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 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 National foreword This British Standard is the official English language version of EN 12014-3:2005. It supers
3、edes DD ENV 12014-3:1998 which is withdrawn.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 responsibility to: A list of organizations represented on this committee can be obtained on request to its secretary. Cros
4、s-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 “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of
5、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 Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; pr
6、esent 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 European developments and promulgate them in the UK. Summary of pages This document comprises a front cover, a
7、n 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. Amendments issued since publication Amd. No. Date CommentsEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORME
8、N 12014-3 May 2005 ICS 67.120.10 Supersedes ENV 12014-3:1998 English version 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 Produits alimentaires - Dter
9、mination de la teneur en nitrates et/ou en nitrites des produits carns - Partie 3: Dtermination 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 Bes
10、timmung des Nitrat- und Nitritgehaltes in Fleischerzeugnissen nach enzymatischer Reduktion 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 Europea
11、n 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 Central Secretariat or to any CEN member. This European Standard exists in three official versions (English, F
12、rench, 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 Central Secretariat has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Re
13、public, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMAL
14、ISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: rue de Stassart, 36 B-1050 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: EEN 12014-3:2005 (E) 2 Contents Page Foreword 3 1 Scope .4 2 Normative
15、 references .4 3 Principle.4 4 Reagents4 5 Apparatus and equipment7 6 Procedure 7 7 Evaluation8 8 Precision9 9 Test report .10 Annex A (informative) Precision data .12 Bibliography.14EN 12014-3:2005 (E) 3 Foreword This European Standard (EN 12014-3:2005) has been prepared by Technical Committee CEN/
16、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 of an identical text or by endorsement, at the latest by November 2005, and conflicting national standards shall be wit
17、hdrawn 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 parts: Part 1: General considerations; Part 2: HPLC/IC method for the determination of nitrate content of vegetables and
18、 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 chromatographic (IC) method for the determination of nitrate and nitrite content of meat products; Part 5: Enzymatic determin
19、ation 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 products after Cadmium reduction. This European Standard includes a Bibliography. According to the CEN/CENELEC Internal Re
20、gulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands,
21、 Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. 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
22、 with a content of 9 mg/kg to 22 mg/kg nitrite calculated as sodium nitrite 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. F
23、or further information on applicability, see 1. 2 Normative references The 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
24、 amendments) applies. EN ISO 3696, Water for analytical laboratory use Specification 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 s
25、pectrometrically at a wavelength of 540 nm 2. Nitrate in an aqueous extract 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
26、 dihydrocloride. The colour intensity of this resulting red compound is measured 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 mater
27、ials of recognized analytical grade and water of at least grade 3 according 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/l 1) 4.3 Carrez solution No. 1 Dissolve 150 g of potassium hexacyanofe
28、rrate(II), K 4 Fe(CN) 6 3 H 2 O in water and dilute to 1 000 ml. Store the solution in a brown bottle and replace it every two months. 4.4 Carrez solution No. 2 Dissolve 230 g of zinc acetate, Zn (CH 3 COO) 2 2 H 2 O, in water and dilute to 1 000 ml. 1) c is the substance concentrationEN 12014-3:200
29、5 (E) 5 4.5 Reagents for the enzymatic determination of nitrate 2) 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 water. 4.5.2 Solution
30、of tetrasodium salt of reduced -nicotinamide adenine dinucleotide phosphate (-NADPH-Na 4 , 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 88 %) in 50 ml of th
31、e 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/mg 3) ) in 10 ml of imidazole buffer solution (4.5.1) and add 50 mg of eth
32、ylenedintrilotetraacetic 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 temperature and filte
33、r 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 dihydrochloride in 250 ml
34、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 solutions 4.7.1 General
35、 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-biopharm, Darms
36、tadt (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 International unit o
37、r standard unit) is defined as the amount of enzyme which catalyses the transformation of 1 mol substrate per minute under standard conditions.EN 12014-3:2005 (E) 6 4.7.2 Sodium nitrite stock solution, (NaNO 2 ) = 400 mg/l Dissolve 200 mg of sodium nitrite weighed to the nearest 0,1 mg in water in a
38、 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 nitrite stock solution (NaNO 2 ) = 20 mg/l Pipette 25 ml of the sodium nitrite stock solution (4.7.2) into a 500 ml volumetric
39、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) containing 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 sodiu
40、m 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 mix the contents of the flask and filter through a fluted filter paper (5.4)
41、. 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 Potassium nitrate stock solution, (KNO 3 ) = 600 mg/l 4) Dissolve 300 mg of potassium nitrate weighed to the nearest 0,1
42、 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, (KNO 3 ) = 30 mg/l Pipette 25 ml of the potassium nitrate stock solution (4.8.2) into a 500 ml volumetric flask and dilute to the mark with water. 4.8.4 Potassium
43、 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, respectively, of nitrite ion into a series of 200 ml volumetric flasks, add sodium hydroxide solution (4.2) to adjust the p
44、H-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 mix the contents of the flask and filter through a fluted filter paper (5.4). Discard the first 20 ml of each of the f
45、iltrates. 4.9 Hydrochloric acid, fuming, w = 37 % 5), nitrite free 4) is the mass concentration 5) w is the mass fraction 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, ca
46、pable of homogenizing the test sample, this includes a high-speed rotational 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 ce
47、ll (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 Preparation of the sample solution Homogenize the labo
48、ratory sample with the appropriate equipment (5.2) Take care that the temperature of the sample material does not rise above 25 C. If a mincer is used, pass the sample at least twice through the equipment. Weigh, to the nearest 10 mg, 10 g of the homogenized sample into e.g. a wide neck conical flas
49、k, add about 50 ml of water and homogenize for 30 s to 60 s. Rinse the shaft of the homogenizer into the flask with 50 ml of hot water. Add sodium hydroxide solution (4.2) to adjust the pH-value to 8,0 to 8,5 by using a pH-meter (5.5). Heat the flask for 15 minutes in a boiling water bath (5.3), shaking several times. NOTE For uncooked meat products the pH should not exceed 8,5 since otherwise it may not be possible to clarify the solutions by filtering afte
