1、BRITISH STANDARD BS EN 1139:1995 Method for Determination of D-isocitric acid content of fruit and vegetable juices: NADPH spectrometric method The European Standard EN1139:1994 has the status of a BritishStandard UDC 663.81/.82:620.1:543.42:543.477BSEN1139:1995 This BritishStandard, having been pre
2、pared under the directionof the Consumer Products and Services Sector Board, was published under theauthority of the StandardsBoard and comesintoeffect on January1995 BSI01-2000 The following BSI references relate to the work on this standard: Committee reference AW/21 Draft for comment90/51494 DC I
3、SBN 0 580 23171 2 Cooperating organizations The European Committee for Standardization (CEN), under whose supervision this European Standard was prepared, comprises the national standards organizations of the following countries: Austria Oesterreichisches Normungsinstitut Belgium Institut belge de n
4、ormalisation Denmark Dansk Standard Finland Suomen Standardisoimisliito, r.y. France Association franaise de normalisation Germany Deutsches Institut fr Normung e.V. Greece Hellenic Organization for Standardization Iceland Technological Institute of Iceland Ireland National Standards Authority of Ir
5、eland Italy Ente Nazionale Italiano di Unificazione Luxembourg Inspection du Travail et des Mines Netherlands Nederlands Normalisatie-instituut Norway Norges Standardiseringsforbund Portugal Instituto Portugus da Qualidade Spain Asociacin Espaola de Normalizacin y Certificacin Sweden Standardisering
6、skommissionen i Sverige Switzerland Association suisse de normalisation United Kingdom BritishStandards Institution Amendments issued since publication Amd. No. Date CommentsBSEN1139:1995 BSI 01-2000 i Contents Page Cooperating organizations Inside front cover National foreword ii Foreword 2 1 Scope
7、 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 Precision 5 10 Test report 5 Annex A (informative) Bibliography 6 Annex B (informative) Statistical results of the interlaboratory test 6 National annex NA (informative)
8、 Committees responsible Inside back cover National annex NB (informative) Cross-references Inside back cover Table 1 6BSEN1139:1995 ii BSI 01-2000 National foreword This BritishStandard has been prepared under the direction of the Consumer Products and Services Sector Board and is the English langua
9、ge version of EN1139:1994 Fruit and vegetable juices Enzymatic determination of D-isocitric acid content NADPH spectrometric method, published by the European Committee for Standardization (CEN). EN1139 was produced as a result of international discussions in which the United Kingdom took an active
10、part. 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 comp
11、rises a front cover, an inside front cover, pagesi andii, theEN title page, pages2 to6, an inside back cover and a back cover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will be indicated in the amendment table on the inside front cover.EUROPEA
12、N STANDARD NORME EUROPENNE EUROPISCHE NORM EN1139 October1994 UDC 663.81/.82:620.1:543.42:543.477 Descriptors: Food products, beverages, fruit and vegetable juices, chemical analysis, determination of content, citric acid, enzymatic methods, spectrophotometric analysis English version Fruit and vege
13、table juices Enzymatic determination of D-isocitric acid content NADPH spectrometric method Jus de fruits et de lgumes Dosage enzymatique de lacide D-isocitrique Mthode spectrophotometrique par le NADPH Frucht und Gemsesfte Enzymatische Bestimmung des Gehaltes an D-Isocitronensure Spektralphotometri
14、sche Bestimmung von NADPH This European Standard was approved by CEN on1994-09-29. 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 alteration. Up-to-date lists and
15、 bibliographical references concerning such national standards may be obtained on application to the Central Secretariat or to any CEN member. The European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibili
16、ty 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, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portu
17、gal, Spain, Sweden, Switzerland and United Kingdom. CEN European Committee for Standardization Comit Europen de Normalisation Europisches Komitee fr Normung Central Secretariat: rue de Stassart 36, B-1050 Brussels 1994 Copyright reserved to CEN members Ref. No. EN1139:1994 EEN1139:1994 BSI 01-2000 2
18、 Foreword This European Standard was prepared by the Technical Committee CEN/TC174, 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 end
19、orsement, at the latest by April1995, and conflicting national standards shall be withdrawn at the latest by April1995. Annexes designated “informative” are given only for information. In this standard Annex A and Annex B are informative. According to the CEN/CENELEC Internal Regulations, the follow
20、ing countries are bound to implement this European Standard: Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland, United Kingdom.EN1139:1994 BSI 01-2000 3 1 Scope This European Standard specifies
21、an enzymatic method for the determination of the total content of D-isocitric acid, present either in the form of the free acid or its salts including esters and lactones in fruit and vegetable juices and related products. 2 Normative references This European Standard incorporates by dated or undate
22、d 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 when in
23、corporated in it by amendment or revision. For undated references the latest edition of the publication referred to applies. ISO5725:1986, Precision of test methods Determination of repeatability and reproducibility for a standard test-method by inter-laboratory tests. ISO3696:1987, Water for analyt
24、ical laboratory useSpecification and test methods. 3 Symbols and abbreviations For the purposes of this standard, the following symbols and abbreviations apply: 4 Principle D-isocitric acid is isolated from the test sample via its barium salt and is determined enzymatically. In this method D-isocitr
25、ate is oxidatively decarboxylated to2-oxo-glutarate by NADP in the presence of the enzyme ICDH: The amount of NADPH formed (measured by the increase in absorbance) is equivalent to the amount of D-isocitrate present. 5 Reagents Use only reagents of recognized analytical grade and only water in accor
26、dance with at least grade3 of ISO3696:1987. 5.1 Activated charcoal, Clarocarbon G 1) . 5.2 Hydrochloric acid, c (HCl)=approximately4mol/l. 5.3 Sodium hydroxide solution, c(NaOH)=approximately4mol/l. 5.4 Ammonia solution, (NH 3 )=25g/100g. 5.5 Acetone 5.6 Barium chloride solution, (BaCl 2 .2H 2 O)=30
27、0g/l Dissolve30g barium chloride (BaCl 2 .2H 2 O) in water and dilute to100ml. 5.7 Sodium sulfate solution (Na 2 SO 4 )=71g/l Dissolve71g sodium sulfate (Na 2 SO 4 ) in water and dilute to1litre. 5.8 Manganese sulfate solution, c(MnSO 4 )=approximately0,075mol/l Dissolve125mg manganese sulfate (MnSO
28、 4 .H 2 O) in10ml water. This solution is stable for at least6months at room temperature. 5.9 Tris buffer solution, pH=7,0 Dissolve2,42g Tris (hydroxymethyl)-aminomethane and35mg ethylenediamine tetra-acetic acid disodium salt (dihydrate) in80ml water, acidify to pH7,0 with hydrochloric acid(5.2) an
29、d dilute with water to100ml. This buffer solution keeps for at least1year at+4 C. ICDH Isocitrate dehydrogenase (EC a 1.1.1.42); NADP -Nicotinamide-adenine-dinucleotide- phosphate; NADPH -Nicotinamide- adenine-dinucleotide- phosphate, reduced; IU 1International Unit (IU) of enzyme activity catalyzes
30、 the conversion of1mol of substrate per minute at25 C under standard conditions; c Substance concentration; Mass concentration; Mass fraction; g Acceleration due to gravity at the surface of the earth. a Enzyme Commission (EC): Classification system. Enzyme Handbook, Springer, Berlin1969. 1) Claroca
31、rbon G is the trade-name of a product supplied by Merck, Bundesrepublik Deutschland. This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN of the products named. Equivalent products may be used if they can be shown to lead the same resu
32、lts.EN1139:1994 4 BSI 01-2000 5.10 Tris buffer solution, pH=7,4 Dissolve2,42g Tris (hydroxymethyl)-aminomethane and35g ethylenediamine tetra-acetic acid disodium salt (dihydrate) in80ml water, acidify to pH7,4 with hydrochloric acid(5.2) and dilute with water to100ml. this buffer solution keeps for
33、atleast1year at+4 C. 5.11 NADP solution Dissolve50mg -nicotinamide adenine dinucleotide phosphate-disodium salt (NADP-Na 2 ) in5ml water. This solution keeps for at least4weeks at+4 C. 5.12 ICDH enzyme solution Dissolve isocitrate-dehydrogenase from pigs heart, (ICDH)=10mg/ml, (approximately20IU/ml)
34、, in glycerol solution, w (glycerol)=50g/100g. This solution is stable for approx.6months at+4 C. 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 equi
35、valent to(6.1) e.g.positive displacement capillary pipettes. 6.3 Cuvettes, made of glass or plastic, of10mm optical path length, and which do not have significant absorption at334,340 and365nm. 6.4 Spectral-line photometer, with mercury lamp filters for measuring at365nm or334nm. 6.5 Spectrometer, (
36、variable wavelength) for measuring at340nm (alternative to6.4). 6.6 Pleated filter paper, pore sizes10m. 6.7 Centrifuge, capable of producing a centrifugal force of3000 g at the base of the centrifuge tubes(6.8) (the value of g is fixed, for the purpose of this standard, at9,81msec 2 ). 6.8 Centrifu
37、ge tubes, 100ml capacity. 7 Procedure 7.1 Preparation of the test sample Normally products shall not be pretreated 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 volumet
38、ric basis, after dilution to a known relative density. 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 high viscosity and/or very high content of cells (for example pulp), determination on
39、 the basis of a weighed test sample is the usual procedure. 7.2 Determination 7.2.1 Isolation of Disocitrate from the test sample Treat10ml of test sample with5ml sodium hydroxide solution(5.3) in100ml centrifuge tube(6.8) and allow to stand for10min at room temperature (approximately20 C to25 C). A
40、fter adding5ml hydrochloric acid(5.2) dilute the solution to25ml with water. Then add consecutively2ml ammonia solution(5.4)3ml barium chloride solution(5.6) and20ml acetone(5.5). Mix thoroughly with a glass rod. Allow to stand for10min and then centrifuge(6.7) for about5min. Carefully decant the su
41、pernatant solution and add20ml sodium sulfate solution(5.7) to the precipitate in the centrifuge tube and stir with a glass rod. Dissolve the clumped precipitate by heating for10min in boiling water-bath with frequent stirring, then after cooling to room temperature transfer quantitively to50ml grad
42、uated flask with tris-buffer solution(5.9) and make up to the mark. Transfer the contents of the graduated flask to a conical flask containing1g activated charcoal(5.1), allow to stand5min and then filter through a pleated filter paper(6.6) The clear, colourless filtrate is used for the enzymatic de
43、termination of isocitrate(7.2.2) 7.2.2 Enzymatic determination of D-isocitrate 7.2.2.1 General The determination shall normally be carried out at constant temperature, between20 C and25 C. A constant temperature in the range25 C to37 C may also be used, providing equivalent results are obtained. The
44、 absorption maximum of NADH is at340nm. When using a variable wavelength spectrophotometer, measure at the absorption maximum only. When using a mercury vapour lamp, spectral-line photometer, measure at a wavelength of334nm or365nm. Do not use single-mark transfer pipettes for pipetting the solution
45、. Solutions of enzyme, coenzyme and buffer may be added from suitable automatic pipettes. Enzyme test pipettes(6.1) or their equivalent(6.2) shall be used for pipetting the sample solution. The determination may also be carried out using a commercially available test-combination kit. If the substanc
46、e to be determined is available in a suitably pure form, it is recommended to include it as a standard solution.EN1139:1994 BSI 01-2000 5 7.2.2.2 Blank test solution Pipette into cuvette(6.3)3,0ml buffer solution(5.10),0,1ml manganese sulfate solution(5.8) and0,1ml NADP solution(5.11). Mix, after ab
47、out3minutes, read the absorbance (A 1Blank ) against air (no cuvette in the light path). 7.2.2.3 Test sample solution Pipette into cuvette(6.3)2,0ml buffer solution(5.10),0,1ml manganese sulfate solution(5.8),0,1ml NADP solution(5.11) and1,00ml test sample (from7.2.2). Mix, after about3minutes, read
48、 the absorbance (A 1Sample ) against air (no cuvette in the light path). 7.2.2.4 Enzyme reaction and quantification Start the reaction by the addition of0,01ml ICDH enzyme solution(5.12) to each of the solutions7.2.2.2 and7.2.2.3. Mix, wait until the reaction has stopped(510min) and read the absorba
49、nces (A 2 ) of the solutions against air. If the reaction has not stopped after10min, continue to read the absorbance at5min intervals until the absorbance increases at a constant rate and extrapolate back to A 2at the time when the enzyme solution (ICDH) was added. 8 Calculation According to the reaction on which the determination is based, there is a linear proportionality between the amount of NADPH formed (and therefore the absorbance difference) and the concentration of D-isocitric acid: A=(A 2
