1、BRITISH STANDARD BS EN 1137:1995 Method for Determination of citric acid (citrate) content of fruit and vegetable juices: NADH spectrometric method The European Standard EN1137:1994 has the status of a BritishStandard UDC663.81/.82:620.1:543.42:547.477.1BSEN1137:1995 This BritishStandard, having bee
2、n prepared under the directionof the Consumer Products and Services Sector Board, was published under theauthority of the Standards Board and comes into effect on 15January1995 BSI01-2000 The following BSI references relate to the work on this standard: Committee reference AW/21 Draft for comment90/
3、51498DC ISBN 0 580 23166 6 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 b
4、elge 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 National Standards Author
5、ity 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 Certificacin Sweden Standa
6、rdiseringskommissionen i Sverige Switzerland Association suisse de normalisation United Kingdom BritishStandards Institution Amendments issued since publication Amd. No. Date CommentsBSEN1137:1995 BSI 01-2000 i Contents Page Cooperating organizations Inside front cover National foreword ii Foreword
7、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 Precision 5 10 Test report 5 Annex A (informative) Bibliography 6 Annex B (informative) Statistical results of the inter-laboratory test 6 National annex NA (in
8、formative) Committees responsible Inside back cover National annex NB (informative) Cross-references Inside back cover Table B.1 6BSEN1137:1995 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 En
9、glish language version of EN1137:1994 Fruit and vegetable juices Enzymatic determination of citric acid (citrate) content NADH spectrometric method, published by the European Committee for Standardization (CEN). EN1137 was produced as a result of international discussions in which the United Kingdom
10、 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 legal obligations. Summary of pages Th
11、is document comprises 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 fro
12、nt cover.EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN1137 October1994 UDC 663.81/.82:620.1:543.42:547.477.1 Descriptors: Food products, beverages, fruit and vegetable juices, chemical analysis, determination of content, citric acid, enzymatic methods, spectrophotometric analysis English vers
13、ion Fruit and vegetable juices Enzymatic determination ofcitric acid (citrate) content NADH spectrometric method Jus de fruits et de lgumes Dosage enzymatique de lcide citrique (citrate) Mthode spectromtrique par le NADH Frucht-und Gemsesfte Enzymatische Bestimmung des Gehaltes an Citronensure (Citr
14、at) Spektralphotometrische Bestimmung von NADH 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.
15、 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 other language made by translation
16、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, Iceland, Ireland, Italy, Luxembourg, Neth
17、erlands, 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 CEN members Ref. No. EN1137:1994EEN11
18、37:1994 BSI 01-2000 2 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 ide
19、ntical 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 informative. According to the CEN/CENELEC Internal Re
20、gulations, 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, United Kingdom.EN1137:1994 BSI 01-2000 3 1 Scope This Europea
21、n standard specifies a method for the enzymatic determination of the total content of citric acid, present either in the form of the free acid or its salts, in fruit and vegetable juices and related products. 2 Normative references This European Standard incorporates by dated or undated reference, p
22、rovisions 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
23、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 analytical laborator
24、y use Specification and test methods. 3 Symbols and abbreviations For the purposes of this standard, the following symbols and abbreviations apply: 4 Principle The method is based on the enzymatic conversion of citrate to oxaloacetate. Pyruvate is formed by spontaneous decarboxylation of oxaloacetat
25、e. Thesubsequent enzymatic reduction of both oxaloacetate and pyruvate by the reduced form ifnicotinamide adenine dinucleotide is followed spectrometrically. The total amount of NADH used, measured by the decrease in absorbance, is equivalent to the amount of citric acid. 4.1 Reactions The following
26、 enzymatic reactions take place atpH=7,8: 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 Glycylglycine buffer, pH7,8. Dissolve7,13g glycylglycine in70ml water, adjust to pH7,8 with about13ml sodium hydro
27、xide, c(NaOH)=5mol/l, add10ml zinc chloride solution, (ZnCl 2 )=0,8g/l and dilute to100ml with water. The buffer is stable for at least four weeks at4 C. 5.3 NADH solution. Dissolve30mg reduced -nicotinamide-adenine-dinucleotide, disodium salt (-NADH-Na 2 ) and60mg sodium hydrogen carbonate (NaHCO 3
28、 ) in6ml water. The solution is stable for at least four weeks at4 C. 5.4 MDL/LDH enzyme suspension. Mix0,1ml malate dehydrogenase, (MDH)=5mg/ml, approximately6000IU/ml with oxaloacetate as substrate,0,4ml ammonium sulfate solution, c (NH 4 ) 2 SO 4 =3,2mol/l and0,5ml lactate dehydrogenase, (LDH)=5m
29、g/ml, approximately2750IU/ml with pyruvate as substrate. The suspension is stable for at least one year at4 C. 5.5 CL enzyme suspension. Dissolve168mg lyophilisate(5mg enzyme protein) of citrate lyase (approximately1,25IU/ml with citrate as substrate) in1ml of ice-cold water. The suspension is stabl
30、e for at least one week at4 C and for at least four weeks when frozen. NAD -Nicotinamide-adenine-dinucleotide; NADH -Nicotinamide-adenine-dinucleotide, reduced form; CL Citrate lyase (EC a 4.1.3.6); MDH Malate dehydrogenase (EC a 1.1.1.37); LDH Lactate dehydrogenase (EC a 1.1.1.27); IU 1Internationa
31、l Unit (IU) of enzyme activity catalyzes the conversion of1mol of substrate per minute at25 C under standard conditions; c Substance concentration; Mass concentration. a Enzyme Commission (EC): Classification System Enzyme Handbook, Springer, Berlin 1969. EN1137:1994 4 BSI 01-2000 6 Apparatus Usual
32、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 to6.1 (alternative to6.1) e.g.positive displacement capillary pipettes. 6.3 Cuvettes, made of glass or plastic,
33、with10mm optical path length, and which do not have significant absorption at334mm,340mm or365nm. 6.4 Spectral-line photometer, with mercury lamp and filters for measuring at334nm or at365nm. 6.5 Spectrometer, (variable wavelength) for measuring at340nm (alternative to6.4). 7 Procedure 7.1 Preparati
34、on 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 relative densi
35、ty. 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), determinatio
36、n on the basis of a weighed test sample is the usual procedure. Dilute the sample to be examined so that the citric acid concentration is between0,02g/l and0,4g/l. This solution is used directly for the determination, even if it is coloured. Mix cloudy juices well and dilute them; they, and also ver
37、y strongly coloured juices, may need to be diluted beyond that required by the citrate content. 7.2 Test procedure 7.2.1 General The determination shall normally be carried out at constant temperature, between20 C and25 C. Aconstant temperature in the range25 C to37 C may also be used, providing equ
38、ivalent results are obtained. The absorption maximum of NADH is at340nm. When using a variable wavelength spectrometer, 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 pipette
39、s for pipetting the solutions. 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-com
40、bination kit. If the substance to be determined is available in a suitably pure form, it is recommended to include it as a standard solution. 7.2.2 Blank test solution Pipette into cuvette1,00ml buffer solution(5.2),0,1ml NADH solution(5.3),2,00ml water and0,02ml MDH/LDH enzyme suspension(5.4). Mix,
41、 read the absorbance (A 1 ) Blankof the solution against air (no cuvette in light path) after approximately5min. 7.2.3 Test sample solution Pipette into cuvette1,00ml buffer solution(5.2),0,1ml NADH solution(5.3),0,20ml of the test sample,1,80ml water and0,02ml MDH/LDH enzyme suspension(5.4). Mix, r
42、ead the absorbance (A 1 ) Sampleof the solution against air (nocuvette in light path) after approximately5min. If the initial extinction value is too high (A 1 1,000) start a new determination, beginning at7.2.2 and using a reduced concentration of NADH. Thecapacity of the test is affected by this m
43、easure, so that the concentration range of the test sample shall also be reduced. If the citrate concentration of the sample solution is less than0,02g/l, the sample volume to be pipetted into the cuvette can be increased to as much as2,0ml. In this case, the volume of water to be added is correspon
44、dingly reduced so that both blank and sample cuvettes contain the same total volume. The volume of sample solution (V 2 ) in the calculation (see clause8) shall then be altered accordingly. 7.2.4 Enzyme reaction and quantification Start the reaction by the addition of0,02ml CL enzyme suspension(5.5)
45、 each of the solutions7.2.2 and7.2.3. Mix, and after the reaction is complete (about5min to10min) read the absorbance (A 2 ) of the solutions against air (no cuvette in light path). Check the completion of reaction by reading after a further2min.EN1137:1994 BSI 01-2000 5 8 Calculation According to t
46、he reactions on which this determination is based, there is a linear proportionality between the amount of NADH used (and hence the absorbance difference, %A) and the concentration of citric acid. %A =(A 1 /A 2 ) Sample (A/A 2 ) Blank The calculation of the concentration of a substance in dilute sol
47、ution by absorptiometric measurement is based on the Beer-Lambert law. The citric acid content, , in grams per litre of sample is calculated from the following equation: When using a commercially available test-combination kit, the numerical factor(3,016) of the above equation is different, due to a
48、 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 density of the single strength sample. Report the citric acid concentrat
49、ion in grams perlitre to two decimal places. 9 Precision Details of the inter-laboratory test on the precision of the method are summarized in Annex B. Thevalues derived from the inter-laboratory test may not be applicable to analyte concentration ranges and matrices other than given in Annex B. 9.1 Repeatability The 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 value r in not
