ISO/TS 4985:2023

TECHNICAL ISO/TS
SPECIFICATION 4985
IDF/RM 255
First edition
2023-07
Milk and milk products —
Determination of alkaline
phosphatase activity — Fluorimetric
microplate method
Reference numbers
IDF/RM 255:2023(E)
IDF/RM 255:2023(E)
© ISO and IDF 2023
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Published in Switzerland
ii
IDF/RM 255:2023(E)
Contents Page
Forewords .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents . 2
6 Apparatus and materials .3
7 Sampling . 4
8 Preparation . 4
8.1 Preparation of alkaline phosphatase-free sample . 4
8.1.1 General . 4
8.1.2 Alkaline phosphatase-free milk . 4
8.1.3 Alkaline phosphatase-free cheese . 4
8.2 Preparation of test sample . 5
8.2.1 Milk samples. 5
8.2.2 Cheese samples . 5
9 Procedure .5
9.1 Instrument . 5
9.1.1 Instrument settings . 5
9.1.2 Test the linearity range of the instrument . 6
9.1.3 Quality control . 6
9.2 Calibration . 6
9.2.1 General . 6
9.2.2 Preparation of the working standard solutions . 6
9.2.3 Preparation of the calibration curve . 7
9.3 Determination . 7
9.3.1 Milk samples. 7
9.3.2 Cheese samples . 8
9.3.3 Plate layout and fluorescence measurement . 9
9.4 Test sample-related controls. 9
9.4.1 Recommended negative and positive controls . 9
9.4.2 Interfering substance test . 10
9.4.3 Heat-stable microbial alkaline phosphatase control test . 10
10 Calculation and expression of results .10
10.1 Calibration curve . 10
10.2 Calculation of ALP activity of unknown samples . 11
10.2.1 Milk samples. 11
10.2.2 Cheese samples .12
10.2.3 Calculation example .13
10.3 Expression of results . . 14
11 Precision .14
11.1 Interlaboratory test . . 14
11.2 Repeatability . 14
11.3 Reproducibility . 14
12 Test report .15
Annex A (informative) Precision data .16
Annex B (informative) Method comparison .17
Annex C (informative) Examples of preparation of test sample .19
iii
IDF/RM 255:2023(E)
Bibliography .21
iv
IDF/RM 255:2023(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
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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 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.
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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). It is being published jointly by ISO
and IDF.
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.
v
IDF/RM 255:2023(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 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 on P20 of the Standing Committee on Analytical
Methods for Processing Aids and Indicators under the aegis of its project leader Dr C. Egger (CH).
vi
TECHNICAL SPECIFICATION
IDF/RM 255:2023(E)
Milk and milk products — Determination of alkaline
phosphatase activity — Fluorimetric microplate method
1 Scope
This document specifies a fluorimetric microplate method for the determination of alkaline phosphatase
[5]
(ALP, EC 3.1.3.1) activity in raw and heat-treated whole milk, semi-skimmed milk, skimmed milk,
cream, flavoured milks and cheeses.
This method is applicable to milk and milk-based drinks from cows, sheep and goats. Although the
method was not tested in milk from other species, it can also be applicable to milk from other species
with a similar composition to cow, sheep or goat milk, such as milk from buffalo and camelids. It is also
applicable to milk powder after reconstitution and soft, 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).
NOTE This method was adapted from Reference [6].
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
amount of enzyme that is capable of catalysing the transformation of 1 μmol of substrate per minute
under the conditions of the specified procedure
Note 1 to entry: The ALP activity in milk is expressed as milliunits of enzyme activity per litre of sample (mU/l)
and as milliunits of enzyme activity per gram of sample (mU/g) in the case of cheese.
4 Principle
The ALP activity of the sample is measured by a continuous fluorimetric direct kinetic assay. A non-
fluorescent aromatic monophosphoric substrate, 4-methylumbelliferone phosphate (4-MUP), in the
presence of any ALP derived from the sample, undergoes hydrolysis of its phosphate, producing the
highly fluorescent product 4-methylumbelliferone (4-MU). Fluorimetric measurement of ALP activity is
measured at 37 °C, over a 15 min period.
The measured fluorescence is proportional to the concentration of the emitted fluorescent product and
is used to calculate the enzyme activity.
IDF/RM 255:2023(E)
5 Reagents
Use only reagents of recognized analytical grade.
5.1 Magnesium chloride solution, substance concentration, c(MgCl ) = 1 mol/l.
Weigh 50,8 g of magnesium chloride hexahydrate (MgCl · 6 H O, molecular mass is 203,3 g/mol) in
2 2
a 50 ml glass beaker (6.17), dissolve with distilled water and transfer in a volumetric flask of 250 ml
(6.18). Adjust the volume with water up to the mark.
This solution can be stored in aliquots (e.g. 10 ml) at below −20 °C for one year.
TM ®1) 2)
5.2 Tergitol 15-S-9 (CAS Registry Number 84133-50-6) solution, c = 100 g/l.
TM
Weigh 25,0 g of Tergitol 15-S-9 in a 150 ml glass beaker (6.17), dissolve by stirring gently with
distilled water at 25 °C ± 2 °C and transfer in a volumetric flask of 250 ml (6.18). Adjust the volume with
water up to the mark.
This solution can be stored in aliquots (e.g. 15 ml) at a temperature between 2 °C and 8 °C for one year.
5.3 2-amino-2-methyl-1-propanol (AMP) buffer solution, c = 0,11 mol/l, pH = 10,1.
Weigh 9,8 g of AMP, purity > 95 %, molecular mass is 89,14 g/mol, in a 600 ml glass beaker (6.17),
add 500 ml of distilled water on a stirrer and adjust pH to 10,1 with a hydrochloric acid solution
c(HCl) = 5 mol/l. Transfer in a 1 000 ml volumetric flask (6.18) and adjust with water up to the mark.
This solution can be stored in aliquots (e.g. 30 ml) at below −20 °C for one year.
2+
5.4 Diethanolamine (DEA) buffer solution, c = 2 mol/l, pH = 9,8, c(Mg ) = 0,5 mmol/l.
Weigh 210,3 g of DEA, molecular mass is 105,14 g/mol, in a 1 000 ml glass beaker (6.17), add 700 ml of
distilled water while stirring and adjust pH to 9,8 with a hydrochloric acid solution c(HCl) = 5 mol/l.
Add 0,5 ml of MgCl solution (5.1). Transfer in a 1 000 ml volumetric flask (6.18) and adjust with water
up to the mark.
This solution can be stored in aliquots (e.g. 50 ml) at below −20 °C for one year.
2+ TM
5.5 DEA extraction buffer, c = 1,5 mol/l, pH = 9,8, c(Mg ) = 1,5 mmol/l, c(Tergitol ) = 0,1 %.
Weigh 157,7 g of DEA in a 600 ml glass beaker (6.17), add 500 ml of distilled water on a stirrer and adjust
pH to 9,8 with a hydrochloric acid solution c(HCl) = 5 mol/l HCl 5 mol/l. Add 1,5 ml of MgCl solution
TM
(5.1) and 10 ml of Tergitol 15-S-9 solution (5.2). Transfer in a 1 000 ml volumetric flask (6.18) and
adjust with water up to the mark.
This solution can be stored in aliquots (e.g. 200 ml) at below −20 °C for one year.
5.6 4-methylumbelliferone sodium salt (4-MU) standard stock solution, c = 2,5 mmol/l.
Weigh 9,9 mg of 4-MU, purity > 98 %, molecular mass is 198,16 g/mol, in a 25 ml glass beaker (6.17),
solubilize and rinse well with the AMP buffer solution (5.3), transfer to a volumetric flask of 20 ml (6.18)
and fill up to the mark with the same buffer. Keep solution in the dark. ®
1) Chemical Abstracts Service (CAS) Registry Number is a trademark of the American Chemical Society (ACS).
This information is given for the convenience of users of this document and does not constitute an endorsement by
ISO of the product named. Equivalent products may be used if they can be shown to lead to the same results.
2) This 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 the product named. Equivalent
products may be used if they can be shown to lead to the same results.
IDF/RM 255:2023(E)
This solution can be stored in aliquots (e.g. 200 µl) at below −20 °C for one year.
5.7 4-methylumbelliferone phosphate (4-MUP) substrate solution, c = 2,5 mmol/l.
Weigh 12,8 mg of 4-MUP, molecular mass is 256,15 g/mol, in a 25 ml glass beaker (6.17), solubilize and
rinse well with the DEA buffer solution (5.4), transfer in a volumetric flask of 20 ml (6.18) and fill up to
the mark with the same buffer.
Prepare the solution freshly and keep in the dark.
6 Apparatus and materials
Usual laboratory equipment and, in particular, the following shall be used.
6.1 Fluorescence microplate reader, capable of reading 96-well microplates at an excitation
wavelength of 365 nm and emission wavelength of 450 nm. Capable of temperature control at
37 °C ± 1 °C and allowing kinetic measurements (e.g. one reading per minute for 15 min).
6.2 Microplates, black, flat bottom, 96 well.
6.3 Pipette, of capacity 20 µl to 100 µl.
6.4 Pipette, of capacity 100 µl to 1 000 µl.
6.5 Multichannel pipettor, capable of dispensing 20 µl to 100 µl.
6.6 Air displacement pipette, of capacity 5 ml.
A glass pipette can also be used.
6.7 Microtubes, of capacity 2 ml.
6.8 Tubes, of capacity 15 ml and 50 ml.
6.9 Glass test tube, of approximately diameter 12 mm and length 10 cm.
6.10 Water bath, heating block or incubator suitable of maintaining a temperature of 37 °C ± 1 °C,
63 °C ± 1 °C and 95 °C ± 1 °C.
6.11 Vortex mixer.
®3)
6.12 Parafilm , or other suitable laboratory-grade film.
6.13 Aluminium foil.
6.14 Grinding device.
®4)
6.15 ULTRA-TURRAX , or other homogenizer provided with a stem of diameter of approximately
6 mm to 8 mm. ®
3) 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. ®
4) ULTRA-TURRAX 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.
IDF/RM 255:2023(E)
6.16 Centrifuge, capable of centrifuging at 1 000g at 4 °C for 10 ml or 15 ml, and 50 ml tubes.
6.17 Glass beaker, of capacity 5 ml (approximately diameter 20 mm and length 30 mm), 10 ml (of
approximately diameter 25 mm and length 30 mm), 25 ml, 50 ml, 150 ml, 600 ml and 1 000 ml.
6.18 One-mark volumetric flasks, of capacity 20 ml, 25 ml, 250 ml and 1 000 ml.
6.19 Analytical balance.
6.20 pH meter.
7 Sampling
A representative sample should have been sent to the laboratory. It should not have been damaged or
changed during transport or storage.
Sampling is not part of the method specified in this document. A recommended sampling method is
[1]
given in ISO 707 | IDF 50 .
[1]
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 C.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
8.1 Preparation of alkaline phosphatase-free sample
8.1.1 General
The ALP-free sample is used as sample blank, for calibration and for sample dilution if necessary.
8.1.2 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. Cover the tube or container containing the milk portion and place it in the water bath or in
the heating block (6.10) 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.
8.1.3 Alkaline phosphatase-free cheese
For each type of cheese to be tested, prepare a phosphatase-free cheese from the supernatant of the
cheese (see 9.3.2.6) by heating a portion of the supernatant as described in 8.1.2, replacing the milk by
the supernatant.
IDF/RM 255:2023(E)
8.2 Preparation of test sample
8.2.1 Milk samples
8.2.1.1 General
Carefully mix all test samples prior to use.
NOTE It is usually not necessary to prewarm test samples.
8.2.1.2 Pasteurized test samples
Use pasteurized test samples as delivered, in amounts as required.
8.2.1.3 Dilution of test samples with high ALP values
Prepare dilutions of the test samples of milk using phosphatase-free milk (see 8.1.2) in order to bring
their ALP levels within the linearity ran
...