1、BRITISH STANDARD BS 4401-14: 1979 ISO 4134:1979 Methods of test for Meat and meat products Part 14: Determination of L-(+)-glutamic acid content (reference method) ISO title: Meat and meat products Determination of L-(+)-glutamic acid content (Reference method) UDC 637.5:664.91/.94:543:547.466.64BS4
2、401-14:1979 This British Standard, having been prepared under the directionof the Food and Agriculture Standards Committee,was published underthe authority of the Executive Board and comes into effect on 31 October1979 BSI 10-1999 The following BSI references relate to the work on this standard: Com
3、mittee reference FAC/6 Draft for comment 79/50880 DC ISBN 0 580 11024 9 Cooperating organizations The Food and Agriculture Standards Committee, under whose direction this British Standard was prepared, consists of representatives from the following Government departments and scientific and industria
4、l organizations. Agricultural Co-operation and Marketing Services Agricultural Research Council, Meat Research Institute British Food Manufacturing Industries Research Association* British Industrial Biological Research Association Ltd Campden Food Preservation Research Association Consumer Standard
5、s Advisory Committee of BSI Department of Agriculture and Fisheries for Scotland Department of Agriculture (Government of Northern Ireland) Department of Industry (Laboratory of the Government Chemist)* Flour Milling and Baking Research Association Food and Drink Industries Council Food Manufacturer
6、s Federation Incorporated Institute of Brewing Local Authority Joint Advisory Committee on Food Standards Ministry of Agriculture, Fisheries and Food* National Farmers Union* National Farmers Union of Scotland Tobacco Advisory Council The organizations marked with an asterisk in the above list, toge
7、ther with the following, were directly represented on the committee entrusted with the preparation of this British Standard: Agricultural Research Council Bacon and Meat Manufacturers Association British Poultry Federation Limited Chemical Society, Analytical Division Institute of Meat Licensed Anim
8、al Slaughterers and Salvage Association Meat and Livestock Commission National Federation of Meat Traders Associations (Inc) Public Health Laboratory Service Society of Chemical Industry Ulster Farmers Union Amendments issued since publication Amd. No. Date of issue CommentsBS4401-14:1979 BSI 10-199
9、9 i Contents Page Cooperating organizations Inside front cover National foreword ii 1 Scope and field of application 1 2 References 1 3 Definition 1 4 Principle 1 5 Reagents 1 6 Apparatus 2 7 Sampling and laboratory sample 2 8 Procedure 2 9 Expression of results 3 10 Test report 4 Annex Example of p
10、lotting and extrapolation of absorbance values 5 Publications referred to Inside back coverBS4401-14:1979 ii BSI 10-1999 National foreword This Part of BS4401 is identical with ISO4134 “Meat and meat products Determination of L-(+)-glutamic acid content (Reference method)” published by the Internati
11、onal Organization for Standardization (ISO). Other Parts of this British Standard are: Part 1: Determination of ash (technically equivalent to ISO/R937); Part 2: Determination of nitrogen content (technically equivalent to ISO/R936); Part 3: Determination of moisture content (technically equivalent
12、to ISO1442); Part 4: Determination of total fat content (technically equivalent to ISO1443-4); Part 5: Determination of free fat content (technically equivalent to ISO/R1444); Part 6: Determination of chloride content (technically equivalent to ISO/R1841); Part 7: Determination of nitrate content; P
13、art 8: Determination of nitrite content; Part 9: Measurement of pH (identical with ISO2917); Part 10: Determination of total phosphorus content (identical with ISO2294); Part 11: Determination of L()-hydroxyproline content (reference method) (identical with ISO3496); Part 12: Determination of starch
14、 content of meat products (reference method) (identical with ISO5554); Part 13: Determination of glucono-delta-lactone content (reference method) (identical with ISO4133). Terminology and conventions. The text of the International Standard has been approved as suitable for publication, without devia
15、tion, as a British Standard. Some terminology and certain conventions are not identical with those used in British Standards; attention is especially drawn to the following: Wherever the words “International Standard” appear, referring to this publication, they should be read as “British Standard”.
16、The comma has been used throughout as a decimal marker. In British Standards it is current practice to use a full point on the baseline as the decimal marker. NOTE 1The general reference to ISO3100 in clause2 and7.1 applies at present only to ISO3100-1:1975 which is related to BS5348-1:1976; subsequ
17、ent Parts of ISO3100 are expected to be published as dual-numbered Parts of BS5348. Cross-references International Standard Corresponding British Standard ISO648:1977 BS1583:1961 One-mark pipettes (Technically equivalent) ISO/R 835:1968 BS700:1976 Specification for graduated pipettes (including blow
18、out pipettes) (Technically equivalent) ISO1042:1975 BS1792:1960 One-mark volumetric flasks (Technically equivalent) ISO1442:1973 BS4401 Methods of test for meat and meat products Part 3:1970 Determination of moisture content (Technically equivalent) ISO 3100 BS5348 Methods for sampling meat and meat
19、 products (see note 1)BS4401-14:1979 BSI 10-1999 iii NOTE 2Textual errors. When the text of the International Standard was adopted, the printing errors, listed below, were noticed. They have been corrected in this British Standard and have been reported to ISO. The International Standard will be ame
20、nded in due course. Clause4, reaction1): “ -cetoglutarate” has been deleted and replaced by “ -cetoglutamate”. Clause4, reaction2): “chlorure diodonitrotetrazolium” has been deleted and replaced by “iodonitrotetrazolium chloride”. Clause5.3, paragraph a), line4: “exemple” has been deleted and replac
21、ed by “example”. A British 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 do
22、cument comprises a front cover, an inside front cover, pages i to iv, pages1to6, 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.iv blankBS4401
23、-14:1979 BSI 10-1999 1 1 Scope and field of application This International Standard specifies a reference method for the determination of the L-(+)-glutamic acid content of meat and meat products. 2 References ISO1442, Meat and meat products Determination of moisture content. ISO3100, Meat and meat
24、products Sampling. 3 Definition L-(+)-glutamic acid content of meat and meat products the L-(+)-glutamic acid content determined according to the procedure described in this International Standard and expressed as a percentage by mass 4 Principle Extraction of the L-(+)-glutamic acid present in a te
25、st portion with ice-cold perchloric acid solution. Centrifuging, decantation and filtration, followed by adjustment of pH and transformation of the L-(+)-glutamate in a portion of the filtrate by the following reactions1) with nicotinamide adenine dinucleotide (NAD), and2), with concomitant oxidatio
26、n of an equivalent amount of nicotinamide adenine dinucleotide (reduced) (NADH): Photometric measurement of the amount of formazane formed. 5 Reagents All reagents shall be of analytical quality. Except for the solutions of inorganic compounds(5.1 and5.2), all solutions shall be stored in stoppered
27、brown glass bottles which have been scrupulously cleaned and steamed or sterilized. The water used shall be double-distilled or demineralized and distilled water, obtained by carrying out the final distillation in an all-glass apparatus. NOTEWater distilled only once may contain metal ion traces, an
28、d demineralized water may contain micro-organisms. Metal ions may decrease the activity of enzymes, while micro-organisms may give rise to an aspecific enzymatic background activity that might adversely affect the results of analysis. 5.1 Perchloric acid solution,1,0M. Dilute8,6ml of perchloric acid
29、,70% (m/m), 20 1,67g/ml, to100ml with water. 5.2 Potassium hydroxide solution,2M. Dissolve56,1g of potassium hydroxide in water and dilute to500ml. 5.3 Triethanolamine-phosphate buffer solution,pH8,5. a) Dissolve1,86g of triethanolamine hydrochloride in water and adjust the pH to8,6 with the potassi
30、um hydroxide solution(5.2) using a pH meter. Add0,68g of octylphenol-decaethyleneglycolether (forexample Triton X-100) and dilute to100ml with water. b) Dissolve0,86g of dipotassium hydrogen phosphate (K 2 HPO 4 ) and0,007g of potassium dihydrogen orthophosphate (KH 2 PO 4 ) in water and dilute to10
31、0ml. Mix20ml of solution a) with5ml of solution b). Keep the solution at4 C. 5.4 Nicotinamide adenine dinucleotide (NAD) solution. Weigh0,025g of NAD in a small, stoppered flask and add5,0ml of water. The solution can be kept for at least4 weeks at4 C. 5.5 Iodonitrotetrazolium (INT) chloride 2-(p-io
32、dophenyl)-3-(p-nitrophenyl) 5-phenyltetrazolium chloride solution. Weigh0,030g of INT in a small, stoppered brown flask and add50ml of water. The solution can be kept for at least2 months at4 C in the dark. 5.6 Diaphorase (lipoamine-dehydrogenase, EC 1) 1.6.4.3) solution. Dissolve0,003g of lyophiliz
33、ed diaphorase in1ml of water. The solution can be kept for at least3 weeks at4 C. 5.7 Glutamate dehydrogenase (GIDH) (EC 1) 1.4.1.2) solution,10mg/ml, free from ammonium sulphate, ethylenedinitrilotetraacetic acid (EDTA) and glutaminase. 1) 2) 1) The EC number refers to the Enzyme Classification num
34、ber as given in: The International Union of Biochemistry, “Enzyme nomenclature”, Elsevier Publ. Co. Amsterdam1965.BS4401-14:1979 2 BSI 10-1999 This solution is supplied as such (for example in quantities of1,0ml) and can be kept for at least12months at4 C. 5.8 L-(+)-glutamic acid, standard solution.
35、 Dissolve0,0500g of L-(+)-glutamic acid in25ml of water. Adjust the pH to7,0 with the potassium hydroxide solution(5.2) and dilute to50ml. Keep this solution at4 C and dilute1+49 shortly before use. 6 Apparatus Usual laboratory equipment not otherwise specified, and the following items: 6.1 Mechanic
36、al meat mincer, laboratory size, fitted with a perforated plate with holes not exceeding4mm in diameter. 6.2 Laboratory mixer 6.3 Laboratory centrifuge with50 or100ml centrifuge tubes. 6.4 pH meter 6.5 Fluted filter papers, diameter about15 cm. 6.6 One-mark volumetric flasks, capacity100 and250ml, c
37、omplying with ISO1042, class A. 6.7 One-mark pipettes, capacity100,50 and25ml, complying with ISO648, class A. 6.8 Graduated pipettes, for delivering2,50,50,2 and0,05ml, complying with ISO/R835, class A. 6.9 Small plastic spatula, bent at90 , for mixing the contents of the photometric cell. 6.10 Pho
38、toelectric colorimeter, provided with a filter having a transmittance maximum at492nm, or spectrophotometer. 6.11 Photometric cells of10mm optical path length. 7 Sampling and laboratory sample 7.1 Sampling See ISO3100. 7.2 Laboratory sample Proceed from a representative sample of at least200g. Store
39、 the sample in such a way that deterioration and change in composition are prevented. 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 it in a completely filled, air-tight, closed container; store it, if
40、 necessary, in such a way that deterioration and change in composition are prevented. Analyse the sample as soon as possible, but always within24h. 8.2 Test portion Weigh, to the nearest10mg, approximately50g of the test sample(8.1) and transfer this test portion to the jar of the laboratory mixer(6
41、.2). 8.3 Preparation of extract 8.3.1 Add100ml of ice-cold perchloric acid solution(5.1) and homogenize. 8.3.2 Transfer a part of the homogenate to a centrifuge tube (see6.3). Centrifuge for10min at3000min 12)and, after having carefully moved aside the fat layer, decant the supernatant liquid throug
42、h a fluted filter paper(6.5) into a200ml conical flask, discarding the first10ml of the filtrate. 8.3.3 Transfer50ml of the solution (which should be only slightly turbid) into a100ml beaker and adjust the pH to10 with the potassium hydroxide solution(5.2). 8.3.4 Transfer the contents of the beaker
43、quantitatively into a100ml volumetric flask, dilute to the mark with water and mix. 8.3.5 Cool the solution in ice for10min, and filter through a fluted filter paper(6.5), discarding the first10ml of the filtrate. 8.3.6 Pipette25ml, or some other appropriate volume (V ml), of the filtrate into a250m
44、l volumetric flask and dilute to the mark with water. NOTEThe volume V should be chosen so that the concentration of L-(+)-glutamic acid is less than30mg/l. 8.4 Determination 8.4.1 Bring solutions5.3 and8.3.6 to a temperature of20 to25 C. Pipette into each of two photometric cells(6.11)2,50ml of the
45、 buffer solution(5.3),0,20ml of the NAD solution(5.4),0,20ml of the INT solution(5.5) and0,05ml of the diaphorase solution(5.6). Into one of the cells pipette0,50ml of the extract(8.3.6); the solution obtained is the test solution. 2) A rotational frequency of3000min 1corresponds to3000 revolutions
46、per minute.BS4401-14:1979 BSI 10-1999 3 Into the other cell pipette0,50ml of water; the solution obtained is the blank solution. Mix with the plastic spatula(6.9) and read the absorbance of each cell at492nm against air. The temperature of the solution should be20 to25 C. Note the absorbances as: 8.
47、4.2 Pipette0,05ml of the GIDH solution(5.7) on the plastic spatula(6.9). Mix with the contents of one of the cells by moving the spatula up and down. Repeat this operation with the second cell. Read the absorbance of each cell at492nm after10 to15min and every2min thereafter until a constant increas
48、e in absorbance is obtained. Plot the absorbance against time. Extrapolate the absorbance values to the moment of start of the reaction (see Annex). Note these extrapolated absorbance values as: 8.4.3 Determine the micromolar absorptivity of the formazane by repeating the operations described in8.4.
49、1 and8.4.2, but replacing the5,0ml of extract in the first photometric cell by0,5ml of the standard L-(+)-glutamic acid solution(5.8). Note the absorbances corresponding to the operations carried out in accordance with8.4.1 as: and the extrapolated absorbance values corresponding to the operations carried out in accordance with8.4.2 as: 8.5 Duplicate determination Carry out two independent determinations starting with different test portions taken from the same test sample(8.1). 9 Expression of results 9.1 Method
