1、March 2014Translation by DIN-Sprachendienst.English price group 12No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 6
2、7.100.10!%,k“2097295www.din.deDDIN EN ISO 11816-1Milk and milk products Determination of alkaline phosphatase activity Part 1: Fluorimetric method for milk and milk-based drinks(ISO 11816-1:2013);English version EN ISO 11816-1:2013,English translation of DIN EN ISO 11816-1:2014-03Milch und Milcherze
3、ugnisse Bestimmung der Aktivitt der alkalischen Phosphatase Teil 1: Fluorimetrisches Verfahren fr Milch und flssige Milchprodukte(ISO 11816-1:2013);Englische Fassung EN ISO 11816-1:2013,Englische bersetzung von DIN EN ISO 11816-1:2014-03Lait et produits laitiers Dtermination de lactivit de la phosph
4、atase alcaline Partie 1: Mthode fluorimtrique pour le lait et les boissons base de lait(ISO 11816-1:2013);Version anglaise EN ISO 11816-1:2013,Traduction anglaise de DIN EN ISO 11816-1:2014-03SupersedesDIN EN ISO 11816-1:2006-07www.beuth.deIn case of doubt, the German-language original shall be cons
5、idered authoritative.Document comprises 20 pages02.14 DIN EN ISO 11816-1:2014-03 2 A comma is used as the decimal marker. National foreword This document (EN ISO 11816-1:2013) has been prepared by Technical Committee ISO/TC 34 “Food products” (Secretariat: AFNOR, France), Subcommittee SC 5 “Milk and
6、 milk products” (Secretariat: NEN, Netherlands) in collaboration with Technical Committee CEN/TC 302 “Milk and milk products Methods of sampling and analysis” (Secretariat: NEN, Netherlands). The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtsch
7、aftliche Produkte (Food and Agricultural Products Standards Committee), Working Committee NA 057-05-13 AA Milch und Milcherzeugnisse Probenahme- und Untersuchungsverfahren. DIN EN ISO 11816 consists of the following parts, under the general title Milk and milk products Determination of alkaline phos
8、phatase activity: Part 1: Fluorimetric method for milk and milk-based drinks Part 2: Fluorimetric method for cheese Another method to determine the alkaline phosphatase activity is given in DIN EN ISO 22160, Determination of alkaline phosphatase activity Enzymatic photo-activated system (EPAS) metho
9、d. The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 707 DIN EN ISO 707 ISO 5725-1 DIN ISO 5725-1 ISO 5725-2 DIN ISO 5725-2 Amendments This standard differs from DIN EN ISO 11816-1:2006-07 as follows: a) Clause 1 “Scope” has been revised
10、to consider phosphatase activities in raw milk higher than 7 000 mU/l; b) subclauses concerning A/D tests and positive and negative control tests have been revised; c) Subclause 8.2 “Reagent controls to test the suitability of ready-to-use working substrate (4.3)” has been added; d) precision data h
11、as been updated; e) the results from two collaborative trials completed in 2008 and 2010 have been incorporated; f) the standard has been technically and editorially revised and brought in line with current rules of presentation. Previous editions DIN EN ISO 11816-1: 2000-07, 2006-07 DIN EN ISO 1181
12、6-1:2014-03 3 National Annex NA (informative) Bibliography DIN EN ISO 707, Milk and milk products Guidance on sampling DIN ISO 5725-1, Accuracy (trueness and precision) of measurement methods and results Part 1: General principles and definitions DIN ISO 5725-2, Accuracy (trueness and precision) of
13、measurement methods and results Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method DIN EN ISO 11816-1:2014-03 4 This page is intentionally blank EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM November 2013 ICS 67.100.10 Supersedes EN I
14、SO 11816-1:2006English Version Milk and milk products - Determination of alkaline phosphatase activity - Part 1: Fluorimetric method for milk and milk-based drinks (ISO 11816-1:2013) Lait et produits laitiers - Dtermination de lactivit de la phosphatase alcaline - Partie 1: Mthode fluorimtrique pour
15、 le lait et les boissons base de lait(ISO 11816-1:2013) Milch und Milcherzeugnisse - Bestimmung der Aktivitt der alkalischen Phosphatase - Teil 1: Fluorimetrisches Verfahren fr Milch und flssige Milchprodukte(ISO 11816-1:2013)This European Standard was approved by CEN on 14 September 2013. CEN membe
16、rs 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 applicatio
17、n 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 Manageme
18、nt 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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, L
19、ithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17
20、, B-1000 Brussels 2013 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 11816-1:2013 EEN ISO 11816-1 Contents Page Foreword . 3 1 Scope . 4 2 Terms and definitions 4 3 Principle 4 4 Reagents . 4 5 Apparatus 5 6 Sampling . 6 7 Pr
21、eparations . 6 7.1 Alkaline phosphatase-free milk 6 7.2 Preparation of test sample 7 8 Procedure 7 8.1 Verification of instrument performance . 7 8.2 Reagent controls to test the suitability of ready-to-use working substrate (4.3) 8 8.3 Calibration . 8 8.4 Determination . 9 8.5 Test sample-related c
22、ontrols 9 9 Calculation and expression of results . 10 9.1 Calibration ratio 10 9.2 Calculation 11 9.3 Expression of test results . 11 10 Precision . 11 10.1 Collaborative test . 11 10.2 Repeatability . 11 10.3 Reproducibility . 11 11 Test report . 12 Annex A (informative) Collaborative trials 13 Bi
23、bliography 16 2DIN EN ISO 11816-1:2014-03EN ISO 11816-1:2013 (E) This document (EN ISO 11816-1:2013) 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 w
24、hich 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 May 2014, and conflicting national standards shall be withdrawn at the latest by May 2014. Attention is drawn to the possibilit
25、y that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. This document supersedes EN ISO 11816-1:2006. According to the CEN-CENELEC Internal Regulations, the national standards or
26、ganizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembour
27、g, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO 11816-1:2013 has been approved by CEN as EN ISO 11816-1:2013 without any modification. ”Foreword3DIN EN ISO 11816-1:2014-03EN IS
28、O 11816-1:2013 (E)EN ISO 11816|IDF 155 consists of the following parts, under the general title Milk and milk products Determination of alkaline phosphatase activity: Part 1: Fluorimetric method for milk and milk-based drinks Part 2: Fluorimetric method for cheese 1 ScopeThis part of ISO 11816|IDF 1
29、55 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 applicab
30、le 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 Terms and definitionsFor th
31、e purposes of this document, the following terms and definitions apply.2.1alkaline phosphatase (ALP) activityactivity of the alkaline phosphatase present in the product, determined by the specified procedureNote 1 to entry: The alkaline phosphatase activity is expressed as milliunits of enzyme activ
32、ity per litre of sample (mU/l).2.2unit of alkaline phosphatase activityamount of alkaline phosphatase enzyme that catalyses the transformation of 1 mol of substrate per minute3 PrincipleThe alkaline phosphatase activity of the sample is measured by a continuous fluorimetric direct kinetic assay. A n
33、on-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 alkalin
34、e phosphatase (ALP) activity is measured at 38 C over a 3-min period when 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
35、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).4 ReagentsUse only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralized water, or water o
36、f equivalent purity.4DIN EN ISO 11816-1:2014-03EN ISO 11816-1:2013 (E) 4.1 Fluorophos substrate,1)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-hydroxybenz
37、othiazole 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.4.2 Substrate buffer solution, diethanolamine (DEA) buffer solution, c(DEA) = 2,4 mol/l, with
38、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.4.3 Working substrateAllow the Fluorophos substrate (4.1) and the substrate buffer solu
39、tion (4.2) to come to room temperature. Add the content of one bottle of substrate buffer solution (240 ml) (4.2) to that of one bottle of Fluorophos substrate (144 mg) (4.1), and mix well by inversion for 3 min to create 1,0 millimolar (pH 10) solution. Use amber glass to protect against light.Allo
40、w 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 8.2 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 (8.2
41、).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.4.4 Calibrator solutions, Fluoroyellow(FY) 2-(2-benzothi
42、azolyl)-6-hydroxybenzothiazole in substrate buffer solution (4.2).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.4.4.1 Calibrator solution A, con
43、taining 0 mol/l of Fluoroyellow.4.4.2 Calibrator solution B, containing 17,24 103mol/l of Fluoroyellow.4.4.3 Calibrator solution C, containing 34,48 103mol/l of Fluoroyellow.4.5 Daily instrument control solution, containing 34,48 x103mol/l of Fluoroyellow.The daily instrument control solution remain
44、s 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 ApparatusUsual laboratory equipment and, in particular, the following.1) The reagents specified in 4.1 to 4.5 and the apparatus s
45、pecified in 5.1 to 5.4 (except 5.3.3) comprise the Fluorophos Test System, which is the trade name of a product supplied by Advanced Instruments, Inc., Two Technology Way, Norwood, Massachusetts 02062, USA. The manufacturer may change the packaging configurations supplied with Fluorophos Test system
46、. The user should refer to the manufacturers 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 endor
47、sement by ISO or IDF of the products named. Equivalent products may be used if they can be shown to lead to the same results.5DIN EN ISO 11816-1:2014-03EN ISO 11816-1:2013 (E)5.1 Filter fluorimeter, with thermostatically controlled cuvette holder, capable of operating at 38 C 1 C and right-angle opt
48、ics, allowing excitation at a wavelength of 440 nm and emission at between 520 nm and 560 nm e.g. Fluorophos instrument1).5.2 Cuvettes, disposable, of non-fluorescent glass.5.3 Pipettes5.3.1 Fixed-volume dispenser, capable of dispensing 2,0 ml.5.3.2 Positive-displacement or air displacement pipette,
49、 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.5.3.3 Pipettes, of capacity 2 ml and 3 ml.5.4 Incubator block, capable of maintaining a temperature of 38 C 1 C, suitable for holding cuvettes.5.5 Suitable laboratory-grade film.5.6 Vortex mix
