EN ISO 11816-2:2016

SLOVENSKI STANDARD
01-november-2016
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SIST EN ISO 11816-2:2003
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)OXRURPHWULMVNDPHWRGD]DVLU,62
Milk and milk products - Determination of alkaline phosphatase activity - Part 2:
Fluorometric method for cheese (ISO 11816-2:2016)
Milch und Milchprodukte - Bestimmung der Aktivität der alkalischen Phosphatase - Teil 2:
Fluorimetrisches Verfahren für Käse (ISO 11816-2:2016)
Lait et produits laitiers - Détermination de l'activité de la phosphatase alcaline - Partie 2:
Méthode fluorimétrique pour le fromage (ISO 11816-2:2016)
Ta slovenski standard je istoveten z: EN ISO 11816-2:2016
ICS:
67.100.30 Sir Cheese
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 11816-2
EUROPEAN STANDARD
NORME EUROPÉENNE
August 2016
EUROPÄISCHE NORM
ICS 67.100.30 Supersedes EN ISO 11816-2:2003
English Version
Milk and milk products - Determination of alkaline
phosphatase activity - Part 2: Fluorimetric method for
cheese (ISO 11816-2:2016)
Lait et produits laitiers - Détermination de l'activité de Milch und Milchprodukte - Bestimmung der Aktivität
la phosphatase alcaline - Partie 2: Méthode der alkalischen Phosphatase - Teil 2: Fluorimetrisches
fluorimétrique pour le fromage (ISO 11816-2:2016) Verfahren für Käse (ISO 11816-2:2016)
This European Standard was approved by CEN on 29 July 2016.

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
© 2016 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 11816-2:2016 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
European foreword
This document (EN ISO 11816-2:2016) 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 February 2017, and conflicting national standards
shall be withdrawn at the latest by February 2017.
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 11816-2:2003.
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 11816-2:2016 has been approved by CEN as EN ISO 11816-2:2016 without any
modification.
INTERNATIONAL ISO
STANDARD 11816-2
IDF
155-2
Second edition
2016-08-15
Milk and milk products —
Determination of alkaline
phosphatase activity —
Part 2:
Fluorimetric method for cheese
Lait et produits laitiers — Détermination de l’activité de la
phosphatase alcaline —
Partie 2: Méthode fluorimétrique pour le fromage
Reference numbers
ISO 11816-2:2016(E)
IDF 155-2:2016(E)
©
ISO and IDF 2016
ISO 11816-2:2016(E)
IDF 155-2:2016(E)
© ISO and IDF 2016, Published in Switzerland
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 at the address below or ISO’s member body in the country of
the requester.
ISO copyright office International Dairy Federation
Ch. de Blandonnet 8 • CP 401 Silver Building • Bd Auguste Reyers 70/B • B-1030 Brussels
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11 Tel. + 32 2 325 67 40
Fax +41 22 749 09 47 Fax + 32 2 325 67 41
copyright@iso.org info@fil-idf.org
www.iso.org www.fil-idf.org
ii © ISO and IDF 2016 – All rights reserved

ISO 11816-2:2016(E)
IDF 155-2:2016(E)
Contents Page
Forewords .iv
1 Scope . 1
2 Normative reference . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents . 2
6 Apparatus . 3
7 Sampling . 4
8 Preparation of test sample . 4
9 Procedure. 4
9.1 Verification of instrument performance . 4
9.1.1 General. 4
9.1.2 Daily instrument tests . 5
9.1.3 Controls . 5
9.2 Reagent controls to test the suitability of ready to use working substrate (5.3) . 5
9.3 Calibration . 6
9.4 Determination . 6
9.5 Test sample related controls . 7
9.5.1 Recommended negative and positive control tests . 7
9.5.2 Interfering substance test . 8
9.5.3 Heat-stable microbial alkaline phosphatase control test . 8
10 Calculation and expression of results . 8
10.1 Calibration ratio . 8
10.2 Calculation . 9
10.2.1 Supernatant . 9
10.2.2 Cheese . 9
10.3 Expression of results .10
11 Precision .10
11.1 Interlaboratory test.10
11.2 Repeatability .10
11.3 Reproducibility .10
12 Test report .10
Annex A (informative) Interlaboratory trial .11
Annex B (informative) Examples of preparation of a test sample .13
Bibliography .15
ISO 11816-2:2016(E)
IDF 155-2:2016(E)
Forewords
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 on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical
Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information
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 second edition of ISO 11816-2|IDF 155-2 cancels and replaces the first edition
(ISO 11816-2|IDF 155-2:2003), 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 2016 – All rights reserved

ISO 11816-2:2016(E)
IDF 155-2:2016(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 and endorsement
ISO 11816|IDF 155 was prepared by the IDF Standing Committee on Analytical Methods for Processing
Aids and Indicators and ISO Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and
milk products.
The work was carried out by the IDF/ISO Project Group on Alkaline phosphatase activity in cheese (P06),
of the Standing Committee on Analytical Methods for Processing Aids and Indicators, under the aegis of
its project leader Mrs. M. Nicolas (FR).
This ISO/IDF International Standard cancels and replaces ISO 11816-2|IDF 155-2:2003, 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 11816-2:2016(E)
INTERNATIONAL STANDARD
IDF 155-2:2016(E)
Milk and milk products — Determination of alkaline
phosphatase activity —
Part 2:
Fluorimetric method for cheese
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 cheese.
This method is applicable to soft cheeses, semi-hard and hard cheeses provided that the mould is only
on the surface of the cheese and not also in the inner part (e.g. blue veined cheeses). For large hard
cheeses, specific conditions of sampling apply (see Clause 7).
The instrument can read activities in the supernatant up to 7 000 milliunits per litre (mU/l).
2 Normative reference
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO 5725-1, Accuracy (trueness and precision) of measurement methods and results — Part 1: General
principles and definitions
ISO 5725-2, Accuracy (trueness and precision) of measurement methods and results — Part 2: Basic method
for the determination of repeatability and reproducibility of a standard measurement method
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
alkaline phosphatase activity
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 gram of
sample (mU/g).
3.2
unit of alkaline phosphatase activity
amount of alkaline phosphatase enzyme that catalyses the transformation of 1 µmol of substrate
per minute
4 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
ISO 11816-2:2016(E)
IDF 155-2:2016(E)
measurement of alkaline phosphatase (ALP) activity is measured at 38 °C over a 3 min period when
1) ®
using the 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).
5 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or
demineralized water or water of equivalent purity.
2)
® ®
5.1 Fluorophos substrate in bottles, each containing 144 mg of Fluorophos substrate powder,
molar mass 580 grams per mole.
This is a non-fluorescent aromatic monophosphoric ester substrate, 2′-[2-benzothiazolyl]-6′-
hydroxybenzothiazole phosphate.
This substrate remains stable for 2 years from the date of manufacture, provided it is stored in
unopened bottles at a temperature between 2 °C and 8 °C. Protect against light.
5.2 Substrate buffer solution, diethanolamine (DEA) buffer solution, c(DEA) = 2,4 mol/l, with
pH 10,0.
The substrate buffer solution remains stable for 2 years from the date of manufacture, provided it is
stored in unopened bottles at a temperature between 2 °C and 8 °C. Protect against light.
5.3 Working substrate. ®
Allow the Fluorophos substrate (5.1) and the substrate buffer solution (5.2) to come to room
temperature. Add the content of one bottle substrate buffer solution (240 ml) (5.2) to that of one bottle ®
Fluorophos substrate (144 mg) (5.1) and mix well by inversion for 3 min. 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 analog-to-digital (A/D) test given in 9.2 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.
The working substrate remains stable for 60 days when protected from light and stored at a temperature
between 2 °C and 8 °C, or for 6 h at 38 °C. ®
5.4 Working calibrator solutions, Fluoroyellow (FY) [2′-(2-benzothiazolyl)-6′-
hydroxybenzothiazole] in substrate buffer solution (5.2).
The working calibrator solutions remain stable for 18 months from manufacturing date when stored in
unopened bottles at a temperature between 2 °C and 8 °C.
1) Fluorophos is a registered trademark. This information is given for the convenience of users of this document
and does not constitute an endorsement by either ISO or IDF of these products. Equivalent products may be used if
they can be shown to lead to the same results.
2) The reagents specified in 5.1 to 5.6 and the apparatus specified in 6.1 to 6.4 (except 6.3.1) are available from
Advanced Instruments, Inc., Two Technology Way, Norwood, Massachussetts 02062, USA. The manufacturer
may change packaging configurations supplied with the 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 registered trademarks of Advanced Instruments Inc. This information is given for the convenience
of users of this document and does not constitute an endorsement by either ISO or IDF of these products.
2 © ISO and IDF 2016 – All rights reserved

ISO 11816-2:2016(E)
IDF 155-2:2016(E)
Mix gently prior to use to ensure optimal results. ®
5.4.1 Calibrator solution A, containing 0 μmol/l of Fluoroyellow .
−3 ®
5.4.2 Calibrator solution B, containing 17,24 × 10 μmol/l of Fluoroyellow .
−3 ®
5.4.3 Calibrator solution C, containing 34,48 × 10 μmol/l of Fluoroyellow .
−3 ®
5.5 Daily instrument control solution, containing 34,48 × 10 μmol/l of Fluoroyellow .
The daily instrument control solution remains stable for 18 months from manufacturing date when
stored in unopened bottles at a temperature between 2 °C and 8 °C.
Mix gently prior to use to ensure optimal results. ®
5.6 Fluorophos cheese extraction buffer, diethanolamine (DEA) buffer, pH 8,0 with magnesium
and Triton X-100.
The cheese extraction buffer remains stable for 3 years from manufacturing date when stored in
unopened bottles at a temperature between 2 °C and 8 °C.
5.7 Positive, negative and Phosphacheck-N™ controls.
6 Apparatus
Usual laboratory equipment and, in particular, the following.
6.1 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 between
® 1)
520 nm and 560 nm [e.g. Fluorophos instrument ].
6.2 Cuvettes, disposable, non-fluorescent glass, of diameter 12 mm and of length 75 mm.
6.3 Pipettes.
6.3.1 Pipette, of capacity 2,0 ml and 3,0 ml.
6.3.2 Positive-displacement or air-displacement pipette, of capacity 0,075 ml.
6.4 Heating block, capable of maintaining a temperature of 38 °C ± 1 °C, suitable for holding cuvettes.
3) ®
6.5 Plastic paraffin film (e.g. Parafilm ) or other suitable laboratory-grade film.
6.6 Vortex mixer
...