1、| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | BRITISH STANDARD BS EN 12138 : 1998 The Eu
2、ropean Standard EN 12138 : 1997 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-malic acid content NAD spectrometric methodThis British Standard, having been prepared und
3、er the direction of the Consumer Products and Services Sector Board, was published under the authority of the Standards Board and comes into effect on 15 January 1998 BSI 1998 ISBN 0 580 28944 3 BS EN 12138 : 1998 Amendments issued since publication Amd. No. Date Text affected National foreword This
4、 British Standard is the English language version of EN 12138 : 1997. 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 international/Europe
5、an 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. ISO
6、 5725 : 1986, to which informative reference is made in the text, has been superseded by ISO 5725-1 : 1994, ISO 5725-2 : 1994, ISO 5725-3 : 1994, ISO 5725-4 : 1994 and ISO 5725-6 : 1994 which are identical with BS ISO 5725 Accuracy (trueness and precision) of measurement methods and results, BS ISO
7、5725-1 : 1994 General principles and definitions, BS ISO 5725-2 : 1994 Basic method for the determination of repeatability and reproducibility of a standard measurement method, BS ISO 5725-3 : 1994 Intermediate measures of the precision of a standard measurement method, BS ISO 5725-4 : 1994 Basic me
8、thod for the determination of the trueness of a standard measurement method, and BS ISO 5725-6 : 1994 Use in practice of accuracy values. Cross-references The British Standards which implement international or European publications referred to in this document may be found in the BSI Standards Catal
9、ogue under the section entitled International Standards Correspondence Index, or by using the Find facility of the BSI Standards Electronic Catalogue. Compliance with a British Standard does not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover,
10、 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 1997 CEN All rights of exploitation
11、in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 12138 : 1997 E EUROPEAN STANDARD EN 12138 NORME EUROPE ENNE EUROPA ISCHE NORM September 1997 ICS 67.160.20 Descriptors: Fruit and vegetable juices, chemical analysis, determination of content, malic acid, enzymatic
12、 methods, spectrophotometric analysis, procedure English version Fruit and vegetable juices Enzymatic determination of D-malic acid content NAD spectrometric method Jus de fruits et de le gumes Dosage enzymatique de lacide D-malique Me thode spectrome trique par le NAD Frucht- und Gemu sesa fte Enzy
13、matische Bestimmung des Gehaltes an D-A pfelsa ure Spektralphotometrische Bestimmung von NAD This European Standard was approved by CEN on 1997-09-06. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status
14、 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 official versions (English, French, German). A ver
15、sion 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, Belgium, Czech Republic, Denmark, Finland, Fra
16、nce, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.Page 2 EN 12138 : 1997 BSI 1998 Foreword This European Standard has been prepared by Technical Committee CEN/TC 174, Fruit and vegetable juices Methods of analysis,
17、 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 March 1998, and conflicting national standards shall be withdrawn at the latest by March 1998. According
18、 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, Italy, Luxembourg, Netherlands, Norway, Portugal, Sp
19、ain, 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 3 7 Procedure 4 8 Calculation 4 9 Precision 5 10 Test report 5 Annexes A (informative) Bibliography 6 B (informative) Info
20、rmation on how to treat creep reactions 6 C (informative) Statistical results of the interlaboratory test 7Page 3 EN 12138 : 1997 BSI 1998 1) Boehringer Mannheim is an example of a suitable product available commercially. This information is given for the convenience of users of this standard and do
21、es not constitute an endorsement by CEN of this product. 1 Scope This European Standard specifies an enzymatic method for the determination of the total content of D-malic acid, in fruit and vegetable juices and related products. 2 Normative references This European Standard incorporates by dated or
22、 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 only
23、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) ISO 5725 : 1986 Precision of test methods Determination of repea
24、tability and reproducibility for a standard test method by inter-laboratory tests 3 Symbols and abbreviations For the purposes of this standard, the following symbols and abbreviations apply: D-MDH D-Malate dehydrogenase decarboxyl NAD -Nicotinamide-adenine-dinucleotide IU 1 International unit (IU)
25、of enzyme activity catalyses the conversion of 1mmol of substance per minute at 25 C under standard conditions c Substance concentration r Mass concentration 4 Principle D-malic acid (D-malate) is oxidized by -nicotinamide- adenine-dinucleotide (NAD) in the presence of D-malate dehydrogenase decarbo
26、xyl (D-MDH) to oxaloacetate which then decomposes to give pyruvate and carbonic acid. D-MDH D-malate + NAD + pyruvate + CO 2 + NADH + H + The amount of NADH formed (measured by the increase in the ultraviolet absorbance of the solution) is equivalent to the amount of D-malate present. The D-malate d
27、ehydrogenase is not 100 % specific to D-malic acid. It reacts with L-tartaric acid at a much lower rate. If it is necessary to assay D-malic acid in the presence of high concentrations of tartaric acid, as in wine, the sample has to be pre-treated to remove the tartaric acid. 5 Reagents 5.1 General
28、Use only reagents of recognized analytical grade and only water in accordance with at least grade 3 of EN ISO 3696 : 1995. NOTE. The test can also be carried out using single reagents. 5.2 Test kit combination The Boehringer Mannheim test kit 1) contains: 5.2.1 bottle 1 with approximately 30 ml of s
29、olution consisting of HEPES-buffer (4-(2-hydroxyethyl)-1-piperazine ethansulfonic acid), pH = 9,0 and stabilizers; 5.2.2 bottle 2 with approximately 210 mg of lyophilized NAD; 5.2.3 three bottles with lyophilized D-MDH, approximately 8 IU each; 5.3 calcium hydroxide; 5.4 ethanol approximately 98 %;
30、5.5 potassium hydroxide. 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 equivalent accuracy to that of 6.1, e.g. positive displacement capillary pipettes. 6.3
31、 Cuvettes, made of quartz, glass or plastics, of 10 mm optical path length, and which do not have a significant absorption at wavelengths of 334 nm, 340 nm or 365 nm. 6.4 Spectral-line photometer, with a mercury lamp and filters for measuring at wavelengths of 365 nm or 334 nm. 6.5 Spectrometer, wit
32、h variable wavelength for measuring at 340 nm (alternative to 6.4). 6.6 Volumetric flasks, of 50 ml capacity. 6.7 Membrane filter, of pore size 0,2mm.Page 4 EN 12138 : 1997 BSI 1998 7 Procedure 7.1 Sample preparation 7.1.1 Normal preparation Normally products shall not be pre-treated, however diluti
33、on may be necessary and their analysis by this method shall be on a volumetric basis, results being expressed per litre of sample. The analysis of concentrated products may also be carried out on a volumetric basis, after dilution to a known relative density. In this case, the relative density shall
34、 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), a determination on the basis of a weighed test sampl
35、e is the usual procedure. Clarify cloudy samples containing low concentrations of D-malic acid by prior centrifugation or membrane filtration through a 0,2 mm filter (6.7). 7.1.2 Preparation of the test samples containing high levels of tartaric acid Take 25 ml of juice and mix with 125 mg of calciu
36、m hydroxide (5.3) and 5 ml of ethanol (5.4) (approximatively 98 %) for 2 min. Adjust the pH of the solution to between 7 and 8 with potassium hydroxide (5.5); transfer quantitatively into a 50 ml volumetric flask (6.6) and dilute to the mark. Clarify solution (as given 7.1.1) and use colourless clea
37、r solution in enzyme assay. 7.2 Test procedure 7.2.1 General The determination shall be carried out between 20 C and 25 C or a constant temperature in the range of 25 C to 37 C providing equivalent results are obtained. The absorption maximum of NADH is at a wavelength of 340 nm. When using a variab
38、le wavelength spectrometer (6.5), measure at the absorption maximum only. When using a mercury vapour lamp, spectral-line photometer (6.4), measure at a wavelength of 334 nm or 365 nm. Do not use single-mark transfer pipettes for pipetting the solutions. Solutions of enzyme, co-enzyme and buffer may
39、 be added from suitable automatic pipettes. Enzyme test pipettes (6.1) or their equivalent (6.2) shall be used for pipetting the sample solution. It is recommended to include D-malic acid as a standard solution in this assay. 7.2.2 Blank test solution Pipette into the cuvette 1,00 ml of the HEPES bu
40、ffer (5.2.1), 0,10 ml of the NAD solution (5.2.2) and 1,80 ml of water. Mix and after 6 min, read the absorbance (A 1Blank ) against air (with no cuvette in the lightpath). 7.2.3 Test sample solution Pipette into the cuvette 1,00 ml of the HEPES buffer (5.2.1), 0,10 ml of the NAD solution (5.2.2), 1
41、,70 ml of water and 0,10 ml of sample solution. Mix and after 6 min, read the absorbance (A 1Sample ) against air (with no cuvette in the lightpath). 7.2.4 Enzyme reaction and quantification Start the reaction by the addition of 0,05 ml of D-MDH solution (5.2.3) to each of the solutions given in 7.2
42、.2 and 7.2.3. Mix and wait until the reaction has stopped (approximately 20 min) and read the absorbances (A 2 ) of the solutions against air. If the reaction has not stopped after 20 min, continue to read the absorbances at 5 min intervals until it increases at a constant rate and extrapolate this
43、back to the time when the final enzyme solution D-MDH was added. (This process allows for side reactions and, the so called creep reaction, which is described in more detail in annex B). 8 Calculation According to the reaction on which the determination is based, there is a linear proportionality be
44、tween the amount of NADH formed (and therefore the absorbance difference) and the concentration of D-malic acid, as given in the following equation: DA =(A 2 2A 1 ) Sample 2 (A 2 2 A 1 ) Blank (1) The calculation of the concentration of a substance in dilute solution by absorptiometric measurement i
45、s based on the BeerLambert law. The D-Malic acid content of the sample (in g/l) is calculated from the following equation: r (D-malic acid) = DA (2) M3 V e3 d3 v3 1000 where M is the molecular mass of D-malic acid (134,09 gmol -1 ) V is the total volume of the test solution within the cuvette (in ml
46、); v is sample volume (in ml); d is the lightpath of the cuvette (in cm); e is the extinction coefficient of NADH ; at 340 nm = 6,3 (lmmol -1 cm -1 ); 365 nm = 3,4 (lmmol -1 cm -1 ); 334 nm = 6,18 (lmmol -1 cm -1 ). If the volumes given in 7.2 are adhered to, the calculation becomes, in g/l of D-mal
47、ic acid: r (D-malic acid) = (3) 3DA 3,956 e Take into account the dilution factor and the relation of the value to mass or volume. If a concentrated product has been diluted to single strength, report the relative density of the single strength sample. The D-malic acid content is reported (in g/l) t
48、o one decimal.Page 5 EN 12138 : 1997 BSI 1998 9 Precision Details of the interlaboratory test on the precision of the method are summarized in Annex C. The values derived from the interlaboratory test may not be applicable to analyte concentration ranges and matrices other than given in Annex C. 9.1
49、 Repeatability The absolute difference between two single test results found on identical test material by one operator using the same apparatus within the shortest feasible time interval will exceed the repeatability limit r in not more than 5 % of the cases. The value is : r = 2,03 + 0,044 r (D-malic acid) mg/l where: r (D-malic acid) is the measured content. 9.2 Reproducibility The absolute difference between two single test results on identical test material reported by two laboratories will exceed t
