1、BS EN ISO20541:2008ICS 67.100.01NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDMilk and milk products Determination ofnitrate content Method by enzymaticreduction andmolecular-absorptionspectrometry afterGriess reaction (ISO20541:2008)This British Standardwas p
2、ublished underthe authority of theStandards Policy andStrategy Committee on 30September 2008 BSI 2008ISBN 978 0 580 56968 5Amendments/corrigenda issued since publicationDate CommentsBS EN ISO 20541:2008National forewordThis British Standard is the UK implementation of EN ISO 20541:2008.The UK partic
3、ipation 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 be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Us
4、ers are responsible for its correct application.Compliance with a British Standard cannot confer immunityfrom legal obligations.EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 20541September 2008ICS 67.100.01English VersionMilk and milk products - Determination of nitrate content -Method by en
5、zymatic reduction and molecular-absorptionspectrometry after Griess reaction (ISO 20541:2008)Lait et produits laitiers - Dtermination de la teneur ennitrates - Mthode par rduction enzymatique etspectromtrie dabsorption molculaire aprs raction deGriess (ISO 20541:2008)Milch und Milcherzeugnisse - Bes
6、timmung desNitratgehaltes - Verfahren mit enzymatischer Reduktionund Moleklabsorptionsspektrometrie nach Griess-Reaktion (ISO 20541:2008)This European Standard was approved by CEN on 6 August 2008.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the condition
7、s for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the CEN Management Centre or to any CEN member.This European Standard exists in three offic
8、ial versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Be
9、lgium, 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
10、STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 2008 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 20541:2008: EBS EN ISO 20541:2008EN ISO 20541:
11、2008 (E) 3 Foreword This document (EN ISO 20541:2008) 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 Europea
12、n Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by March 2009, and conflicting national standards shall be withdrawn at the latest by March 2009. Attention is drawn to the possibility that some of the elements o
13、f 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. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European
14、 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 the United Kin
15、gdom. Endorsement notice The text of ISO 20541:2008 has been approved by CEN as a EN ISO 20541:2008 without any modification. BS EN ISO 20541:2008ISO 20541:2008(E) IDF 197:2008(E) ISO and IDF 2008 All rights reserved iiiContents Page Foreword iv Foreword. v 1 Scope . 1 2 Normative references . 1 3 T
16、erms and definitions. 1 4 Principle. 2 5 Reagents 2 6 Apparatus 4 7 Sampling 5 8 Preparation of test sample. 5 8.1 Dried milk, dried whey and milk protein concentrates . 5 8.2 Caseins and caseinates . 5 8.3 Cheese . 6 8.4 Whey cheese . 6 9 Procedure 6 9.1 Preparation of the test portion 6 9.2 Remova
17、l of fat and protein 6 9.3 Reagent blank test 7 9.4 Determination 7 9.5 Calibration . 9 10 Calculation and expression of results 9 10.1 Nitrite (matrix blank) content (see Clause 4, Note 3) 9 10.2 Total nitrite/nitrate content 9 10.3 Nitrate content. 10 11 Precision 10 11.1 Interlaboratory testing 1
18、0 11.2 Repeatability 10 11.3 Reproducibility 11 12 Test report . 11 Annex A (informative) Interlaboratory trials 12 Bibliography . 15 BS EN ISO 20541:2008ISO 20541:2008(E) IDF 197:2008(E) iv ISO and IDF 2008 All rights reservedForeword ISO (the International Organization for Standardization) is a wo
19、rldwide 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 o
20、n 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
21、 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 Standa
22、rd 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 20541IDF 197 was prepa
23、red 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. BS EN ISO 20541:2008ISO 20541:2008(E) IDF 197:2008(E) ISO and IDF 2008 All rights reserved vForeword IDF (the In
24、ternational Dairy Federation) is a non-profit organization representing the dairy sector worldwide. IDF membership comprises National 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
25、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
26、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 r
27、esponsible for identifying any or all such patent rights. ISO 20541IDF 197 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 t
28、he Joint ISO-IDF Action Team Minor compounds of the Standing Committee on Minor components and characterization of physical properties under the aegis of its project leaders, Mr. M. Carl (DE) and Mrs. C. Bckman (FL). BS EN ISO 20541:2008BS EN ISO 20541:2008INTERNATIONAL STANDARD ISO 20541:2008(E)IDF
29、 197:2008(E) ISO and IDF 2008 All rights reserved 1Milk and milk products Determination of nitrate content Method by enzymatic reduction and molecular-absorption spectrometry after Griess reaction WARNING The use of this International Standard may involve hazardous materials, operations and equipmen
30、t. This standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this standard to establish safety and health practices and determine the applicability of regulatory limitations prior to use. 1 Scope This International Standard spe
31、cifies a method for the determination of the nitrate content of milk and milk products by molecular-absorption spectrometry after Griess reaction (preceded by enzymatic reduction). The method is, in particular, applicable to whole, partly skimmed, skimmed and dried milk, hard, semi-hard and soft che
32、eses, processed cheese, whey cheese, caseins, caseinates, dried whey and milk protein concentrates. The method can be used at contents corresponding to a measured concentration in the sample solution (with blank subtracted) of more than 0,2 mg/l. 2 Normative references The following referenced docum
33、ents are indispensable for the application of this document. For dated references only the edition cited applies. For undated references the last edition of the referenced document (including any amendments) applies. ISO 565, Test sieves Metal wire cloth, perforated metal plate and electroformed she
34、et Nominal sizes of openings ISO 648, Laboratory glassware Single-volume pipettes ISO 835, Laboratory glassware Graduated pipettes ISO 1042, Laboratory glassware One-mark volumetric flasks ISO 3696, Water for analytical laboratory use Specification and test methods 3 Terms and definitions For the pu
35、rposes of this document, the following terms and definitions apply. 3.1 nitrite content mass fraction of nitrite compounds determined by the procedure specified in this International Standard BS EN ISO 20541:2008ISO 20541:2008(E) IDF 197:2008(E) 2 ISO and IDF 2008 All rights reserved3.2 nitrate cont
36、ent mass fraction of nitrate compounds determined by the procedure specified in this International Standard NOTE The nitrate content is expressed as the mass fraction in milligrams of nitrate ion (NO3) per kilogram of product. 4 Principle A test portion is dispersed in warm water. The fat and protei
37、ns are removed either by precipitation using Carrez reagents and filtering or by centrifugal ultra-filtration using membrane cones (see Notes 1 and 2). The nitrate is reduced to nitrite in a portion of the filtrate by means of nitrate reductase. A red-violet azo dye is developed, in portions of both
38、 the unreduced filtrate (for nitrite) and the reduced solution (for nitrate), by addition of sulfanilamide and N-(1-naphthyl)ethylenediamine dihydrochloride, and the absorbance measured at a wavelength of 540 nm (or Hg 546 nm). The nitrite content of the sample and the total nitrite content after re
39、duction of nitrate are calculated by comparing the measured absorbances with those of a set of sodium nitrite calibration solutions. The nitrate content is calculated from the difference between these two contents. NOTE 1 The two alternative procedures for removal of fat and protein are described in
40、 9.2.1 and 9.2.2. NOTE 2 For whey powder, whey protein concentrate and similar products, ultra-filtration is used in preference to Carrez precipitation as the latter often leads to turbidity problems and, as a consequence, to poor precision. NOTE 3 The low endogenous nitrite level is not reported bu
41、t taken into account in the matrix blank solution. 5 Reagents Unless otherwise specified, use only reagents of recognized analytical grade, free of nitrate and nitrite, and water complying with ISO 3696 grade 3 at least, free from nitrate and nitrite. Water used for preparation of enzyme or coenzyme
42、 solutions shall be freshly double-distilled or of equivalent purity. 5.1 Sodium hydroxide solution, c(NaOH) = 1 mol/l. 5.2 Sodium chloride solution, c(NaCI) = 0,9 g/100 ml. 5.3 Hydrochloric acid, 20(HCI) = 1,19 g/ml. 5.4 Hydrochloric acid solution, c(HCI) = 2 mol/l. Carefully add 160 ml of hydrochl
43、oric acid (5.3) to about 700 ml of water in a 1 000 ml one-mark volumetric flask (6.4) while regularly swirling the contents. Cool the contents to room temperature. Dilute to the mark with water and mix carefully. 5.5 Carrez reagents, as follows: 5.5.1 Carrez reagent I: Potassium hexacyanoferrate(II
44、) solution, c(K4Fe(CN)6.3H2O) = 150 g/l. Dissolve 15,0 g of potassium hexacyanoferrate(II) trihydrate in water in a 100 ml one-mark volumetric flask (6.4). Dilute to the mark with water and mix. 5.5.2 Carrez reagent II: Zinc sulfate solution, c(ZnSO4.7H2O) = 300 g/l. Dissolve 30,0 g of zinc sulfate
45、heptahydrate in water in a 100 ml one-mark volumetric flask (6.4). Dilute to the mark with water and mix. BS EN ISO 20541:2008ISO 20541:2008(E) IDF 197:2008(E) ISO and IDF 2008 All rights reserved 35.6 Standard solutions, as follows: 5.6.1 Sodium nitrite (NaNO2) stock solution. Accurately weigh (75,
46、0 0,1) mg of pre-dried (at 102 C for 2 h) sodium nitrite into a 100 ml one-mark volumetric flask. Dissolve it in a suitable amount of water. Dilute to the mark with water and mix. The nitrite stock solution obtained contains 500 mg of nitrite per litre. Prepare calibration solutions by diluting the
47、stock solution with water to give several solutions with different nitrite concentrations in the range from 0,05 mg/I to 5,0 mg/I. When stored at room temperature, the sodium nitrite stock solution remains stable for 1 day. 5.6.2 Potassium nitrate (KNO3) stock solution. Accurately weigh (81,5 0,1) m
48、g of pre-dried (at 102 C for 2 h) potassium nitrate into a 100 ml one-mark volumetric flask. Dissolve it in a suitable amount of water. Dilute to the 100 ml mark with water and mix. The obtained nitrate stock solution contains 500 mg of nitrate per litre. Prepare calibration solutions by diluting th
49、e stock solution with water to give several solutions with different nitrate concentrations in the range from 0,05 mg/I to 5,0 mg/I. When stored at 4 C, the potassium nitrate stock solution remains stable for 1 week. 5.7 Potassium phosphate buffer solution, pH = 7,5. Accurately weigh (57,6 0,1) mg of dipotassium hydrogen phosphate (K2HPO4.3H2O) into a 100 ml one-mark volumetric flask. Dissolve it in a suitable amount of water. Dilute to the mark with water and mix. Accurately weigh (17,0 0,1) mg of potassium dihyd
