1、| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | BRITISH STANDARD BS EN 12014-5 : 1997 The
2、European Standard EN 12014-5 : 1997 has the status of a British Standard ICS 67.080.20 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW Foodstuffs Determination of nitrate and/or nitrite content Part 5. Enzymatic determination of nitrate content of vegetable-containing food for
3、 babies and infantsBS EN 12014-5 : 1997 This British Standard, having been prepared under the direction of the Consumer Products and Services Sector Board, was published under the authority of the Standards Board and comes into effect on 15 August 1997 BSI 1997 The following BSI references relate to
4、 the work on this standard: Committee reference AW/-/3 Draft for comment 95/501747 DC ISBN 0 580 28102 7 Amendments issued since publication Amd. No. Date Text affected Committees responsible for this British Standard The preparation of this British Standard was entrusted to Technical Panel AW/-/3,
5、Food analysis Horizontal methods, upon which the following bodies were represented: Association of Public Analysts Food and Drink Federation Institute of Food Science and Technology Laboratory of the Government Chemist Ltd. Ministry of Agriculture, Fisheries and Food Royal Society of ChemistryBS EN
6、12014-5 : 1997 BSI 1997 i Contents Page Committees responsible Inside front cover National foreword ii Foreword 2 Text of EN 12014-5 3ii BSI 1997 BS EN 12014-5 : 1997 National foreword This Part of BS EN 12014 has been prepared by Technical Panel AW/-/3 and is the English language version of EN 1201
7、4-5 : 1997 Foodstuffs Determination of nitrate and/or nitrite content Part 5: Enzymatic determination of nitrate content of vegetable-containing food for babies and infants, published by the European Committee for Standardization (CEN). Cross-references Publication referred to Corresponding British
8、Standard EN 12014-1 : 1997 BS EN 12014-1 : 1997 Foodstuffs Determination of nitrate and/or nitrite content Part 1: General considerations EN ISO 3696 : 1995 BS EN ISO 3696 : 1995 Water for analytical laboratory use. Specification and test methods Compliance with a British Standard does not of itself
9、 confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, pages i and ii, the EN title page, pages 2 to 8, an inside back cover and a back cover.CEN European Committee for Standardization Comite Europe en de Normalisation Europa isches Ko
10、mitee fu r Normung Central Secretariat: rue de Stassart 36, B-1050 Brussels 1997 All rights of exploitation in any form and by any means reserved worldwide for CEN national members. Ref. No. EN 12014-5 : 1997 E EUROPEAN STANDARD EN 12014-5 NORME EUROPE ENNE EUROPA ISCHE NORM April 1997 ICS 67.080.20
11、 Descriptors: Food products, infant foods, vegetables, chemical analysis, determination of content, nitrates, nitrites, enzymatic methods English version Foodstuffs Determination of nitrate and/or nitrite content Part 5: Enzymatic determination of nitrate content of vegetable-containing food for bab
12、ies and infants Produits alimentaires Determination de la teneur en nitrates et/ou en nitrites Partie 5: De termination enzymatique de la teneur en nitrates des aliments a base de le gumes pour be be s et petits enfants Lebensmittel Bestimmung des Nitrat- und/oder Nitritgehaltes Teil 5: Enzymatische
13、 Bestimmung des Nitratgehaltes in gemu sehaltiger Sa uglings- und Kleinkindernahrung This European Standard was approved by CEN on 1997-02-28. 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 na
14、tional 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
15、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, Denmark, Finland, France, Germany, Greece, Ic
16、eland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.Page 2 EN 12014-5 : 1997 BSI 1997 Foreword This European Standard has been prepared by CEN/TC 275, Food analysis Horizontal methods, the secretariat of which is held by DIN. This series Fo
17、odstuffs Determination of nitrate and/or nitrite content consists 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 pro
18、ducts after enzymatic reduction of nitrate to nitrite Part 4: 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
19、 nitrate content of vegetables and vegetable products after cadmium reduction 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 October 1997 and conflicting national standards shall be withdrawn at
20、the latest by October 1997. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands,
21、 Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Contents Page Foreword 2 1 Scope 3 2 Normative references 3 3 Principle 3 4 Reagents 3 5 Apparatus and equipment 3 6 Procedure 4 7 Calaculation 5 8 Precision 5 9 Test report 5 Annexes A (informative) Precision data 6 B (informativ
22、e) Bibliography 7Page 3 EN 12014-5 : 1997 BSI 1997 1) c is the substance concentration. 2) These reagents are included in commercially available test kits. If these test kits are used, follow the manufacturers instructions. 3) U. This unit (often called the international unit or standard unit) is de
23、fined as the amount of enzyme which catalyses the transformation of 1 mmol substrate per minute under standard conditions. 1 Scope This European Standard specifies an enzymatic method for the determination of vegetable-containing food for babies and infants 1, 2. This method is applicable to nitrate
24、 contents in the range of 50 mg/kg to 200 mg/kg. 2 Normative references This European Standard incorporates by dated or undated reference, provisions from other publications. These normative references are cited at the appropriate places in the text and the publications are listed hereafter. For dat
25、ed references, subsequent amendments to or revisions of any of these publications apply to this European Standard only when incorporated in it by amendment or revision. For undated references the latest edition of the publication referred to applies. EN 12014-1 Foodstuffs Determination of nitrate an
26、d/or nitrite content Part 1: General considerations EN ISO 3696 Water for analytical laboratory use Specification and test methods 3 Principle Enzymatic determination in an aqueous sample extract by measuring the amount of NADPH used up in the following reaction: Nitrate + NADPH + H + Nitrite + NADP
27、 + +H 2 O nitrate reductase where the amount of NADPH used up is equivalent to the quantity of nitrate 3. 4 Reagents During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and water of at least grade 3 as defined in ISO 3696. When preparing solutions, the puri
28、ties of the reagents available shall be taken into account. 4.1 Carrez solution No.1 Dissolve 150 g of potassium hexacyanoferrate(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. 4.2 Carrez solution No.2 Dissolve 300 g
29、of zinc sulfate, ZnSO 4 .7 H 2 O in water and dilute to 1 000 ml. 4.3 Sodium hydroxide solution, c 1) (NaOH) = 2mol/l 4.4 Imidazole buffer solution 2) , pH = 7,3 Dissolve 0,68 g of imidazole (C 3 H 4 N 2 ) in 80 ml of water, adjust the pH to 7,3 with 2 mol/l hydrochloric acid solution and dilute to
30、100 ml with water. The solution will be stable for at least 1 year at 4 C. 4.5 FAD solution 2) Prepare a solution of 0,17 mg of the disodium salt of flavin adenine dinucleotide, FAD-Na 2 per millilitre by using FAD-Na 2 with a content by mass of not less than 88 %. The solution will be stable for at
31、 least 1 day if stored at 4 C. 4.6 NADPH solution 2) Prepare a solution of 5,6 mg of tetrasodium salt of reducedb-nicotinamide adenine dinucleotide phosphate,b-NADPH-Na 4 , per millilitre by using b-NADPH-Na 4 with a content by mass of not less than 98 %. The solution will be stable for at least 1 d
32、ay if stored at 4 C but for only 2 h at room temperature. 4.7 Nitrate reductase solution 2) Dissolve 65 mg of freeze-dried nitrate reductase having an activity of about 0,4 U/mg 3) and obtained from Aspergillus speices (EC 1.6.6.2) 4 in 5 ml of water. The solution will be stable for at least 2 weeks
33、 of storage at 4 C. 5 Apparatus and equipment Usual laboratory apparatus and, in particular, the following. 5.1 Homogenizer (disperser, electric mixer or similar equipment). Ensure that the equipment ensures thorough dispersion or homogenization of the sample and does not release heavy metals which
34、would inhibit the enzymes used. 5.2 Combined hot plate and magnetic stirrer. 5.3 Fluted filter paper. 5.4 Graduated pipettes for enzymatic analysis, set for partial discharge, in 0,05 ml, 0,1 ml, 1 ml and 2 ml, or piston-type pipettes. 5.5 Glass or plastics cells having an optical path length of 1 c
35、m and no significant absorption at wavelengths of 334 nm, 340 nm or 365 nm. 5.6 Spectrometer, for measurement at a wavelength of 340 nm, with a spectral bandwidth of not more than 5 nm. NOTE. Spectral-line filter photometers fitted with a mercury vapour lamp, for measurement at 365 nm or 334 nm, may
36、 also be used.Page 4 EN 12014-5 : 1997 BSI 1997 Table 1. Pipetting plan Fluid pipetted into the cells Blank Test solution Buffer solution (4.4) 1,00 ml 1,00 ml FAD solution (4.5) 0,05 ml 0,05 ml NADPH solution (4.6) 0,10 ml 0,10 ml Sample solution (6.2.1) 1,00 ml Water 2,00 ml 1,00 ml Mix, and after
37、 about 5 min measure the absorbances (A 1 ) with no cell (5.5) in the reference path, then start the reaction by adding the following Nitrate reductase solution (4.7) 0,05 ml 0,05 ml Mix and, after 40 min measure the absorbances of the solutions in quick succession, with no cell in the reference pat
38、h (A 2 ). Repeat this measurement after a further 20 min (A 3 ). 5.7 Centrifuge, capable of producing a centrifugal acceleration of 2 900 g at the base of centrifuge tubes (5.8), optional. 5.8 Centrifuge tubes, of suitable capacity. 5.9 Refrigerator, capable of being maintained at 4 C. 6 Procedure 6
39、.1 Sample preparation Bring sample material which has been stored in a cool place to room temperature. Mix the entire contents of the container in a suitable mixer (5.1), if necessary grinding and homogenizing it. Ensure that the temperature of the sample does not exceed 60 C. For fluid samples, if
40、necessary, dilute the sample to be analysed until the nitrate concentration is 30 mg/l to 300 mg/l. The resultant sample solution can be used for the determination without further preparation even if it is coloured, a volume (V 2 ) of 0,1 ml being taken for the test. 6.2 Determination 6.2.1 Sample s
41、olution preparation Weigh out about 5 g or another suitable amount of the sample prepared as specified in 6.1 to the nearest milligram into a 100 ml conical flask, and add 50 ml of boiling water. Cover the flask with a watch glass, place it in a boiling water bath (e.g. glass beaker) for 20 min to 3
42、0 min, stirring to prevent the formation of lumps (see 5.2). Prevent overheating the bottom of the conical flask by avoiding direct contact with the bottom of the water bath. Remove the magnetic stirrer, add 5,0 ml of Carrez solution No. 1 (4.1), and shake the mixture for 5 s to 10 s. Then add 5,0 m
43、l of Carrez solution No. 2 (4.2) and shake again for 5 s to 10 s. Adjust the pH of the mixture to a value of 8 to 9 (but not greater than 9) with sodium hydroxide solution (4.3) and shake again. Cool the contents of the conical flask to room temperature and filter quantitatively into a 100 ml volume
44、tric flask, V 1 , rinsing out the last portions with water. If the initial sample mass is high, for example more than 10 g, it is advisable to centrifuge the clarified sample mixture for 10 min at 2 900g before filtering it quantitatively into the 100 ml volumetric flask. Wash the settled matter twi
45、ce with water and centrifuge again, collect each of the supernatants in the 100 ml volumetric flask and then dilute the solution to the mark with water. Shake the volumetric flask and place it in a refrigerator (5.9) at approximately 4 C for 30 min to allow any fat to separate. Then filter the conte
46、nts of the flask through a fluted filter paper (5.3), discarding the first few millilitres of the filtrate. Collect the filtrate (sample solution) in a conical flask having a ground glass stopper. Use the sample solution to determine nitrate content employing 1,0 ml (V 2 )i n the test. Any slight cl
47、oudiness will not normally interfere with the determination. 6.2.2 Measurement of the enzymatic reaction Carry out the determination at about 20 C. The absorption maximum of NADPH occurs at a wavelength of 340 nm. If a spectrometer (5.6) is used, carry out the measurements at the absorption maximum,
48、 only, but if a spectral-line filter photometer fitted with a mercury vapour lamp is used, work at a wavelength of 365 nm or 334 nm. The change (decrease) in the absorbance of the test solution (A 1 2 A 2 ) test solution should be between 0,1 and 0,5. If the change in the absorbance is too small, th
49、e volume of the sample solution pipetted into the cell may be increased (up to 2,00 ml). In such cases, reduce the quantity of water used so that the cells for the test solution and the blank contain the same total volume. If the change in absorbance is too great, dilute the sample solution. If such steps are necessary, allow for the change in the dilution factor (F) in the calculation. Only one blank per series is necessary.Page 5 EN 12014-5 : 1997 BSI 1997 7 Calculation In the reaction which fo
