prEN ISO 11816-2
(Main)Milk and milk products - Determination of alkaline phosphatase activity - Part 2: Fluorimetric method for cheese (ISO/DIS 11816-2:2022)
Milk and milk products - Determination of alkaline phosphatase activity - Part 2: Fluorimetric method for cheese (ISO/DIS 11816-2:2022)
ISO 11816-2:2016|IDF 155-2:2016 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).
Milch und Milchprodukte - Bestimmung der Aktivität der alkalischen Phosphatase - Teil 2: Fluorimetrisches Verfahren für Käse (ISO/DIS 11816-2:2022)
Lait et produits laitiers - Détermination de l'activité de la phosphatase alcaline - Partie 2: Méthode fluorimétrique pour le fromage (ISO/DIS 11816-2:2022)
L'ISO 11816-2 :2016|FIL 155-2 :2016 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 permet de détecter des activités dans le surnageant de l'ordre de 7 000 milliunités par litre (mU/l).
Mleko in mlečni proizvodi - Določanje aktivnosti alkalne fosfataze - 2. del: Fluorometrijska metoda za sir (ISO/DIS 11816-2:2022)
General Information
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Standards Content (Sample)
SLOVENSKI STANDARD
oSIST prEN ISO 11816-2:2023
01-februar-2023
Mleko in mlečni proizvodi - Določanje aktivnosti alkalne fosfataze - 2. del:
Fluorometrijska metoda za sir (ISO/DIS 11816-2:2022)
Milk and milk products - Determination of alkaline phosphatase activity - Part 2:
Fluorimetric method for cheese (ISO/DIS 11816-2:2022)
Milch und Milchprodukte - Bestimmung der Aktivität der alkalischen Phosphatase - Teil
2: Fluorimetrisches Verfahren für Käse (ISO/DIS 11816-2:2022)
Lait et produits laitiers - Détermination de l'activité de la phosphatase alcaline - Partie 2:
Méthode fluorimétrique pour le fromage (ISO/DIS 11816-2:2022)
Ta slovenski standard je istoveten z: prEN ISO 11816-2
ICS:
67.100.30 Sir Cheese
oSIST prEN ISO 11816-2:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN ISO 11816-2:2023
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oSIST prEN ISO 11816-2:2023
DRAFT INTERNATIONAL STANDARD
ISO/DIS 11816-2
ISO/TC 34/SC 5 Secretariat: NEN
Voting begins on: Voting terminates on:
2022-12-06 2023-02-28
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
ICS: 67.100.30
This document is circulated as received from the committee secretariat.
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
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THEREFORE SUBJECT TO CHANGE AND MAY
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NATIONAL REGULATIONS.
ISO/DIS 11816-2:2022(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
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oSIST prEN ISO 11816-2:2023
ISO/DIS 11816-2:2022(E)
DRAFT INTERNATIONAL STANDARD
ISO/DIS 11816-2
ISO/TC 34/SC 5 Secretariat: NEN
Voting begins on: Voting terminates on:
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
ICS: 67.100.30
This document is circulated as received from the committee secretariat.
COPYRIGHT PROTECTED DOCUMENT
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
© ISO 2022
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NATIONAL REGULATIONS.
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ii
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PROVIDE SUPPORTING DOCUMENTATION. © ISO 2022
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oSIST prEN ISO 11816-2:2023
ISO/DIS 11816-2:2022(E)
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 .4
9.1 Verification of instrument performance . 4
9.1.1 General . 4
9.1.2 Daily instrument tests . 5
9.1.3 Using FLM200 . 5
9.1.4 Using FLM300 . 5
9.1.5 Controls . 5
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 . 6
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 alkaline phosphatase control test . 9
10 Calculation and expression of results . 9
10.1 Calibration ratio . 9
10.2 Calculation . 9
10.2.1 Supernatant . 9
10.2.2 Cheese . 10
10.3 Expression of results . . 10
11 Precision .10
11.1 Interlaboratory test . . 10
11.2 Repeatability . 10
11.3 Reproducibility . 11
12 Test report .11
Annex A (informative) Interlaboratory trial.12
Annex B (informative) Examples of preparation of a test sample.14
Bibliography .16
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oSIST prEN ISO 11816-2:2023
ISO/DIS 11816-2:2022(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 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 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.
This third edition cancels and replaces the second edition (ISO 11816-2|IDF 155-2:2016), which has
been technically revised. The main changes compared with the previous edition are as follows:
The FLM200 required updating of the electronics to meet world regulatory requirements. Therefore,
the instrument was discontinued, and we introduce the FLM300 version of the instrument. This version
upgraded the electronics and the user interface. The changes in the user interfaced had an impact on
some of the instructions for use and the flow of those instructions which are detailed here in the new
standard. There was no change to the assay or the test procedure with the changes to the software and
interface. 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
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ISO/DIS 11816-2:2022(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.
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 IDF and ISO.
The work was carried out by the IDF/ISO Action Team P05 of the Standing Committee on Analytical
Methods for Processing Aids and Indicators, under the aegis of its project leader Mr Rick Zampa (US)
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oSIST prEN ISO 11816-2:2023
DRAFT INTERNATIONAL STANDARD ISO/DIS 11816-2:2022(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 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 terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at https:// www .electropedia .org/
— ISO Online browsing platform: available at https:// www .iso .org/ obp
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
measurement of alkaline phosphatase (ALP) activity is measured at 38 °C over a 3 min period when
1
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oSIST prEN ISO 11816-2:2023
ISO/DIS 11816-2:2022(E)
®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 9.1.3 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 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 5.7 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
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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.
6.7 Grinding device.
6.8 Glass beaker, of capacity 5 ml (of approximately diameter 20 mm and length 30 mm) and 10 ml
(of approximately diameter 25 mm and length 30 mm).
3) Parafilm® and Ultra turrax® are examples 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 either ISO or IDF of
these products.
3
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®3)
6.9 High-speed homogenizer (e.g. Ultra turrax ) provided with the stem of diameter of
approximately 6 mm to 8 mm.
6.10 One-mark volumetric flasks, of capacity 25 ml.
6.11 Centrifuge, capable of centrifuging at 1 000 g at 4 °C.
6.12 Glass test tube, of approximately diameter 12 mm and length 10 cm.
6.13 Glass Pasteur pipette, an air-displacement pipette can also be used.
6.14 Water bath, capable of maintaining a temperature of 63 °C ± 1 °C
7 Sampling
A representative sample should be sent to the laboratory. It should not have been damaged or altered
during transport or storage.
Sampling is not part of the method specified in this part of ISO 11816|IDF 155. A recommended sampling
[1]
method is given in ISO 707|IDF 50 .
However, ISO 707|IDF 50 is not suitable for large hard cheeses where the whey curd mixture has been
scalded at temperatures above 50 °C. If the cheese is made from raw milk, the ALP activity is not
homogenously distributed within these cheeses. The activity is high in the outer layer of the cheese
wheel, between 0 cm to 4 cm below, the rind of the round side, but very low or even undetectable in the
core.
Samples of large hard cheeses, therefore, shall be sampled by taking a portion of 1 cm, taken at 0,5 cm
below the rind of the round side (see Figure B.1).
In case of doubt regarding the type of cheese, between a hard and a semi-hard cheese, proceed to the
sampling as described for large hard cheeses.
8 Preparation of test sample
Remove the rind or the surface from the test sample with a clean knife. Ensure that the test sample
is not contaminated with surface microflora during its preparation. Especially for soft cheese with
moulded surface, remove all the rind but in a layer as thin as possible, so as to avoid eliminating the
fat layer under the mould surface (see Figure B.2). For large hard cheeses, proceed as described under
Clause 7. Grind the test sample by means of a grinding mill or other appropriate device (6.7) and mix
thoroughly. Keep the prepared sample in an airtight container.
9 Procedure
9.1 Verification of instrument performance
9.1.1 General
It is important to check instrument performance for drift, stray light and stability prior to analysing
test samples. Follow good laboratory practice standards when operating the filter fluorimeter (6.1).
Quality control tests includes the following:
a) The daily A/D (analog-to-digital) test, used to check the proper functioning of the equipment.
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b) The daily instrument control test, using the daily instrument control solution (5.5) to monitor any
electronic or optical drift in the fluorimeter.
c) The use of external positive, negative and normal controls, described in 9.1.3, is recommended for
monitoring daily instrument precision parameters
9.1.2 Daily instrument tests
®
9.1.2.1 When using the Fluorophos instrument, perform the A/D test daily before testing
commences.
9.1.3 Using FLM200
9.1.3.1 Access the A/D test according to the instrument’s User Guide through “SETUP” menu. Press
“SETUP” key, then select menu items “A/D Test” by pressing < or >. With nothing in the cuvette holder,
press “START”. Allow the figures appearing on the display screen to stabilize. The display should read
302 ± 4. If the reading is outside that range, clean the excitation and emission filters and repeat A/D
test.
9.1.3.2 Dispense 2,0 ml of daily instrument control solution (4.5) into labelled cuvette. Place the
cuvette in the incubator block (5.4) set at 38 °C for 20 min. Insert the prewarmed cuvette into the
cuvette holder. Close the lid. When the display is stable, record the displayed value, which should be
602 ± 12. If outside hat range, use the small screwdriver supplied to slowly turn the potentiometer
screw on the left-hand side of the instrument clockwise or anticlockwise, as necessary, until the display
reads 602. Allow the numbers to equilibrat
...
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