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 methodThis British Standard, having bee
3、n 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 06-1999 ISBN 0 580 32214 9 BS EN 12631:1999 Amendments issued since publication Amd. No. Date Comments Nationa
4、l 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, which has the responsibility to: aid enquirers to understand the text; present to the responsible Europea
5、n 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 of organizations represented on this committee can be obtained on request to its secretary. Cros
6、s-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 Standards Correspondence Index”, or by using the “Find” facility of the BSI Standards Electroni
7、c 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 Standard does not of itself confer immunity from legal obligations. Summary of pages This docume
8、nt comprises 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
9、All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 12631:1999 E EUROPEAN STANDARD EN 12631 NORME EUROPE ENNE EUROPA ISCHE NORM February 1999 ICS 67.160.20 Descriptors: fruit and vegetable juices, chemical analysis, determination of conten
10、t, lactic acid, enzymatic methods, spectrophotometric analysis, procedure English version Fruit 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 thod
11、e spectrome trique par le NAD Frucht- und Gemu sesa fte Enzymatische Bestimmung des Gehaltes 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 Regulati
12、ons which stipulate the conditions 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 Europ
13、ean Standard exists in three official 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 natio
14、nal standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.Page 2 EN 12631:1999 BSI 06-1999 Foreword This European Standard has been prepared by
15、 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 1999, and conflic
16、ting 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, Finland, France, Germa
17、ny, Greece, Iceland, Ireland, Italy, 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 5 9 Precis
18、ion 6 10 Test report 6 Annex A (informative) 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 12631:1999 BSI 06-1999 1 Scope This European Standard specifies an enzymatic method for
19、 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 or undated reference, provisions from other publications. These normative references are cited at
20、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 revision. For undated references the latest edition of t
21、he 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. c substance concentration; r mass concentration; f volume
22、fraction; g acceleration due to gravity at the surface of the earth (9,81 m/s 2 ). 3.2 Abbreviations For the purposes of this standard, the following abbreviations apply. GPT Glutamate-pyruvate-transaminase; L-LDH L-lactate dehydrogenase; D-LDH D-lactate dehydrogenase; NAD b-Nicotinamide-adenine-din
23、ucleotide; NADH b-Nicotinamide-adenine-dinucleotide, reduced form; IU 1 International Unit (IU) of enzyme activity catalyses the conversion of 1mmol of substance per minute at 258C under standard conditions. 4 Principle L-lactic acid (lactate) is oxidized by nicotinamide-adenine-dinucleotide (NAD) i
24、n 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 + (1) L-LDH D-lactate + NAD + pyruvate + NADH + H + (2) D-LDH The equilibrium of the re
25、actions (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 (equation 3), the equilibrium can be displaced in favour of pyruvate and NADH. Py
26、ruvate + L-glutamate L-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 sample. 5 Reagents 5.1 General Use only reagents of recognized analytical grad
27、e 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 combination kit. 5.2 Glycylglycine 5.3 L-glutamic acid 5.4 Sodium hydroxide solutions 5.4.1 Sodium hydroxide solution, c(NaOH) = 10 mol/l. 5.4.
28、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 dehydrogenase, solution in glycerol f(L-LDH) = 50 %, specific activity of approximatel
29、y 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-lactate, lithium salt 5.11 Buffer solution, pH = 10,0 Dissolve 4,75 g of glycylglyci
30、ne (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 months at +48C. 5.12 Nicotinamide-adenine dinucleotide solution Dissolve 420 m
31、g of NAD (5.5) in 12 ml of water. The solution is stable for at least 4 weeks at +48C.Page 4 EN 12631:1999 BSI 06-1999 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 supernata
32、nt, shake the suspension well prior to use. The suspension is stable for at least one year at +48C. 5.14 L-lactate dehydrogenase solution Use the L-LDH solution (5.7) undiluted. The solution is stable for at least one year at +48C. 5.15 D-lactate dehydrogenase suspension Use the D-LDH suspension (5.
33、8) undiluted. The suspension is stable for at least one year at +48C. 5.16 L-lactic acid standard solution, c(CH 3 CHOHCOOH) = 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,
34、 c(CH 3 CHOHCOOLi) = 0,5 mmol/l. Dissolve 48 mg of D-lactate, Li-salt (5.10) in 100 ml water. Dilute this solution 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, grad
35、uated 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 displacement capillary pipettes. 6.3 Cuvettes, made of quartz, glass or plastic, of 1 cm optical path length, which do not have a significant abso
36、rption 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, (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
37、 the base of the centrifuge tube (6.8). NOTE The rotational frequency required to give correct centrifugal acceleration can be calculated from the following equation: a = 11,183 r3 (n/1 000) 2 (4) where a is the centrifugal acceleration; r is the radius of the centrifuge in centimetres, measured fro
38、m 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, 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 D- and L-lacti
39、c acid content is between 2mg and 35mg per cuvette. This is equivalent to 20 mg to 350 mg of lactic acid per litre 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.
40、 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, 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 an
41、alysis 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 shall be indicated. Based on a weighed sample and taking the dilution factor for analysis into account, the results may also be expressed per k
42、ilogram 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 sample is the usual procedure. Mix cloudy samples well before dilution. Clarify cloudy samples containing low contents of D- and L-lactic acid by
43、 membrane filtration through a 0,45mm filter with possibly a prior centrifugation. 7.2 Test procedure 7.2.1 General The determination shall normally 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 equivalen
44、t 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 vapour lamp, spectral-line photometer, measure at a wavelength of 334 nm or 365 nm. Do not use single-mark trans
45、fer pipettes for pipetting the solutions. Solutions of enzyme, coenzyme and buffer may be added from suitable 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 ru
46、n. 7.2.2 Blank test solution See also the pipetting schemes under 7.2.3 and 7.2.4. Pipette into cuvettes 1,00 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 A 1 . Add 0,02 ml of
47、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 determination respectively). Mix and after 20 min read the absorbance A 2 .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.
48、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 A 1 . Add 0,02 ml of the L-LDH-solution (5.14). Mix and after 20 min read the absorbance A 2 . Check for completion of the rea
49、ction 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 L-LDH (see annex C). Table 1 Pipette into cuvettes Blank Sample Buffer solution (5.11) 1,00 ml 1,00 ml NAD-solution (5.12) 0,20 ml 0,20 ml GPT-suspension (5.13) 0,02 ml 0,02 ml Sample solution (7.1) 0,10 ml Water 1,50 ml 1,40 ml Mix and read absorbances A 1 of the solution, after approximately 5 min Start reaction
