1、BRITISH STANDARD BS EN 1140:1995 Method for Determination of D-glucose and D-fructose content of fruit and vegetable juices: NADPH:Spectrometric method The European Standard EN1140:1994 has the status of a BritishStandard UDC 663.81/.82:620.1:543.42:547.455.623:547.455.633BSEN1140:1995 This British
2、Standard, having been prepared under the directionof the Consumer Products and Services Sector Board, was published under theauthority of the Standards Boardand comes into effect on January1995 BSI 01-2000 The following BSI references relate to the work on this standard: Committee reference AW/21 Dr
3、aft for comment 90/51493 DC ISBN 0 580 23173 9 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
4、 Belgium Institut belge de normalisation 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 Nation
5、al Standards Authority of Ireland 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 Certifi
6、cacin Sweden Standardiseringskommissionen i Sverige Switzerland Association suisse de normalisation United Kingdom British Standards Institution Amendments issued since publication Amd. No. Date CommentsBSEN1140:1995 BSI 01-2000 i Contents Page Cooperating organizations Inside front cover National f
7、oreword ii Foreword 2 1 Scope 3 2 Normative references 3 3 Symbols and abbreviations 3 4 Principle and reactions 3 5 Reagents 3 6 Apparatus 4 7 Procedure 4 8 Calculation 5 9 Precision 5 10 Test report 5 Annex A (informative) Bibliography 7 Annex B (informative) Statistical results of the interlabora
8、tory test 7 National annex NA (informative) Committees responsible Inside back cover National annex NB (informative) Cross-references Inside back cover Table B.1 7BSEN1140:1995 ii BSI 01-2000 National foreword This BritishStandard has been prepared under the direction of the Consumer Products and Se
9、rvices Sector Board and is the English language version of EN1140:1994 Fruit and vegetable juices Enzymatic determination of D-glucose and D-fructose content NADPH spectrometric method, published by the European Committee for Standardization(CEN). EN1140 was produced as a result of international dis
10、cussions in which the United Kingdom took an active 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 le
11、gal obligations. Summary of pages This document comprises a front cover, an inside front cover, pages i and ii, theEN title page, pages2 to8, 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
12、 the amendment table on the inside front cover.EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN1140 October1994 UDC 663.81/.82:620.1:543.42:547.455.623:547.455.633 Descriptors: Food products, beverages, fruit and vegetable juices, chemical analysis, determination of content, glucose, fructose, e
13、nzymatic methods English version Fruit and vegetables juices Enzymatic determination of D-glucose and D-fructose content NADPHspectrometricmethod Jus de fruits et de lgumes Dosage enzymatique du glucose-D et du fructose-D Mthode spectromtrique par le NADPH Frucht- und Gemsesfte Enzymatische Bestimmu
14、ng der Gehalte an D-Glucose und D-Fructose Spektralphotometrische 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 n
15、ational 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. The European Standard exists in three official versions(English, French, German). A version in a
16、ny 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, Denmark, Finland, France, Germany, Greece, Ice
17、land, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, 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
18、 CEN members Ref No. EN1140:1994 EEN1140:1994 BSI 01-2000 2 Foreword This European Standard has been 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 Nationa
19、l Standard, either by publication of an identical text or by endorsement, 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 informativ
20、e. According to the CEN/CENELEC Internal Regulations, the following 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 and United Kingdom.
21、EN1140:1994 BSI 01-2000 3 1 Scope This European Standard specifies an enzymatic method for the determination of the D-glucose and D-fructose content of fruit and vegetable juices and related products. 2 Normative references This European Standard incorporates by dated or undated reference, provision
22、s 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 incorporated in it by am
23、endment 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 analytical laboratory use Sp
24、ecification and test methods. 3 Symbols and abbreviations For the purpose of this standard, the following symbols and abbreviations apply: 4 Principle and reactions 4.1 Principle D-glucose and D-fructose contained in a diluted test sample are phosphorylated in the C-6 position in an enzyme-catalysed
25、 reaction involving ATP and HK. In a concomitant reaction, glucose6-phosphate is converted stoichiometrically to6-phosphogluconate in the presence of NADP, the reaction being catalysed by the enzyme G-6-P-DH and an amount of NADPH equivalent to the amount of D-glucose present in the test sample bein
26、g formed(4.2). The D-fructose content may be determined by making use of a further reaction in which PGI catalyses the isomerization of F-6-P to G-6-P(4.3). The quantification of NADPH formed and hence the content of D-glucose and/or D-fructose is performed by spectrometry. 4.2 Reactions: D-glucose
27、determination 4.3 Reactions: D-fructose determination 5 Reagents 5.1 General Use only reagents of recognized analytical grade and only water in accordance with at least grade3 of ISO3696:1987. 5.2 Triethanolamine buffer, pH=7,6 Dissolve14,0g triethanolamine hydrochloride and0,25g magnesium sulfate M
28、gSO 4 .7H 2 O in80ml water, adjust to pH7,6 with about5ml sodium hydroxide solution, c(NaOH)=5mol/l and make up to100ml with water. The buffer is stable for at least four weeks at4 C. 5.3 NADP solution Dissolve60mg -Nicotinamide-adeninedinucletoide phosphate-disodium salt (-NADP-Na 2 ) in6ml water.
29、The solution is stable for at least four weeks at4 C. 5.4 ATP solution Dissolve300mg adenosine-5-triphosphate-disodium salt (ATP-Na 2 H 2 .3H 2 O) and300mg sodium hydrogen carbonate (NaHCO 3 ) in6ml water. The solution is stable for at least four weeks at4 C. ATP Adenosine-5-triphosphate; ADP Adenos
30、ine-5-diphosphate; NADP -Nicotinamide-adenine- dinucleotidephosphate; NADPH -Nicotinamide-adenine- dinucleotidephosphate (reducedform); G-6-P Glucose6-phosphate; F-6-P Fructose6-phosphate; HK Hexokinase(EC a 2.7.1.1); G6P-DH Glucose-6-phosphate dehydrogenase (EC a1.1.1.49); PGI Phosphoglucose-isomer
31、ase (EC a 5.3.1.9); IU One International Unit of enzyme activity, which catalyzes the conversion of1mol of substrate per min at25 C; c Substance concentration; Mass concentration. a Enzyme Commission(EC): Classification System. Enzyme Handbook, Springer, Berlin1969.EN1140:1994 4 BSI 01-2000 5.5 HK/G
32、6P-DH enzyme suspension Suspend hexokinase, (HK)=2mg/ml, about280IU/ml(with D-glucose serving as the substrate in the presence of ATP) and glucose-6-phosphate-dehydrogenase, (G6P-DH)=1mg/ml, about140IU/ml with glucose6-phosphate as substrate in ammonium sulfate solution, c (NH 4 ) 2 SO 4 )=3,2mol/l.
33、 The suspension is stable for at least one year at4 C. 5.6 PGI enzyme suspension Suspend phosphoglucose isomerase, (PGI)=2mg/ml, about700IU/ml (withfructose 6-phosphate as substrate) in ammonium sulfate solution, c (NH 4 ) 2 SO 4 )=3,2mol/l. The suspension is stable for at least one year at4 C. 6 Ap
34、paratus 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 accuracy equivalent to 6.1 (alternative to6.1) e.g.positive displacement capillary pipettes. 6.3 Cuvettes made of glas
35、s or plastic, of10mm optical path length, and which do not have significant absorption at334nm,340nm and365nm. 6.4 Spectral-line photometer, with mercury lamp and filters for measuring at334nm or365nm. 6.5 Spectrometer, (variable wavelength) for measuring at340nm(alternative to6.4). 7 Procedure 7.1
36、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 volumetric basis, after dilution to a known relat
37、ive 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 product. In products with high viscosity and/or very high content of cells(for example pulp), dete
38、rmination on the basis of a weighed test sample is the usual procedure. Mix cloudy juices well. Dilute the test sample so that the D-glucose or the D-glucose+D-fructose concentration is between0,1g/1 to1,0g/1. This solution is to be used for the determination even if it is coloured. 7.2 Test procedu
39、re 7.2.1 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 absorption maximum of NADPH is at340nm. When using a variable wavelength spect
40、rometer, 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 solutions. Solutions of enzyme, coenzyme and buffer may be added from suitable automatic pi
41、pettes. 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 substance to be determined is available in a suitably pure form, it is recommended to incl
42、ude it as a standard solution. 7.2.2 Blank test solution Pipette into cuvettes1,00ml buffer solution(5.2),0,10ml NADP solution(5.3),0,10ml ATP solution(5.4), and2,00ml water. Mix, and after about3min, read the absorbance(A 1 ) of the solution against air(no cuvette in lightpath). 7.2.3 Test sample s
43、olution Pipette into cuvettes1,00ml buffer solution(5.2),0,10ml NADP solution(5.3),0,10ml ATP solution(5.4),0,10ml test sample and1,90ml water. Mix and after about3min, read the absorbance(A 1 ) of the solution against air (nocuvette in lightpath). 7.2.4 Enzyme reaction and quantification The follow
44、ing procedure is carried out in each solution(7.7.2 and7.2.3) individually: Add0,02ml enzyme suspension(HK/G6P-DH;5.5). Mix, wait until the reaction has stopped(10min to15min) and read the absorbances(A 2 ) of the solutions. Check for completion of reaction by reading absorbance A 2at2min intervals.
45、 If the reaction is not complete after15min, extrapolate the absorbance back to the time of addition of enzyme suspension(HK/G6P-DH)(5.5).EN1140:1994 BSI 01-2000 5 Then add0,02ml enzyme suspension(PGI)(5.6). Mix, wait until the reaction has stopped(10min to15min) and read the absorbances(A 3 ) of th
46、e solutions. Check the, completion of reaction by reading absorbance A 3at2min intervals. If the reaction is not complete after15min and the absorbance increase remains constant, extrapolate the absorbance back to the time of addition of enzyme suspension(PGI)(5.6). 8 Calculation According to the re
47、actions on which this determination is based, there is a linear proportionality between the amount of NADPH formed(and hence the absorbance difference%A) and the concentration of D-glucose and D-fructose. %A D-glucose =(A 2 A 1 ) Sample (A 2 A 1 ) Blank %A D-fructose =(A 3 A 2 ) Sample (A 3 A 2 ) Bl
48、ank The calculation of the concentration of a substance in dilute solution by absorptiometric measurement is based on the Beer-Lambert law. The D-glucose and/or D-fructose content in grams per litre of sample is calculated from the following equation: If the volumes given in7.7.2 and7.2.3 are not al
49、tered, then the D-glucose and D-fructose contents in grams per litre of sample are calculated from the following equations: and: When using a commercially available test-combinations kit, the numerical factors(5,801 and5,837) in the above equations may be different, due to a different total assay volume(V 1 ). During calculation, take into account any 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 densit
