1、BRITISH STANDARD CONFIRMED OCTOBER 1992 BS 4401-7: 1976 Methods of test Meat and meat products Part 7: Determination of nitrate content UDC 637.5:664.91/.94:543:546.17BS4401-7:1976 This British Standard, having been prepared under the directionof the Farming, Food andAgriculture Standards Committee,
2、 was published underthe authority of the Executive Board on 30 July1976 BSI 08-1999 The following BSI references relate to the work on this standard: Committee reference FAC/6 Draft for comment 72/52092 ISBN 0 580 09321 2 Co-operating organizations The Farming, Food and Agriculture Standards Committ
3、ee, under whose supervision this British Standard was prepared, consists of representatives from the following Government departments and consumer, scientific and industrial organizations. Agricultural Co-operative Company and Market Services British Food Manufacturing Industries Research Associatio
4、n* British industrial Biology Research Association Central Council for Agricultural and Horticultural Co-operation Consumer Standards Advisory Committee of BSI Department of Agriculture and Fisheries for Scotland Department of Industry Laboratory of the Government Chemist* Flour Milling and Baking R
5、esearch Association Food Manufacturers Federation Inc.* Campden Food Preservation Research Association Institute of Brewing Local Authorities Joint Advisory Committee on Food Standards Ministry of Agriculture, Fisheries and Food* Ministry of Agriculture, Government of Northern Ireland National Farme
6、rs Union* National Farmers Union of Scotland Tobacco Advisory Committee The organizations marked with an asterisk in the above list, together with the following, were directly represented on the committee entrusted with the preparation of this British Standard: Agricultural Research Council British
7、Bacon Curers Federation British Poultry Federation Institute of Meat Licensed Animal Slaughterers and Salvage Association Meat and Livestock Commission Meat Manufacturers Association National Council of Associations of Fresh Meat Wholesalers National Federation of Meat Traders Associations Public He
8、alth Laboratory Service Sausage and Meat Pie Manufacturers Association Society of Analytical Chemistry Society of Chemical Industry Ulster Farmers Union Amendments issued since publication Amd. No. Date of issue CommentsBS4401-7:1976 BSI 08-1999 i Contents Page Co-operating organizations Inside fron
9、t cover Foreword ii 1 Scope 1 2 References 1 3 Definition 1 4 Principle 1 5 Reagents 1 6 Apparatus 2 7 Sample 2 8 Procedure 2 9 Expression of result 3 10 Test report 4 Figure 1 Reduction column 4 Publications referred to Inside back coverBS4401-7:1976 ii BSI 08-1999 Foreword The separate Parts of th
10、is British Standard describe chemical methods prepared by Sub-committee 6 Meat and Meat Products, of ISO/TC34 Agricultural Food Products. Apart from editorial modifications and the differences described in the note to clause 1 and note 2 to 8.6, this Part is in technical agreement with ISO3091. A Br
11、itish Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. Summary of pages This document comprises a fr
12、ont cover, an inside front cover, pages i and ii, pages1 to4, an inside back cover and a back cover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will be indicated in the amendment table on the inside front cover.BS4401-7:1976 BSI 08-1999 1 1 Sco
13、pe This Part of BS4401 describes a reference method for the determination of the nitrate content of meat and meat products. The method is applicable to all meat products, other than those having an unusually high content of strongly reducing substances. Strongly reducing substances (e.g. ascorbic ac
14、id, ascorbates) tend to reduce nitrite under the acidic conditions obtained in this method. If the content of reducing substances is much higher than that which has normally resulted from the manufacturing processes hitherto applied, the results obtained by this method could be misleading. NOTEISO30
15、91 describes the method as suitable for meat and meat products, without indicating any limitation. 2 References The titles of the British Standards referred to in this standard are listed on the inside back cover. 3 Definition For the purposes of this British Standard the following definition applie
16、s. nitrate content the quantity of nitrate determined by the procedure described. Nitrate content is expressed as milligrams of potassium nitrate per kilogram (partsper million) 4 Principle The method consists of the extraction of a test portion with hot water, precipitation of the proteins, filtrat
17、ion and reduction of the extracted nitrate to nitrite by metallic cadmium. After development ofared colour by addition of sulphanilamide and N-1-naphthylethylenediamine dihydrochloride to the filtrate, photometric measurement is carried out at a wavelength of 538nm. 5 Reagents 5.1 General. The reage
18、nts described in 5.2 to 5.8 inclusive are required and shall be of analytical quality. The water used shall comply with the requirements of BS3978. 5.2 Zinc rods, length about 15cm and diameter5mm to 7mm. 5.3 Solutions for precipitation of proteins 5.3.1 Reagent I. Dissolve 106g of potassium ferrocy
19、anide trihydrate K 4 Fe(CN) 6 .3H 2 O in water and dilute to 1000ml. 5.3.2 Reagent II. Dissolve 220g of zinc acetate dihydrate Zn(CH 3 COO) 2 .2H 2 O and 30ml of glacial acetic acid in water and dilute to 1000ml. 5.3.3 Borax solution saturated. Dissolve 50g ofdisodium tetraborate decahydrate (Na 2 B
20、 4 O 7 .1OH 2 O) in 1000ml of tepid water and cool to room temperature. 5.4 Cadmium sulphate solution, 30g/l. Dissolve 37g of cadmium sulphate (3CdSO 4 .8H 2 O) in water and dilute to 1000ml. 5.5 Hydrochloric acid, dilute, about 0.1N. Dilute8ml of concentrated hydrochloric acid( 20 1.19g/ml) to 1000
21、ml with water. 5.6 Ammonia buffer solution, pH 9.6 to 9.7. Dilute20ml of concentrated hydrochloric acid( 20 1.19g/ml) with 500ml of water. After mixing, add10g of disodium dihydrogen ethylenediamine-NNN N-tetra-acetate dihydrate, CH 2 N(CH 2 COOH)CH 2 COONa 2 .2H 2 O, and 55ml of concentrated ammoni
22、a solution ( 200.88g/ml). Dilute to 1000ml with water and mix. Check the pH. 5.7 Standard sodium nitrite solutions. Dissolve1.000g of sodium nitrite (NaNO 2 ) in water and dilute to 100ml in a one-mark volumetric flask. Pipette 5ml of the solution into a 1000ml one-mark volumetric flask. Dilute to t
23、he mark. Prepare a series of standard solutions by pipetting5ml, 10ml and 20ml respectively of this solution into 100ml one-mark volumetric flasks and diluting to the mark with water. These standard solutions contain respectively, 2.54g, 5.04g and10.04g of sodium nitrite per millilitre. The standard
24、 solutions and the dilute (0.05g/l) sodium nitrite solution from which they are prepared shall be made up on the day of use. 5.8 Solutions necessary for colour development 5.8.1 Solution I. Dissolve 2g of sulphanilamide (NH 2 C 6 H 4 SO 2 NH 2 ) in 800ml of water by heating on a water bath. Cool, fi
25、lter if necessary, and add100ml of concentrated hydrochloric acid( 20 1.19g/ml), while stirring. Dilute to1000ml with water. 5.8.2 Solution II. Dissolve 0.25g of N-1-naphthylethylenediamine dihydrochloride (C 10 H 7 NHCH 2 CH 2 NH 2 .2HCl) in water. Dilute to250ml with water. Store the solution in a
26、 well stoppered brown bottle and keep it in a refrigerator, for not longer than one week. 5.8.3 Solution III. Dilute 445ml of concentrated hydrochloric acid ( 201.19g/ml) to 1000ml with water.BS4401-7:1976 2 BSI 08-1999 5.9 Standard potassium nitrate solution. Dissolve1.465g of potassium nitrate (KN
27、O 3 ) in water and dilute to 100ml in a one-mark volumetric flask. Pipette 5ml of the solution into a 1000ml volumetric flask and dilute to the mark. Prepare this solution on the day of use. This solution contains 73.254g of potassium nitrate per millilitre. 6 Apparatus Usual laboratory equipment is
28、 required and, in particular, the following items. 6.1 Mechanical meat mincer, laboratory size, fitted with a perforated plate with holes not greater than4mm in diameter. 6.2 Analytical balance 6.3 One-mark volumetric flasks, 100ml, 200ml and1000ml sizes, class B complying with the requirements of B
29、S1792. 6.4 One-mark pipettes, 10ml and 20ml sizes and, if necessary, another size according to the aliquot of filtrate (see 8.9.1). The pipettes shall comply with the requirements for class A of BS1583. 6.5 Boiling water bath 6.6 Fluted filter paper, diameter about 15cm, free of nitrite and nitrate.
30、 6.7 Reduction column, glass, of the form and dimensions shown in Figure 1. A flexible connection is desirable between the bottom of the column and the exit capillary tube so that the position of the outlet, and hence the flow rate, may be regulated. 6.8 Photoelectric colorimeter, or spectrophotomet
31、er, with cells of 1cm optical path length. 6.9 Conical flask, 300ml. 7 Sample 7.1 Proceed from a representative sample of at least200g. (See BS 1)“Methods for sampling meat and meat products”.) 7.2 Prepare the test sample (see 8.1) immediately or, if this cannot be done, store the sample at a temper
32、ature of 0 to 5 C, for not longer than 4days. 8 Procedure 8.1 Preparation of test sample. Make the sample homogeneous by passing it at least twice through the meat mincer (6.1) and mixing. Keep the homogenized sample in a completely filled, air-tight, closed container under refrigeration. Analyse un
33、cooked products immediately after homogenization. For other samples analyse the test sample as soon as possible, but always within 24h. 8.2 Preparation of the cadmium column 8.2.1 Place 3 to 5 zinc rods (5.2) in the cadmium sulphate solution (5.4) contained in a beaker (1litre of cadmium sulphate so
34、lution is sufficient for preparing one cadmium column). 8.2.2 Remove the spongy metallic cadmium deposit from the zinc rods every 1h or 2h by swirling them in the solution or rubbing them against each other. 8.2.3 After 6h to 8h, decant the solution and wash the deposit twice with 1litre of water, t
35、aking care that the cadmium is continuously covered with a layer of liquid. 8.2.4 Transfer the cadmium with 400ml of 0.1N hydrochloric acid (5.5) to a laboratory mixer and blend for 10s. Return the contents of the mixer to the beaker. 8.2.5 Occasionally stir up the cadmium with a glass rod. After le
36、aving the cadmium for a night under the hydrochloric acid, stir once more to remove all bubbles of gas from the cadmium. 8.2.6 Decant the solution and wash the cadmium twice, each time with 1litre of water. 8.2.7 Fit a glass wool plug to the bottom of the glass column (see Figure 1). 8.2.8 Wash the
37、cadmium into the glass column withwater until the height of the cadmium bed is about 17cm. Drain the column occasionally during filling, taking care not to allow the level of the liquid to fall below the top of the cadmium bed. Eliminate inclusions of gas (for example with a plastics knitting needle
38、). The liquid should flow out at a rate not exceeding 3ml/min. 8.3 Duplicate procedure. Carry out in duplicate the procedure described in 8.4, 8.5, 8.8 and 8.9. 8.4 Test portion. Weigh, to the nearest milligram,10g of the test sample. 8.5 Precipitation of protein 8.5.1 Transfer the test portion quan
39、titatively into the conical flask (6.9) and add, successively, 5ml of saturated borax solution (5.3.3) and 100ml of water at a temperature not below 70 C. 8.5.2 Heat the flask and its contents for 15min on the boiling water bath (6.5) and shake the flask repeatedly. 8.5.3 Allow the flask and its con
40、tents to cool to roomtemperature and add, successively, a 2ml of reagent I (5.3.1) and 2ml of reagent II (5.3.2). Mix thoroughly after each addition. 1) In course of preparation.BS4401-7:1976 BSI 08-1999 3 8.5.4 Transfer the contents to a 200ml one-mark volumetric flask (6.3). Dilute to the mark wit
41、h water and mix. Allow the flask to stand for 30min at room temperature. 8.5.5 Carefully decant the supernatant liquid and filter it through the fluted filter paper (6.6) so as to obtain a clear solution. NOTEAfter protein precipitation the same filtrate can be used to determine both the nitrate and
42、 the nitrite contents. 8.6 Pre-treatment of the cadmium column. Wash the cadmium column successively with 25ml of dilute hydrochloric acid (5.5), 50ml of water, and25ml of the ammonia buffer solution (5.6) diluted 1 + 9. Do not permit the level of the liquid in the funnel to fall below the top of th
43、e capillary inlet tube of the cadmium column. NOTE 1If the column is in frequent use, the pre-treatment described in 8.6 need not be repeated each time the column is used. NOTE 2ISO3091 requires that, during use, the column is washed with water in place of the diluted buffer solution and that the pr
44、etreatment should be done as part of every determination. 8.7 Checking the reducing capacity of the cadmium column 8.7.1 Pipette 20ml of the potassium nitrate standard solution (5.9) and simultaneously add 5ml of the ammonia buffer solution (5.6), into the reservoir on top of the cadmium column. Col
45、lect the effluent in a 100ml one-mark volumetric flask (6.3). 8.7.2 When the reservoir is nearly empty, wash the walls with about 15ml of the ammonia buffer solution (5.6) diluted 1 + 9. Repeat the same treatment with another 15ml portion of the diluted buffer solution. After this portion has run in
46、to the column as well, completely fill the reservoir with the diluted (1 + 9) buffer solution. (See note 2 to 8.6.) 8.7.3 After nearly 100ml of effluent has been collected, remove the flask from under the column, dilute to the mark with water and mix. 8.7.4 Pipette 10ml of the effluent into a 100ml
47、one-mark volumetric flask (6.3) and proceed as specified in 8.9.2 to 8.9.4. 8.7.5 If the nitrite concentration of the effluent, as determined from the calibration curve (see 8.10), isbelow 0.904g of sodium nitrite per millilitre (i.e.90% of the theoretical value), the cadmium column should be reject
48、ed and prepared afresh. 8.8 Reduction of nitrate to nitrite 8.8.1 Pipette into the reservoir on top of the column20ml of the filtrate (8.5.5) and simultaneously add 5ml of the ammonia buffer solution (5.6). Collect the effluent from the column in a 100ml one-mark volumetric flask (6.3). 8.8.2 Procee
49、d as described in 8.7.2 and 8.7.3. 8.9 Colour measurement 8.9.1 Pipette an aliquot portion (V ml) of the effluent, but not more than 25ml, into a 100ml one-mark volumetric flask (6.3) and add water to obtain a volume of about 60 ml. 8.9.2 Add 10ml of solution I (5.8.1), followed by 6ml of solution III (5.8.3), mix and leave the solution for5min at room temperature in the dark. 8.9.3 Add 2ml of solution II (5.8.2), mix and leavethe solution for 3min to 10min at room temperature in the dark. Dilute to the mark with water. 8.9.4 Measure the absorbance
