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1、| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | BRITISH STANDARD BS EN 12631:1999 The Euro
2、pean Standard EN 12631:1999 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 D- and L-lactic acid (lactate) content NAD spectrometric method Licensed Copy: sheffieldun sheff
3、ieldun, na, Sat Oct 28 16:08:00 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 June 1999 BSI 06
4、-1999 ISBN 0 580 32214 9 BS EN 12631:1999 Amendments issued since publication Amd. No.DateComments National foreword This British Standard is the English language version of EN 12631:1999. The UK participation in its preparation was entrusted to Technical Committee AW/21, Fruit and vegetable juices,
5、 which has 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 U
6、K. A list 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 Inte
7、rnational 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 Br
8、itish Standard 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 16:08:00 GMT+00:00 2
9、006, Uncontrolled 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 nationa
10、l Members. Ref. No. EN 12631:1999 E EUROPEAN STANDARDEN12631 NORME EUROPE ENNE EUROPA ISCHE NORM February 1999 ICS 67.160.20 Descriptors: fruit and vegetable juices, chemical analysis, determination of content, lactic acid, enzymatic methods, spectrophotometric analysis, procedure English version Fr
11、uit and vegetable juices Enzymatic determination of D- and L-latic acid (lactate) content NAD spectrometric method Jus de fruits et de le gumes Dosage enzymatique des acides D- et L-latiques (lactate) Me thode spectrome trique par le NAD Frucht- und Gemu sesa fte Enzymatische Bestimmung des Gehaltes
12、 an D- und L-Milchsa ure (Lactat) 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 nationa
13、l 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 official versions (English, French, German). A version in any o
14、ther 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, Belgium, Czech Republic, Denmark, Finland, France, Germany,
15、 Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. Licensed Copy: sheffieldun sheffieldun, na, Sat Oct 28 16:08:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Page 2 EN 12631:1999 BSI 06-1999 Foreword This European Standard has
16、been prepared by Technical Committee 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 1
17、999, and conflicting national standards 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, Finlan
18、d, France, Germany, Greece, Iceland, 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 8Calculation5 9Precisio
19、n6 10Test report6 Annex A (informative) 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 16:08:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI
20、 Page 3 EN 12631:1999 BSI 06-1999 1 Scope This European Standard specifies an enzymatic method for the determination of the total content of D- and L-lactic acid and lactate salts in fruit and vegetable juices and related products. 2 Normative references This European Standard incorporates by dated
21、or undated reference, provisions from other 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 onl
22、y when incorporated in it by amendment or 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
23、 standard, the following symbols apply. csubstance concentration; rmass concentration; fvolume fraction; 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. GPTGlutamate-pyruvate-transaminase; L
24、-LDHL-lactate dehydrogenase; D-LDHD-lactate dehydrogenase; NADb-Nicotinamide-adenine-dinucleotide; NADHb-Nicotinamide-adenine-dinucleotide, reduced form; IU1 International Unit (IU) of enzyme activity catalyses the conversion of 1 mmol of substance per minute at 25 8C under standard conditions. 4 Pr
25、inciple L-lactic acid (lactate) is oxidized by nicotinamide-adenine-dinucleotide (NAD) in the presence of L-lactate dehydrogenase (L-LDH) to produce pyruvate. D-lactic acid (lactate) is oxidized in the same way in the presence of D-lactate dehydrogenase (D-LDH). L-lactate + NAD+pyruvate + NADH + H+(
26、1) L-LDH D-lactate + NAD+pyruvate + NADH + H+(2) D-LDH The equilibrium of the reactions (1) and (2) lies almost completely on the side of lactate. However, by trapping the pyruvate in a subsequent reaction catalysed by the enzyme glutamate-pyruvate transaminase (GPT) in the presence of L-glutamate (
27、equation 3), the equilibrium can be displaced in favour of pyruvate and NADH. Pyruvate + L-glutamateL-alanine GPT (3)+ a-ketoglutarate The amount of NADH formed (as measured by the increase in absorbance at 334 nm, 340 nm or 365 nm) is equivalent to the amounts of D- and L-lactic acid present in the
28、 sample. 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 combination kit. 5.2 Glycylglycine 5.3 L-glutamic acid 5.4 Sodium
29、 hydroxide solutions 5.4.1 Sodium hydroxide solution, c(NaOH) = 10 mol/l. 5.4.2 Sodium hydroxide solution, c(NaOH) = 10 mmol/l. 5.5 Nicotinamide-adenine dinucleotide 5.6 Glutamate-pyruvate transaminase, suspension, r(GPT) = 10 mg/l, specific activity of approximately 80 lU/mg. 5.7 L-lactate dehydrog
30、enase, solution in glycerol f(L-LDH) = 50 %, specific activity of approximately 550 IU/mg. 5.8 D-lactate dehydrogenase, suspension in ammonium sulfate, c(D-LDH) = 3,2 mol/l, specific activity of approximately 300 IU/mg. 5.9 L-lactic acid standard solution, c(L-lactic acid) = 1,0 mol/l. 5.10 D-lactat
31、e, lithium salt 5.11 Buffer solution, pH = 10,0 Dissolve 4,75 g of glycylglycine (5.2) and 0,88 g of L-glutamic acid (5.3) in approximately 50 ml of water. Adjust to pH 10,0, with approximately 4,6 ml of sodium hydroxide (5.4.1) and make up to 60 ml with water. The solution is stable for at least 3
32、months at +4 8C. 5.12 Nicotinamide-adenine dinucleotide solution Dissolve 420 mg of NAD (5.5) in 12 ml of water. The solution is stable for at least 4 weeks at +4 8C. Licensed Copy: sheffieldun sheffieldun, na, Sat Oct 28 16:08:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Page 4 EN 12631:1999 BSI 0
33、6-1999 5.13 Glutamate-pyruvate transaminase suspension Centrifuge 2 ml of the GPT-suspension (5.6) for 10 min at approximately 4 000 rpm. Aspirate and discard 1,0 ml of the clear supernatant, shake the suspension well prior to use. The suspension is stable for at least one year at +4 8C. 5.14 L-lact
34、ate dehydrogenase solution Use the L-LDH solution (5.7) undiluted. The solution is stable for at least one year at +4 8C. 5.15 D-lactate dehydrogenase suspension Use the D-LDH suspension (5.8) undiluted. The suspension is stable for at least one year at +4 8C. 5.16 L-lactic acid standard solution, c
35、(CH3CHOHCOOH) = 0,5 mmol/l. Dilute L-lactic acid standard solution (5.9) 1 to 2 000 with sodium hydroxide (5.4.2). Prepare fresh solution prior to use. 5.17 D-lactic acid standard solution, c(CH3CHOHCOOLi) = 0,5 mmol/l. Dissolve 48 mg of D-lactate, Li-salt (5.10) in 100 ml water. Dilute this solutio
36、n 1 volume part to 9 volume parts with water. Prepare fresh solution prior to 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 (
37、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 334 nm, 340 nm and 365 nm. 6.4 Spectral-line photometer, with mercury lamp and filters for measuring at
38、 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 the centrifuge tube (6.8). NOTEThe rotational frequency required to give correct centrifugal acceler
39、ation can be calculated from the following equation: a = 11,18 3 r 3 (n/1 000)2(4) where ais the centrifugal acceleration; ris 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; nis the rotational freque
40、ncy per minute. 6.7 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 D- and L-lactic acid content is between 2 mg and 35 mg per cuvette. This is equivalent to 20 mg to 350 mg of lactic acid per litre
41、 sample (solution). If the concentration of the lactic acid in the sample (solution) is less than 20 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 cu
42、vette. 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 concentrated samples may also be carried out on a volumetric basis, after dilution to a known relative d
43、ensity. 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 product. In products with a high viscosity and/or a very high content of cells (for example pulp), dete
44、rmination on the basis of a weighed test sample is the usual procedure. Mix cloudy samples well before dilution. Clarify cloudy samples containing low contents of D- and L-lactic acid by membrane filtration through a 0,45 mm filter with possibly a prior centrifugation. 7.2 Test procedure 7.2.1 Gener
45、al The determination shall normally be carried out at a constant temperature between 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 spectrop
46、hotometer (6.5), measure at the absorption 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
47、automatic pipettes. Use only enzyme test pipettes (6.1) or their equivalent (6.2) for pipetting the sample solution. Include a standard solution of D- and L-lactic acid in each analytical run. 7.2.2 Blank test solution See also the pipetting schemes under 7.2.3 and 7.2.4. Pipette into cuvettes 1,00
48、ml of the buffer solution (5.11), 0,20 ml of the NAD-solution (5.12), 1,50 ml of water and 0,02 ml of the GPT-suspension (5.13). Mix and after 5 min read the absorbance A1. Add 0,02 ml of the L-LDH-solution (5.14) or 0,05 ml of the D-LDH-solution (5.15) (for L-lactic acid or D-lactic acid determinat
49、ion respectively). Mix and after 20 min read the absorbance A2. Licensed Copy: sheffieldun sheffieldun, na, Sat Oct 28 16:08:00 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Page 5 EN 12631:1999 BSI 06-1999 7.2.3 L-lactic acid test solution Pipette into cuvettes 1,00 ml of the buffer solution (5.11), 0,20 ml of the NAD-solution (5.12), 1,40 ml of water, 0,02 ml of the GPT-suspension (5.13) and 0,10 ml of sample solution (7.1). Mix and after 5 min read the absorbance A1. Add 0,02 ml of the L-LDH-solution (5.14). Mix and after 20 min read