1、BRITISH STANDARD BS EN 1138:1995 Method for Determination of L-malic acid (L-malate) content of fruit and vegetable juices: NADH spectrometric method The European Standard EN1138:1994 has the status of a BritishStandard UDC 663.81/.82:620.1:543.42:543.476BSEN 1138:1995 This BritishStandard, having b
2、een prepared under the directionof the Consumer Products and Services Sector Board, was published under theauthority of the Standards Board and comesinto effect on 15January1995 BSI 02-2000 The following BSI references relate to the work on this standard: Committee reference AW/21 Draft for comment9
3、0/51499DC ISBN 0 580 23167 4 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
4、 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 National Standards Auth
5、ority 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 Stan
6、dardiseringskommissionen i Sverige Switzerland Association suisse de normalisation United Kingdom BritishStandards Institution Amendments issued since publication Amd. No. Date CommentsBSEN1138:1995 BSI 02-2000 i Contents Page Cooperating organizations Inside front cover National foreword ii Forewor
7、d 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 6 Annex B (informative) Statistical results of the inter-laboratory test 6 Natio
8、nal annex NA (informative) Committees responsible Inside back cover National annex NB (informative) Cross-references Inside back cover Table 1 6BSEN1138:1995 ii BSI 02-2000 National foreword This BritishStandard has been prepared under the direction of the Consumer Products and Services Sector Board
9、 and is the English language version of EN1138:1994 Fruit and vegetable juices Enzymatic determination of L-malic acid (L-malate) content NADH spectrometric method, published by the European Committee for Standardization (CEN). EN1138 was produced as result of international discussions in which the
10、UnitedKingdom took an activepart. 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
11、 of pages This 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 th
12、e inside front cover.EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 1138 October 1994 UDC 663.81/.82:620.1:543.42:543.476 Descriptors: Food products, beverages, fruit and vegetable juices, chemical analysis, determination of content, malic acid, enzymatic methods, spectrophotometric analysis E
13、nglish version Fruit and vegetables juices Enzymatic determination of L-malic acid (L-malate) content NADH spectrometric method Jus de fruits et de lgumes Dosage enzymatique de lcide L-malique (L-malate) Mthode spectrophotomtrique par le NADH Frucht- und Gemsesfte Enzymatische Bestimmung des Gehalte
14、s an L-pfelsure (L-Malat) 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 w
15、ithout 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 version in any other languag
16、e 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, Iceland, Ireland, I
17、taly, 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 CEN members Ref
18、. No. EN1138:1994 EEN1138:1994 BSI 02-2000 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 National Standard, eit
19、her 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 informative. According to th
20、e 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 UnitedKingdom.EN1138:1994 BSI 02-
21、2000 3 1 Scope This European standard specifies an enzymatic method for the determination of the total content of L-malic 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
22、dated or 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 Standa
23、rd only when incorporated in it by amendment or revision. For undated references the latest edition of the publication referred to applies. ISO 5725:1986, Precision of test methods Determination of repeatability and reproducibility for a standard test method by inter-laboratory tests. ISO 3696:1987,
24、 Water for analaytical laboratory use Specification and test methods. 3 Symbols and abbreviations For the purposes of this standard, the following symbols and abbreviations apply: 4 Principle and reactions The method is based on the enzymatic conversion ofL-malate to oxaloacetate and spectrometric m
25、easurement of the silmultaneous reduction of nicotinamide adenine dinucleotide at pH10,0 (reaction1): The equilibrum of reaction1) lies almost completely in the direction of L-malate. However, by trapping the oxaloacetate in a subsequent reaction2) catalysed by the enzyme GOT in the presence of L-gl
26、utamate, the equilibrum can be displaced in favour of oxaloacetate and NADH: The amount of NADH formed, measured by the increase in absorbance, is equivalent to the amount of L-malic acid. 5 Reagents 5.1 General. Use only reagents of recognized analytical grade and only water with at least grade3 of
27、 ISO3696:1987. 5.2 Glycylglycine buffer, pH10,0. Dissolve4,75g glycylglycine and0,88g L-glutamic acid in50ml water, adjust to pH10,0 with approximately4,6ml sodium hydroxide solution, c(NaOH)=10mol/l, and make up to60ml with water. The solution is stable for at least3 months at4 C. 5.3 NAD solution.
28、 Dissolve420mg NAD in12ml water. The solution is stable for at least4weeks at4 C. 5.4 GOT Enzyme suspension. Glutamateoxaloacetate transaminase, (GOT)=2mg/ml, suspended in a solution of ammonium sulfate, c(NH 4 ) 2 SO 4 =3,2mol/l. Thisresults in an enzyme activity of approximately400IU/ml with L-asp
29、artate and2-oxoglutarate as substrates. The suspension is stable for at least1year at4 C. 5.5 L-MDH Enzyme suspension. L-malate dehydrogenase,1(L-MDH)=5mg/ml suspended in a solution of ammonium sulfate, c(NH 4 ) 2 SO 4 =3,2mol/l. This results in an enzyme activity of approximately6000 IU/ml with oxa
30、loacetate as substrate. The suspension is stable for at least1 year at4 C. NAD -nicotinamide-adenine-dinucleotide; NADH -nicotinamide-adenine-dinucleotide (reduced form); GOT glutamate-oxaloacetate-transaminase (EC a 2.6.1.1); L-MDH L-malate-dehydrogenase (EC a 1.1.1.37); IU 1International Unit (IU)
31、 of enzyme activity catalyzes the conversion of1 4mol of substrate per minute at25 C under standard conditions; c Substance concentration; Mass concentration. a Enzyme Commission (EC): Classification System. Enzyme Handbook, Springer, Berlin1969.EN1138:1994 4 BSI 02-2000 6 Apparatus Usual laboratory
32、 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, of10mm opt
33、ical path length, and which do not have significant absorption at334,340 or365nm. 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 Preparation of the test samp
34、le 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 density. In this case, t
35、he relative density shall be indicated. Based in 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 vicosity and/or very high content of cells (for example pulp), determination on the basis of a
36、weighed test sample is the usual procedure. Mix the cloudy samples well before dilution; these, and also very strongly coloured samples, may need to be diluted beyond that required by the malate content. Clarify cloudy samples containing very small concentrations of malic acid by prior centrifugatio
37、n. Dilute the sample to be examined so that the L-malic concentration is between0,02g/l and0,35g/l. Thissolution is normally used directly for the determination, even if it is coloured. 7.2 Test procedure 7.2.1 General The determination shall normally be carried out at a constant temperature, betwee
38、n20 C and25 C. Aconstant temperature in the range25 C to37 C may also be used, providing equivalent 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
39、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 pipettes. Enzyme test pipettes(6.1), or their equivalent(6.2) shall be used for pipetting the sampl
40、e 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 include it as a standard solution. 7.2.2 Blank test solution Pipette into cuvette1,00ml buffer solut
41、ion(5.2)0,20ml NAD solution(5.3),1,50ml water and0,01ml GOT enzyme suspension(5.4). Mix and after approximately3min read the absorbance (A 1 ) Blankof the solution against air (nocuvette in light-path). 7.2.3 Test sample solution Pipette into cuvette1,00ml buffer solution(5.2),0,20ml NAD solution(5.
42、3),1,40ml water,0,01ml GOT enzyme suspension(5.4) and0,1ml test sample. Mix and after approximately3min read the absorbance (A 1 ) Sampleof the solution against air (no cuvette in light-path). If the concentration of L-malic acid in the test sample is less than0,02g/l, the test sample volume may be
43、increased to as much as1,50ml with a corresponding reduction in the amount of water added, so that the total assay volume remains the same(2,71ml). 7.2.4 Enzyme reaction and quantification Start the reaction by addition of0,01ml L-MDH enzyme suspension(5.5) to each of the solutions7.2.2 and7.2.3. Mi
44、x, and on completion of the reaction (about5min to10min) read the absorbances of the solutions (A 2 ). Check the completion of reaction by reading again after a further2min.EN1138:1994 BSI 02-2000 5 8 Calculation According to the reactions on which this determination is based, there is a linear prop
45、ortionality between the amount of NADH formed (and hence the absorbance difference, %A) and the concentration of malic acid. %A = (A 2 A 1 ) Sample (A 2 A 2 ) Blank The calculation of the concentration of a substance in dilute solution by absorptiometric measurement is based on th Beer-Lambert law.
46、The L-malic acid content ing/l of sample is calculated from the following equation: When using a commercially available test-combination kit, the numerical factor(3,647) in the above equation may be different, due to a different total assay volume. During calculation, take into account any dilution
47、factor and the relationship 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 L-malic acid content in grams per litre to two decimal places. 9 Precision Details of the inter-laboratory
48、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 absolute difference between two single test results found on identical
49、 test material by one operator using the same apparatus within the shortest feasible time interval will exceed the repeatability value r in not more than5% of the cases. The value is: r=0,014+0,030 g/l where: is the measured content, calculated as mean value from the two single test results. 9.2 Reproducibility The absolute difference between two single test results on identical test material reported by two laboratories will exceed the reproducibility value R in not more than5% of the cases. The value is: R =
