1、December 2008DEUTSCHE NORM English price group 12No part of this standard 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 67.100.01!$Tq7“149782
2、0www.din.deDDIN EN ISO 20541Milk and milk products Determination of nitrate content Method by enzymatic reduction and molecular-absorption spectrometryafter Griess reaction (ISO 20541:2008)English version of DIN EN ISO 20541:2008-12Milch und Milcherzeugnisse Bestimmung des Nitratgehaltes Verfahren m
3、it enzymatischer Reduktion und Moleklabsorptionsspektrometrie nachGriess-Reaktion (ISO 20541:2008)Englische Fassung DIN EN ISO 20541:2008-12SupersedesDIN 10476:2001-11www.beuth.deDocument comprises 22 pagesDIN EN ISO 20541:2008-12 2 National foreword This standard falls in the domain of Technical Co
4、mmittee CEN/TC 302 “Milk and milk products Methods of sampling and analysis” (Secretariat: NEN, Netherlands). It is identical with International Standard ISO 20541:2008 which was prepared by Technical Committee ISO/TC 34 “Food products” (Secretariat: AFNOR, France), Subcommittee SC 5 “Milk and milk
5、products” (Secretariat: NEN, Netherlands). The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und land-wirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Technical Committee Milch und Milchprodukte Probenahme- und Untersuchungsver
6、fahren. Based on the results of the parallel voting procedure, ISO 20541:2008 has been adopted as a European Standard. The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 565 DIN ISO 565 ISO 707 DIN EN ISO 707 ISO 835 DIN EN ISO 835 ISO 104
7、2 DIN EN ISO 1042 ISO 3696 DIN ISO 3696 ISO 5725-2 DIN ISO 5725-2 Amendments This standard differs from DIN 10476:2001-11 as follows: a) Ultra-filtration of fat and proteins has been included as an alternative procedure to Carrez precipitation. b) The results of a German study carried out in 1998 an
8、d an international study carried out in 2004 have been included; the precision of the method has been derived from the results of these interlaboratory tests. c) Appendix A “Alternative procedure using macro cells” has been deleted. d) The standard has been revised in form and substance and harmoniz
9、ed with current rules of presentation. e) The Bibliography has been updated. Previous editions DIN 10476: 2001-11 3 National Annex NA (informative) Bibliography DIN ISO 565, Test sieves Metal wire cloth, perforated metal plate and electroformed sheet Nominal sizes of openings DIN EN ISO 707, Milk an
10、d milk products Guidance on sampling DIN EN ISO 835, Laboratory glassware Graduated pipettes DIN EN ISO 1042, Laboratory glassware One-mark volumetric flasks DIN ISO 3696, Water for analytical laboratory use; specification and test methods DIN ISO 5725-2, Accuracy (trueness and precision) of measure
11、ment methods and results Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method DIN EN ISO 20541:2008-12 DIN EN ISO 20541:2008-12 4 This page is intentionally blank EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 20541September 2008ICS
12、67.100.01English VersionMilk and milk products - Determination of nitrate content -Method by enzymatic reduction and molecular-absorptionspectrometry after Griess reaction (ISO 20541:2008)Lait et produits laitiers - Dtermination de la teneur ennitrates - Mthode par rduction enzymatique etspectromtri
13、e dabsorption molculaire aprs raction deGriess (ISO 20541:2008)Milch und Milcherzeugnisse - Bestimmung desNitratgehaltes - Verfahren mit enzymatischer Reduktionund Moleklabsorptionsspektrometrie nach Griess-Reaktion (ISO 20541:2008)This European Standard was approved by CEN on 6 August 2008.CEN memb
14、ers are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions 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
15、 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 translationunder the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the s
16、ame status as theofficial 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,Rom
17、ania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR 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 reservedwo
18、rldwide for CEN national Members.Ref. No. EN ISO 20541:2008: E2 DIN EN ISO 20541:2008-12 EN ISO 20541:2008 (E) Contents Page Foreword .3 1 Scope4 2 Normative references4 3 Terms and definitions .4 4 Principle .5 5 Reagents 5 6 Apparatus.7 7 Sampling 8 8 Preparation of test sample .8 8.1 Dried milk,
19、dried whey and milk protein concentrates8 8.2 Caseins and caseinates8 8.3 Cheese9 8.4 Whey cheese9 9 Procedure.9 9.1 Preparation of the test portion.9 9.2 Removal of fat and protein .9 9.3 Reagent blank test.10 9.4 Determination 10 9.5 Calibration12 10 Calculation and expression of results 12 10.1 N
20、itrite (matrix blank) content (see Clause 4, Note 3) 12 10.2 Total nitrite/nitrate content.12 10.3 Nitrate content .13 11 Precision 13 11.1 Interlaboratory testing 13 11.2 Repeatability 13 11.3 Reproducibility 14 12 Test report14 Annex A (informative) Interlaboratory trials.15 Bibliography18 3 Forew
21、ord 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 European Standard shall
22、 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 of this document
23、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 Standard: Austr
24、ia, 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 Kingdom. Endorsemen
25、t notice The text of ISO 20541:2008 has been approved by CEN as a EN ISO 20541:2008 without any modification. EN ISO 20541:2008 (E) DIN EN ISO 20541:2008-12 WARNING The use of this International Standard may involve hazardous materials, operations and equipment. This standard does not purport to add
26、ress 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 specifies a method for the determination of
27、 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 cheeses, processed cheese, whey cheese, cas
28、eins, 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 documents are indispensable for the applicati
29、on 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 sheet Nominal sizes of openings ISO 648, La
30、boratory 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 purposes of this document, the following t
31、erms and definitions apply. 3.1 nitrite content mass fraction of nitrite compounds determined by the procedure specified in this International Standard 4 DIN EN ISO 20541:2008-12 EN ISO 20541:2008 (E) 3.2 nitrate content mass fraction of nitrate compounds determined by the procedure specified in thi
32、s 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 proteins are removed either by precipitation using Carrez reagents and filtering or by ce
33、ntrifugal 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 the unreduced filtrate (for nitrite) and the reduced solution (for nitrate), by ad
34、dition 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 reduction of nitrate are calculated by comparing the measured absorbances with those
35、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 9.2.1 and 9.2.2. NOTE 2 For whey powder, whey protein concentrate and similar prod
36、ucts, 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 but taken into account in the matrix blank solution. 5 Reagents Unless otherwise spec
37、ified, 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 solutions shall be freshly double-distilled or of equivalent purity. 5.1 Sodium hy
38、droxide 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 hydrochloric acid (5.3) to about 700 ml of water in a 1 000 ml one-mark volumetric flask (6
39、.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) solution, c(K4Fe(CN)6.3H2O) = 150 g/l. Dissolve 15,0 g of potassium hexacyanoferr
40、ate(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 heptahydrate in water in a 100 ml one-mark volumetric flask (6.4). Dilute to the ma
41、rk with water and mix. 5 EN ISO 20541:2008 (E) DIN EN ISO 20541:2008-12 5.6 Standard solutions, as follows: 5.6.1 Sodium nitrite (NaNO2) stock solution. Accurately weigh (75,0 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 am
42、ount 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 stock solution with water to give several solutions with different nitrite concentrations in the range from 0,05 mg/I to 5,0 m
43、g/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) mg 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
44、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 the stock solution with water to give several solutions with different nitrate concentrations in the range from 0,05 mg/I to 5,0
45、 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 wa
46、ter. Dilute to the mark with water and mix. Accurately weigh (17,0 0,1) mg of potassium dihydrogen phosphate (KH2PO4) into a 50 ml one-mark volumetric flask. Dissolve it in a suitable amount of water. Dilute to the mark with water and mix. Using the pH-measurement unit (6.18), adjust the pH of the d
47、ipotassium hydrogen phospate solution to pH 7,5 by addition of potassium dihydrogen phosphate solution. When stored at 4 C, the potassium phosphate buffer solution remains stable for 2 weeks. 5.8 NADPH/FAD solution. Weigh accurately (5,6 0,1) mg of 3-nicotinamide-adenine dinucleotide phosphate (redu
48、ced form), tetrasodium salt (-NADPH-Na4, with a mass fraction of at least 98 %), and (80,0 0,1) mg flavine-adenine dinucleotide, disodium salt (FAD-Na2, with a mass fraction of at least 88 %), into a 25 ml one-mark volumetric flask. Dissolve them in a suitable amount of potassium phosphate buffer so
49、lution (5.7). Dilute to the mark with the buffer solution (5.7) and mix. Freshly prepare the NADPH/FAD solution immediately before use. 5.9 Nitrate reductase (NR) solution. Weigh 65 mg of nitrate reductase (NR) from Aspergillus niger (EC 1.6.6.2, Iyophilizate containing approximately 0,4 U/mg) into a 10 ml measuring tube. Add 5 ml of water and mix. When stored at 4 C, the nitrate reductase solution remains stable for 2 weeks. 6 DIN EN ISO 20541:2008-12 EN ISO 20541:2008 (E) 5.10 Colour reagents, as follows
