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1、| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | BRITISH STANDARD BS EN 1988-2:1998 The Eur
2、opean Standard EN 1988-2:1998 has the status of a British Standard ICS 67.040 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW Foodstuffs Determination of sulfite Part 2: Enzymatic method Licensed Copy: sheffieldun sheffieldun, na, Mon Oct 30 01:44:52 GMT+00:00 2006, Uncontroll
3、ed Copy, (c) BSI This British Standard, having been 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 June 1998 BSI 1998 ISBN 0 580 29240 1 BS EN 1988-2:1998 Amendments issued since pu
4、blication Amd. No.DateText affected National foreword This British Standard is the English language version of EN 1988-2:1998 published by the European Committee for Standardization (CEN). The UK participation in its preparation was entrusted to Technical Panel AW/-/3, Food analysis Horizontal metho
5、ds, which has the responsibility to: aid enquirers to understand the text; present to the responsible European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in th
6、e UK. A list of organizations represented on this panel can be obtained on request to its secretary. Cross-references The British Standards which implement international or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled Inter
7、national Standards Correspondence Index, or by using the Find facility of the BSI Standards Electronic Catalogue. 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 Bri
8、tish Standard does not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, the EN title page, pages 2 to 8, an inside back cover and a back cover. Licensed Copy: sheffieldun sheffieldun, na, Mon Oct 30 01:44:52 GMT+00:00 20
9、06, Uncontrolled Copy, (c) BSI CEN European Committee for Standardization Comite Europe en de Normalisation Europa isches Komitee fu r Normung Central Secretariat: rue de Stassart 36, B-1050 Brussels 1998 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national
10、 Members. Ref. No. EN 1988-2:1998 E EUROPEAN STANDARDEN 1988-2 NORME EUROPE ENNE EUROPA ISCHE NORM February 1998 ICS 67.040 Descriptors: Food products, chemical analysis, determination of content, sulfites, enzymatic methods English version Foodstuffs Determination of sulfite Part2: Enzymatic method
11、 Produits alimentaires Dosage des sulfites Partie 2: Me thode enzymatique Lebensmittel Bestimmung von Sulfit Teil 2: Enzymatisches Verfahren This European Standard was approved by CEN on 1 January 1998. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the con
12、ditions 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 in thre
13、e 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 of Aus
14、tria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. Licensed Copy: sheffieldun sheffieldun, na, Mon Oct 30 01:44:52 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Page
15、 2 EN 1988-2:1998 BSI 1998 Foreword This European Standard has been prepared by Technical Committee CEN/TC 275, Food analysis Horizontal methods, the secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication of an identical te
16、xt or by endorsement, at the latest by August 1998, and conflicting national standards shall be withdrawn at the latest by August 1998. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Au
17、stria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. This European Standard Foodstuffs Determination of sulfite, consists of the following parts: Part 1: Optim
18、ized Monier-Williams method; Part 2: Enzymatic method. Contents Page Foreword2 Introduction3 1Scope3 2Normative references3 3Principle3 4Reagents3 5Apparatus4 6Procedure4 7Calculation5 8Precision5 9Test report6 Annex A (informative) Bibliography7 Annex B (informative) Precision data7 Licensed Copy:
19、sheffieldun sheffieldun, na, Mon Oct 30 01:44:52 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Page 3 EN 1988-2:1998 BSI 1998 1) It has been shown that the analysis of isolated soy protein leads to false positive results in the range of 20 mg/kg to 30 mg/kg expressed as sulfur dioxide. Therefore, when
20、analysing foodstuffs containing isolated soy proteins a proportional enhancement of the result may be obtained and is taken into account. 2) These reagents are included in commercially available test kits. If these test kits are used, the manufacturers instructions should be followed. 3) This unit (
21、often called the International unit or Standard unit) is defined as the amount of enzyme which will catalyse the transformation of 1 mmol substrate per minute under standard conditions. Introduction Sulfite can be used as a preservative in foodstuffs. In order to minimize possible negative health ef
22、fects, many countries have regulated the use of sulfite in foods. This has resulted in the development of several methods of analysis to detect the presence and quantity of sulfite in a great variety of foods. 1 Scope This European Standard specifies an enzymatic method for the determination of the
23、sulfite content, expressed as sulfur dioxide, in foodstuffs. Other sulfur-containing substances such as sulfate, sulfide or thiosulfate do not interfere with the determination. Carbonyl-sulfite complexes react as free sulfites. Isothiocyanates, occurring in, e.g. mustard, interfere with the determin
24、ation. The method is not applicable to cabbages, dried garlic, dried onions, ginger, leeks and soy protein1). It has been shown that the analysis of isolated soy protein leads to false positive results. Specific products, for which European Standards for the determination of the sulfites exist, are
25、excluded from the scope of this horizontal European Standard. 2 Normative references This European Standard incorporates by 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 herea
26、fter. 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 publication referred to applies. EN ISO 3696, Water for analytical la
27、boratory use Specification and test methods. (ISO 3696:1987) 3 Principle Oxidation of sulfite to sulfate in the presence of sulfite oxidase with the liberation of hydrogen peroxide at the same time. SO322+ O2+ H2OSO422+ H2O2 sulfite oxidase Reduction of hydrogen peroxide and conversion of NADH to NA
28、D+in the presence of NADH peroxidase. H2O2+ NADH + H+2H2O + NAD+ NADH peroxidase Conversion of NADH to NAD+is determined spectrometrically and is proportional to the concentration of sulfite, see 1 to 6 in annex A. 4 Reagents During the analysis, unless otherwise stated, use only reagents of recogni
29、zed analytical grade and only water of at least grade 3 as defined in EN ISO 3696. 4.1 Ammonium sulfate. 4.2 EthylenediamineN,N,N9,N9tetraacetic acid (EDTA). 4.3 Sodium hydrogen carbonate. 4.4 Sodium sulfite. 4.5 Ammonium sulfate solution, substance concentration c(NH4)2SO4 = 2 mol/l. 4.6 Sodium hyd
30、roxide solution, c(NaOH) = 0,1 mol/l. 4.7 Sodium hydroxide solution, c(NaOH) = 2 mol/l. 4.8 Triethanolamine buffer solution2), c(C6H15NO3) = 0,6 mol/l, pH 8,0. Dissolve 5,57 g of triethanolamine hydrochloride in 40 ml of water in a beaker. Adjust to pH 8,0 with the sodium hydroxide solution (4.6). T
31、ransfer the solution to a 50 ml volumetric flask and dilute to the mark with water and mix. The buffer is stable for 1 year at +4 C. 4.9 NADH solution2)(Reduced nicotinamide-adenine dinucleotide), c(NADH) = 7 3 1023mol/l. Dissolve 25 mg of b-nicotinamide-adenine dinucleotide disodium salt (b-NADH-Na
32、2) and 50 mg of sodium hydrogen carbonate (4.3) in 5,0 ml of water and mix. The solution is stable for at least 4 weeks at +4 C. 4.10 NADH peroxidase suspension2)(EC 1.11.1.1), (see 7 of annex A). Make a suspension of 10 enzyme units/ml (U/ml)3)in the ammonium sulfate solution (4.5), pH approximatel
33、y 7. The suspension is stable for 1 year at +4 C. 4.11 Sulfite oxidase suspension2)(EC 1.8.3.1), (see 7 of annex A). Prepare a suspension of 2,5 enzyme units/ml in ammonium sulfate solution (4.5), pH approximately 7. The suspension is stable for 1 year at +4 C. Licensed Copy: sheffieldun sheffieldun
34、, na, Mon Oct 30 01:44:52 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Page 4 EN 1988-2:1998 BSI 1998 4.12 Reference solution. Weigh 0,6 g of sodium sulfite (4.4) (equivalent to about 300 mg of sulfur dioxide), to the nearest 0,1 mg, and 37 mg of EDTA (4.2) and dissolve in water. Transfer the solution
35、 quantitatively to a 1 000 ml volumetric flask, dilute to the mark with water and mix. Take 100 ml of this solution as reference sample and analyse the sulfite content within 30 min. The coefficient of variation for the reference values shall not exceed 0,06. 4.13 Polyvinylpyrrolidone, cross linked
36、(polyvinylpolypyrrolidone). 4.14 Ascorbate oxidase, e.g. as ascorbate oxidase spatula, of defined activity. 4.15 Bentonite. 5 Apparatus Usual laboratory apparatus and in particular the following: 5.1 Water bath, capable of being controlled at 60 C2 C. 5.2 Homogenizer. 5.3 Graduated micropipettes, 10
37、 ml, 20 ml, 50 ml and 100 ml. If mechanical pipettes with disposable ends/capillaries are used, it is of the utmost importance that they are calibrated. 5.4 pH-meter. 5.5 Spectrometer, suitable for measurements at a wavelength of 340 nm. 5.6 Quartz cells, with an optical path length of 1 cm. Disposa
38、ble cells/cuvettes can also be used. 5.7 Centrifuge, suitable for centripetal acceleration of 2 000 g with blender cups or glass tubes of suitable capacity. 6 Procedure 6.1 Preparation of the sample test solutions 6.1.1 General Remove high concentrations of ascorbic acid of more than 100 mg/kg or 10
39、0 mg/l of sample (see 6.1.2.3). If the concentration of sulfite in the sample test solution is higher than 0,3 g/l, dilute the sample test solution prior to the determination or take a smaller sample volume. 6.1.2 Liquid samples 6.1.2.1 White wine, brandy and beer Analyse white wine and brandy direc
40、tly. Beer should be filtered to remove carbon dioxide. It may be necessary to decolorize beer. For the decolorization, add not more than about 0,7 g of bentonite (4.15) to 10 ml of filtered beer in a 50 ml glass beaker, stir the mixture for 2 min using a magnetic stirrer and then filter the solution
41、 into another 50 ml glass beaker. For the enzymatic determination (6.2) take 100 ml to 200 ml of wine or 500 ml of brandy or beer respectively. 6.1.2.2 Red wines Adjust 25 ml of red wine to pH 7,5 to 8,0 with the sodium hydroxide solution (4.7) in a beaker. Transfer the solution into a 50 ml volumet
42、ric flask and dilute to the mark with water and mix. It is often necessary to decolourize red wine. This can be done as described in 6.1.2.3 for fruit juices. 6.1.2.3 Fruit juices Centrifuge cloudy juices at approximately 2 000 g. Add 5 ml of juice into a beaker and adjust the pH to 5 to 6 with the
43、sodium hydroxide solution (4.7). Remove ascorbic acid by adding approximately 40 units of ascorbate oxidase (4.14) in solution to the juice and leave the sample for 10 min, or remove the ascorbic units by stirring for 3 min with an ascorbate oxidase spatula (4.14). Then adjust the pH to 7,5 to 8,0 w
44、ith the sodium hydroxide solution (4.7). In the case of coloured juices, add approximately 0,25 g of polyvinylpolypyrrolidone (4.13) and stir the mixture for 1 min. Transfer the mixture to a 10 ml volumetric flask and dilute with water. Filter the solution and take 200 ml for enzymatic determination
45、 (6.2). 6.1.3 Solid foodstuffs Homogenize the sample thoroughly and extract with water at 60 C for 5 min. Shake occasionally. Cool the sample to ambient temperature before analysing. Vary the sample size depending on the amount of sulfite. In the case of e.g. potato flakes, take 5,0 g of homogenized
46、 material into a 50 ml volumetric flask. Add 40 ml of water. Close the flask and extract in a water bath (5.1) at 60 C for 5 min. Shake occasionally. Cool the volumetric flask, either by letting it stand for at least 15 min, to ambient temperature, or in a water bath of 20 C, and dilute to the mark
47、with water (V3= 50 ml). If necessary centrifuge the solution. The following sample quantities of some other foods are suggested: Dried fruit1,0 g of sample/50 ml of water Jam5,0 g of sample/50 ml of water Spices0,1 g of sample/50 ml of water Dried potato products2,0 g of sample/50 ml of water Take 1
48、00 ml to 500 ml of these solutions for enzymatic determination (see 6.2). Licensed Copy: sheffieldun sheffieldun, na, Mon Oct 30 01:44:52 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Page 5 EN 1988-2:1998 BSI 1998 Table 1 Fluids pipetted into the cellsSample cellBlank cell Triethanolamine buffer solut
49、ion (4.8) 1,00 ml1,00 ml NADH solution (4.9)0,10 ml0,10 ml NADH peroxidase suspension (4.10)0,01 ml0,01 ml Sample test solution0,10 ml Water1,90 ml2,00 ml Mix gently. Measure the absorbance A1of the sample cell and of the blank cell after 5 min (without a cell in the reference path), then start the reaction by adding the following: Sulfite oxidase suspension (4.11)0,05 ml0,05 ml Mix and read the absorbance A2after approximately 30 min. Take an additional reading after 5 min to check that no further change in the ab
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