1、BSI Standards PublicationBS EN ISO 11816-1:2013Milk and milk products Determination of alkalinephosphatase activityPart 1: Fluorimetric method for milk andmilk-based drinksBS EN ISO 11816-1:2013 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN ISO11816-1:2013. It
2、 supersedes BS EN ISO 11816-1:2006 which iswithdrawn.The UK participation in its preparation was entrusted to TechnicalCommittee AW/5, Chemical analysis of milk and milk products.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does no
3、t purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2013. Published by BSI StandardsLimited 2013ISBN 978 0 580 76891 0ICS 67.100.10Compliance with a British Standard cannot confer immunity fromlegal oblig
4、ations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 30 November 2013.Amendments issued since publicationDate Text affectedEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 11816-1 November 2013 ICS 67.100.10 Supersedes EN ISO 11816-1
5、: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 le lait e
6、t 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 members are
7、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 th
8、e 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 Management Cent
9、re 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, Lithuani
10、a, 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, B-100
11、0 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 EBS EN ISO 11816-1:2013EN ISO 11816-1:2013 (E) 3 Foreword This document (EN ISO 11816-1:2013) has been prepared by Technical Committee ISO/TC 34 “Food
12、 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,
13、at the latest by May 2014, and conflicting national standards shall be withdrawn at the latest by May 2014. Attention is drawn to the possibility 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 al
14、l such patent rights. This document supersedes EN ISO 11816-1:2006. 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, Croatia, Cyprus, Czech Republic, Denmark,
15、Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endorsement notice
16、The text of ISO 11816-1:2013 has been approved by CEN as EN ISO 11816-1:2013 without any modification. BS EN ISO 11816-1:2013ISO 11816-1:2013(E)IDF 155-1:2013(E) ISO and IDF 2013 All rights reserved iiiContents PageForeword v1 Scope . 12 Terms and definitions . 13 Principle 14 Reagents 15 Apparatus
17、. 26 Sampling 37 Preparations . 37.1 Alkaline phosphatase-free milk 37.2 Preparation of test sample 48 Procedure. 48.1 Verification of instrument performance . 48.2 Reagent controls to test the suitability of ready-to-use working substrate (4.3) . 58.3 Calibration 58.4 Determination . 68.5 Test samp
18、le-related controls 69 Calculation and expression of results . 79.1 Calibration ratio . 79.2 Calculation 89.3 Expression of test results . 810 Precision . 810.1 Collaborative test 810.2 Repeatability . 810.3 Reproducibility . 811 Test report . 9Annex A (informative) Collaborative trials 10Bibliograp
19、hy .13BS EN ISO 11816-1:2013ISO 11816-1:2013(E)ForewordISO (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 mem
20、ber 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 El
21、ectrotechnical 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
22、 of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2. www.iso.org/directivesAttention 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 res
23、ponsible 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. www.iso.org/patentsAny trade name used in this document is information given for
24、the convenience of users and does not constitute an endorsement.The committee responsible for this document is 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.This third edition of ISO 118
25、16-1|IDF 155-1 cancels and replaces the second edition (ISO 11816-1:2006), which has been technically revised.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 mil
26、k-based drinks Part 2: Fluorimetric method for cheeseIDF 155-1:2013(E)iv ISO and IDF 2013 All rights reservedBS EN ISO 11816-1:2013ISO 11816-1:2013(E)ForewordIDF (the International Dairy Federation) is a non-profit organization representing the dairy sector worldwide. IDF membership comprises Nation
27、al Committees in every member country as well as regional dairy associations having signed a formal agreement on cooperation with IDF. All members of IDF have the right to be represented on the IDF Standing Committees carrying out the technical work. IDF collaborates with ISO in the development of s
28、tandard methods of analysis and sampling for milk and milk products.The main task of Standing Committees is to prepare International Standards. Draft International Standards adopted by the Standing Committees are circulated to the National Committees for endorsement prior to publication as an Intern
29、ational Standard. Publication as an International Standard requires approval by at least 50 % of 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 identify
30、ing 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.ISO 11816-1|IDF 155-1 was prepared by the International Dairy Federation (IDF) and Technical Committee ISO/TC 34, Food products, Subcommitte
31、e 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 Project Group on Determination of alkaline phosphatase activity fluorimetric method, of the Standing Committee on Analytical Methods for Processing Aids and Indicators, under th
32、e aegis of its project leader, Ms. Eileen Garry (USA). This third edition of ISO 11816-1|IDF 155-1 cancels and replaces IDF 155-1:2006, which has been technically revised.ISO 11816|IDF 155 consists of the following parts, under the general title Milk and milk products Determination of alkaline phosp
33、hatase activity: Part 1: Fluorimetric method for milk and milk-based drinks Part 2: Fluorimetric method for cheeseIDF 155-1:2013(E) ISO and IDF 2013 All rights reserved vBS EN ISO 11816-1:2013BS EN ISO 11816-1:2013Milk and milk products Determination of alkaline phosphatase activity Part 1: Fluorime
34、tric method for milk and milk-based drinks1 ScopeThis 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
35、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
36、obtain a measurement not higher than 7 000 mU/l.2 Terms and definitionsFor the 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:
37、 The alkaline phosphatase activity is expressed as milliunits of enzyme activity 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
38、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
39、, producing a highly fluorescent product. Fluorimetric measurement of alkaline 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
40、 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).4 ReagentsUse only reagents of recognized analytical grade,
41、 unless otherwise specified, and distilled or demineralized water, or water of equivalent purity.INTERNATIONAL STANDARDISO 11816-1:2013(E)IDF 155-1:2013(E) ISO and IDF 2013 All rights reserved 1BS EN ISO 11816-1:2013ISO 11816-1:2013(E)4.1 Fluorophos substrate,1)in bottles, each containing 144 mg of
42、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 sto
43、red 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 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
44、 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 solution (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
45、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.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 8.
46、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).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 a
47、t 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-benzothiazolyl)-6-hydroxybenzothiazole in substrate buffer solution (4.2).The calibrator solutions remain stable for 18 months from the date of
48、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, containing 0 mol/l of Fluoroyellow.4.4.2 Calibrator solution B, containing 17,24 103mol/l of Fluoroyellow.4.4.3 Calibrator solution C, cont
49、aining 34,48 103mol/l of Fluoroyellow.4.5 Daily instrument control solution, containing 34,48 x103mol/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.5 ApparatusUsual laboratory equipment and, in particular, the following.1) The reagents specified in 4.1 to 4.5 and the apparatus specified in 5.1 to 5.4 (except 5.3.3)
