SIST EN ISO 22160:2007

SLOVENSKI STANDARD
SIST EN ISO 22160:2007
01-julij-2007
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0HWRGD(3$6(Q]\PDWLFSKRWRDFWLYDWHGV\VWHP,62
Milk and milk-based drinks - Determination of alkaline phosphatase activity - Enzymatic
photo-activated system (EPAS) method (ISO 22160:2007)
Milch und flüssige Milcherzeugnisse - Bestimmung der Aktivität der alkalischen
Phosphatase - Verfahren mit einem enzymatisch photoaktivierten System (EPAS) (ISO
22160:2007)
Lait et boissons a base de lait - Détermination de l'activité de la phosphatase alcaline -
Méthode par un systeme de photoactivation enzymatique (ISO 22160:2007)
Ta slovenski standard je istoveten z: EN ISO 22160:2007
ICS:
67.100.10 0OHNRLQSUHGHODQLPOHþQL Milk and processed milk
SURL]YRGL products
SIST EN ISO 22160:2007 en;fr;de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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EUROPEAN STANDARD
EN ISO 22160
NORME EUROPÉENNE
EUROPÄISCHE NORM
April 2007
ICS 67.100.10 Supersedes EN ISO 11816-1:2000
English Version
Milk and milk-based drinks - Determination of alkaline
phosphatase activity - Enzymatic photo-activated system
(EPAS) method (ISO 22160:2007)
Lait et boissons à base de lait - Détermination de l'activité Milch und flüssige Milcherzeugnisse - Bestimmung der
de la phosphatase alcaline - Méthode par un système de Aktivität der alkalischen Phosphatase - Verfahren mit
photoactivation enzymatique (ISO 22160:2007) einem enzymatisch photoaktivierten System (EPAS) (ISO
22160:2007)
This European Standard was approved by CEN on 6 March 2007.
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 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 Management Centre has the same status as the
official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2007 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 22160:2007: E
worldwide for CEN national Members.

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EN ISO 22160:2007 (E)





Foreword


This document (EN ISO 22160:2007) has been prepared by Technical Committee ISO/TC 34
"Agricultural 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 October 2007, and conflicting national
standards shall be withdrawn at the latest by October 2007.

This document supersedes EN ISO 11816-1:2000.

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, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United
Kingdom.


Endorsement notice

The text of ISO 22160:2007 has been approved by CEN as EN ISO 22160:2007 without any
modifications.


2

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INTERNATIONAL ISO
STANDARD 22160
IDF
209
First edition
2007-04-01

Milk and milk-based drinks —
Determination of alkaline phosphatase
activity — Enzymatic photo-activated
system (EPAS) method
Lait et boissons à base de lait — Détermination de l'activité de la
phosphatase alcaline — Méthode par un système de photoactivation
enzymatique




Reference numbers
ISO 22160:2007(E)
IDF 209:2007(E)
©
ISO and IDF 2007

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ISO 22160:2007(E)
IDF 209:2007(E)
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©  ISO and IDF 2007
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO or IDF at the respective
address below.
ISO copyright office International Dairy Federation
Case postale 56 • CH-1211 Geneva 20 Diamant Building • Boulevard Auguste Reyers 80 • B-1030 Brussels
Tel. + 41 22 749 01 11 Tel. + 32 2 733 98 88
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Published in Switzerland

ii © ISO and IDF 2007 – All rights reserved

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ISO 22160:2007(E)
IDF 209:2007(E)
Contents Page
Foreword. iv
1 Scope . 1
2 Terms and definitions. 1
3 Principle. 1
4 Reagents. 2
5 Apparatus . 3
6 Sampling. 3
7 Preparations . 4
7.1 Test samples . 4
7.2 Alkaline phosphatase-free milk. 4
8 Procedure (see Annex A) . 4
8.1 Calibration . 4
8.2 Calibration adjustment. 6
8.3 Control tests and calibration check. 9
8.4 Determination. 10
8.5 Heat-resistant microbial alkaline phosphatase control. 10
9 Calculation and expression of results. 10
9.1 General. 10
9.2 Manual calculation. 10
9.3 Expression of results . 11
10 Precision. 11
10.1 Interlaboratory test . 11
10.2 Repeatability. 11
10.3 Reproducibility. 12
11 Test report . 12
Annex A (informative) Procedure flowchart . 13
Annex B (informative) Results of interlaboratory trials. 14
Bibliography . 16

© ISO and IDF 2007 – All rights reserved iii

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ISO 22160:2007(E)
IDF 209:2007(E)
Foreword
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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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.
ISO 22160⎪IDF 209 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.
iv © ISO and IDF 2007 – All rights reserved

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ISO 22160:2007(E)
IDF 209:2007(E)
Foreword
IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National
Committee in every member country. Every National Committee has the right to be represented on the IDF
Standing Committees carrying out the technical work. IDF collaborates with ISO in the development of
standard methods of analysis and sampling for milk and milk products.
Draft International Standards adopted by the Action Teams and Standing Committees are circulated to the
National Committees for voting. Publication as an International Standard requires approval by at least 50 % of
the IDF National Committees casting a vote.
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.
ISO 22160⎪IDF 209 was prepared by the International Dairy Federation (IDF) and Technical Committee
ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published jointly by IDF
and ISO.
All work was carried out by the Joint ISO-IDF Action Team on Heat treatment, of the Standing Committee on
Minor components & characterization of physical properties, under the aegis of its project leader, Mr R. Salter
(US).

© ISO and IDF 2007 – All rights reserved v

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ISO 22160:2007(E)
INTERNATIONAL STANDARD
IDF 209:2007(E)

Milk and milk-based drinks — Determination of alkaline
phosphatase activity — Enzymatic photo-activated system
(EPAS) method
1 Scope
This International Standard specifies a method for the determination of the alkaline phosphatase activity in
pasteurized whole milk, semi-skimmed milk, skimmed milk, cream and flavoured milks using a
chemiluminescent (EPAS) method.
The method is applicable to milk and milk-based drinks from cows, sheep, buffalo and goats.
The method is also suitable for any liquid-based sample if diluted in such a way that the diluted alkaline
phosphatase activity has less than 7 000 milliunits per litre.
NOTE There has been a successful collaborative trial with cow, sheep, buffalo, and goat whole milk, as well as
skimmed cow milk (< 0,5 % fat), 20 % fat cream and 2 % fat chocolate milk (all mass fractions).
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1
alkaline phosphatase activity
ALP
activity of the alkaline phosphatase present in the product, determined according to the procedure described
in this International Standard
[4], [5]
NOTE The alkaline phosphatase activity is expressed as milliunits of enzyme activity per litre (mU/l) .
2.2
unit of alkaline phosphatase activity
amount of alkaline phosphatase enzyme that catalyses the transformation of 1 µmol of stable aromatic
substrate per minute
3 Principle
The alkaline phosphatase activity is measured by photo-activation of the hydrolysed product followed by an
instrumental measurement of photo-activation. In the presence of alkaline phosphatase, a stable aromatic
dioxetane-phosphate substrate is hydrolysed at 35 °C ± 1 °C to produce a photo-activated (chemiluminescent)
product. The photo-activation of the product is amplified by a macromolecular enhancing component. The
hydrolysis reaction is stopped after a specified incubation time (3 min). The amount of chemiluminescent
product thus produced is measured and converted to enzyme units by a luminometer. Luminometer calibration
is based on calibration using tablets with known enzyme activity.
© ISO and IDF 2007 – All rights reserved 1

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ISO 22160:2007(E)
IDF 209:2007(E)
4 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralized
water or water of equivalent purity.
4.1 Non-chemiluminescent dioxetane ester substrate [0,2 mol/l 3-(2'-spiroadamantanane)-4-methoxy-4-
(3''-phosphate phenyl-1,2 dioxetane disodium salt in DEAE buffer with 1 % fluorosine], is commercially
®
1)
available [e.g. as Charm reagent AP liquid].
It is recommended to store the substrate at between 0 °C and 7 °C. If stored in amber plastic vials and at 4 °C,
the substrate remains stable for 6 months. If stored at 30 °C, the substrate is stable for 24 h only.
When using the substrate in the assay, keep it at between 0 °C and 7 °C or on ice.
4.2 Stopping solution
Prepare the stopping solution by mixing the same amount of 0,15 mol/l 2-amino-2-methyl-1-propanol and
0,02 % benzalkonium chloride, pH 10,7. The stopping solution shall be at room temperature (18 °C to 24 °C)
prior to use. For assay consistency when not using a thermoprobe, record and maintain the temperature to
within 0,5 °C of the solution temperature used at calibration.
NOTE The stopping solution is used to stop enzymatic hydrolysis of the dioxetane ester substrate (4.1).
Commercially supplied stopping solution has a shelf life of 1 year when kept at 4 °C, or of 2 months when kept
at room temperature. For daily use, room temperature storage is recommended.
4.3 Working calibrators, for example, calibration tablets (dried raw milk with measured phosphatase
content in tablet form to rehydrate in milk) with a phosphatase activity of 875 µU/l ± 26 µU/l. Rehydrate the
tablets in three different volumes of milk-based drink presenting no phosphatase activity or negative test
sample (7.2) to create a standard calibration curve.
Commercially supplied calibration tablets may be stored at 4 °C for 2 years.
4.3.1 Fluid white milk calibrators
In each of three 50 ml test tubes (5.9), marked A , B and C , respectively, dissolve one calibrator tablet in
1 1 1
100 µl of distilled water.
Add 20 ml to tube A , 5 ml to tube B and 2,5 ml to tube C of fluid white milk products (presenting no
1 1 1
phosphatase activity) or negative test sample (7.2). This makes calibration standards A , B and C with a

1 1 1
phosphatase activity of A = 44 mU/l, B = 175 mU/l and C = 350 mU/l, respectively. Cap the tubes and
1 1 1
shake their contents vigorously. Allow the tube contents to rehydrate under refrigeration for 10 min. Mix well
before use.
4.3.2 Cream and flavoured-milk calibrators
In each of three 50 ml test tubes (5.9), marked A , B and C , respectively, dissolve one calibrator tablet in
2 2 2
100 µl of distilled water.
Add 10 ml to tube A , 5 ml to tube B and 2,5 ml to tube C of cream or flavoured-milk products (presenting no
2 2 2
phosphatase activity) or negative test sample (7.2), making calibration standards A , B , C with phosphatase
2 2 2
activities of A = 88 mU/l, B = 175 mU/l and C = 350 mU/l, respectively. Cap the tubes and shake their

2 2 2
contents vigorously. Allow the tube contents to rehydrate under refrigeration for 10 min. Mix well before use.

1) The reagents specified in Clause 4 and the apparatus specified in 5.1 are available from Charm Sciences Inc., 659
Andover St., Lawrence, MA 01843, USA. These are examples of suitable products 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. Equivalent products may be used if they can be shown to produce the same results.
2 © ISO and IDF 2007 – All rights reserved

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ISO 22160:2007(E)
IDF 209:2007(E)
4.4 Positive control
As positive control, use freeze-dried alkaline phosphatase in a 15 ml amber bottle. Rehydrate the positive
control with 10 ml of a representative dairy drink presenting no phosphatase activity, or with a prepared
negative test sample (7.2). Rehydrated positive control contains 450 mU/l of phosphatase enzyme.
Allow the control to stand for 10 min to rehydrate. Vigorously shake before use.
The rehydrated positive control is stable when stored at between 0 °C and 7 °C for 48 h. Positive control
rehydrated with fluid milk is stable frozen at or below −15 °C for 2 months. Thaw the positive control in water
at room temperature. Vigorously shake the control to homogenize before use. Do not re-freeze.
5 Apparatus
Usual laboratory equipment and, in particular, the following.
5.1 Luminometer, capable of operating at a wavelength of 540 nm, with linear outputs being converted by
®
1)
internal software into enzyme activity [e.g. Charm Luminometer models NovaLum, Luminator K or T ].
A vial adapter is needed with NovaLum and Lum-T model. NovaLum and Lum-T should be used in the
propped up position. A temperature probe supported by NovaLum should be used to measure the stopping
solution (4.2) temperature.
Measurements should be optimized according to the manufacturer's recommendations for the apparatus used.
5.2 Mini-vials, disposable, made of non-luminescent plastic, with caps, and of capacity 2 ml.
5.3 Fixed volume pipette, of capacity 100 µl.
5.4 Fixed-volume dispenser, capable of dispensing 1,0 ml. Check that the volume of water dispensed is
accurate to 1,00 g ± 0,05 g before use.
5.5 One-mark volumetric flasks, of capacity 100 ml.
5.6 Analytical balance, capable of weighing to the nearest 1 mg.
5.7 Incubator block or dry well bath, capable of operating at 35 °C ± 1 °C and 63 °C ± 0,2 °C with wells of
mini-vial dimensions.
5.8 Water bath, adjustable, capable of operating at 63 °C ± 0,2 °C and 95 °C ± 2 °C.
5.9 Test tubes, of capacity 50 ml, of diameter 13 mm and length 100 mm, with leakproof caps.
5.10 Printer, with connection cables for printout of results.
6 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 International Standard. A recommended sampling method
is given in ISO 707⎪IDF 50.
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ISO 22160:2007(E)
IDF 209:2007(E)
7 Preparations
7.1 Test samples
7.1.1 General
Carefully mix all test samples prior to use. The ambient temperature of the room should be between 18 °C and
24°C.
Samples should be tested at refrigeration temperature (0 to 7) °C or on ice during assay.
7.1.2 Pasteurized samples
Use the test samples as obtained in amounts as required.
7.1.3 Raw milk
Pipette 1 ml (or an appropriate amount such that the activity after dilution is less than 7 000 mU/l) of test
sample into a 100 ml one-mark volumetric flask (5.5). Dilute to the mark with alkaline phosphatase-free milk
(7.2). Mix carefully.
7.1.4 Flavoured-milk products and cream products
Use the test samples as obtained in amounts as required. Test samples of viscous products may require a
pipetting accuracy determination using mass (100 µl = 100 mg). Wipe the pipette tips after drawing the sample.
Possibly, modification of the pipette tip (cutting wider opening) might be required for accurate dispensing.
7.2 Alkaline phosphatase-free milk
Prepare alkaline phosphatase-free milk (negative test sample) by heating 35 ml or necessary volume of a test
portion (7.1.2, 7.1.3 or 7.1.4) in a tube in a water bath (5.8) set at 95 °C. Allow the test portion to reach 95 °C
and keep it at this temperature for 1 min. Then cool rapidly.
The negative sample is used as the 0 (zero) calibrator and shall have a mean value of less than 5 mU/l (or
less than 15 mU/l with flavoured-milk products and cream products) in the appropriately calibrated
luminometer channel. If stored at 4 °C, the negative sample may be kept for 48 h.
Fluid white milks may be kept for up to 6 months if stored frozen at or below −15 °C. Thaw the negative
sample in water at room temperature. Vigorously shake the sample to make it homogeneous before use. Do
not re-freeze.
It should be noted that some milk products, such as sheep milk, will precipitate and separate when the
negative test sample is prepared at 95 C for 1 min. In this case, a lower temperature for a longer time is
required, e.g. 63 °C for 30 min.
8 Procedure (see Annex A)
8.1 Calibration
8.1.1 Establish a calibration curve for each type of product to be tested. Begin by setting the luminometer
background (B ) to 100 and the correction (C ) to 100.
g r
Consult the luminometer manual for instructions on making adjustments. Calibration curves are stable and
need to be run when new batch lots/lot numbers of the dioxetane ester substrate (4.1) and stopping buffer
(4.2) are used. A flowchart for the calibration is given in Annex A.
4 © ISO and IDF 2007 – All rights reserved

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ISO 22160:2007(E)
IDF 209:2007(E)
NOTE The calibration mode on some luminometers automatically guides users through steps 8.1.1 to 8.2.2. (The
calibration menu is item 8 of the main menu.) Select alkaline phosphatase calibration. Select channel to calibrate. Select
calibrate. Select test portion to calibrate, for example milk, cream or chocolate (flavoured-milk product) or other. The
luminometer prompts the user for the next steps.
8.1.2 Limit calibration assays to three tubes per assay. Use the fixed-volume pipette (5.3), provided with a
clean tip, to add 100 µl of the dioxetane ester substrate (4.1) to the bottom of three mini-vials (5.2).
8.1.3 For the test portions obtained in 7.1.2 to 7.1.4, add, with the fixed-volume pipette (5.3) provided with a
clean tip, 100 µl of negative test sample (7.2) to each vial. Deliver the contents of the sample to the bottom of
the tube to ensure all of the sample contacts the substrate. Mix the contents of the vials in the vial rack with a
back and forth motion for about 10 times in 5 s.
8.1.4 Place the mini-vials (8.1.3) in the incubator block (5.7) set at 35 °C for 3 min. At the end of incubation,
add, with the fixed-volume dispenser (5.4), within 15 s, 1,0 ml of stopping solution (4.2) to the contents of all
mini-vials.
8.1.5 Remove the mini-vials from the incubator. Cap each vial and shake each vigorously for 5 s.
If necessary unscrew the cap and attach the vial to the luminometer adapter. Insert the vial into the
luminometer. Read and record the readings of the luminometer (5.1) at count completion (beep). Repeat with
each vial. Do not touch the liquid solution with the adapter to avoid contaminating the next vial. If the solution
does contact the vial, rinse it with water and dry before re-use. Calculate the mean negative value (N). Repeat
the determination specified in 8.1.2 to 8.1.5 if any one value varies by more than 30 % of the mean.
8.1.6 For the test portions obtained in 7.1.2 to 7.1.3, repeat steps 8.1.2 through 8.1.5 with calibrator solution
C (4.3.1), each in triplicate. For the test portion obtained in 7.1.4, repeat steps 8.1.2 through 8.1.5 with
1
calibrator solution C (4.3.2), each in triplicate. Calculate the mean calibrator C or C value (C ). Repeat this
2 1 2 x
determination if any one value varies by more than 30 % of the mean.
8.1.7 Calculate the correction value, C , using the mean calibration value, C (see 8.1.6), and the mean
r x
negative value, N (see 8.1.5) in Equation (1). Enter C in the luminometer. Consult the luminometer manual for
r
instructions on how to change the luminometer correction to the new correction value number C :
r
CC=−( N)× 0,286 (1)
r x
NOTE Automated luminometers will print N and C and automatically calculate and adjust the values of C and B .

x r g
8.1.8 Calculate the background value using C and the mean negative value N in Equation (2), and enter
r
the value B in the luminometer. Consult the luminometer manual for instructions on how to change the
g
luminometer background to the new background value number B :
g
BN=+[ /(C /100)] 100 (2)
gr
NOTE Automated luminometers will print N and C and automatically calculate, adjust and print C and B .
x r g
8.1.9 Repeat steps 8.1.2 to 8.1.5 with the negative test sample (7.2), in triplicate. Verify whether the mean
value with milk (in mU/l) is less than 5 mU/l, or in the case of flavoured-milk products and creams, less than
15 mU/l. If the mean value is out of range, go to step 8.1.1.
8.1.10 Repeat step 8.1.6 with calibrator C or C . The mean reading target range is between 320 mU/l and
1 2
400 mU/l for calibrator C (either C or C ). Repeat this determination if any one value varies by more than
1 2
30 % of the mean. If that value is out of range, go to step 8.1.1.
8.1.11 Perform the calibration assay (8.1.2 to 8.1.5) substituting in step 8.1.3 working calibrator A (A or A
1 2
depending the test portion mentioned in 4.3) in place of the negative sample. Determine the mean value for
calibrator A or A . Repeat this determination if any one value varies by more than 40 % of the mean value.
1 2
© ISO and IDF 2007 – All rights reserved 5

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ISO 22160:2007(E)
IDF 209:2007(E)
8.1.12 Perform the calibration assay (8.1.6) substituting working calibrator B (B or B depending the test
1 2
portion mentioned in 4.3) in place of calibrator C in step 8.1.6. Determine the mean value for calibrator B.
Repeat thi
...