1、 - STD-BSI BS EN 1788-2-ENGL 1798 1b24bb7 0708355 452 BS EN 1988-2:1998 BRITISH STANDARD Foodstuffs - Determination of sulfite - Part 2: Enzymatic method The European Standard EN 19882: 1998 has the status of a British Standard ICs 67.040 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPY
2、RIGHT LAW BS EN 19-21998 direction of the Consumer AmdNo. Products and SeMces Sector Board, was published under the authority of the Standards Board and comes into effect on 15 June 1998 0 BSI 1998 National foreword Date Textaffected This British Standard is the English language version of EN 19882:
3、1998 published by the European Committee for StanarWon (CEN). The UK participation in its preparation was entrusted to Technical Panel AWIJ3, Food analysis - Horizontal methods, which has the responsibility to: - aid enquirers to understand the te* - present to the responsible European committee any
4、 enquiries on the intepretaton, or proposa3s for change, and keep the K interests informed; - monitor related internationai and European developments and promugate them in the UK. A list of oankations represented on this panel can be obtained on request to its SeCretaly. Cross-references The British
5、 Standards which implement international or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “Internationai Standards Correspondence index”, or by using the “Fnd” facility of the BSI standards Electronic Catalogue. A British St
6、andard does not purport to inciude all the necessary provisions of a contract Users of British standards are responsible for their correct appiication. Compliance with a British Standard does not of itself confer immunity from legai obligations. Summary of pages This document comprises a front cover
7、, an inside fiont cover, the EN title page, pages 2 to 8, an inside back cover and a back cover. ISBN O 580 29240 1 EXJROPIWN STANDARD NORME EUR0PEE”E EuRopAIScHEI NORM EN 1988-2 February 1998 ICs 67.040 Descriptors: Food products, chemical analysis, determination of content, sulfites, enzymatic met
8、hods English version FoodsMk - Detemination of sult - Part 2: Emymatc method Produits alimentaires - Dosage des suifites - Partie 2: Mthode enzymatique TeilzEnzymatJSc hesverfahren kbensmitkl - Bestimmung von Sulfit - This European Standard was approved by CEN on 1 January 1998. CEN members are boun
9、d to comply with the CENKENELEC Internal Regulations which stipuae the conditions for giving this Europecui Standard the status of a national standard without any alteration. Up-to-date iists and bibliogcaphical references concerning such national standards may be obtained on application to the Cent
10、ral 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 the same status as
11、the officiai versions. CENmembers are the national standards bodies of Aus* Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. CEN European Committee for Standardizatio
12、n Comit Europen de Normalisation Europaisches Komitee fiir Normung Central Secretariat: rue de Stassart 36, B-1060 Brussels 01998 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 19882:1998 E - STD.BS1 BS EN 1788-2-ENGL 1778 LbZLibb
13、S 0708358 Lbl Page 2 EN 19882:1998 Foreword This European Standard has been prepared by Technical Committee CENRC 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 ide
14、ntical text or by endorsement, at the latest by August 1998, and conflictllig nalionai standards shail be withdrawn at the latest by August 1998. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the foliowing countries are bound to implement this European St
15、andank Austria, Belgium, Czech Republic, Denmark, Finland, France, Gem Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norwax Portugal, Spain, Sweden, Switzerland and the United Kingdom. of sulfite, consists of the foliowing parts: Part 1: Optimized Mmier-Wzuiams method; Part 2: Enm/mah me
16、thod. This European standard Foodscus - Det4mninut.ion Contents Foreword Introduction 1 Scope 2 Nonnative references 3 Principle 4 Reagents 5 Apparatus 6 Procedure 7 Calculation 8 Precision 9 Testreport Annex A (informative) Bibliography Annex B (informative) Precision data page 2 3 3 3 3 3 4 4 5 5
17、6 7 7 O BSI 1998 STD*BSI BS EN 1988-2-ENGL 1778 Lb24bb7 0708357 OTB * * B Introduction Suifite can be used as a preservative in foodstuffs. In order to minimize possible negative heaith effects, many countries have regulated the use of sulfite in foods. This has resulted in the development of severa
18、l methods of anaysis 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 suite content, expressed as sulfur dioxide, in foodstuffs. other SulfurcontaUUng substances such as sulfate, sul
19、fide 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 protein). It has
20、been shown that the analysis of isolated soy protein leads to falce 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 Standard incorpomt
21、es 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 to this European
22、Standard only when incorporated in it by amendment or revision. For undated references the latest edition of the publication referred to applies. EN IS0 3696, Water for analyt2cal laboratory use - Specif2cation and test methods. (IS0 3696: 1987) 3 Principle Oxidation of suifite to sulfate in the pre
23、sence of sulfite oxidase with the liberation of hydrogen peroxide at the same time. SO$- + O2 + H20 - sulfite Oxid= S042- + H202 Reduction of hydrogen peroxide and conversion of NADH to NAD+ in the presence of NADH peroxidase. NADH peroxidase H202 + NADH + H+ 2H2O + NAD+ Page 3 EN 198821998 Conversi
24、on of NADH to NAD+ is determined spectrometricaliy and is proportional to the concentration of sulfite, see i to 16) in annex k 4 Reagents During the analysis, unless otherwise stated, use oniy reagents of recognized anaiyticai grade and only water of at least grade 3 as defined in EN IS0 3696. 4.1
25、Ammonium sulfate. 4.2 Ethylened.iamine-N,N,N“-tetmacetic acid (EDTA). 4.3 Sodium hydrogen carbonate. 4.4 Sodium sulfite. 4.6 Ammonium sulfate solution, substance concentration c(“q)2SO4 = 2 moll. 4.6 Sodium hydmxide solution, c(Na0H) = 0,I mom. 4.7 Sodium hydmride solution, c(Na0H) = 2 moll. 4.8 Fre
26、thamiamine trqfim solution?), c(CgH15N03) = 0,6 moll, pH 8,O. Dissolve 5,57 g of triethanolamine hydrochloride in 40 ml of water in a beaker. Actjust to pH 8,O with the sodium hydroxide solution (4.6). Transfer the solution to a 50 ml volumetric flask and dilute to the mark with water and mix. The b
27、uffer is stable for 1 year at +4 “C. 4.9 NADH solution2) (Reduced nicotinamide-adenine dinucleotide), c(NADH) = 7 X 10 -3 moM. Dissohre 25 mg of -nicotinamide-adenine dinucleotide disodium salt (-NADH-Na2) and 50 mg of sodium hydrogen carbonate (4.3) in 5,O ml of water and mix. The solution is stabl
28、e for at least 4 weeks at +4 “C. 4.10 NADH peroxidme suspension2) (EC l.ll.l.l), (see 7 of annex A). Make a suspension of 10 enzyme unitdml mi) in the ammonium sulfate solution (4.Q pH approximately 7. The suspension is stable for 1 year at +4 “C. 4.11 Sulfite oxidase suspension2) (EC 1.8.3.1), (see
29、 7 of annex A). Prepare a suspension of 2,5 enzyme units/ml in ammonium sulfate solution (4.Q pH approximately 7. The suspension is stable for 1 year at +4 “C. It has been shown that the analysis of isolated soy protein leads to false positive results in the range of 20 mgkg to 30 mgkg expressed as
30、sulfur dioxide. Therefore, when 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 sho
31、uld be followed. 3, This unit (often called the International unit or Standard unit) is defined as the amount of enzyme which will catalyse the transformation of 1 pmol substrate per minute under standard conditions. O BSI 1998 STD=BSI BS EN 1788-2-ENGL 1478 Lb24bb9 07083b0 81“ Page 4 EN 198821998 4
32、.12 Refme solution. Weigh 0,6 g of sodium sulfite (4.4) (equivalent to about 300 mg of sulfur dioxide), to the nearest 0,l mg, and 37 mg of EDTA (4.2) and dissolve in water. n-anSfer the solution quantitatively to a 1 O00 ml volumetric flask, dilute to the mark with water and mix We 100 pl of this s
33、olution as reference sample and analyse the suite content within 30 min. The coefficient of variation for the reference values shall not exceed 0,M. 4.13 Polyvinyllpymol, cross linked olyvinylpolypyrroIidone). 4.14 As V2 is the sample volume taken for the enzymatic analysis, in millilitres, (here: 0
34、,l ml up to 0,5 mi); V3 is the total volume of sample test solution for solid samples, in millilitres (here: 50 mi); Fluids pipetted into the cells ?iiethanolamine buffer solution (4.8) NADH solution (4.9) NADH peroxidase suspension (4.10) Sample test solution Water Page 6 EN 19882:1998 M is the rel
35、ative molecular mass of sulfur dioxide (M,1 g/mol; d is the light path of the cell, in centimetres (here: 1 cm); E is the absorption coefficient of NADH at 340 nm (6,3 lmmol-l.cm-l); m is the sample mass, in grams of solid samples (6.1.3); F is the dilution factor if the sample has been diluted duri
36、ng the sample preparation (see 6.1, 6.1.2.2 or 6.1.2.3). 8 Precision 8.1 General Details of the interlaboratory test according to IS0 5725 1986 (see 8 of annex A) of the precision of the method are summarized in annex B. The values derived from the interlaboratory test may not be applicable to analy
37、te concentsation ranges and matrices other than given in annex B. 8.2 Repeatability The absolute difference between two singie test results found on identical test material by one operator using the same apparatus within the shortest feasible lime interval wili exceed the repeatability limit r in no
38、t more than 5 % of the cases. The values are: - Wie z=75mg/l r=8mg/l Dried apples Z = 800 mgkg r = 298 mgkg Dried apples Z = 960 mgkg r = 358 mgkg Lemon juice Z = 270 mg4 r = 37 mg/i Sultanas 5=260mgkg r=45mglkg Beer Z = 4,9 mg/i r = 0,8 mgl O BSI 1998 Page 6 EN 1988-21998 8.3 Reproducibility The ab
39、solute difference between two single test results on identical test mariai reported by two laboratorieS wii exceed the reproducjbiliity limit R in not more than 5 % Of the cases. The vaiues are: Wine Z=75mg/l R=16rn Driedapple Z=soOmg/kg R=311mg/kg Driedapples Z=96omg/kg R=374mg/kg Lemon juice Z = 2
40、70 m R = 79 mg/i Sultanas Z = 260 mg/kg R = 129mg/kg Beer Z = 4,9 mg/i R = 1,6 mg/i 9 %t report The test report shall contain at least the following: - a31 information necessary for the identification of the sample; - a reference to this European Standard or to the method used; - the results and the
41、 units in which the resuits have been expressed; - date and type of sampling procedure (if known); - date of receipt; -dateoftest; - any particuiar points observed in the course of the test; - any operations not specified in the method or regarded as optional which might have affected thg results. Q
42、 BSI 1998 STD.BS1 BS EN 1788-2-ENGL 1778 = 1b2qbbS 07083b3 529 Annex A (informative) Bibliography i Nordic Committee on Food Analysis, No 135 (1990). 2 Methods for the enzymatic food analysis, published by Boehringer, Mannheim. 3 Beutler, H O Food Chemistry 15,157-164 (1984). 4 Official collection o
43、f methods of food analysis accordmg to 0 35 LMBG (German food and commodities regulations), Beuth, Berlin, 1993, Method L 30.001. 5 Official collection of methods of food analysis according to 0 35 LMBG (German food and commodities regulations), Beuth, Berlin 1993, Method L 36.008. 6 Jod of the Inst
44、itute of Brewing, European Brewery Convention, Vol. 98, 1992, Method 9.12.3. 7 Enzyme Commission (EC): Classification System. Enzyme Handbook, Springer, Berlin 1969. 8 IS0 57251986, Precision of test methods - Determination of repeatubity and reproducibility for a standard test method by inter-labom
45、tory tests. 9 Edberg, U.: Journal of AOAC International, 76 (1993) M. Page 7 EN 19882:1998 Annex B (informative) Precision data The precision of the method has been established by inter-laboratory tests carried out in accordance with IS0 5725 1986 (see 8 of annex A of this standard) especially on sa
46、mples with a low sulfite concentration (see i, 4 and 5 of annex A of this standard). The study samples consisted of potato flakes, wine, lemon juice, dried apples, sultanas and beer having a sulfite content between O mgkg and 960 mgkg. Eleven laboratories participated in the full study analysing twe
47、lve samples. Six laboratories analysed eight samples in a complementary study. The results of the coilaborative studies show that the method is well suited for the quantitative analysis of sullite at levels well below 100 mg SO2/kg. For very low concenxations of sulfite the type of food is of great
48、importance. In the anaysis of solid samples or when the sulfite has adhered to particles (e.g. in juice), a high coefficicnt of variation may be expected, especially if the analyst has little experience with enzymatic methos. Concentrations in wine of between 1 mg/l and 10 mg/i can be determined wit
49、h good reliabiiity The limit of detection of the method, expressed as absorbance, is O,. For a sample of 1 ml the limit of detection, calculated as the mean value of a representative number of blanks (n 20) plus three times the coefficient of vanation of the mean value (according to recommenations of the European Community) is 42 mg S02/kg. in accordance with IS0 57251986, the parameters given in Table B.l (matched palls/blind duplicates) and B.2 have been identified in inter-laboratory tes
