1、BS EN ISO 11816-2:2016Milk and milk products Determination of alkalinephosphatase activityPart 2: Fluorimetric method for cheese(ISO 11816-2:2016)BSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06BS EN ISO 11816-2:2016 BRITISH STANDARDNational forewordThis British Stan
2、dard is the UK implementation of EN ISO11816-2:2016. It supersedes BS EN ISO 11816-2:2003 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 beobtain
3、ed on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2016.Published by BSI Standards Limited 2016ISBN 978 0 580 76892 7ICS 67.100.30Compliance with a
4、British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 30 September 2016.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dEUROPEAN STANDARD NORME EUROPENNE EUROPIS
5、CHE NORM EN ISO 11816-2 August 2016 ICS 67.100.30 Supersedes EN ISO 11816-2:2003English Version Milk and milk products - Determination of alkaline phosphatase activity - Part 2: Fluorimetric method for cheese (ISO 11816-2:2016) Lait et produits laitiers - Dtermination de lactivit de la phosphatase a
6、lcaline - Partie 2: Mthode fluorimtrique pour le fromage (ISO 11816-2:2016) Milch und Milchprodukte - Bestimmung der Aktivitt der alkalischen Phosphatase - Teil 2: Fluorimetrisches Verfahren fr Kse (ISO 11816-2:2016) This European Standard was approved by CEN on 29 July 2016. CEN members are bound t
7、o 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-C
8、ENELEC 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 Centre has
9、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, Lithuania, Luxe
10、mbourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey andUnited Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brusse
11、ls 2016 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 11816-2:2016 EBS EN ISO 11816-2:2016EN ISO 11816-2:2016 (E) 3 European foreword This document (EN ISO 11816-2:2016) has been prepared by Technical Committee ISO/TC 34 “Foo
12、d 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 February 2017, and conflicting national standards shall be withdrawn at the latest by February 2017. 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 identifyin
14、g any or all such patent rights. This document supersedes EN ISO 11816-2:2003. 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
15、, Denmark, 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. Endorsem
16、ent notice The text of ISO 11816-2:2016 has been approved by CEN as EN ISO 11816-2:2016 without any modification. BS EN ISO 11816-2:2016ISO 11816-2:2016(E)IDF 155-2:2016(E)Forewords .iv1 Scope . 12 Normative reference . 13 Terms and definitions . 14 Principle 15 Reagents 26 Apparatus . 37 Sampling 4
17、8 Preparation of test sample . 49 Procedure. 49.1 Verification of instrument performance . 49.1.1 General 49.1.2 Daily instrument tests . 59.1.3 Controls 59.2 Reagent controls to test the suitability of ready to use working substrate (5.3) 59.3 Calibration 69.4 Determination . 69.5 Test sample relat
18、ed controls . 79.5.1 Recommended negative and positive control tests .79.5.2 Interfering substance test 89.5.3 Heat-stable microbial alkaline phosphatase control test . 810 Calculation and expression of results . 810.1 Calibration ratio . 810.2 Calculation 910.2.1 Supernatant 910.2.2 Cheese . 910.3
19、Expression of results . 1011 Precision 1011.1 Interlaboratory test 1011.2 Repeatability 1011.3 Reproducibility 1012 Test report 10Annex A (informative) Interlaboratory trial .11Annex B (informative) Examples of preparation of a test sample 13Bibliography .15 ISO and IDF 2016 All rights reserved iiiC
20、ontents PageBS EN ISO 11816-2:2016ISO 11816-2:2016(E)ForewordsISO (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. E
21、ach 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 Internati
22、onal 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 differen
23、t 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 no
24、t 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 inf
25、ormation given for the convenience of users and does not constitute an endorsement.For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about ISOs adherence to the WTO principles in the Technical Barriers to Trade (TBT) see
26、the following URL: Foreword - Supplementary informationThe 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 second edition of ISO 11816-2|IDF
27、155-2 cancels and replaces the first edition (ISO 11816-2|IDF 155-2:2003), 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
28、k-based drinks Part 2: Fluorimetric method for cheeseIDF 155-2:2016(E)iv ISO and IDF 2016 All rights reservedBS EN ISO 11816-2:2016ISO 11816-2:2016(E)IDF (the International Dairy Federation) is a non-profit private sector organization representing the interests of various stakeholders in dairying at
29、 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
30、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 t
31、hat 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
32、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 and endorsementISO 11816|IDF 155 was prepared by the IDF Standing Committee on Analytical Methods for Processing Aids and Indicators and ISO
33、Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products.The work was carried out by the IDF/ISO Project Group on Alkaline phosphatase activity in cheese (P06), of the Standing Committee on Analytical Methods for Processing Aids and Indicators, under the aegis of its p
34、roject leader Mrs. M. Nicolas (FR).This ISO/IDF International Standard cancels and replaces ISO 11816-2|IDF 155-2:2003, 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:
35、 Part 1: Fluorimetric method for milk and milk-based drinks Part 2: Fluorimetric method for cheeseIDF 155-2:2016(E) ISO and IDF 2016 All rights reserved vBS EN ISO 11816-2:2016BS EN ISO 11816-2:2016Milk and milk products Determination of alkaline phosphatase activity Part 2: Fluorimetric method for
36、cheese1 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 cheese.This method is applicable to soft cheeses, semi-hard and hard cheeses provided that the mould is only on the surface of the cheese and not al
37、so in the inner part (e.g. blue veined cheeses). For large hard cheeses, specific conditions of sampling apply (see Clause 7).The instrument can read activities in the supernatant up to 7 000 milliunits per litre (mU/l).2 Normative referenceThe following documents, in whole or in part, are normative
38、ly referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.ISO 5725-1, Accuracy (trueness and precision) of measurement method
39、s and results Part 1: General principles and definitionsISO 5725-2, Accuracy (trueness and precision) of measurement methods and results Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method3 Terms and definitionsFor the purposes of this doc
40、ument, the following terms and definitions apply.3.1alkaline phosphatase activityALP activityactivity 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 gram of
41、 sample (mU/g).3.2unit of alkaline phosphatase activityamount of alkaline phosphatase enzyme that catalyses the transformation of 1 mol of substrate per minute4 PrincipleThe alkaline phosphatase activity of the sample is measured by a continuous fluorimetric direct kinetic assay. A non-fluorescent a
42、romatic 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 INTERNATIONAL STANDARDISO 11816-2:2016
43、(E)IDF 155-2:2016(E) ISO and IDF 2016 All rights reserved 1BS EN ISO 11816-2:2016ISO 11816-2:2016(E)measurement of alkaline phosphatase (ALP) activity is measured at 38 C over a 3 min period when using the Fluorophos1). This includes pre-incubation of substrate and sample, followed by multiple kinet
44、ic 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 Reagen
45、tsUse only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralized water or water of equivalent purity.5.1 Fluorophossubstrate2)in bottles, each containing 144 mg of Fluorophossubstrate powder, molar mass 580 grams per mole.This is a non-fluorescent aromat
46、ic monophosphoric ester substrate, 2-2-benzothiazolyl-6-hydroxybenzothiazole phosphate.This substrate remains stable for 2 years from the date of manufacture, provided it is stored in unopened bottles at a temperature between 2 C and 8 C. Protect against light.5.2 Substrate buffer solution, diethano
47、lamine (DEA) buffer solution, c(DEA) = 2,4 mol/l, with pH 10,0.The substrate buffer solution remains stable for 2 years from the date of manufacture, provided it is stored in unopened bottles at a temperature between 2 C and 8 C. Protect against light.5.3 Working substrate.Allow the Fluorophossubstr
48、ate (5.1) and the substrate buffer solution (5.2) to come to room temperature. Add the content of one bottle substrate buffer solution (240 ml) (5.2) to that of one bottle Fluorophossubstrate (144 mg) (5.1) and mix well by inversion for 3 min. Use amber glass to protect against light.Allow the obtai
49、ned solution to stand at room temperature for at least 30 min prior to use.Use the analog-to-digital (A/D) 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 a temperature between 2 C and 8 C, or for 6 h at 38 C.5.4 Working calibrator solutions, Fluoroyellow(FY) 2-(2-benzothiazolyl)-6-hydroxybenzothiazole
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