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1、BRITISH STANDARD BS EN 12014-3:2005 Foodstuffs Determination of nitrate and/or nitrite content Part 3: Spectrometric determination of nitrate and nitrite content of meat products after enzymatic reduction of nitrate to nitrite The European Standard EN 12014-3:2005 has the status of a British Standar
2、d ICS 67.120.10 ? Licensed Copy: sheffieldun sheffieldun, na, Sat Oct 28 16:12:24 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS EN 12014-3:2005 This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 June 2005 BSI 30 June 2005 ISBN 0 580 46249 8 N
3、ational foreword This British Standard is the official English language version of EN 12014-3:2005. It supersedes DD ENV 12014-3:1998 which is withdrawn. The UK participation in its preparation was entrusted to Technical Committee AW/-/3, Food analysis horizontal methods, which has the responsibilit
4、y to: A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international or European publications referred to in this document may be found in the BSI Catalogue under the section entitled “Internatio
5、nal Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Standards Online. This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British
6、 Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and E
7、uropean developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the EN title page, pages 2 to 14, an inside back cover and a back cover. The BSI copyright notice displayed in this document indicates when the document was last issued.
8、 Amendments issued since publication Amd. No. DateComments Licensed Copy: sheffieldun sheffieldun, na, Sat Oct 28 16:12:24 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 12014-3 May 2005 ICS 67.120.10Supersedes ENV 12014-3:1998 English version Foodstu
9、ffs - Determination of nitrate and/or nitrite content - Part 3: Spectrometric determination of nitrate and nitrite content of meat products after enzymatic reduction of nitrate to nitrite Produits alimentaires - Dtermination de la teneur en nitrates et/ou en nitrites des produits carns - Partie 3: D
10、termination spectromtrique de la teneur en nitrates et en nitrites aprs rduction enzymatique des nitrates en nitrites Lebensmittel - Bestimmung des Nitrat- und/oder Nitritgehaltes - Teil 3: Spektralphotometrische Bestimmung des Nitrat- und Nitritgehaltes in Fleischerzeugnissen nach enzymatischer Red
11、uktion von Nitrat zu Nitrit This European Standard was approved by CEN on 1 April 2005. CEN members 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 list
12、s and bibliographical references concerning such national standards may be obtained on application to the Central Secretariat 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 respon
13、sibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Irelan
14、d, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: rue de Stassart, 36 B-1050
15、 Brussels 2005 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 12014-3:2005: E Licensed Copy: sheffieldun sheffieldun, na, Sat Oct 28 16:12:24 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EN 12014-3:2005 (E) 2 Contents Page Foreword
16、3 1Scope.4 2Normative references .4 3Principle.4 4Reagents4 5Apparatus and equipment7 6Procedure7 7Evaluation8 8Precision9 9Test report.10 Annex A (informative) Precision data .12 Bibliography.14 Licensed Copy: sheffieldun sheffieldun, na, Sat Oct 28 16:12:24 GMT+00:00 2006, Uncontrolled Copy, (c) B
17、SI EN 12014-3:2005 (E) 3 Foreword This European Standard (EN 12014-3:2005) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication
18、 of an identical text or by endorsement, at the latest by November 2005, and conflicting national standards shall be withdrawn at the latest by November 2005. This document supersedes ENV 12014-3:1998. This series Foodstuffs - Determination of nitrate and/or nitrite content consist of the following
19、parts: Part 1: General considerations; Part 2: HPLC/IC method for the determination of nitrate content of vegetables and vegetable products; Part 3: Spectrometric determination of nitrate and nitrite content of meat products after enzymatic reduction of nitrate to nitrite; Part 4: Ion-exchange chrom
20、atographic (IC) method for the determination of nitrate and nitrite content of meat products; Part 5: Enzymatic determination of nitrate content of vegetable-containing food for babies and infants; Part 7: Continuous flow method for the determination of nitrate content of vegetables and vegetable pr
21、oducts after Cadmium reduction. This European Standard includes a Bibliography. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia
22、, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. Licensed Copy: sheffieldun sheffieldun, na, Sat Oct 28 16:12:24 GMT+00:00 2006, Uncont
23、rolled Copy, (c) BSI EN 12014-3:2005 (E) 4 1 Scope This European Standard specifies a spectrometric method for the determination of nitrate and nitrite content of meat products and has been validated for different meat products with a content of 9 mg/kg to 22 mg/kg nitrite calculated as sodium nitri
24、te and 23 mg/kg to 48 mg/kg nitrate calculated as sodium nitrate. NOTE Experiences have shown that the method is also applicable for total nitrite and nitrate content from 5 mg/kg up to 125 mg/kg calculated as sodium nitrite. For further information on applicability, see 1. 2 Normative references Th
25、e following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN ISO 3696, Water for analytical laboratory use Sp
26、ecification and test methods (ISO 3696:1987). 3 Principle Nitrite in an aqueous extract of the test sample is treated with sulfanilamide and N-(1-naphthyl)- ethylenediamine dihydrochloride. The formed red compound is measured spectrometrically at a wavelength of 540 nm 2. Nitrate in an aqueous extra
27、ct of the analytical sample is converted into nitrite by nitrate reductase. This converted nitrite together with the nitrite which is already in the analytical sample reacts with sulfanilamide and N-(1-naphthyl)ethylenendiamine dihydrocloride. The colour intensity of this resulting red compound is m
28、easured in a spectrometer at 540 nm. The nitrate content is calculated from the difference between the spectrometric measurements. 4 Reagents 4.1 General During the analysis, unless otherwise stated, use only reagents and materials of recognized analytical grade and water of at least grade 3 accordi
29、ng to EN ISO 3696. When preparing solutions, the purities of the reagents available shall be taken into account. 4.2 Sodium hydroxide solution c(NaOH) 1 mol/l1) 4.3 Carrez solution No. 1 Dissolve 150 g of potassium hexacyanoferrate(II), K4Fe(CN)6 3 H2O in water and dilute to 1 000 ml. Store the solu
30、tion in a brown bottle and replace it every two months. 4.4 Carrez solution No. 2 Dissolve 230 g of zinc acetate, Zn (CH3COO)2 2 H2O, in water and dilute to 1 000 ml. 1)c is the substance concentration Licensed Copy: sheffieldun sheffieldun, na, Sat Oct 28 16:12:24 GMT+00:00 2006, Uncontrolled Copy,
31、 (c) BSI EN 12014-3:2005 (E) 5 4.5 Reagents for the enzymatic determination of nitrate2) 4.5.1 Imidazole buffer solution, pH = 7,8 Dissolve 0,68 g of imidazole in an 100 ml volumetric flask in 80 ml of water, adjust the pH to 7,8 with hydrochloric acid (c(HCl) = 2 mol/l) and dilute to the mark with
32、water. 4.5.2 Solution of tetrasodium salt of reduced -nicotinamide adenine dinucleotide phosphate (-NADPH-Na4, mass fraction of at least 98 %) Dissolve 125 mg of reduced -nicotinamide adenine dinucleotide phosphate and 2,5 mg of disodium salt of flavin adenin dinucleotide (mass fraction of at least
33、88 %) in 50 ml of the imidazole buffer solution (4.5.1). Prepare this solution immediately before use. 4.5.3 Nitrate reductase buffer solution Dissolve 11 mg of nitrate reductase lyophilisate from aspergillus sp. (EC 1.6.6.2, approximately 0,4 U/mg3) in 10 ml of imidazole buffer solution (4.5.1) and
34、 add 50 mg of ethylenedintrilotetraacetic acid disodium salt- dihydrate. The solution will be stable for about 14 days if stored at 4 C. 4.6 Colour reagents 4.6.1 Colour reagent No. 1 Dissolve 8 g of sulfanilamide in 500 ml of water while heating on a water bath (5.3). Cool the solution to room temp
35、erature and filter if necessary. Add 330 ml of hydrochloric acid (4.9) while stirring continuously and dilute the solution to 1 000 ml with water. The solution will be stable for several weeks at room temperature. 4.6.2 Colour reagent No. 2 Dissolve 0,330 g of N-(1-naphthyl)ethylenediamine dihydroch
36、loride in 250 ml of water. Keep the solution in a brown bottle and replace it every week. 4.6.3 Colour reagent mixture Prepare the required amount of colour reagent mixture by mixing equal parts by volume of colour reagents No. 1 (4.6.1) and No. 2 (4.6.2) on the day of use. 4.7 Sodium nitrite soluti
37、ons 4.7.1 General It is recommended to prepare sodium nitrite standard solutions of sodium nitrite on the day of use. 2)The Nitrat test kit for the enzymatic determination of nitrate and nitrite is the trade name of a product produced by Boehringer Mannheim/Roche Diagnostics, Mannheim, supplied by r
38、-biopharm, Darmstadt (Germany) This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results. 3)U, this unit (often called the Inter
39、national unit or standard unit) is defined as the amount of enzyme which catalyses the transformation of 1 mol substrate per minute under standard conditions. Licensed Copy: sheffieldun sheffieldun, na, Sat Oct 28 16:12:24 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EN 12014-3:2005 (E) 6 4.7.2 Sodium
40、 nitrite stock solution, ?(NaNO2) = 400 mg/l Dissolve 200 mg of sodium nitrite weighed to the nearest 0,1 mg in water in a 500 ml volumetric flask and dilute the solution to the mark with water. The stock solution may be used for 2 weeks if stored in a refrigerator at 4 C. 4.7.3 Diluted sodium nitri
41、te stock solution ?(NaNO2) = 20 mg/l Pipette 25 ml of the sodium nitrite stock solution (4.7.2) into a 500 ml volumetric flask and dilute to the mark with water. 4.7.4 Sodium nitrite standard solutions Pipette 10 ml, 20 ml, 30 ml and 40 ml, respectively, of the diluted stock solution (4.7.3) contain
42、ing 0,13 mg, 0,27 mg, 0,40 mg, and 0,53 mg, respectively, of nitrite into a series of 200 ml volumetric flasks, add sodium hydroxide solution (4.2) to adjust the pH-value at 8,0 to 8,5 by using a pH-meter (5.5). Add 4 ml each of Carrez solution No. 1 (4.3) and No. 2 (4.4), shaking after each additio
43、n, dilute to the mark with water, thoroughly mix the contents of the flask and filter through a fluted filter paper (5.4). Discard the first 20 ml of each of the filtrates. 4.8 Potassium nitrate solutions 4.8.1 General It is recommended to prepare potassium nitrate solutions on the day of use. 4.8.2
44、 Potassium nitrate stock solution, ?(KNO3) = 600 mg/l4) Dissolve 300 mg of potassium nitrate weighed to the nearest 0,1 mg in water in a 500 ml volumetric flask and dilute the solution to the mark with water. 4.8.3 Diluted potassium nitrate stock solution, ?(KNO3) = 30 mg/l Pipette 25 ml of the pota
45、ssium nitrate stock solution (4.8.2) into a 500 ml volumetric flask and dilute to the mark with water. 4.8.4 Potassium nitrate standard solutions Pipette 10 ml, 20 ml, 30 ml and 40 ml, respectively, of the diluted stock solution (4.8.3), corresponding to 0,14 mg, 0,27 mg, 0,41 mg, and 0,55 mg, respe
46、ctively, of nitrite ion into a series of 200 ml volumetric flasks, add sodium hydroxide solution (4.2) to adjust the pH-value at 8,0 to 8,5 by using a pH-meter (5.5). Add 4 ml each of Carrez solution No. 1 (4.3) and No. 2 (4.4), shaking after each addition, dilute to the mark with water, thoroughly
47、mix the contents of the flask and filter through a fluted filter paper (5.4). Discard the first 20 ml of each of the filtrates. 4.9 Hydrochloric acid, fuming, w = 37 %5) , nitrite free 4)? is the mass concentration 5)w is the mass fraction Licensed Copy: sheffieldun sheffieldun, na, Sat Oct 28 16:12
48、:24 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EN 12014-3:2005 (E) 7 5 Apparatus and equipment 5.1 General Usual laboratory apparatus and, in particular, the following: 5.2 Homogenizing equipment, mechanical or electrical, capable of homogenizing the test sample, this includes a high-speed rotationa
49、l cutter, or a mincer fitted with a plate with holes not exceeding 4,5 mm in diameter. 5.3 Water bath, capable of being maintained at 100 C 5.4 Fluted filter papers, nitrate and nitrite free 5.5 pH meter, with pH measuring cell (glass and reference electrodes) 5.6 Spectrometer, for carrying out measurements at a wavelength of 540 nm, with suitable cells, made of glass or plastics which have no significant optical absorption at a wavelength of 540 nm. 6 Procedure 6.1 P