1、| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | BRITISH STANDARD BS EN 12632:1999 The Euro
2、pean Standard EN 12632:1999 has the status of a British Standard ICS 67.160.20 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW Fruit and vegetable juices Enzymatic determination of acetic acid (acetate) content NAD spectrometric methodThis British Standard, having been prepare
3、d under the direction of the Consumer Products and Services Sector Committee, was published under the authority of the Standards Committee and comes into effect on 15 July 1999 BSI 07-1999 ISBN 0 580 32212 2 BS EN 12632:1999 Amendments issued since publication Amd. No. Date Comments National forewor
4、d This British Standard is the English language version of EN 12632:1999. The UK participation in its preparation was entrusted to Technical Committee AW/21, Fruit and vegetable juices, which has the responsibility to: aid enquirers to understand the text; present to the responsible European committ
5、ee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. A list of organizations represented on this committee can be obtained on request to its secretary. Cross-referen
6、ces The British Standards which implement international or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Find” facility of the BSI Standards Electronic Catalog
7、ue. 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 document compri
8、ses a front cover, an inside front cover, 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 Komitee fu r Normung Central Secretariat: rue de Stassart 36, B-1050 Brussels 1999 CEN All right
9、s of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 12632:1999 E EUROPEAN STANDARD EN 12632 NORME EUROPE ENNE EUROPA ISCHE NORM February 1999 ICS 67.160.20 Descriptors: fruit and vegetable juices, chemical analysis, determination of content, acetic
10、 acid, enzymatic methods, spectrophotometric analysis, procedure English version Fruit and vegetable juices Enzymatic determination of acetic acid (acetate) content NAD spectrometric method Jus de fruits et de le gumes Dosage enzymatique de lacide ace tique (ace tate) Me thode spectrome trique par l
11、e NAD Frucht- und Gemu sesa fte Enzymatische Bestimmung des Gehaltes an Essigsa ure (Acetat) Spektralphotometrische Bestimmung von NAD This European Standard was approved by CEN on 8 January 1999. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the condition
12、s 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 offi
13、cial 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,
14、Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.Page 2 EN 12632:1999 BSI 07-1999 Foreword This European Standard has been prepared by Technical Committee CEN/TC 174,
15、Fruit and vegetable juices Methods of analysis, the Secretariat of which is held by AFNOR. 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 August 1999, and conflicting national standards shall be
16、withdrawn at the latest by August 1999. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Ita
17、ly, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Contents Page Foreword 2 1 Scope 3 2 Normative references 3 3 Symbols and abbreviations 3 4 Principle 3 5 Reagents 3 6 Apparatus 4 7 Procedure 4 8 Calculation 6 9 Precision 6 10 Test report 6 Annex A (i
18、nformative) Bibliography 7 Annex B (informative) Statistical results of the inter-laboratory tests 7 Annex C (informative) Information on how to treat “creep” reactions 8Page 3 EN 12632:1999 BSI 07-1999 1 Scope This European Standard specifies an enzymatic method for the determination of the total c
19、ontent of acetic acid or acetate salts in fruit and vegetable juices and related products. 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
20、publications are listed hereafter. For dated 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 ISO
21、3696:1995, Water for analytical laboratory use Specification and test methods (ISO 3696:1987) 3 Symbols and abbreviations 3.1 Symbols For the purposes of this standard, the following symbols apply: c substance concentration; r mass concentration; g acceleration due to gravity at the surface of the e
22、arth (9,81 m/s 2 ). 3.2 Abbreviations For the purposes of this standard, the following abbreviations apply: ACS Acetyl Coenzyme A synthetase; CoA Coenzyme A; ATP Adenosine-59-Tri-phosphate; AMP Adenosine-Mono-phosphate; CS Citrate synthase; NAD b-Nicotinamide-adenine-dinucleotide; NADH b-Nicotinamid
23、e-adenine-dinucleotide, reduced form; MDH Malate-dehydrogenase; IU 1 International Unit (IU) of enzyme activity catalyses the conversion of 1mmol of substance per minute at 25 8C under standard conditions. 4 Principle Acetic acid (acetate) is converted in the presence of the enzyme acetyl-coenzyme A
24、-synthetase (ACS) with adenosine-59-triphosphate (ATP) and coenzyme A (CoA) to acetyl-CoA (reaction 1): ACS Acetate + ATP + CoA, acetyl2 CoA + AMP + pyrophosphate (1) Acetyl-CoA reacts with oxaloacetate to produce citrate in the presence of citrate synthase (CS) (reaction 2): CS Acetyl-CoA + oxaloac
25、etate + H 2 O, citrate + CoA (2) The oxaloacetate required for reaction (2) is formed from malate and nicotinamide-adenine dinucleotide (NAD) in the presence of malate dehydrogenase (MDH) (reaction 3). In this reaction NAD is reduced to NADH. MDH Malate + NAD + , oxaloacetate + NADH + H + (3) The de
26、termination is based on the formation of NADH measured by the increase in absorbance at 340 nm, 334 nm or 365 nm. Since a preceding indicator reaction is used, the amount of NADH formed is related to the acetic acid concentration but is not linearly proportional. 5 Reagents 5.1 General Use only reag
27、ents of recognized analytical grade and only water in accordance with at least grade 3 of EN ISO 3696:1995. NOTE The determination can also be carried out using a commercially available test kit. 5.2 Triethanolamine hydrochloride. 5.3 L-malic acid. 5.4 Magnesium chloride, MgCl 2 .6H 2 O. 5.5 Potassi
28、um hydroxide solution, c(KOH) = 2 mol/l. 5.6 Nicotinamide-adenine dinucleotide (ca. 98 %). 5.7 Coenzyme A. 5.8 Adenosine-59-triphosphate, disodium salt ATP-Na 2 H 2 . 5.9 Sodium hydrogen carbonate, NaHCO 3 . 5.10 Malate dehydrogenase, suspension in ammonium sulfate, c(MDH) = 3,2 mol/l, specific acti
29、vity approximately 1 200 IU/mg.Page 4 EN 12632:1999 BSI 07-1999 5.11 Citrate synthase, suspension in ammonium sulfate, c(CS) = 3,2 mol/l, specific activity approximately 110 IU/mg. 5.12 Acetyl-CoA synthetase, lyophilized. 5.13 Ammonium sulfate, (NH 4 ) 2 SO 4 . 5.14 Sodium acetate,C H 3 COONa.3H 2 O
30、. 5.15 Buffer solution, pH = 8,4. Dissolve 7,59 g of triethanolamine hydrochloride (5.2), 420 mg of malic acid (5.3) and 210 mg of magnesium chloride (5.4) in approximately 70 ml of water. Adjust the solution pH to 8,4 with approximately 21 ml of the potassium hydroxide solution (5.5) and make up to
31、 100 ml with water. The buffer is stable for 4 weeks at +48C. 5.16 Nicotanimide-adenine dinucleotide/Coenzyme A solution Dissolve 144 mg of NAD (5.6) and 30 mg of CoA (5.7) in 6 ml of water. The solution is stable for 1 week at +48C. 5.17 Adenosine-59-triphosphate solution Dissolve 300 mg of ATP-Na
32、2 H 2 (5.8) and 300 mg of sodium hydrogen carbonate (5.9) in 6 ml of water. The solution is stable for 4 weeks at +48C. 5.18 Malate dehydrogenase/Citrate synthase suspension Mix 0,6 ml of the MDH suspension (5.10) and 0,6 ml of the CS suspension (5.11). The suspension is stable for 1 year at +48C. 5
33、.19 Acetyl-CoA synthetase suspension Dissolve 20 mg of the ACS lyophilizate (5.12) in 0,5 ml of the ammonium sulfate solution (5.20). The suspension is stable for 2 weeks at +48C. 5.20 Ammonium sulfate solution, c(NH 4 ) 2 SO 4 ) = 1 mol/l. Dissolve 13,2 g of ammonium sulfate (5.13)i n approximately
34、 80 ml of water, adjust to pH 7,3 with approximately 0,2 ml of potassium hydroxide solution (5.5) and make up to 100 ml with water. The solution is stable for 1 year at 208Ct o2 58 C. 5.21 Acetate standard solution, c(CH 3 COO 2 ) = 5 mmol/l. Dissolve 68 mg of sodium acetate (5.14) in 100 ml of wate
35、r. Prepare fresh solution before use. 6 Apparatus Usual laboratory apparatus and, in particular, the following: 6.1 Enzyme test pipettes, graduated along the stem only, with long ungraduated delivery tip. 6.2 Pipettes, with an accuracy equivalent to 6.1 (alternative to 6.1) for example positive disp
36、lacement capillary pipettes. 6.3 Cuvettes, made of quartz, glass or plastic, of 1 cm optical path length, which do not have a significant absorption at 334 nm, 340 nm and 365 nm. 6.4 Spectral-line photometer, with mercury lamp and filters for measuring at 334 nm or 365 nm. 6.5 Spectrophotometer (var
37、iable wavelength), for measuring at 340 nm (alternative to 6.4). 6.6 Centrifuge, capable of producing a centrifugal acceleration of 3 000 g at the base of the centrifuge tube (6.8). NOTE The rotational frequency required to give correct centrifugal acceleration can be calculated from the following e
38、quation: a = 11,183 r3 (4) n (1 000) 2 where a is the centrifugal acceleration; r is the radius of the centrifuge in centimetres, measured from the mid point (the centrifuge axis) to the bottom of the centrifuge tube when swung out; n is the rotational frequency per minute. 6.7 Membrane filter, memb
39、rane filter with a pore size of 0,45mm. 6.8 Centrifuge tubes 7 Procedure 7.1 Preparation of the test sample Dilute the fruit juice so that the acetic acid content is between 1mg and 30mg per cuvette. This is equivalent to 10 mg to 300 mg acetic acid per litre sample (solution). If the concentration
40、of acetic acid in the sample (solution) is less than 10 mg/l the sample volume to be pipetted into the cuvette can be increased up to 1,5 ml. If this occurs, the volume of water to be added must be reduced in order to obtain the same final volume in the cuvette. Use the (diluted) sample directly, ev
41、en if it is slightly coloured. The analysis by this method shall be on a volumetric basis, results being expressed per litre of sample. The analysis of concentrated samples may also be carried out on a volumetric basis, after dilution to a known relative density. In this case, the relative density s
42、hall be indicated. Based on a weighed sample and taking the dilution factor for analysis into account, the results may also be expressed per kilogram of product. In products with a high viscosity and/or a very high content of cells (for example pulp), determination on the basis of a weighed test sam
43、ple is the usual procedure. Mix cloudy samples well before dilution. Clarify cloudy samples containing low concentrations of acetic acid by membrane filtration through a 0,45mm filter (6.7) and centrifuge.Page 5 EN 12632:1999 BSI 07-1999 7.2 Test procedure 7.2.1 General The determination shall norma
44、lly be carried out at a constant temperature between 208C and 258C. A constant temperature in the range 258Ct o3 78 C may also be used, providing equivalent results are obtained. The absorption maximum of NADH is at 340 nm. When using a variable wavelength spectrophotometer (6.5), measure at the abs
45、orption maximum only. When using a mercury vapour lamp, spectral-line photometer, measure at a wavelength of 334 nm or 365 nm. Do not use single-mark transfer pipettes for pipetting the solutions. Solutions of enzyme, coenzyme and buffer may be added from suitable automatic pipettes. Enzyme test pip
46、ettes (6.1) or their equivalent (6.2) shall be used for pipetting the sample solution. Include a standard solution of acetic acid in each analytical run. 7.2.2 Blank test solution Also see the pipetting scheme given in 7.2.3. Pipette into cuvettes 1,00 ml of the buffer solution (5.15), 0,10 ml of th
47、e NAD/CoA solution (5.16), 0,10 ml of the ATP solution (5.17) and 2,00 ml of water. Mix and after 3 min read the absorbance A 0 . Add 0,02 ml of the MDH/CS-solution (5.18), mix and after 2 min read the absorbance A 1 . Add 0,01 ml of the ACS suspension (5.19), mix and after 10 min to 15 min read the
48、 absorbance A 2 . 7.2.3 Acetic acid test solution Pipette into cuvettes 1,00 ml of the buffer solution (5.15), 0,10 ml of the NAD/CoA solution (5.16), 0,10 ml of the ATP solution (5.17), 0,10 ml of the sample solution (7.1) and 1,90 ml of water. Mix and after 3 min read the absorbance A 0 . Add 0,02
49、 ml of the MDH/CS-solution (5.18), mix and after 2 min read the absorbance A 1 . Add 0,01 ml of the ACS suspension (5.19), mix and after the reaction has stopped (10 min to 15 min) read the absorbance A 2 . Check for completion of the reaction by reading the final absorbances at 2 min intervals for 10 min. If necessary (a constantly increasing value) correct for the “creep” reaction by extrapolating the absorbance back to the time of the addition of MDH/CS (see annex C). Table 1 Pipette into cuvettes Blank Sample ml ml B
