1、| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | BRITISH STANDARD BS EN 12146 : 1997 The Eu
2、ropean Standard EN 12146 : 1996 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 sucrose content NADP spectrometric methodBS EN 12146 : 1997 This British Standard, having be
3、en prepared under 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 May 1997 BSI 1997 The following BSI references relate to the work on this standard: Committee reference AW/21 Draft for comment 90/
4、51492 DC ISBN 0 580 27063 7 Amendments issued since publication Amd. No. Date Text affected Committees responsible for this British Standard The preparation of this British Standard was entrusted to Technical Committee AW/21, Fruit and vegetable juices, upon which the following bodies were represent
5、ed: British Fruit Juice Importers Association British Retail Consortium British Soft Drinks Association Limited Campden and Chorleywood Food Research Association Laboratory of the Government Chemist Letherhead Food Research Association Ministry of Agriculture, Fisheries and Food Royal Society of Che
6、mistryBS EN 12146 : 1997 BSI 1997 i Contents Page Committees responsible Inside front cover National foreword ii Foreword 2 Text of EN 12146 3ii BSI 1997 BS EN 12146 : 1997 National foreword This British Standard has been prepared by Technical Committee AW/21, and is the English language version of
7、EN 12146 : 1996 Fruit and vegetable juices Enzymatic determination of sucrose content NADP spectrometric method, published by the European Committee for Standardization (CEN). EN 12146 was produced as a result of international discussions in which the United Kingdom took an active part. Cross-refere
8、nces Publication referred to Corresponding British Standard ISO 3696 : 1987 BS EN ISO 3696 : 1995 Specification for water for laboratory use ISO 5725 : 1986, to which informative reference is made in the text, has been superseded by ISO 5725-1 : 1994, ISO 5725-2 : 1994, ISO 5725-3 : 1994, ISO 5725-4
9、 : 1994 and ISO 5725-6 : 1994 which are identical with BS ISO 5725 Accuracy (trueness and precision) of measurement methods and results, BS ISO 5725-1 : 1994 General principles and definitions, BS ISO 5725-2 : 1994 Basic methods for the determination of repeatability and reproducibility of a standar
10、d measurement method, BS ISO 5725-3 : 1994 Intermediate measures of the precision of a standard measurement method, BS ISO 5725-4 : 1994 Basic methods for the determination of the trueness of a standard measurement method, and BS ISO 5725-6 : 1994 Use in practice of accuracy values. Compliance with
11、a British Standard does not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, pages i and ii, the EN title page, pages 2 to 10, an inside back cover and a back cover.CEN European Committee for Standardization Comite Europ
12、e en de Normalisation Europa isches Komitee fu r Normung Central Secretariat: rue de Stassart 36, B-1050 Brussels 1996 Copyright reserved to CEN members Ref. No. EN 12146 : 1996 E EUROPEAN STANDARD EN 12146 NORME EUROPE ENNE EUROPA ISCHE NORM September 1996 ICS 67.160.20 Descriptors: Food products,
13、beverages, fruit and vegetable juices, chemical analysis, determination, sucrose, enzymatic methods, spectrometric analysis English version Fruit and vegetable juices Enzymatic determination of sucrose content NADP spectrometric method Jus de fruits et de le gumes Dosage enzymatique du saccharose Me
14、 thode spectrome trique par le NADP Frucht- und Gemu sesa fte Enzymatische Bestimmung des Saccharosegehaltes Spektralphotometrisches Verfahren mit NADP This European Standard was approved by CEN on 1996-05-09. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate
15、the conditions for giving this European Standard the status 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
16、in three official versions (English, French, German). A version 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
17、 of Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.Page 2 EN 12146 : 1996 BSI 1997 Foreword This European Standard has been prepared by Technical Committee CEN/TC 174, Frui
18、t and vegetable juices Method of analysis, of which the secretariat 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 March 1997, and conflicting national standards shall be withdrawn
19、 at the latest by March 1997. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherland
20、s, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. Contents Page 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 6 10 Test report 6 Annexes A (informative) Bi
21、bliography 7 B (informative) Information on how to treat creep reactions 7 C (informative) Statistical results of the interlaboratory test 8Page 3 EN 12146 : 1996 BSI 1997 1) Enzyme Commission (EC); Classification System Enzyme Handbook, Springer, Berlin 1969. 1 Scope This European Standard specifie
22、s an enzymatic method for the determination of the content of sucrose in fruit and vegetable juices and related products. 2 Normative references This European Standard incorporates by dated or undated reference, provisions from other publications. These normative references are cited at the appropri
23、ate 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 amendment or revision. For undated references the latest edition of the publicati
24、on referred to applies. EN ISO 3696 : 1995 Water for analytical laboratory use Specification and test methods. ISO 5725 : 1986 Precision of test methods Determination of repeatability and reproducibility for a standard test method by inter-laboratory tests. 3 Symbols and abbreviations For the purpos
25、es of this standard, the following symbols and abbreviations apply: ATP Adenosine-59-triphosphate ADP Adenosine-59-diphosphate NADP b-Nicotinamide-adenine- dinucleotidephosphate NADPH b-Nicotinamide-adenine- dinucleotidephosphate (reduced form) G-6-P Glucose-6-phosphate HK Hexokinase (EC 2.7.1.1) 1)
26、 G6P-DH Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) 1) BF b-Fructosidase (EC 3.2.1.26) 1) IU One International Unit of enzyme activity, which catalyses the conversion of 1mmol of substrate per min at 25 C c Substance concentration r Mass concentration 4 Principle and reactions 4.1 Principle Sucr
27、ose is enzymatically hydrolysed (inverted) by the action of BF in a diluted test sample to give equal amounts of D-glucose and D-fructose. The D-glucose formed is then phosphorylated in the C-6 position in an enzyme-catalysed reaction involving ATP and HK. In a concomitant reaction, G-6-P is convert
28、ed stoichiometrically to 6-phosphogluconate in the presence of NADH, the reaction being catalysed by the enzyme G6P-DH and an amount of NADPH equivalent to the amount of D-glucose present in the test sample being formed (4.2). The quantification of the NADPH formed and hence the content of D-glucose
29、 and sucrose is performed by spectrometry. In juices where there is a low level of sucrose (less than 5 g/l) and a higher level of glucose it is impossible to obtain an accurate quantification of sucrose via the conventional enzymatic determination. In these circumstances the glucose should be remov
30、ed, by reaction with iodine at an alkaline pH, prior to quantification of the sucrose. 4.2 Reactions sucrose + H 2 0 D-glucose + D-fructose (1) BF glucose + ATP G-6-P + ADP (2) HK G-6-P + NADP G6P - DH 6-phosphogluconate + NADPH + H + (3) 5 Reagents 5.1 General Use only reagents of recognized analyt
31、ical grade and only water in accordance with at least grade 3 of EN ISO 3696 : 1995. 5.2 Sodium hydroxide solutions Sodium hydroxide solutions are prepared at c (NaOH) = 5 mol/l, c (NaOH) = 4 mol/l and c (NaOH) = 2 mol/l 5.3 Citrate buffer pH = 4,6 Dissolve 6,9 g of citric acid monohydrate (C 6 H 8
32、O 7 H 2 O) and 9,1 g of trisodium citrate dihydrate (C 6 H 5 Na 3 O 7 2H 2 O) in 150 ml of water, adjust to pH = 4,6 with sodium hydroxide solution (c (NaOH) = 2,0 mol/l) (5.2) and dilute to 200 ml with water. The buffer is stable for at least 1 year at 4 C.Page 4 EN 12146 : 1996 BSI 1997 5.4 b-Fruc
33、tosidase solution Dissolve 10 mg of b-fructosidase,r = 5 mg/ml, about 750 IU/ml, in 2 ml of citrate buffer (5.3). The solution is stable for at least 1 week at 4 C. 5.5 Triethanolamine buffer pH = 7,6 Dissolve 14,0 g of triethanolamine hydrochloride and 0,25 g of magnesium sulfate heptahydrate (MgSO
34、 4 7H 2 O) in 80 ml of water, adjust to pH = 7,6 with approximately 5 ml of sodium hydroxide solution (c (NaOH) = 5 mol/l) and make up to 100 ml with water. The buffer is stable for at least 4 weeks at 4 C. 5.6 NADP solution Dissolve 60 mg of -nicotinamide-adenine-dinucleotide phosphate-disodium sal
35、t (-NADP-Na 2 ) in 6 ml of water. The solution is stable for at least 4 weeks at 4 C. 5.7 ATP solution Dissolve 300 mg of adenosine-59-triphosphate-disodium salt (ATP-Na 2 H 2 3H 2 O) and 300 mg of sodium hydrogen carbonate (NaHCO 3 ) in 6 ml of water. The solution is stable for at least 4 weeks at
36、4 C. 5.8 HK/G6P-DH enzyme suspension Suspend hexokinase,r(HK) = 2 mg/ml, about 280 IU/ml (with D-glucose serving as the substrate in the presence of ATP) and G6P-DH, r(G6P-DH) = 1 mg/ml, about 140 IU/ml (with G-6-P as substrate) in ammonium sulfate solution, c(NH 4 ) 2 SO 4 ) = 3,2 mol/l. The suspen
37、sion is stable for at least 1 year at 4 C. 5.9 Iodine solution Dissolve 130 g of iodine and 150 g of potassium iodide in sufficient quantity of water in a 1 l volumetric flask, when dissolved dilute to volume with water. 5.10 Sulfuric acid Prepare a solution of sulfuric acid at c(H 2 S0 4 ) = 0,5 mo
38、l/l from an appropriate concentrated standard solution. 5.11 Sodium sulfite solutions Prepare a saturated solution of sodium sulfite in water (solubility r(Na 2 SO 3 ) = 12,54 g/100ml at 0 C and 28,3 g/100ml at 80 C). From this concentrated solution prepare a dilute solution by carrying out a 1 to 1
39、0 dilution in water. 5.12 Phenolphthalein solution Prepare a solution of phenolphthalein (r = 0,5 g/100 ml) in ethanol. 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,
40、 with accuracy equivalent to 6.1 (alternative to 6.1) e.g. positive displacement capillary pipettes. 6.3 Cuvettes, made of quartz, glass or plastics of 10 mm path length, and which do not have a significant absorption at the wavelengths of interest (e.g. 334 nm, 340 nm or 360 nm). 6.4 Spectral-line
41、photometer, with mercury lamp and filters for measuring at wavelengths of 334 nm or 365 nm. 6.5 Spectrometer, (variable wavelength) for measuring at a wavelength of 340 nm (alternative to 6.4). 7 Procedure 7.1 Preparation of the test sample 7.1.1 Normal sample preparation The fruit juice shall be di
42、luted so that the sucrose/glucose concentration is between 0,1 g/l and 1,5 g/l. This solution shall be used directly and will normally not require any pre-treatment. The analysis by this method shall be on a volumetric basis, results being expressed per litre of sample. The analysis of concentrated
43、products may also be carried out on a volumetric basis, after dilution to a known relative 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
44、 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 sample is the usual procedure. Mix cloudy samples well before dilution; these, and also very strongly coloured samples, may need to be diluted beyond that required b
45、y the sucrose content. Clarify cloudy samples containing very low concentrations of sucrose by prior centrifugation or membrane filtration through a 0,2mm filter. 7.1.2 Modified sample preparation for sucrose quantification in presence of high concentration of glucose Prepare a 1 in 5 dilution of th
46、e fruit juice (clarified by filtration or centrifugation as given above in 7.1.1). To 10 ml of the diluted/clarified fruit juice, in a 50 ml volumetric flask, add 10 ml of iodine solution (5.9) and 2,5 ml of 4 mol/l sodium hydroxide solution (5.2) (solutions shall be added in this sequence). Leave t
47、he flask for 10 min in the dark. Add to this solution 10 ml sulfuric acid solution (5.10). The excess iodine is destroyed by addition of saturated and diluted sodium sulfite solutions (5.11) and shaking until no yellow/brown colour remains. Adjust the pH of the solution to approximately pH = 8 to 9
48、by titration with 4 mol/l sodium hydroxide solution (5.2), using phenolphthalein indicator solution (5.12) until a pale pink colour persists. This solution shall be made up to 50 ml, then used for the determination of sucrose.Page 5 EN 12146 : 1996 BSI 1997 7.2 Test procedure 7.2.1 General The deter
49、mination shall be carried out at a constant temperature between 20 C to 25 C, or a constant temperature up to 37 C, providing equivalent results are obtained. The absorption maximum of NADPH is at a wavelength of 340 nm. 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 of 334 nm or 365 nm. Do not use single-mark transfer pipettes for pipetting the solutions. Solutions of enzyme, coenzy
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