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 methodThis British Standard, having been prepared under the direction of the Consumer Products an
3、d 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 publication Amd. No. Date Text affected National foreword This British Standard is the English language versio
4、n 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 methods, which has the responsibility to: aid enquirers to understand the text; present to the responsible Euro
5、pean 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 the UK. A list of organizations represented on this panel can be obtained on request to its secretary. Cross
6、-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 “International Standards Correspondence Index”, or by using the “Find” facility of the BSI Standards Electronic
7、 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 British Standard does not of itself confer immunity from legal obligations. Summary of pages This documen
8、t comprises a front cover, an inside front cover, the EN title page, pages 2 to 8, an inside back cover and a back cover.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 A
9、ll rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 1988-2:1998 E EUROPEAN STANDARD EN 1988-2 NORME EUROPE ENNE EUROPA ISCHE NORM February 1998 ICS 67.040 Descriptors: Food products, chemical analysis, determination of content, sulfites, en
10、zymatic methods English version Foodstuffs Determination of sulfite Part 2: Enzymatic method 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 m
11、embers 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. Up-to-date lists and bibliographical references concerning such national standards may be obtained on applic
12、ation 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 under the responsibility of a CEN member into its own language and notified to the Central Secretariat has th
13、e same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.Page 2 EN 1988-2:199
14、8 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 text or by endorse
15、ment, 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: Austria, Belgium,
16、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: Optimized Monier-Will
17、iams method; Part 2: Enzymatic method. Contents Page Foreword 2 Introduction 3 1 Scope 3 2 Normative references 3 3 Principle 3 4 Reagents 3 5 Apparatus 4 6 Procedure 4 7 Calculation 5 8 Precision 5 9 Test report 6 Annex A (informative) Bibliography 7 Annex B (informative) Precision data 7Page 3 EN
18、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 analysing foodstuffs containing isolated soy proteins a proportional enhancement of the result may be
19、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 (often called the International unit or Standard unit) is defined as the amount of enzyme which will ca
20、talyse the transformation of 1mmol substrate per minute under standard conditions. Introduction Sulfite can be used as a preservative in foodstuffs. In order to minimize possible negative health effects, many countries have regulated the use of sulfite in foods. This has resulted in the development
21、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 sulfite content, expressed as sulfur dioxide, in foodstuffs. Other sulfur-containing substances such as
22、 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 determination. The method is not applicable to cabbages, dried garlic, dried onions, ginger, leeks and soy prot
23、ein 1) . 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 excluded from the scope of this horizontal European Standard. 2 Normative references This European St
24、andard 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 hereafter. For dated references, subsequent amendments to or revisions of any of these publications apply
25、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 laboratory use Specification and test methods. (ISO 3696:1987) 3 Principle Oxidation of sulfite to sulf
26、ate in the presence of sulfite oxidase with the liberation of hydrogen peroxide at the same time. SO 3 22 +O 2 +H 2 OS O 4 2 2 +H 2 O 2 sulfite oxidase Reduction of hydrogen peroxide and conversion of NADH to NAD + in the presence of NADH peroxidase. H 2 O 2 + NADH + H + 2H 2 O + NAD + NADH peroxida
27、se 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 recognized analytical grade and only water of at least grade 3 as defined in EN ISO
28、 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(NH 4 ) 2 SO 4 = 2 mol/l. 4.6 Sodium hydroxide solution, c(NaOH) = 0,1 mol/l. 4.7 Sodium hydroxide solution, c(
29、NaOH) = 2 mol/l. 4.8 Triethanolamine buffer solution 2) , c(C 6 H 15 NO 3 ) = 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). Transfer the solution to a 50 ml volumetric flask and dilute to
30、the mark with water and mix. The buffer is stable for 1 year at +4 C. 4.9 NADH solution 2) (Reduced nicotinamide-adenine dinucleotide), c(NADH) = 73 10 23 mol/l. Dissolve 25 mg ofb-nicotinamide-adenine dinucleotide disodium salt (b-NADH-Na 2 ) and 50 mg of sodium hydrogen carbonate (4.3) in 5,0 ml o
31、f water and mix. The solution is stable for at least 4 weeks at +4 C. 4.10 NADH peroxidase suspension 2) (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 approximately 7. The suspension is stable for 1 year at +4 C. 4.11
32、Sulfite oxidase suspension 2) (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.Page 4 EN 1988-2:1998 BSI 1998 4.12 Reference solution. Weigh 0,6 g of sodium sulfite (4.4)
33、(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 quantitatively to a 1 000 ml volumetric flask, dilute to the mark with water and mix. Take 100ml of this solution as reference sample and analyse the sulfite co
34、ntent within 30 min. The coefficient of variation for the reference values shall not exceed 0,06. 4.13 Polyvinylpyrrolidone, cross linked (polyvinylpolypyrrolidone). 4.14 Ascorbate oxidase, e.g. as ascorbate oxidase spatula, of defined activity. 4.15 Bentonite. 5 Apparatus Usual laboratory apparatus
35、 and in particular the following: 5.1 Water bath, capable of being controlled at 60 C 2 C. 5.2 Homogenizer. 5.3 Graduated micropipettes,1 0m l, 20ml, 50ml and 100ml. If mechanical pipettes with disposable ends/capillaries are used, it is of the utmost importance that they are calibrated. 5.4 pH-mete
36、r. 5.5 Spectrometer, suitable for measurements at a wavelength of 340 nm. 5.6 Quartz cells, with an optical path length of 1 cm. Disposable 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 Proce
37、dure 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 100 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 det
38、ermination 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 directly. 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
39、.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 into another 50 ml glass beaker. For the enzymatic determination (6.2) take 100ml to 200ml of wine or 500ml of brandy or beer respectively. 6.1.2.2 Red wines Adjus
40、t 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 volumetric 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
41、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 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
42、 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 with 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. Tr
43、ansfer the mixture to a 10 ml volumetric flask and dilute with water. Filter the solution and take 200ml for enzymatic determination (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 bef
44、ore 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 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 volumetri
45、c 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 with water (V 3 = 50 ml). If necessary centrifuge the solution. The following sample quantities of some other foods are suggested: Dried fruit 1,0 g of sample/50 ml of
46、water Jam 5,0 g of sample/50 ml of water Spices 0,1 g of sample/50 ml of water Dried potato products 2,0 g of sample/50 ml of water Take 100ml to 500ml of these solutions for enzymatic determination (see 6.2).Page 5 EN 1988-2:1998 BSI 1998 Table 1 Fluids pipetted into the cells Sample cell Blank cel
47、l Triethanolamine buffer solution (4.8) 1,00 ml 1,00 ml NADH solution (4.9) 0,10 ml 0,10 ml NADH peroxidase suspension (4.10) 0,01 ml 0,01 ml Sample test solution 0,10 ml Water 1,90 ml 2,00 ml Mix gently. Measure the absorbance A 1 of the sample cell and of the blank cell after 5 min (without a cell
48、 in the reference path), then start the reaction by adding the following: Sulfite oxidase suspension (4.11) 0,05 ml 0,05 ml Mix and read the absorbance A 2 after approximately 30 min. Take an additional reading after 5 min to check that no further change in the absorbance has taken place. 6.2 Determ
49、ination Perform the determination according to Table 1 at a temperature of 20 C to 25 C in a quartz cell (5.6) usually with a sample volume of 100ml. If the sample volume is different from 100ml, adjust the volume of added water so that the final volume of water and sample is 2,00 ml. If the reaction has not stopped, continue to read the absorbance at intervals of 2 min until the change in absorbance is constant. If the absorbance decreases constantly, extrapolate the absorbance back to the time of addition
