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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 method Licensed Copy: sheffieldun sheffieldun, n
3、a, Sat Oct 28 16:08:16 GMT+00:00 2006, Uncontrolled Copy, (c) BSI This British Standard, having been prepared 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 ISB
4、N 0 580 32212 2 BS EN 12632:1999 Amendments issued since publication Amd. No.DateComments National foreword 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 ha
5、s the responsibility to: aid enquirers to understand the text; present to the responsible European committee 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
6、 of organizations represented on this committee can be obtained on request to its secretary. Cross-references 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
7、 Standards Correspondence Index, or by using the Find facility of the BSI Standards Electronic Catalogue. 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 Sta
8、ndard does not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, the EN title page, pages 2 to 8, an inside back cover and a back cover. Licensed Copy: sheffieldun sheffieldun, na, Sat Oct 28 16:08:16 GMT+00:00 2006, Unco
9、ntrolled Copy, (c) BSI 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 rights of exploitation in any form and by any means reserved worldwide for CEN national Members
10、. Ref. No. EN 12632:1999 E EUROPEAN STANDARDEN 12632 NORME EUROPE ENNE EUROPA ISCHE NORM February 1999 ICS 67.160.20 Descriptors: fruit and vegetable juices, chemical analysis, determination of content, acetic acid, enzymatic methods, spectrophotometric analysis, procedure English version Fruit and
11、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 le NAD Frucht- und Gemu sesa fte Enzymatische Bestimmung des Gehaltes an Essigsa ure (Acetat
12、) 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 conditions for giving this European Standard the status of a national standard without any alteratio
13、n. 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 any other language made by translatio
14、n 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, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy
15、, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. Licensed Copy: sheffieldun sheffieldun, na, Sat Oct 28 16:08:16 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Page 2 EN 12632:1999 BSI 07-1999 Foreword This European Standard has been prepared by Technical Commi
16、ttee CEN/TC 174, 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 st
17、andards shall be 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, Icel
18、and, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Contents Page Foreword2 1Scope3 2Normative references3 3Symbols and abbreviations3 4Principle3 5Reagents3 6Apparatus4 7Procedure4 8Calculation6 9Precision6 10Test report6 Annex A (infor
19、mative) Bibliography7 Annex B (informative) Statistical results of the inter-laboratory tests7 Annex C (informative) Information on how to treat creep reactions8 Licensed Copy: sheffieldun sheffieldun, na, Sat Oct 28 16:08:16 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Page 3 EN 12632:1999 BSI 07-199
20、9 1 Scope This European Standard specifies an enzymatic method for the determination of the total content 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 othe
21、r publications. These normative references are cited at the appropriate places in the text and the 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
22、revision. For undated references the latest edition of the publication referred to applies. EN ISO 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: csu
23、bstance concentration; rmass concentration; gacceleration due to gravity at the surface of the earth (9,81 m/s2). 3.2 Abbreviations For the purposes of this standard, the following abbreviations apply: ACSAcetyl Coenzyme A synthetase; CoACoenzyme A; ATPAdenosine-59-Tri-phosphate; AMPAdenosine-Mono-p
24、hosphate; CSCitrate synthase; NADb-Nicotinamide-adenine-dinucleotide; NADHb-Nicotinamide-adenine-dinucleotide, reduced form; MDHMalate-dehydrogenase; IU1 International Unit (IU) of enzyme activity catalyses the conversion of 1 mmol of substance per minute at 25 8C under standard conditions. 4 Princi
25、ple Acetic acid (acetate) is converted in the presence of the enzyme acetyl-coenzyme A-synthetase (ACS) with adenosine-59-triphosphate (ATP) and coenzyme A (CoA) to acetyl-CoA (reaction 1): ACS Acetate + ATP + CoA , acetyl 2 CoA + AMP + pyrophosphate (1) Acetyl-CoA reacts with oxaloacetate to produc
26、e citrate in the presence of citrate synthase (CS) (reaction 2): CS Acetyl-CoA + oxaloacetate + H2O , 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 reac
27、tion NAD is reduced to NADH. MDH Malate + NAD+, oxaloacetate + NADH + H+(3) The determination 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 co
28、ncentration but is not linearly proportional. 5 Reagents 5.1 General Use only reagents of recognized analytical grade and only water in accordance with at least grade 3 of EN ISO 3696:1995. NOTEThe determination can also be carried out using a commercially available test kit. 5.2 Triethanolamine hyd
29、rochloride. 5.3 L-malic acid. 5.4 Magnesium chloride, MgCl2.6H2O. 5.5 Potassium 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-Na2H2. 5.9 Sodium hydrogen carbonate, NaHCO3. 5.10 Malate dehydroge
30、nase, suspension in ammonium sulfate, c(MDH) = 3,2 mol/l, specific activity approximately 1 200 IU/mg. Licensed Copy: sheffieldun sheffieldun, na, Sat Oct 28 16:08:16 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Page 4 EN 12632:1999 BSI 07-1999 5.11 Citrate synthase, suspension in ammonium sulfate, c(
31、CS) = 3,2 mol/l, specific activity approximately 110 IU/mg. 5.12 Acetyl-CoA synthetase, lyophilized. 5.13 Ammonium sulfate, (NH4)2SO4. 5.14 Sodium acetate, CH3COONa.3H2O. 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
32、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 100 ml with water. The buffer is stable for 4 weeks at +4 8C. 5.16 Nicotanimide-adenine dinucleotide/Coenzyme A solution Dissolve
33、144 mg of NAD (5.6) and 30 mg of CoA (5.7) in6ml of water. The solution is stable for1week at+48C. 5.17 Adenosine-59-triphosphate solution Dissolve 300 mg of ATP-Na2H2(5.8) and 300 mg of sodium hydrogen carbonate (5.9) in 6 ml of water. The solution is stable for 4 weeks at +4 8C. 5.18 Malate dehydr
34、ogenase/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 +4 8C. 5.19 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
35、 suspension is stable for 2 weeks at +4 8C. 5.20 Ammonium sulfate solution, c(NH4)2SO4)=1mol/l. Dissolve 13,2 g of ammonium sulfate (5.13) in approximately 80 ml of water, adjust to pH 7,3 with approximately0,2ml of potassium hydroxide solution(5.5) and make up to 100 ml with water. The solution is
36、stable for 1 year at 20 8C to 25 8C. 5.21 Acetate standard solution, c(CH3COO2)=5mmol/l. Dissolve 68 mg of sodium acetate (5.14) in 100 ml of water. Prepare fresh solution before use. 6 Apparatus Usual laboratory apparatus and, in particular, the following: 6.1 Enzyme test pipettes, graduated along
37、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 displacement 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 33
38、4 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 (variable 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
39、 the centrifuge tube (6.8). NOTEThe rotational frequency required to give correct centrifugal acceleration can be calculated from the following equation: a = 11,18 3 r 3(4) n (1 000)2 where ais the centrifugal acceleration; ris the radius of the centrifuge in centimetres, measured from the mid point
40、 (the centrifuge axis) to the bottom of the centrifuge tube when swung out; nis the rotational frequency per minute. 6.7 Membrane filter, membrane filter with a pore size of 0,45 mm. 6.8 Centrifuge tubes 7 Procedure 7.1 Preparation of the test sample Dilute the fruit juice so that the acetic acid co
41、ntent is between 1 mg and 30 mg per cuvette. This is equivalent to 10 mg to 300 mg acetic acid per litre sample (solution). If the concentration 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 occur
42、s, 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, even 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 con
43、centrated samples may also be carried out on a volumetric basis, after dilution to a known relative density. In this case, the relative density shall 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 pr
44、oduct. 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 sample is the usual procedure. Mix cloudy samples well before dilution. Clarify cloudy samples containing low concentrations of acetic acid by membrane filtra
45、tion through a 0,45 mm filter (6.7) and centrifuge. Licensed Copy: sheffieldun sheffieldun, na, Sat Oct 28 16:08:16 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Page 5 EN 12632:1999 BSI 07-1999 7.2 Test procedure 7.2.1 General The determination shall normally be carried out at a constant temperature b
46、etween 20 8C and 25 8C. A constant temperature in the range 25 8C to 37 8C 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 absorption maximum only. When using a mercury
47、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 pipettes (6.1) or their equivalent (6.2) shall
48、 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 cuvettes1,00ml of the buffer solution(5.15), 0,10 ml of the NAD/CoA solution (5.16), 0,10 ml of the ATP
49、solution (5.17) and 2,00 ml of water. Mix and after 3 min read the absorbance A0. Add 0,02 ml of the MDH/CS-solution (5.18), mix and after 2 min read the absorbance A1. Add 0,01 ml of the ACS suspension (5.19), mix and after 10 min to 15 min read the absorbance A2. 7.2.3 Acetic acid test solution Pipette into cuvettes1,00ml 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