EN ISO 11816-2:2024
(Main)Milk and milk products - Determination of alkaline phosphatase activity - Part 2: Fluorimetric method for cheese (ISO 11816-2:2024)
Milk and milk products - Determination of alkaline phosphatase activity - Part 2: Fluorimetric method for cheese (ISO 11816-2:2024)
This document 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 used for the determination of ALP can read activities in the supernatant up to 7 000 milliunits per litre (mU/kg).
Milch und Milchprodukte - Bestimmung der Aktivität der alkalischen Phosphatase - Teil 2: Fluorimetrisches Verfahren für Käse (ISO 11816-2:2024)
Dieses Dokument legt ein fluorimetrisches Verfahren für die Bestimmung der Aktivität der alkalischen Phosphatase (ALP) (EC 3.1.3.1) in Käse fest.
Dieses Verfahren ist anwendbar für Weichkäse, Schnittkäse und Hartkäse unter der Voraussetzung, dass der Schimmel nur an der Käseoberfläche und nicht auch im Innern des Käses vorhanden ist (z. B. Blauschimmelkäse). Für große Hartkäselaibe gelten spezifische Probenahmebedingungen (siehe Abschnitt 7).
Das Gerät zur Bestimmung von ALP kann Aktivitäten im Überstand von bis zu 7 000 Milliunits je Liter (mU/kg) auslesen.N1
Lait et produits laitiers - Détermination de l'activité de la phosphatase alcaline - Partie 2: Méthode fluorimétrique pour le fromage (ISO 11816-2:2024)
Le présent document spécifie une méthode fluorimétrique de détermination de l’activité de la phosphatase alcaline (ALP) (EC 3.1.3.1) dans le fromage.
La présente méthode est applicable aux fromages à pâte molle, pâte pressée non cuite et pâte pressée cuite, à condition que la moisissure ne soit présente que sur la surface du fromage et ne se soit pas propagée à l’intérieur, comme dans le cas des fromages veinés de bleu, par exemple. Pour les gros fromages à pâte pressée cuite, des conditions d’échantillonnage spécifiques s’appliquent (voir l’Article 7).
L’instrument utilisé pour la détermination de l’ALP peut lire des activités jusqu’à 7 000 mU/kg (milliunités par kilogramme).
Mleko in mlečni izdelki - Določanje aktivnosti alkalne fosfataze - 2. del: Fluorometrijska metoda za sir (ISO 11816-2:2024)
Ta dokument določa fluorimetrijsko metodo za določanje aktivnosti alkalne fosfataze (ALP)
(EC 3.1.3.1) v siru.
Ta metoda se uporablja za topljene, poltrde in trde sire pod pogojem, da je plesen samo na površini sira, in ne tudi na notranji strani (npr. sir z modrimi plesnimi). Za velike trde sire veljajo posebni pogoji vzorčenja (glej točko 7).
Instrument, ki se uporablja za določanje alkalne fosfataze, lahko beleži aktivnosti v supernatantu do 7000 milienot na liter (mU/kg).
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
01-marec-2024
Nadomešča:
SIST EN ISO 11816-2:2016
Mleko in mlečni izdelki - Določanje aktivnosti alkalne fosfataze - 2. del:
Fluorometrijska metoda za sir (ISO 11816-2:2024)
Milk and milk products - Determination of alkaline phosphatase activity - Part 2:
Fluorimetric method for cheese (ISO 11816-2:2024)
Milch und Milchprodukte - Bestimmung der Aktivität der alkalischen Phosphatase - Teil
2: Fluorimetrisches Verfahren für Käse (ISO 11816-2:2024)
Lait et produits laitiers - Détermination de l'activité de la phosphatase alcaline - Partie 2:
Méthode fluorimétrique pour le fromage (ISO 11816-2:2024)
Ta slovenski standard je istoveten z: EN ISO 11816-2:2024
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
January 2024
EUROPÄISCHE NORM
ICS 67.100.30 Supersedes EN ISO 11816-2:2016
English Version
Milk and milk products - Determination of alkaline
phosphatase activity - Part 2: Fluorimetric method for
cheese (ISO 11816-2:2024)
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 11816-2:2024) Verfahren für Käse (ISO 11816-2:2024)
This European Standard was approved by CEN on 29 March 2023.
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 11816-2:2024 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
European foreword
This document (EN ISO 11816-2:2024) 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 July 2024, and conflicting national standards shall be
withdrawn at the latest by July 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 11816-2:2016.
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 11816-2:2024 has been approved by CEN as EN ISO 11816-2:2024 without any
modification.
International
Standard
ISO 11816-2
IDF 155-2
Third edition
Milk and milk products —
2024-01
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 11816-2:2024(en)
IDF 155-2:2024(en) © ISO and IDF 2024
ISO 11816-2:2024(en)
IDF 155-2:2024(en)
© ISO and IDF 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 International Dairy Federation
CP 401 • Ch. de Blandonnet 8 Silver Building • Bd Auguste Reyers 70/B
CH-1214 Vernier, Geneva B-1030 Brussels
Phone: +41 22 749 01 11 Phone: +32 2 325 67 40
Fax: +32 2 325 67 41
Email: copyright@iso.org Email: info@fil-idf.org
Website: www.iso.org Website: www.fil-idf.org
Published in Switzerland
ii
ISO 11816-2:2024(en)
IDF 155-2:2024(en)
Contents Page
Forewords .iv
1 Scope . 1
2 Normative references . 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 . 5
9.1 Verification of instrument performance .5
9.1.1 General .5
9.1.2 Daily instrument tests .5
9.1.3 Using FLM200 .5
9.1.4 Using FLM300 .5
9.1.5 Controls .6
9.2 Reagent controls to test the suitability of ready-to-use working substrate (5.3) .6
9.3 Calibration .6
9.3.1 General .6
9.3.2 Using FLM200 .7
9.3.3 Using FLM300 .7
9.4 Determination .7
9.5 Test-sample-related controls .8
9.5.1 Recommended negative and positive control tests .8
9.5.2 Interfering substance test .9
9.5.3 Heat-stable microbial ALP control test .9
10 Calculation and expression of results . 9
10.1 Calibration ratio .9
10.2 Calculation .10
10.2.1 Supernatant .10
10.2.2 Cheese .10
10.3 Expression of test results.10
11 Precision .11
11.1 Interlaboratory study .11
11.2 Repeatability .11
11.3 Reproducibility .11
12 Test report .11
Annex A (informative) Interlaboratory study .12
Annex B (informative) Examples of preparation of a test sample . 14
Bibliography .16
iii
ISO 11816-2:2024(en)
IDF 155-2:2024(en)
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 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
https://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 5,
Milk and milk products, and the International Dairy Federation (IDF), in collaboration with the European
Committee for Standardization (CEN) Technical Committee CEN/TC 302, Milk and milk products — Methods
of sampling and analysis, in accordance with the Agreement on technical cooperation between ISO and CEN
(Vienna Agreement). It is being published jointly by ISO and IDF.
This third edition cancels and replaces the second edition (ISO 11816-2 | IDF 155-2:2016), which has been
technically revised.
The main changes are as follows:
— the FLM200 instrument (which has been discontinued) has been replaced by the FLM300 version;
— the instructions for use of the instrument and the flow of those instructions have been revised in
accordance with FLM300, which has an upgraded user interface and electronics (there has been no
change to the assay or the test procedure with the changes to the interface and software);
— the instrument now includes the heater block which was a separate item previously.
A list of all parts in the ISO 11816 | IDF 155 series can be found on the ISO website.
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 11816-2:2024(en)
IDF 155-2:2024(en)
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.
IDF draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). IDF 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, IDF 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. 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.
This document 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. It is being published jointly by ISO and IDF.
The work was carried out by the IDF/ISO Action Team P19 of the Standing Committee on Analytical Methods
for Processing Aids and Indicators under the aegis of its project leader Mr Rick Zampa (US).
v
ISO 11816-2:2024(en)
International Standard
IDF 155-2:2024(en)
Milk and milk products — Determination of alkaline
phosphatase activity —
Part 2:
Fluorimetric method for cheese
1 Scope
This document 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 used for the determination of ALP can read activities in the supernatant up to 7 000 milliunits
per litre (mU/kg).
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
alkaline phosphatase activity
ALP activity
activity of the enzyme present in the product, determined by the specified procedure
Note 1 to entry: The ALP activity is expressed as milliunits of enzyme activity per gram of sample (mU/kg).
4 Principle
The ALP 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 ALP derived from the sample, undergoes hydrolysis of its phosphate
radical, producing a highly fluorescent product. Fluorimetric measurement of ALP activity is measured at
ISO 11816-2:2024(en)
IDF 155-2:2024(en)
®1)
38 °C over a 3 min period when using a 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.
The reagents specified in 5.1 to 5.7 and the apparatus specified in 6.1 to 6.4 (except 6.3.1) comprise the
2) ®
Fluorophos Test System . The manufacturer can 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.
® ®
5.1 Fluorophos substrate, in bottles, each containing 144 mg of Fluorophos substrate powder, molar
mass of 580 g/mol.
This is a non-fluorescent aromatic monophosphoric ester substrate, 2′-[2-benzothiazolyl]-6′-
hydroxybenzothiazole phosphate.
This 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.
5.2 Substrate buffer solution, diethanolamine (DEA) buffer solution, c(DEA) = 2,4 mol/l, with pH-
value 10,0.
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.
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 of substrate buffer solution (240 ml) (5.2) to that of one bottle of 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 analogue-to-digital (A/D) test given in 9.1.3 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 between 2 °C and
8 °C, or for 6 h when stored at 38 °C. ®
1) Fluorophos is the registered trademark of a product supplied by Advanced Instruments, LLC. This information is
given for the convenience of users of this document and does not constitute an endorsement by ISO or IDF of the product
named. Equivalent products may be used if they can be shown to lead to the same results. ®
2) The Fluorophos Test System is the trade name of a product supplied by Advanced Instruments, LLC. This information
is given for the convenience of users of this document and does not constitute an endorsement by ISO or IDF of the
product named. Equivalent products may be used if they can be shown to lead to the same results.
ISO 11816-2:2024(en)
IDF 155-2:2024(en)
®3)
5.4 Calibrator solutions, Fluoroyellow (FY) [2′-(2-benzothiazolyl)-6′-hydroxybenzothiazole] in
substrate buffer solution (5.2).
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. ®
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 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. ®
5.6 Fluorophos cheese extraction buffer, diethanolamine (DEA) buffer, pH-value 8,0 with magnesium
and Triton X-100.
The cheese extraction buffer remains stable for three years from the date of manufacture, provided it is
stored in unopened bottles at between 2 °C and 8 °C.
4)
5.7 Positive, negative and PhosphaCheck-N™ controls .
6 Apparatus
Usual laboratory equipment and, in particular, the following shall be used.
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 ®
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.
®5)
6.5 Plastic paraffin film (e.g. Parafilm ) or other suitable laboratory-grade film. ®
3) Fluoroyellow is the registered trademark of a product supplied by Advanced Instruments, LLC. 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.
4) The controls and instrument performance check instructions are products supplied by Advanced Instruments, LLC.
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. ®
5) Parafilm 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 or IDF of this product.
ISO 11816-2:2024(en)
IDF 155-2:2024(en)
6.6 Vortex mixer.
6.7 Grinding device.
6.8 G
...
SLOVENSKI STANDARD
01-marec-2024
Nadomešča:
SIST EN ISO 11816-2:2016
Mleko in mlečni proizvodi - Določanje aktivnosti alkalne fosfataze - 2. del:
Fluorometrijska metoda za sir (ISO 11816-2:2024)
Milk and milk products - Determination of alkaline phosphatase activity - Part 2:
Fluorimetric method for cheese (ISO 11816-2:2024)
Milch und Milchprodukte - Bestimmung der Aktivität der alkalischen Phosphatase - Teil
2: Fluorimetrisches Verfahren für Käse (ISO 11816-2:2024)
Lait et produits laitiers - Détermination de l'activité de la phosphatase alcaline - Partie 2:
Méthode fluorimétrique pour le fromage (ISO 11816-2:2024)
Ta slovenski standard je istoveten z: EN ISO 11816-2:2024
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
January 2024
EUROPÄISCHE NORM
ICS 67.100.30 Supersedes EN ISO 11816-2:2016
English Version
Milk and milk products - Determination of alkaline
phosphatase activity - Part 2: Fluorimetric method for
cheese (ISO 11816-2:2024)
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 11816-2:2024) Verfahren für Käse (ISO 11816-2:2024)
This European Standard was approved by CEN on 29 March 2023.
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 11816-2:2024 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
European foreword
This document (EN ISO 11816-2:2024) 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 July 2024, and conflicting national standards shall be
withdrawn at the latest by July 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 11816-2:2016.
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 11816-2:2024 has been approved by CEN as EN ISO 11816-2:2024 without any
modification.
International
Standard
ISO 11816-2
IDF 155-2
Third edition
Milk and milk products —
2024-01
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 11816-2:2024(en)
IDF 155-2:2024(en) © ISO and IDF 2024
ISO 11816-2:2024(en)
IDF 155-2:2024(en)
© ISO and IDF 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 International Dairy Federation
CP 401 • Ch. de Blandonnet 8 Silver Building • Bd Auguste Reyers 70/B
CH-1214 Vernier, Geneva B-1030 Brussels
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Published in Switzerland
ii
ISO 11816-2:2024(en)
IDF 155-2:2024(en)
Contents Page
Forewords .iv
1 Scope . 1
2 Normative references . 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 . 5
9.1 Verification of instrument performance .5
9.1.1 General .5
9.1.2 Daily instrument tests .5
9.1.3 Using FLM200 .5
9.1.4 Using FLM300 .5
9.1.5 Controls .6
9.2 Reagent controls to test the suitability of ready-to-use working substrate (5.3) .6
9.3 Calibration .6
9.3.1 General .6
9.3.2 Using FLM200 .7
9.3.3 Using FLM300 .7
9.4 Determination .7
9.5 Test-sample-related controls .8
9.5.1 Recommended negative and positive control tests .8
9.5.2 Interfering substance test .9
9.5.3 Heat-stable microbial ALP control test .9
10 Calculation and expression of results . 9
10.1 Calibration ratio .9
10.2 Calculation .10
10.2.1 Supernatant .10
10.2.2 Cheese .10
10.3 Expression of test results.10
11 Precision .11
11.1 Interlaboratory study .11
11.2 Repeatability .11
11.3 Reproducibility .11
12 Test report .11
Annex A (informative) Interlaboratory study .12
Annex B (informative) Examples of preparation of a test sample . 14
Bibliography .16
iii
ISO 11816-2:2024(en)
IDF 155-2:2024(en)
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 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
https://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 5,
Milk and milk products, and the International Dairy Federation (IDF), in collaboration with the European
Committee for Standardization (CEN) Technical Committee CEN/TC 302, Milk and milk products — Methods
of sampling and analysis, in accordance with the Agreement on technical cooperation between ISO and CEN
(Vienna Agreement). It is being published jointly by ISO and IDF.
This third edition cancels and replaces the second edition (ISO 11816-2 | IDF 155-2:2016), which has been
technically revised.
The main changes are as follows:
— the FLM200 instrument (which has been discontinued) has been replaced by the FLM300 version;
— the instructions for use of the instrument and the flow of those instructions have been revised in
accordance with FLM300, which has an upgraded user interface and electronics (there has been no
change to the assay or the test procedure with the changes to the interface and software);
— the instrument now includes the heater block which was a separate item previously.
A list of all parts in the ISO 11816 | IDF 155 series can be found on the ISO website.
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 11816-2:2024(en)
IDF 155-2:2024(en)
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.
IDF draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). IDF 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, IDF 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. 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.
This document 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. It is being published jointly by ISO and IDF.
The work was carried out by the IDF/ISO Action Team P19 of the Standing Committee on Analytical Methods
for Processing Aids and Indicators under the aegis of its project leader Mr Rick Zampa (US).
v
ISO 11816-2:2024(en)
International Standard
IDF 155-2:2024(en)
Milk and milk products — Determination of alkaline
phosphatase activity —
Part 2:
Fluorimetric method for cheese
1 Scope
This document 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 used for the determination of ALP can read activities in the supernatant up to 7 000 milliunits
per litre (mU/kg).
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
alkaline phosphatase activity
ALP activity
activity of the enzyme present in the product, determined by the specified procedure
Note 1 to entry: The ALP activity is expressed as milliunits of enzyme activity per gram of sample (mU/kg).
4 Principle
The ALP 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 ALP derived from the sample, undergoes hydrolysis of its phosphate
radical, producing a highly fluorescent product. Fluorimetric measurement of ALP activity is measured at
ISO 11816-2:2024(en)
IDF 155-2:2024(en)
®1)
38 °C over a 3 min period when using a 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.
The reagents specified in 5.1 to 5.7 and the apparatus specified in 6.1 to 6.4 (except 6.3.1) comprise the
2) ®
Fluorophos Test System . The manufacturer can 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.
® ®
5.1 Fluorophos substrate, in bottles, each containing 144 mg of Fluorophos substrate powder, molar
mass of 580 g/mol.
This is a non-fluorescent aromatic monophosphoric ester substrate, 2′-[2-benzothiazolyl]-6′-
hydroxybenzothiazole phosphate.
This 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.
5.2 Substrate buffer solution, diethanolamine (DEA) buffer solution, c(DEA) = 2,4 mol/l, with pH-
value 10,0.
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.
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 of substrate buffer solution (240 ml) (5.2) to that of one bottle of 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 analogue-to-digital (A/D) test given in 9.1.3 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 between 2 °C and
8 °C, or for 6 h when stored at 38 °C. ®
1) Fluorophos is the registered trademark of a product supplied by Advanced Instruments, LLC. This information is
given for the convenience of users of this document and does not constitute an endorsement by ISO or IDF of the product
named. Equivalent products may be used if they can be shown to lead to the same results. ®
2) The Fluorophos Test System is the trade name of a product supplied by Advanced Instruments, LLC. This information
is given for the convenience of users of this document and does not constitute an endorsement by ISO or IDF of the
product named. Equivalent products may be used if they can be shown to lead to the same results.
ISO 11816-2:2024(en)
IDF 155-2:2024(en)
®3)
5.4 Calibrator solutions, Fluoroyellow (FY) [2′-(2-benzothiazolyl)-6′-hydroxybenzothiazole] in
substrate buffer solution (5.2).
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. ®
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 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. ®
5.6 Fluorophos cheese extraction buffer, diethanolamine (DEA) buffer, pH-value 8,0 with magnesium
and Triton X-100.
The cheese extraction buffer remains stable for three years from the date of manufacture, provided it is
stored in unopened bottles at between 2 °C and 8 °C.
4)
5.7 Positive, negative and PhosphaCheck-N™ controls .
6 Apparatus
Usual laboratory equipment and, in particular, the following shall be used.
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 ®
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.
®5)
6.5 Plastic paraffin film (e.g. Parafilm ) or other suitable laboratory-grade film. ®
3) Fluoroyellow is the registered trademark of a product supplied by Advanced Instruments, LLC. 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.
4) The controls and instrument performance check instructions are products supplied by Advanced Instruments, LLC.
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. ®
5) Parafilm 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 or IDF of this product.
ISO 11816-2:2024(en)
IDF 155-2:2024(en)
6.6 Vortex mixer.
6.7 Grinding device.
6.8
...
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