodecapotassium salt (K-PA), phytic acid hexamagnesium salt n-hydrate (Mg-PA);
— ≤ 0,1 % mass fraction of inorganic phosphorus;
— assay ≥ 90 % phosphorus (P) basis (dry basis);
— the substrate should have a percentage of IP6 (= hexaphosphate inositol containing six phosphate groups)
of more than 95.
Information about IP6 ratio or percentage should be available from the supplier upon request.
As phytic acid salt hydrates are supplied with different contents of crystallization water, ensure that the
crystallization water is in the stoichiometric range of 10 mol to 13 mol. In cases of deviation, see 10.3.
For control of the phytate, the blank OD from the standard curve (see 9.4) shall be lower than 0,2. A higher
OD value indicates phosphate or phytase contamination of used reagents.
5.8 Sodium acetate trihydrate, CH COONa·3H O.
3 2
1)
5.9 Polysorbate 20 .
5.10 Diluted nitric acid.
Dilute one volume of nitric acid 65 % mass fraction (5.5) with two volumes water. Store at room temperature.
The maximum storage time is two years.
5.11 Ammonium heptamolybdate reagent.
Dissolve 100,0 g of ammonium heptamolybdate tetrahydrate (5.2) in approximately 800 ml water (hot water
at 50 °C to 60 °C may be used to facilitate salt dissolution). Add 10 ml 25 % mass fraction ammonia solution
1) Tween® 20 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 of this product.

ISO 30024:2024(en)
(5.1) and make up with water to 1 000 ml. Store at room temperature in the dark. The maximum storage
time is two months.
5.12 Ammonium vanadate reagent.
Dissolve completely 2,35 g of ammonium monovanadate (5.3) in approximately 400 ml of water (hot water
at 50 °C to 60 °C may be used to facilitate salt dissolution). Add 20 ml diluted nitric acid (5.10) and make up
with water to 1 000 ml. Store at room temperature in the dark. The maximum storage time is two months.
5.13 Molybdate/vanadate STOP reagent.
Mix one volume of ammonium vanadate reagent (5.12) with one volume of ammonium heptamolybdate
reagent (5.11) and add two volumes diluted nitric acid (5.10). Mix and store at room temperature. The
maximum storage time is one day.
5.14 Polysorbate 20, 10 % mass fraction.
Dissolve 10,0 g of polysorbate 20 (5.9) with water and make up to 100 ml. Store at room temperature. The
maximum storage time is six months.
5.15 Acetate buffer, pH 5,5; 0,25 mol/l.
Dissolve 34,0 g of sodium acetate trihydrate (5.8) in approximately 900 ml water. Adjust the pH with 25 %
mass fraction hydrochloric acid (5.4) to 5,50 ± 0,02 and make up to 1 000 ml with water. Store at room
temperature. The maximum storage time is two weeks.
5.16 Acetate buffer with 0,01 % mass fraction polysorbate 20, pH 5,5; 0,25 mol/l.
Dissolve 34,0 g of sodium acetate trihydrate (5.8) in approximately 900 ml water. Adjust the pH with 25 %
mass fraction hydrochloric acid (5.4) to 5,50 ± 0,02. Add 1 ml 10 % mass fraction polysorbate 20 (5.14) and
make up to 1 000 ml with water. Store at room temperature. The maximum storage time is two weeks.
5.17 Acetate buffer with 0,01 % mass fraction polysorbate 20, pH 5,5; 0,50 mol/l.
Dissolve 68,0 g of sodium acetate trihydrate (5.8) in approximately 900 ml water. Adjust the pH with 25 %
mass fraction hydrochloric acid (5.4) to 5,50 ± 0,02. Add 1 ml 10 % mass fraction polysorbate 20 (5.14) and
make up to 1 000 ml with water. Store at room temperature. The maximum storage time is two weeks.
5.18 Phytate substrate solution, 7,5 mmol/l (3 mmol/l end-concentration in the reaction).
Dissolve 2,00 g of phytate (5.7) in approximately 200 ml acetate buffer (5.15). Depending of the purity and/
or the water content of phytic acid (see 10.3), if necessary, adjust slightly the 2,00 g mass. Adjust the pH to
5,50 ± 0,02, e.g. with 25 % mass fraction hydrochloric acid (5.4), and make up with acetate buffer (5.15) to
250 ml. The maximum storage time is two weeks at 4 °C.
5.19 Phosphate stock standard solution, 50 mmol/l.
Dry approximately 10 g of potassium dihydrogenphosphate (5.6) at 105 °C for 2 h and store it in a desiccator.
Weigh approximately 682 mg of dried potassium dihydrogenphosphate, transfer it quantitatively to a
100 ml volumetric flask and make up to 100 ml with 0,25 mol/l acetate buffer with 0,01 % mass fraction
polysorbate 20 (5.16). Calculate the exact concentration of the phosphate stock standard solution. Store at
4 °C. The maximum storage time is two weeks.
5.20 Phytase standard, as a quality control sample, with phytase activity not less than 3 500 U/g.

ISO 30024:2024(en)
5.21 Phytase stock standard solution.
Weigh 100,0 mg to 300,0 mg of a phytase standard (5.20), transfer it quantitatively to a 100 ml volumetric
flask and dissolve it in approximately 80 ml 0,25 mol/l acetate buffer with 0,01 % mass fraction polysorbate
20 (5.16). Stir it for 15 min to 45 min. After removing the magnetic stirrer, fill up to the mark with 0,25 mol/l
acetate buffer with 0,01 % mass fraction polysorbate 20 (5.16). The maximum storage time is one day at
room temperature, or if aliquoted, the maximum storage time at –18 °C is six months.
5.22 Maize meal
Unprocessed maize grain/broken maize is ground smaller than 1 mm or 2 mm and used as a dilution matrix
for the analysis of mineral feeds and premixtures (see 8.2). Conventional maize flour can also be used.
The phytase activity in maize meal/flour is in itself negligible, but to exclude it completely and to prevent
a significant influence by multiplying the dilutions in the result calculation, heating the meal overnight at
130 °C is recommended.
Equivalent matrix to maize, such as soy protein concentrate, without phytase activity, is possible but should
be validated in-house.
6 Apparatus
Usual laboratory apparatus, in particular, the following shall be used.
6.1 Water bath, thermostatically controlled at 37 °C ± 0,2 °C (with inserts for 2 ml tubes).
6.2 pH-meter, capable of being read to at least two decimal places.
6.3 Magnetic stirrers (≥ 20 W power).
6.4 Egg-shaped stirring bars (40 mm × 20 mm) or cylindrical stirring bars (60 mm × 10 mm) or
equivalent.
6.5 Analytical balance, capable of being read to at least 0,1 mg.
6.6 Balance, capable of being read to at least 0,01 g.
6.7 Vortex mixer.
6.8 Centrifuge, for microcentrifuge tubes (6.12), capable of 11 000g to 20 000g.
6.9 Electronic dispenser or mechanical dispenser.
6.10 Pipettes (electronic and manual), in the range 10 µl to 2 000 µl.
6.11 Spectrophotometer, double beam or microplate reader.
6.12 Microcentrifuge tubes, capacity 2 ml.
7 Sampling and sample preparation
A representative sample should have been sent to the laboratory. It should not have been damaged or
changed during transport or storage.
Sampling is not part of the method specified in this document. A recommended sampling procedure is given
in ISO 6497.
ISO 30024:2024(en)
Sample preparation is not part of the method specified in this document. A recommended sample procedure
is given in ISO 6498 with the following clarifications:
— grinding of compound feed and feed raw materials < 1 mm if the C is too high;
V,r
— grinding of mineral compound feed and premixture samples < 0,5 mm (see Reference [8]);
— coated feed additive products or products with a larger particle size can be pestled (see Reference [9]).
8 Sample extractions
8.1 For compound feeds (excluding mineral feeds)
Perform two weighings for each sample.
Weigh two portions of pellets or mash compound feed, of about 50 g each, into containers (500 ml conical
flasks or 800 ml beakers or equivalent). Add 500 ml water and 0,5 ml of 10 % mass fraction polysorbate 20
(5.14) to the compound feed and mix vigorously for 45 min on a magnetic stirrer (6.3) with stirring bars (6.4).
Inhomogeneity, among other causes, in the sample can lead to high coefficients of variation of repeatability
(C , see 11.3). For compound feed samples showing high C , such inhomogeneity can derive from
V,r V,r
inhomogeneous particle size distribution in products or inhomogeneous compound feed preparation. If
compound feed samples show high C , grind the compound feed samples as described in ISO 6498 or by
V,r
2)
using an Ultra Centrifugal Mill with a sieve of nominal size of openings 1 mm. Grind 150 g of compound
feed. Repeat the extraction with the ground samples as described in this clause.
8.2 For mineral feeds and premixtures
For phytase activities higher than 10 000 U/kg, in mineral feeds and premixtures, but lower than
2 000 000 U/kg:
— weigh 0,5 g ± 0,001 g of ground sample (which should be ground smaller than 0,5 mm) and 50 g ± 0,5 g of
maize meal (5.22) in duplicate into containers (500 ml conical flasks or 800 ml beakers or equivalent);
— add 500 ml acetate buffer (5.16) in each container and mix vigorously for 1 h on a magnetic stirrer (6.3).
NOTE Ratio of mixture: In practice, mineral feed is added to complete feed at a rate of 1 % to 4 %, whereas
premixtures are added at a rate of 0,2 % to 1 %. The sample mass of 0,5 g and 50 g maize correspond to a mixture rate
of 1 %. This mixture rate is used as a convention for the comparability of the results of different laboratories, even if
the declared mixture rate of mineral feed/premixture is lower or higher.
8.3 For feed additives
For phytase activities higher than 2 000 U/g (2 000 000 U/kg), in solid or liquid feed additives:
— for liquid feed additives samples:
— weigh the samples (0,5 g or 1 g ± 0,001 g according to Table 6) in duplicate into a 100 ml volumetric flask;
— add approximately 80 ml acetate buffer (5.16) into each flask and mix for 30 min on a magnetic
stirrer (6.3);
— remove the magnetic stirrer and fill up to 100,0 ml with acetate buffer (5.16);
— for solid feed additives samples:
— weigh the samples (0,5 g or 1 g ± 0,001 g according to Table 6) in duplicate into a 250 ml beaker;
2) Ultra Centrifugal Mill 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 of this product.

ISO 30024:2024(en)
— add accurately 100,0 ml acetate buffer (5.16) in each beaker and mix for 30 min on a magnetic
stirrer (6.3).
NOTE If inhomogeneities or poor repeatabilities occur, an increase of sample mass and adequate adaptation of
the extraction volume can be made.
9 Procedure
9.1 General
The volumes of reaction described in this clause can be multiplied if a photometer with a 1 cm cuvette or
flow cell is used. For feeding stuff samples, a correspondingly larger volume of suspension (e.g. 10 ml) shall
be centrifuged for 15 min at near 3 000g or 3 500g using a conventional laboratory centrifuge. Alternatively,
larger sample volumes can also be filtered through folded filter. The first 5 ml should be then rejected. The
supernatant, filtrate, phosphate standard solutions or phytase level control solution is then used with larger
volumes of acetate buffer, phytate substrate solution and stop reagent.
For sample extracts and phytase level control, if the net measured absorption (absorption minus blank) is
not within the calibration range (absorption/phosphate concentration) the determination shall be repeated
with a suitable dilution.
Products containing phytase feed additives which were not included in the validation studies in the annexes
shall be checked, especially on their linearity of response (release of phosphorus),
9.2 Blank solution
Inorganic phosphate in the sample contributes to colour formation. Therefore, blanks are included for each
sample. For calculation of phytase activity, subtract blank values from the test values.
9.3 Standards
9.3.1 Phosphate standard solution
The phosphate stock standard solution (5.19) shall be diluted with 0,25 mol/l acetate buffer containing
0,01 % mass fraction polysorbate 20 (5.16) according to Table 1.
Table 1 — Dilution steps to obtain standard colorimetric solutions for the phosphate curve
Volume of 0,25 mol/l acetate
Concentration
Standard Volume of phosphate stock Dilution
buffer with 0,01 % mass frac-
a
solution standard solution (5.19) factor
µmol/ml
tion polysorbate 20 (5.16)
A 1 1 2 25
B 1 3 4 12,5
C 1 7 8 6,25
D 1 15 16 3,125
a
Calculate the exact concentrations (5.19).
9.3.2 Phytase level control
For each incubation of samples, a phytase level control is included. A phytase stock standard solution
(5.21) with known activity is diluted to a final activity of 0,15 U/ml to 0,25 U/ml and the exact activity is
determined as specified in 9.5.
9.4 Standard curve
Perform three determinations for each phosphate dilution and two blanks, and average the results. The
procedure is specified in Table 2.

ISO 30024:2024(en)
The deviation between the maximum and minimum OD values observed for the three determinations of
each phosphate dilution, expressed as maximum value minus minimum value divided by maximum value,
does not exceed 15 %. If so, it is possible to discard one value out of the three. If the deviation between the
maximum and the minimum on the two remaining OD values is still higher than 15 %, then proceed to three
new determinations.
In order to keep phytase extracts in a linear range, the calibration curve should cover not more than 0,5 of OD.
For the phosphate standard solutions, pipette 360 µl 0,25 mol/l acetate buffer with 0,01 % mass fraction
polysorbate 20 (5.16) into a 2 ml tube (6.12). Add 40 µl phosphate standard solution (see Table 2).
For the phosphate standard blanks, pipette 400 µl 0,25 mol/l acetate buffer with 0,01 % mass fraction
polysorbate 20 (5.16) into a 2 ml tube (6.12).
In both cases, add 0,8 ml phytate substrate solution (5.18) and 0,8 ml STOP reagent (5.13). Mix the contents
of the tubes and maintain them for 10 min at room temperature. Centrifuge (6.8) the tubes and their contents
for 3 min at 11 000g to 20 000g and measure the OD of the clear supernatant at 415 nm, D(415).
Table 2 — Procedure for standard curve
Assay steps Standard colorimetric solutions Blank
Acetate buffer 0,25 mol/l with 0,01 %
360 µl 400 µl
mass fraction polysorbate 20 (5.16)
Phosphate standard solution (9.3.1) 40 µl 0 µl
Phytate substrate solution (5.18) 0,8 ml 0,8 ml
STOP reagent (5.13) 0,8 ml 0,8 ml
Mix Yes Yes
Time at room temperature 10 min 10 min
Centrifugation 3 min at 11 000g to 20 000g 3 min at 11 000g to 20 000g
Spectrophotometer (6.11) 415 nm (against water) 415 nm (against water)
9.5 Phytase level control
Perform three determinations and two blanks, and average the results. The procedure is specified in Table 3.
For the phytase level control determination solutions, pipette 360 µl 0,25 mol/l acetate buffer with 0,01 %
mass fraction polysorbate 20 (5.16) into a 2 ml tube (6.12). Add 40 µl dilute phytase level control solution
(see 9.3.2). Mix the sample. Pre-incubate the solutions for 5 min at 37 °C. Add 0,8 ml phytate substrate
solution (5.18) preheated to 37 °C. Incubate for exactly 30 min at 37 °C. After 30 min, add 0,8 ml STOP
reagent (5.13) and mix. Maintain the solutions for 10 min at room temperature and then centrifuge them for
3 min at 11 000g to 20 000g. Measure the D(415) of the clear supernatant.
For the phytase level control blank solutions, pipette 360 µl acetate buffer (5.15) into a 2 ml tube (6.12). Add
40 µl dilute phytase level control solution (see 9.3.2). The order of addition of solutions differs from that
used for the determinations. Pre-incubate blanks for 5 min at 37 °C. Then, as step 1, add STOP reagent (5.13).
As step 2, add phytate substrate solution (5.18) preheated to 37 °C. Then proceed with Table 3, column 3,
row 9, onwards.
ISO 30024:2024(en)
Table 3 — Procedure for level control
Assay steps Level control samples Blank
Acetate buffer 0,25 mol/l with 0,01 %
360 µl 360 µl
mass fraction polysorbate 20 (5.16)
Dilute phytase level control solution (see
40 µl 40 µl
9.3.2)
Mix Yes Yes
Pre-incubation at 37 °C 5 min 5 min
Phytate substrate solution (5.18) at 37 °C 0,8 ml 0,8 ml: Step 2
Mix No No
Incubation at 37 °C 30 min No
STOP reagent (5.13) 0,8 ml 0,8 ml: Step 1
Mix Yes Yes
Time at room temperature 10 min 10 min
Centrifugation 3 min at 11 000g to 20 000g 3 min at 11 000g to 20 000g
Spectrophotometer (6.11) 415 nm (against water) 415 nm (against water)
9.6 For compound feed (excluding mineral feeds)
Transfer 2 ml of the compound feed extract to a microcentrifuge tube (6.12) and centrifuge (6.8) for 3 min at
11 000g to 20 000g.
Perform three determinations for each extraction (see Clause 8) and two blanks, and average the results.
The procedure is specified in Table 4.
For the determinations, pipette 300 µl 0,25 mol/l acetate buffer with 0,01 % mass fraction polysorbate 20
(5.16) into a 2 ml tube (6.12). Add 100 µl feed extract (see 8.1) as the test portion. Mix the contents of the
tube. Pre-incubate for 5 min at 37 °C. Add 0,8 ml phytate substrate solution (5.18) preheated to 37 °C.
Incubate for exactly 30 min at 37 °C. After 30 min, add 0,8 ml STOP reagent (5.13) and mix. Maintain the
tube and its contents for 10 min at room temperature and then centrifuge for 3 min at 11 000g to 20 000g.
Measure the D(415) of the clear supernatant.
For the blanks, pipette 300 µl 0,25 mol/l acetate buffer with 0,01 % mass fraction polysorbate 20 (5.16)
into a 2 ml tube (6.12). Add 100 µl feed extract (see 8.1). The order of addition of solutions differs from that
used for the test portion. Pre-incubate blanks for 5 min at 37 °C. Then, as step 1, add STOP reagent (5.13). As
step 2, add phytate substrate solution (5.18) preheated to 37 °C. Then proceed with Table 4, column 3, row 9,
onwards.
Table 4 — Procedure for compound feed (excluding mineral feeds)
Assay steps Feed samples Blank
Acetate buffer 0,25 mol/l with 0,01 %
300 µl 300 µl
mass fraction polysorbate 20 (5.16)
Test portion 100 µl 100 µl
Mix Yes Yes
Pre-incubation at 37 °C 5 min 5 min
Phytate substrate solution (5.18) at 37 °C 0,8 ml 0,8 ml: Step 2
Mix No No
Incubation at 37 °C 30 min No
STOP reagent (5.13) 0,8 ml 0,8 ml: Step 1
Mix Yes Yes
ISO 30024:2024(en)
TTabablele 4 4 ((ccoonnttiinnueuedd))
Assay steps Feed samples Blank
Time at room temperature 10 min 10 min
Centrifugation 3 min at 11 000g to 20 000g 3 min at 11 000g to 20 000g
Spectrophotometer (6.11) 415 nm (against water) 415 nm (against water)
For test portions with ≤ 200 phytase U/kg feed, 200 µl sample extract and 200 µl 0,50 mol/l acetate buffer
with 0,01 % mass fraction polysorbate 20 (5.17) are taken for the assay (1:2 dilution).
Samples with > 2 000 phytase U/kg feed shall be appropriately diluted with 0,25 mol/l acetate buffer with
0,01 % mass fraction polysorbate 20 (5.16).
9.7 For mineral feeds and premixtures
For phytase activities higher than 10 000 U/kg, in mineral feeds and premixtures, but lower than
2 000 000 U/kg:
— centrifuge (6.8) 2 ml of the extract coming from 8.2 for 3 min at 11 000g to 20 000g.
— the supernatant is used following Table 5.
Table 5 — Volumes for mineral feeds and premixtures
Expected activity of Volume of extract Volume of buffer in Activity in the reac- Dilution factor D for
the sample for the reaction the reaction tion calculation
U/kg µl µl U
a
10 000 to 25 000 300 100 0,003 to 0,007 5 1,33
25 000 to 50 000 200 200 0,005 to 0,01 2
50 000 to 200 000 100 300 0,005 to 0,02 4
> 200 000 100 300 Suitable dilution
a
To increase the difference of absorption, due to high phosphate concentrations in the sample, in the range of 10 000 U/kg to
25 000 U/kg, the volume of extract can be increased to 400 μl (without an addition of buffer).
EXAMPLE Suitable dilution: samples with an activity of 400 000 U/kg are diluted 1:1 with extraction buffer
(= two-fold predilution) before 100 μl feed extract is mixed with 300 μl buffer for the reaction (= four-fold dilution).
Hence, the total dilution factor is 8.
Continue the procedure according to 9.6 with the selected volumes as given in Table 5.
9.8 For feed additives
For phytase activities higher than 2 000 U/g (2 000 000 U/kg), in solid or liquid feed additives:
— the clear extraction solution is diluted with acetate buffer (5.16) to a final activity of between 0,06 U/ml
to 0,16 U/ml;
— 100 µl f
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