oSIST prEN ISO 11816-1:2023
(Main)Milk and milk products - Determination of alkaline phosphatase activity - Part 1: Fluorimetric method for milk and milk-based drinks (ISO/DIS 11816-1:2022)
Milk and milk products - Determination of alkaline phosphatase activity - Part 1: Fluorimetric method for milk and milk-based drinks (ISO/DIS 11816-1:2022)
ISO 11816-1|IDF 155-1:2013 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 so as to obtain a measurement not higher than 7 000 mU/l.
Milch und Milcherzeugnisse - Bestimmung der Aktivität der alkalischen Phosphatase - Teil 1: Fluorimetrisches Verfahren für Milch und flüssige Milchprodukte (ISO/DIS 11816-1:2022)
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/DIS 11816-1:2022)
L'ISO 11816-1|FIL 155-1:2013 spécifie une méthode fluorimétrique pour la détermination de l'activité de la phosphatase alcaline (ALP, EC 3.1.3.1) dans les laits crus et traités thermiquement entiers, demi-écrémés, écrémés et dans les laits aromatisés. La méthode est applicable au lait et aux boissons à base de lait de vache, de brebis et de chèvre. Elle est également applicable au lait en poudre après reconstitution.
L'appareillage peut lire des activités jusqu'à 7 000 milliunités par litre (mU/l). Si l'activité est supérieure à 7 000 mU/l, une dilution avec du lait exempt de phosphatase alcaline (7.1) est effectuée de façon à obtenir un mesurage ne dépassant pas 7 000 mU/l.
Mleko in mlečni proizvodi - Določanje aktivnosti alkalne fosfataze - 1. del: Fluorimetrijska metoda za mleko in pijače na osnovi mleka (ISO/DIS 11816-1:2022)
General Information
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Standards Content (Sample)
SLOVENSKI STANDARD
oSIST prEN ISO 11816-1:2023
01-februar-2023
Mleko in mlečni proizvodi - Določanje aktivnosti alkalne fosfataze - 1. del:
Fluorimetrijska metoda za mleko in pijače na osnovi mleka (ISO/DIS 11816-1:2022)
Milk and milk products - Determination of alkaline phosphatase activity - Part 1:
Fluorimetric method for milk and milk-based drinks (ISO/DIS 11816-1:2022)
Milch und Milcherzeugnisse - Bestimmung der Aktivität der alkalischen Phosphatase -
Teil 1: Fluorimetrisches Verfahren für Milch und flüssige Milchprodukte (ISO/DIS 11816-
1:2022)
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/DIS 11816-1:2022)
Ta slovenski standard je istoveten z: prEN ISO 11816-1
ICS:
67.100.10 Mleko in predelani mlečni Milk and processed milk
proizvodi products
oSIST prEN ISO 11816-1:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN ISO 11816-1:2023
DRAFT INTERNATIONAL STANDARD
ISO/DIS 11816-1
ISO/TC 34/SC 5 Secretariat: NEN
Voting begins on: Voting terminates on:
2022-12-02 2023-02-24
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
ICS: 67.100.10
This document is circulated as received from the committee secretariat.
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oSIST prEN ISO 11816-1:2023
ISO/DIS 11816-1:2022(E)
DRAFT INTERNATIONAL STANDARD
ISO/DIS 11816-1
ISO/TC 34/SC 5 Secretariat: NEN
Voting begins on: Voting terminates on:
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
ICS: 67.100.10
This document is circulated as received from the committee secretariat.
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NATIONAL REGULATIONS.
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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 . 3
8 Preparations . 4
8.1 Alkaline phosphatase-free milk . 4
8.2 Preparation of the test sample . 4
8.2.1 General . 4
8.2.2 Pasteurized test samples . 4
8.2.3 Dilution of test samples with high ALP values . 4
9 Procedure .4
9.1 Verification of instrument performance . 4
9.1.1 General . 4
9.1.2 Daily instrument tests . 4
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 . 6
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 . 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
10.3 Expression of test results. 9
11 Precision . 9
11.1 Collaborative test . 9
11.2 Repeatability . 9
11.3 Reproducibility . 9
12 Test report .10
Annex A (informative) Collaborative trials .11
Bibliography .14
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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 fourth edition cancels and replaces the third edition (ISO 11816-1|IDF 155-1:2013), 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.
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.
iv
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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 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 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 11816-1:2023
DRAFT INTERNATIONAL STANDARD ISO/DIS 11816-1:2022(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 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
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).
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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1)
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).
5 Reagents
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and
distilled or demineralized water or water of equivalent purity.
2)
5.1 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.
5.2 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.
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 to create a
~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 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 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.
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.
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 5.5 and the apparatus specified in 6.1 to 6.4 (except 6.3.3) comprise the Fluorophos
Test System, which is the trade name of a product supplied by Advanced Instruments, LLC, 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.
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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 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.
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 at
1)
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 Fixed-volume dispenser, capable of dispensing 2,0 ml.
6.3.2 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.
6.3.3 Pipettes, of capacity 2 ml and 3 ml.
6.4 Incubator block, capable of maintaining a temperature of 38 °C ± 1 °C, suitable for holding
cuvettes.
6.5 Suitable laboratory-grade film.
6.6 Vortex mixer.
6.7 Water bath, capable of maintaining a temperature of 63 °C ± 1 °C and 95 °C ± 1 °C.
6.8 One-mark volumetric flasks, of capacity 100 ml.
7 Sampling
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 .
A presentative sample should have been sent to the laboratory. It should not have been damaged or
changed during transport or storage.
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8 Preparations
8.1 Alkaline phosphatase-free milk
Prepare phosphatase-free milk of the type to be tested by carefully dispensing the desired portion of
milk into a test tube or suitable container, ensuring that no milk touches the rim or sides of the container.
Place the tube or container with the milk portion in the water bath (6.7) set at 95 °C. Preheat the milk
portion to 95 °C, before starting its 5 min heating period at that temperature. Check the temperature by
using a thermometer or thermistor probe placed in the centre of the tube or container. When the milk
portion reaches 95 °C, immediately start its 5 min heating period. Cool the whole portion rapidly after
the heating period.
Test the thus-treated milk portion to ensure that its ALP activity is less than 10 mU/l.
8.2 Preparation of the test sample
8.2.1 General
Carefully mix all test samples prior to use.
NOTE It is usually not necessary to prewarm test samples.
8.2.2 Pasteurized test samples
Use pasteurized test samples as delivered, in amounts as required.
8.2.3 Dilution of test samples with high ALP values
Prepare dilutions of the test samples of milk using phosphatase-free milk (8.1) in order to bring their
ALP levels within the analytical range of assay (<7 000 mU/l). Mix the diluted solutions well.
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 principles when operating the filter fluorimeter (6.1).
Quality control tests include
a) the daily A/D test, used to check the proper functioning of the equipment;
b) the daily instrument control test, using the daily instrument control solution (5.5) to monitor any
electronic or optical drift in the fluorimeter, and;
c) the use of external positive, negative and normal controls, described in 9.1.3, which are
recommended for monitoring daily instrument precision parameters.
9.1.2 Daily instrument tests
When using the Fluorophos® instrument, perform the A/D tests daily before testing commences.
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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 (5.5) into labelled cuvette. Place the
cuvette in the incubator block (6.4) 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 equilibrate for 15 min.
9.
...
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