SIST EN 12014-3:2005

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung des Nitrat- und/oder Nitritgehaltes - Teil 3: Spektralphotometrische Bestimmung des Nitrat- und Nitritgehaltes in Fleischerzeugnissen nach enzymatischer Reduktion von Nitrat zu NitritProduits alimentaires - Détermination de la teneur en nitrates et/ou en nitrites - Partie 3 : Détermination spectrométrique de la teneur en nitrates et en nitrites des produits carnés apres réduction enzymatique des nitrates en nitritesFoodstuffs - 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 nitrite67.120.10Meso in mesni proizvodiMeat and meat products67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 12014-3:2005SIST EN 12014-3:2005en01-september-2005SIST EN 12014-3:2005SLOVENSKI
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SIST EN 12014-3:2005



EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 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 - Détermination de la teneur en nitrates et/ou en nitrites des produits carnés - Partie 3: Détermination spectrométrique de la teneur en nitrates et en nitrites après réduction enzymatique des nitrates en nitrites
Lebensmittel - Bestimmung des Nitrat- und/oder Nitritgehaltes -Teil 3: Spektralphotometrische Bestimmung 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 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 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 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 Republic, 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.
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EN 12014-3:2005 (E) 2 Contents Page Foreword.3 1 Scope.4 2 Normative references.4 3 Principle.4 4 Reagents.4 5 Apparatus and equipment.7 6 Procedure.7 7 Evaluation.8 8 Precision.9 9 Test report.10 Annex A (informative)
Precision data.12 Bibliography.14
SIST EN 12014-3:2005



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 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 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 chromatographic (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 products 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, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. SIST EN 12014-3:2005



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 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. For 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 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 spectrometrically 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 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 materials 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/l1)
4.3 Carrez solution No. 1 Dissolve 150 g of potassium hexacyanoferrate(II), K4[Fe(CN)6] ⋅ 3 H2O 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 (CH3COO)2 ⋅ 2 H2O, in water and dilute to 1 000 ml.
1) c is the substance concentration SIST EN 12014-3:2005



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 water. 4.5.2 Solution of tetrasodium salt of reduced ß-nicotinamide adenine dinucleotide phosphate (ß-NADPH-Na4, mass fraction of at least 98 %)
Dissolve 125 mg of reduced ß-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 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 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 temperature 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 dihydrochloride 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 solutions 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-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 International unit or standard unit) is defined as the amount of enzyme which catalyses the transformation of 1 µmol substrate per minute under standard conditions. SIST EN 12014-3:2005



EN 12014-3:2005 (E) 6 4.7.2 Sodium 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 nitrite 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) 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 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 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 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 p
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