1、BRITISH STANDARD BS 4401-15: 1981 ISO 5553:1981 Methods of test for Meat and meat products Part 15: Detection of polyphosphates ISO title: Meat and meat products Detection of polyphosphates UDC 637.5:664.91/.94:543.061:546.185BS4401-15:1981 This British Standard, having been prepared under the direc
2、tionof the Food and Agriculture Standards Committee,was published underthe authority of the Executive Board and comes into effect on 30 October1981 BSI 10-1999 The following BSI references relate to the work on this standard: Committee reference FAC/6 Draft for comment 79/53563 ISBN 0 580 12325 1 Co
3、operating organizations The Food and Agriculture Standards Committee, under whose direction this British Standard was prepared, consists of representatives from the following: Agricultural Cooperation and Marketing Services Agricultural Research Council (Meat Research Institute)* British Food Manufa
4、cturing Industries Research Association* British Industrial Biological Research Association Campden Food Preservation Research Association Consumer Standards Advisory Committee of BSI Department of Agriculture and Fisheries for Scotland Department of Agriculture (Government of Northern Ireland) Depa
5、rtment of Industry (Laboratory of the Government Chemist)* Flour Milling and Baking Research Association Food Manufacturers Federation Incorporated Institute of Brewing Local Authorities Co-ordinating Body of Trading Standards Ministry of Agriculture, Fisheries and Food* National Farmers Union Natio
6、nal Farmers Union of Scotland Tobacco Advisory Council The organizations marked with an asterisk in the above list, together with the following, were directly represented on the Technical Committee entrusted with the preparation of this British Standard: Agricultural Research Council (Food Research
7、Institute) Agricultural Research Council (Institute for Research on Animal Diseases) Bacon and Meat Manufacturers Association British Poultry Federation Limited Institute of Meat Licensed Animal Slaughterers and Salvage Association Meat and Livestock Commission National Federation of Meat Traders As
8、sociations Incorporated Public Health Laboratory Service Royal Society of Chemistry (Analytical Division) Society of Chemical Industry Amendments issued since publication Amd. No. Date of issue CommentsBS4401-15:1981 BSI 10-1999 i Contents Page Cooperating organizations Inside front cover National f
9、oreword ii 1 Scope 1 2 Field of application 1 3 Reference 1 4 Principle 1 5 Reagents 1 6 Apparatus 1 7 Sample 2 8 Procedure 2 9 Interpretation 2 10 Test report 3 Publication referred to Inside back coverBS4401-15:1981 ii BSI 10-1999 National foreword This Part of this British Standard, which has bee
10、n prepared under the direction of the Food and Agriculture Standards Committee, is identical with ISO5553:1980 “Meat and meat products Detection of polyphosphates”, published by the International Organization for Standardization (ISO). Terminology and conventions. The text of the International Stand
11、ard has been approved as suitable for publication as a British Standard without deviation. Some terminology and certain conventions are not identical with those used in British Standards; attention is especially drawn to the following. The comma has been used throughout as a decimal marker. In Briti
12、sh Standards it is current practice to use a full point on the baseline as the decimal marker. Wherever the words “International Standard” appear, referring to this publication, they should be read as “British Standard”. NOTEThe general reference to ISO3100 in clauses3 and7.1 applies at present only
13、 to ISO3100-1:1975 which is related to BS5348-1:1976; subsequent Parts of ISO3100 are expected to be published as dual-numbered Parts of BS5348. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct app
14、lication. Compliance with a British Standard does not of itself confer immunity from legal obligations. Cross-reference International Standard Corresponding British Standard ISO 3100 (see note) BS 5348 Methods for sampling meat and meat products Summary of pages This document comprises a front cover
15、, an inside front cover, pagesi andii, 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.BS4401-15:1981 BSI 10-1999 1 1 Scope This In
16、ternational Standard specifies a method for the detection of linear condensed phosphates in meat and meat products by thin layer chromatographic separation. 2 Field of application Since polyphosphates are gradually hydrolyzed by enzymes present in the meat or meat product and during heat treatment o
17、f the meat or meat product, this International Standard only applies to the detection of added polyphosphates that are still present in the sample at the time of investigation. 3 Reference ISO 3100, Meat and meat products Sampling. 4 Principle Extraction of the meat or meat product with trichloroace
18、tic acid. Clearing of the serum obtained with ethanol/diethyl ether mixture. Separation of the phosphates by thin layer chromatography and detection of polyphosphates by spraying with reagents for colour development. 5 Reagents All reagents shall be of recognized analytical quality. Distilled water
19、or water of at least equivalent purity shall be used. WARNING All appropriate safety precautions shall be observed when carrying out the procedures specified in this International Standard. 5.1 Trichloroacetic acid 5.2 Diethyl ether 5.3 Ethanol, 95% (V/V). 5.4 Cellulose powder, for thin layer chroma
20、tography. 5.5 Soluble starch 5.6 Reference mixture Dissolve in100ml of water. 200 mg of sodium dihydrogen phosphate monohydrate (NaH 2 PO 4 .H 2 O), 300 mg of tetrasodium diphosphate decahydrate (Na 4 P 2 O 7 .10H 2 O), 200 mg of pentasodium triphosphate (Na 5 P 3 O 10 ), and 200 mg of sodium hexame
21、taphosphate (NaPO 3 ) x x 10. The reference mixture is stable at4 C for at least4weeks. 5.7 Developing solvent Mix 140ml of isopropyl alcohol, 40ml of a135g/l solution of trichloroacetic acid, and0,6ml of ammonium hydroxide, 20=0,90g/ml, about25% (m/m) solution. Keep the solvent in a tightly closed
22、bottle. 5.8 Spray reagent I Mix equal volumes of a 75g/l solution of ammonium molybdate tetrahydrate (NH 4 ) 6 Mo 7 O 24 .4H 2 O and concentrated nitric acid ( 20=1,40g/ml) and dissolve10g of tartaric acid in100ml of this mixture. Prepare the reagent on the day of use. 5.9 Spray reagent II Dissolve
23、0,5 g of 1-amino-2-naphthol-4-sulphonic acid in a mixture of195ml of a150g/l solution of sodium disulphite (sodium metabisulphite;Na 2 S 2 O 5 ) and5ml of a200g/l solution of sodium sulphite(Na 2 SO 3 ). Dissolve40g of sodium acetate trihydrate (NaOOCCH 3 .3H 2 O) in this mixture. Store the reagent
24、in a tightly closed brown bottle in the refrigerator. Discard the solution after1week. 6 Apparatus Usual laboratory equipment not otherwise specified and the following items: 6.1 Glass plates, thoroughly degreased,10cm 20cm. 6.2 Spreading device, for preparing layers of0,25mm thickness. If such a de
25、vice is not available, ready-to-use thin-layer plates with layer thicknesses of0,25mm can be used provided that starch is used as the binder. Plates containing gypsum (calcium sulphate) are not suitable. 6.3 Laboratory mixer 6.4 Desiccator 6.5 Mechanical meat mincer, laboratory size, fitted with a p
26、late with holes of diameter not exceeding4mm. 6.6 Fluted filter paper, of diameter15cm. 6.7 Micro-pipette, 14l, or micro-syringe with micrometer screw and bent glass tip. 6.8 Paper-lined glass tank, of appropriate dimensions, with tightly fitting lid, for development of thin-layer chromatograms. 6.9
27、 Hair-dryer, capable of providing either an air stream at room temperature or a warm air stream. 6.10 Sprayer 6.11 Oven, capable of being controlled at60 C.BS4401-15:1981 2 BSI 10-1999 7 Sample 7.1 Proceed from a laboratory sample of at least200g. See ISO3100. 7.2 Prepare the test sample on the day
28、of its receipt in the laboratory. 8 Procedure 8.1 Preparation of thin-layer plates Dissolve 0,3g of starch (5.5) in90ml of boiling water. Cool, add15g of cellulose powder (5.4) and homogenize in the laboratory mixer (6.3) for1min. Apply this slurry onto glass plates (6.1) with the spreading device (
29、6.2) adjusted to obtain a layer of0,25mm. Air-dry the plates undisturbed for60min at room temperature and heat them finally for10min at100 C. Store the plates in the desiccator (6.4). Alternatively, ready-to-use thin-layer plates may be used (see6.2). 8.2 Preparation of the test sample Homogenize th
30、e sample by passing it at least twice through the meat mincer (6.5) and by mixing. Keep it in a completely filled, air-tight, closed container and store it, if necessary, in a refrigerator. Analyse the sample as soon as possible, but in any case within5h. 8.3 Preparation of serum 8.3.1 Macerate 50g
31、of the test sample (8.2) with15ml of water at40 to60 C in a beaker by means of a spatula or a flattened stirring rod until a homogeneous mass is obtained, but taking no more than5min. 8.3.2 Add 10 g of the trichloroacetic acid (5.1) and again mix thoroughly. 8.3.3 Immediately place in a refrigerator
32、 for1h and then collect the separated serum by decanting through the fluted filter paper (6.6). 8.3.4 If the filtrate is turbid, shake once with an equal volume of the diethyl ether (5.2). Remove the ether layer with a small pipette and add an equal volume of the ethanol (5.3) to the aqueous phase.
33、Shake for1min. Allow the mixture to stand for a few minutes and filter through a fluted filter paper(6.6). 8.4 Chromatographic separation 8.4.1 Pour developing solvent (5.7) into the developing tank (6.8) to a depth of5 to10mm and close the tank with its lid. Allow to stand for at least30min at ambi
34、ent temperature, protected from sunlight and draughts. 8.4.2 Apply 34l of the serum, or64l if the clearing procedure of8.3.4 was carried out, to the cellulose layer (8.1) on a pencil line drawn at about2cm from the bottom. Keep the spots small by applying14l at a time. Use a warm air stream from the
35、 hair-dryer (6.9) for drying. NOTEHot air should be avoided because of the danger of hydrolysis of phosphates. 8.4.3 In the same way, apply34l of the reference mixture (5.6) to the plate at a distance of1 to1,5cm from the sample spot, but at exactly the same distance from the bottom. 8.4.4 Remove th
36、e lid from the tank and quickly but carefully place the cellulose plate in the tank. Replace the lid immediately. Develop the plate at ambient temperature, protected from sunlight and draughts. 8.4.5 Continue the development until the solvent front has ascended approximately10cm from the pencil line
37、. Remove the plate from the tank and dry for10min in the oven (6.11) controlled at60 C, or alternatively, for30min at ambient temperature, or in a stream of cold air. 8.5 Detection of phosphates 8.5.1 Place the plate vertically under a fume hood and spray the plate lightly but uniformly with spray r
38、eagent I (5.8). Yellow spots appear immediately. 8.5.2 Dry the plate in a stream of warm air from the hair dryer (6.9). Subsequently heat in an oven for at least1h at100 C to remove the last traces of nitric acid. Remove the plate from the oven and verify the absence of the pungent smell of nitric a
39、cid. 8.5.3 Allow the plate to cool to room temperature and then replace it under the fume hood. Spray the plate lightly but uniformly with spray reagentII(5.9). Blue spots appear immediately. NOTESpraying with reagent II is not an absolute necessity. However, the intense blue spots produced by this
40、reagent improve the detection considerably. 9 Interpretation Compare the migration distances of the phosphate spots from the sample with those of the phosphates from the reference mixture. An orthophosphate spot is always present. If the sample contained condensed phosphates, a diphosphate spot and/
41、or spots of more highly polymerized phosphates are visible.BS4401-15:1981 BSI 10-1999 3 The R Fvalues of the phosphates in the reference mixture are: Generally the R Fvalues of the polyphosphates in extracts of meat and meat products are somewhat lower. NOTECorrections for the differences in R Fvalu
42、es of the phosphates in the sample extract and in the reference mixture can be obtained by placing an extract of the fresh meat sample on the same plate. As fresh meat only contains monophosphates, the percentage correction can be obtained by comparison of the migration distances of this standard sp
43、ot with the corresponding spot from the reference mixture. 10 Test report The test report shall show the method used and the results obtained. It shall also mention all operating conditions not specified in this International Standard or regarded as optional, as well as any circumstances that may ha
44、ve influenced the results. The report shall include all details necessary for complete identification of the sample. orthophosphate from 0,80 to 0,90 diphosphate (pyrophosphate) from 0,50 to 0,60 triphosphate from 0,25 to 0,35 hexametapolyphosphate (Grahams salt) 0,04 blankBS4401-15:1981 BSI 10-1999
45、 Publication referred to See national foreword.BS 4401-15: 1981 ISO5553:1981 BSI 389 Chiswick High Road London W4 4AL BSIBritishStandardsInstitution BSI is the independent national body responsible for preparing BritishStandards. It presents the UK view on standards in Europe and at the internationa
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