EN ISO 11816-1:2013

SLOVENSKI STANDARD
01-januar-2014
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SIST EN ISO 11816-1:2006
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Milk and milk products - Determination of alkaline phosphatase activity - Part 1:
Fluorimetric method for milk and milk-based drinks (ISO 11816-1:2013)
Milch und Milchprodukte - Bestimmung der Aktivität der alkalischen Phosphatase - Teil 1:
Fluorimetrisches Verfahren für Milch und flüssige Milchprodukte (ISO 11816-1:2013)
Lait et produits laitiers - Détermination de l'activité de la phosphatase alcaline - Partie 1:
Méthode fluorimétrique pour le lait et les boissons à base de lait (ISO 11816-1:2013)
Ta slovenski standard je istoveten z: EN ISO 11816-1:2013
ICS:
67.100.10 0OHNRLQSUHGHODQLPOHþQL Milk and processed milk
SURL]YRGL products
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 11816-1
NORME EUROPÉENNE
EUROPÄISCHE NORM
November 2013
ICS 67.100.10 Supersedes EN ISO 11816-1:2006
English Version
Milk and milk products - Determination of alkaline phosphatase
activity - Part 1: Fluorimetric method for milk and milk-based
drinks (ISO 11816-1:2013)
Lait et produits laitiers - Détermination de l'activité de la Milch und Milcherzeugnisse - Bestimmung der Aktivität der
phosphatase alcaline - Partie 1: Méthode fluorimétrique alkalischen Phosphatase - Teil 1: Fluorimetrisches
pour le lait et les boissons à base de lait (ISO 11816- Verfahren für Milch und flüssige Milchprodukte (ISO 11816-
1:2013) 1:2013)
This European Standard was approved by CEN on 14 September 2013.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, 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: Avenue Marnix 17, B-1000 Brussels
© 2013 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 11816-1:2013 E
worldwide for CEN national Members.

Contents Page
Foreword .3
Foreword
This document (EN ISO 11816-1:2013) 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 May 2014, and conflicting national standards shall be withdrawn at the
latest by May 2014.
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.
This document supersedes EN ISO 11816-1:2006.
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, Former Yugoslav Republic of Macedonia, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Endorsement notice
The text of ISO 11816-1:2013 has been approved by CEN as EN ISO 11816-1:2013 without any modification.
INTERNATIONAL ISO
STANDARD 11816-1
IDF
155-1
Third edition
2013-11-01
Milk and milk products —
Determination of alkaline
phosphatase activity —
Part 1:
Fluorimetric method for milk and
milk-based drinks
Lait et produits laitiers — Détermination de l’activité de la
phosphatase alcaline —
Partie 1: Méthode fluorimétrique pour le lait et les boissons à base de lait
Reference numbers
ISO 11816-1:2013(E)
IDF 155-1:2013(E)
©
ISO and IDF 2013
ISO 11816-1:2013(E)
IDF 155-1:2013(E)
© ISO/IDF 2013
All rights reserved. Unless otherwise specified, 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 or IDF at the respective address below or ISO’s member body in the
country of the requester.
ISO copyright office International Dairy Federation
Case postale 56 • CH-1211 Geneva 20 Silver Building • Bd Auguste Reyers 70/B • B-1030 Brussels
Tel. + 41 22 749 01 11 Tel. + 32 2 733 98 88
Fax + 41 22 749 09 47 Fax + 32 2 733 04 13
E-mail copyright@iso.org E-mail info@fil-idf.org
Web www.iso.org Web www.fil-idf.org
Published in Switzerland
ii © ISO and IDF 2013 – All rights reserved

ISO 11816-1:2013(E)
IDF 155-1:2013(E)
Contents Page
Foreword .v
1 Scope . 1
2 Terms and definitions . 1
3 Principle . 1
4 Reagents . 1
5 Apparatus . 2
6 Sampling . 3
7 Preparations . 3
7.1 Alkaline phosphatase-free milk . 3
7.2 Preparation of test sample . 4
8 Procedure. 4
8.1 Verification of instrument performance . 4
8.2 Reagent controls to test the suitability of ready-to-use working substrate (4.3) . 5
8.3 Calibration . 5
8.4 Determination . 6
8.5 Test sample-related controls . 6
9 Calculation and expression of results . 7
9.1 Calibration ratio . 7
9.2 Calculation . 8
9.3 Expression of test results . 8
10 Precision . 8
10.1 Collaborative test . 8
10.2 Repeatability . 8
10.3 Reproducibility . 8
11 Test report . 9
Annex A
(informative)
Collaborative trials .10
Bibliography .13
ISO 11816-1:2013(E)
IDF 155-1:2013(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. 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. 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.
The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 5, Milk and
milk products, and the International Dairy Federation (IDF). It is being published jointly by ISO and IDF.
This third edition of ISO 11816-1|IDF 155-1 cancels and replaces the second edition (ISO 11816-1:2006),
which has been technically revised.
ISO 11816|IDF 155 consists of the following parts, under the general title Milk and milk products —
Determination of alkaline phosphatase activity:
— Part 1: Fluorimetric method for milk and milk-based drinks
— Part 2: Fluorimetric method for cheese
iv © ISO and IDF 2013 – All rights reserved

ISO 11816-1:2013(E)
IDF 155-1:2013(E)
Foreword
IDF (the International Dairy Federation) is a non-profit organization representing the dairy sector
worldwide. IDF membership comprises National Committees in every member country as well as
regional dairy associations having signed a formal agreement on cooperation with IDF. All members of
IDF have the right to be represented on the IDF Standing Committees carrying out the technical work.
IDF collaborates with ISO in the development of standard methods of analysis and sampling for milk and
milk products.
The main task of Standing Committees is to prepare International Standards. Draft International
Standards adopted by the Standing Committees are circulated to the National Committees for
endorsement prior to publication as an International Standard. Publication as an International Standard
requires approval by at least 50 % of IDF National Committees casting a vote.
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.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
ISO 11816-1|IDF 155-1 was prepared by the International Dairy Federation (IDF) and Technical
Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published
jointly by IDF and ISO.
All work was carried out by the Joint ISO-IDF Project Group on Determination of alkaline phosphatase
activity – fluorimetric method, of the Standing Committee on Analytical Methods for Processing Aids and
Indicators, under the aegis of its project leader, Ms. Eileen Garry (USA).
This third edition of ISO 11816-1|IDF 155-1 cancels and replaces IDF 155-1:2006, which has been
technically revised.
ISO 11816|IDF 155 consists of the following parts, under the general title Milk and milk products —
Determination of alkaline phosphatase activity:
— Part 1: Fluorimetric method for milk and milk-based drinks
— Part 2: Fluorimetric method for cheese
ISO 11816-1:2013(E)
INTERNATIONAL STANDARD
IDF 155-1:2013(E)
Milk and milk products — Determination of alkaline
phosphatase activity —
Part 1:
Fluorimetric method for milk and milk-based drinks
1 Scope
This part of ISO 11816|IDF 155 specifies a fluorimetric method for the determination of alkaline
phosphatase (ALP, EC 3.1.3.1) activity in raw and heat-treated whole milk, semi-skimmed milk, skimmed
milk and flavoured milks. This method is applicable to milk and milk-based drinks from cows, sheep and
goats. It is also applicable to milk powder after reconstitution.
The instrument can read activities up to 7 000 milliunits per litre (mU/l). If the activity is higher than
7 000 mU/l, it is diluted with alkaline phosphatase-free milk (7.1) so as to obtain a measurement not
higher than 7 000 mU/l.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1
alkaline phosphatase (ALP) activity
activity of the alkaline phosphatase present in the product, determined by the specified procedure
Note 1 to entry: The alkaline phosphatase activity is expressed as milliunits of enzyme activity per litre of
sample (mU/l).
2.2
unit of alkaline phosphatase activity
amount of alkaline phosphatase enzyme that catalyses the transformation of 1 µmol of substrate per minute
3 Principle
The alkaline phosphatase activity of the sample is measured by a continuous fluorimetric direct
kinetic assay. A non-fluorescent aromatic monophosphoric ester substrate, 2’-[2-benzothiazolyl]-6’-
hydroxybenzothiazole phosphate, in the presence of any alkaline phosphatase derived from the sample,
undergoes hydrolysis of its phosphate radical, producing a highly fluorescent product. Fluorimetric
measurement of alkaline phosphatase (ALP) activity is measured at 38 °C over a 3-min period when
using Fluorophos®. This includes pre-incubation of substrate and sample, followed by multiple kinetic
readings of the reaction rate.
NOTE Although this is a 3-min test, the first minute is an equilibration period to ensure that the sample is at
38 °C. Measurements of activity are actually made from the beginning of the second minute to the end of the third
minute (i.e. over a 2-min period).
4 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or
demineralized water, or water of equivalent purity.
ISO 11816-1:2013(E)
IDF 155-1:2013(E)
1)
4.1 Fluorophos® substrate, in bottles, each containing 144 mg of Fluorophos® substrate powder,
molecular weight 580 grams per mole.
This is a non-fluorescent aromatic monophosphoric ester substrate, 2’-[2-benzothiazolyl]-6’-
hydroxybenzothiazole phosphate (Fluorophos®). The Fluorophos® substrate remains stable for
two years from the date of manufacture, provided it is stored in unopened bottles at between 2 °C and
8 °C; protect against light.
4.2 Substrate buffer solution, diethanolamine (DEA) buffer solution, c(DEA) = 2,4 mol/l, with pH
10,0, in bottles of 240 ml each. The substrate buffer solution remains stable for two years from the date
of manufacture, provided it is stored in unopened bottles at between 2 °C and 8 °C; protect against light.
4.3 Working substrate
Allow the Fluorophos® substrate (4.1) and the substrate buffer solution (4.2) to come to room
temperature. Add the content of one bottle of substrate buffer solution (240 ml) (4.2) to that of one bottle
of Fluorophos® substrate (144 mg) (4.1), and mix well by inversion for 3 min to create ~1,0 millimolar
(pH 10) solution. Use amber glass to protect against light.
Allow the obtained solution to stand at room temperature for at least 30 min prior to use.
Use the A/D (analogue-to-digital) test given in 8.2 to test the suitability of the ready-to-use working substrate.
Do not use the working substrate if a reading above 1 200 FLU (fluorescence units) is obtained (8.2).
The working substrate remains stable for 60 days when protected from light and stored at between 2 °C
and 8 °C, or for 8 h when stored at 38 °C.
NOTE The volume of the working substrate (240 ml) obtained is sufficient for approximately 115 tests.
4.4 Calibrator solutions, Fluoroyellow®(FY) [2’-(2-benzothiazolyl)-6’-hydroxybenzothiazole] in
substrate buffer solution (4.2).
The calibrator solutions remain stable for 18 months from the date of manufacture, provided they are
stored in unopened bottles at between 2 °C and 8 °C. Mix gently prior to use to ensure optimal results.
4.4.1 Calibrator solution A, containing 0 μmol/l of Fluoroyellow®.
−3
4.4.2 Calibrator solution B, containing 17,24 × 10 μmol/l of Fluoroyellow®.
−3
4.4.3 Calibrator solution C, containing 34,48 × 10 μmol/l of Fluoroyellow®.
−3
4.5 Daily instrument control solution, containing 34,48 x10 µmol/l of Fluoroyellow®.
The daily instrument control solution remains stable for 18 months from the date of manufacture,
provided it is stored in unopened bottles at between 2 °C and 8 °C. Mix gently prior to use to ensure
optimal results.
5 Apparatus
Usual laboratory equipment and, in particular, the following.
1) The reagents specified in 4.1 to 4.5 and the apparatus specified in 5.1 to 5.4 (except 5.3.3) comprise the Fluorophos
Test System, which is the trade name of a product supplied by Advanced Instruments, Inc., Two Technology Way,
Norwood, Massachusetts 02062, USA. The manufacturer may change the packaging configurations supplied with
Fluorophos Test system. The user should refer to the manufacturer’s instructions for preparing reagents if different
from those specified herein. Fluorophos and Fluoroyellow are trademarks of Advanced Instruments, Inc. This
information is given for the convenience of users of this document and does not constitute an endorsement by ISO
or IDF of the products named. Equivalent products may be used if they can be shown to lead to the same results.
2 © ISO and IDF 2013 – All rights reserved

ISO 11816-1:2013(E)
IDF 155-1:2013(E)
5.1 Filter fluorimeter, with thermostatically controlled cuvette holder, capable of operating at
38 °C ± 1 °C and right-angle optics, allowing excitation at a wavelength of 440 nm and emission at between
1)
520 nm and 560 nm [e.g. Fluorophos® instrument ].
5.2 Cuvettes, disposable, of non-fluorescent glass.
5.3 Pipettes
5.3.1 Fixed-volume dispenser, capable of dispensing 2,0 ml.
5.3.2 Positive-displacement or air displacement pipette, of capacity 0,075 ml.
Follow strict instructions for pipetting technique as this is a critical step in generating accurate results.
Ensure that piston of pipette bore is tightly secured prior to use.
5.3.3 Pipettes, of capacity 2 ml and 3 ml.
5.4 Incubator block, capable of maintaining a temperature
...