1、November 2016 English price group 10No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 67.200.10!%v+“2588308www.din.de
2、DIN EN ISO 11702Animal and vegetable fats and oils Enzymatic determination of total sterols content (ISO 11702:2016);English version EN ISO 11702:2016,English translation of DIN EN ISO 11702:2016-11Tierische und pflanzliche Fette und le Enzymatische Bestimmung des GesamtsterinGehaltes (ISO 11702:201
3、6);Englische Fassung EN ISO 11702:2016,Englische bersetzung von DIN EN ISO 11702:2016-11Corps gras dorigines animale et vgtale Dtermination enzymatique de la teneur en strols totaux (ISO 11702:2016);Version anglaise EN ISO 11702:2016,Traduction anglaise de DIN EN ISO 11702:2016-11SupersedesDIN EN IS
4、O 11702:201004www.beuth.deDocument comprises sx pagesDTranslation by DIN-Sprachendienst.In case of doubt, the German-language original shall be considered authoritative.11.16 DIN EN ISO 11702:2016-11 2 A comma is used as the decimal marker. National foreword This document (EN ISO 11702:2016) has bee
5、n prepared by Technical Committee ISO/TC 34 “Food products” (Secretariat: AFNOR, France) in collaboration with Technical Committee CEN/TC 307 “Oil seeds, vegetable and animal fats and oils and their by-products Methods of sampling and analysis” (Secretariat: AFNOR, France). The responsible German bo
6、dies involved in its preparation were DIN-Normenausschuss Lebensmittel und landwirtschaftliche Produkte (DIN Standards Committee Food and Agricultural Products) and the Deutsche Gesellschaft fr Fettwissenschaft (German Fat Research Society), Joint Working Committee NA 057-05-05 AA Gemeinschaftsaussc
7、huss fr die Analytik von Fetten, len, Fettprodukten, verwandten Stoffen und Rohstoffen. The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 648 DIN EN ISO 648 ISO 661 DIN EN ISO 661 ISO 1042 DIN EN ISO 1042 ISO 3696 DIN ISO 3696 ISO 5555 DI
8、N EN ISO 5555 ISO 5725-1 DIN ISO 5725-1 ISO 5725-2 DIN ISO 5725-2 ISO 7550 DIN ISO 7550 Amendments This standard differs from DIN EN ISO 11702:2010-04 as follows: a) milk and milk products (or fat coming from milk and milk products) are now excluded from the scope of the standard; b) the standard ha
9、s been editorially revised and brought in line with the current rules of presentation. Previous editions DIN EN ISO 11702: 2010-04 DIN EN ISO 11702:2016-11 3 National Annex NA (informative) Bibliography DIN EN ISO 648, Laboratory glassware Single-volume pipettes DIN EN ISO 661, Animal and vegetable
10、fats and oils Preparation of test sample DIN EN ISO 1042, Laboratory glassware One-mark volumetric flasks DIN EN ISO 5555, Animal and vegetable fats and oils Sampling DIN ISO 3696, Water for analytical laboratory use Specification and test methods DIN ISO 5725-1, Accuracy (trueness and precision) of
11、 measurement methods and results Part 1: General principles and definitions DIN ISO 5725-2, Accuracy (trueness and precision) of measurement methods and results Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method DIN ISO 7550, Laboratory g
12、lassware Disposable micropipettes DIN EN ISO 11702:2016-11 4 This page is intentionally blank EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 11702 August 2016 ICS 67.200.10 Supersedes EN ISO 11702:2009English Version Animal and vegetable fats and oils Enzymatic determination of total stero
13、ls content (ISO 11702:2016) Corps gras dorigines animale et vgtale Dtermination enzymatique de la teneur en strols totaux (ISO 11702:2016) Tierische und pflanzliche Fette und le Enzymatische Bestimmung des Gesamtsterin-Gehaltes (ISO 11702:2016) This European Standard was approved by CEN on 9 August
14、2016. 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 without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtaine
15、d on application to the CEN-CENELEC Management Centre 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 CEN-C
16、ENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, I
17、taly, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: A
18、venue Marnix 17, B-1000 Brussels 2016 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 11702:2016 EEN ISO 11702:2016 (E) 2 Contents Page European foreword . 3 Foreword 4 1 Scope 5 2 Normative references 5 3 Terms and definitions
19、 . 5 4 Principle . 5 5 Reagents . 6 6 Apparatus . 6 7 Sampling . 7 8 Preparation of the test sample . 7 9 Procedure. 7 9.1 Saponification . 7 9.2 Enzymatic determination of the sterols content . 8 10 Result of the determination 8 11 Precision of the method 9 11.1 Interlaboratory test 9 11.2 Repeatab
20、ility limit 9 11.3 Reproducibility limit 9 12 Test report 10 Annex A (informative) Results of an interlaboratory test 11 Bibliography . 12 DIN EN ISO 11702:2016-11 EN ISO 11702:2016 (E) 3 European foreword This document (EN ISO 11702:2016) has been prepared by Technical Committee ISO/TC 34 “Food pro
21、ducts” in collaboration with Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats and oils and their by-products Methods of sampling and analysis” the secretariat of which is held by AFNOR. This European Standard shall be given the status of a national standard, either by publication
22、of an identical text or by endorsement, at the latest by February 2017, and conflicting national standards shall be withdrawn at the latest by February 2017. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall
23、 not be held responsible for identifying any or all such patent rights. This document supersedes EN ISO 11702:2009. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulg
24、aria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Tu
25、rkey and the United Kingdom. Endorsement notice The text of ISO 11702:2016 has been approved by CEN as EN ISO 11702:2016 without any modification. DIN EN ISO 11702:2016-11 ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO mem
26、ber bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmen
27、tal and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.The procedures used to develop this document and those intended for its further maintenance
28、are described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).Attention is
29、 drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any patent rights identified during the development of the document will be in the Introduction and/or
30、 on the ISO list of patent declarations received (see www.iso.org/patents).Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement.For an explanation on the meaning of ISO specific terms and expressions related to conformity asses
31、sment, as well as information about ISOs adherence to the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following URL: www.iso.org/iso/foreword.html.The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 11, Animal and ve
32、getable fats and oils.This second edition cancels and replaces the first edition (ISO 11702:2009), of which it constitutes a minor revision. The scope has been revised to state that the document is not applicable to milk and milk fat products.4EN ISO 11702:2016 (E) DIN EN ISO 11702:2016-11 1 ScopeTh
33、is International Standard specifies a method for the quantitative determination of the total sterols content by means of an enzymatic staining test. The method is applicable to free and esterified sterols in animal and vegetable fats and oils, fatty foods and related products. The determination is a
34、pplicable to sample quantities of 1 g to 2 g of fat.The method is not applicable to dark coloured fats and oils. The enzyme is not specific for cholesterol, but also oxidizes other 3-hydroxysterols. The method has not been tested for products fortified with sterols at higher levels.Milk and milk pro
35、ducts (or fat coming from milk and milk products) are excluded from the scope of this International Standard.NOTE The method is technically equivalent to IUPAC method 2.4048and DGF standard method F-III 2 (91).72 Normative referencesThe following documents, in whole or in part, are normatively refer
36、enced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.ISO 661, Animal and vegetable fats and oils Preparation of test sampleISO 3
37、696, Water for analytical laboratory use Specification and test methods3 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.3.1total sterols contentwsterolsmass fraction of sterols determined by the method specified in this International StandardNote 1
38、to entry: For vegetable fats and oils, the sterols content is expressed as -sitosterol; for animal fats, as cholesterol.Note 2 to entry: The total sterols content is expressed in milligrams per 100 g of fat.4 PrincipleThe test sample is saponified and the sterols in the unsaponifiable matter are det
39、ermined enzymatically. Cholesterol is oxidized by cholesterol oxidase to cholestenone. The equimolar amount of hydrogen peroxide produced in the process oxidizes in the presence of catalase methanol to formaldehyde. In the presence of ammonium ions, with acetylacetone, it forms a yellow lutidine dye
40、 (3,5-diacetyl-1,4-dihydrolutidine). The latter is determined spectrophotometrically in the visible range at 405 nm. The concentration of the dye is equivalent to the amount of sterols.NOTE Cholesterol oxidase oxidizes cholesterol and other sterols having a hydroxy group in the 3-position. Therefore
41、, phytosterols like stigmasterol and sitosterol are also determined.5 EN ISO 11702:2016 (E) DIN EN ISO 11702:2016-11 5 ReagentsWARNING Attention is drawn to the regulations which specify the handling of hazardous substances. Technical, organizational and personal safety measures shall be followed.Un
42、less otherwise stated, use only reagents of recognized analytical grade.5.1 Water, complying with ISO 3696, grade 3 or better.5.2 Isopropanol.5.3 Acetone.5.4 Acetylacetone.5.5 Suspension of cholesterol oxidase,1)(EC 1.1.3.6) from Nocardia erythropolis, 15 U/ml.5.6 Suspension of catalase, (hydrogen p
43、eroxide oxido-reductase)1)(EC 1.11.1.6) from bovine liver.5.7 Hydrochloric acid, c(HCl) = 8 mol/l.5.8 Methanolic potassium hydroxide solution, c(KOH) = 0,5 mol/l.Dissolve 2,8 g potassium hydroxide in a small amount of hot methanol, cool, and dilute with methanol to 100 ml.5.9 Ammonium phosphate buff
44、er solution, adjusted to pH 7.5.10 Solution 1.Add 19,1 ml acetone (5.3) and 230 000 U catalase (5.6) to 50 ml buffer solution (5.9) in a 100 ml one-mark volumetric flask (6.4), and make up to the mark with water (5.1).5.11 Solution 2.Add 0,26 ml acetylacetone (5.4) and 1,10 ml acetone (5.3) to 25 ml
45、 water (5.1) in a 50 ml one-mark volumetric flask (6.4), and make up to the mark with water.5.12 Solution 3.Prior to use, mix three volumes of solution 1 (5.10) with two volumes of solution 2 (5.11).NOTE Solution 3 can be kept in amber bottles for three months at 4 C, provided it is prepared under s
46、terile conditions.6 Apparatus6.1 Test tubes, of diameter 18 mm.6.2 Filter funnel.1) A suitable ready-made test kid for the colorimetric determination of cholesterol in foodstuffs and other materials is available from R-Biopharm. This information is given for the convenience of users of this document
47、 and does not constitute an endorsement by ISO of this product. Equivalent products may be used if they can be shown to lead to the same results.6EN ISO 11702:2016 (E) DIN EN ISO 11702:2016-11 6.3 Fluted filter, suitable for the filter funnel (6.2).6.4 One-mark volumetric flasks, of capacities 25 ml
48、, 50 ml, and 100 ml, ISO 10422class A.6.5 Enzyme pipettes, of capacities 0,02 ml, ISO 7550,6to 1 ml, ISO 6481class A.6.6 Pipette, of capacity 5 ml, ISO 6481class A.6.7 Round bottomed flask, standard ground joint, of capacity 50 ml.6.8 Test tubes with ground stoppers.6.9 Spectrophotometer, set to 405
49、 nm.6.10 Glass cuvettes, pathlength 1 cm, suitable for the spectrophotometer (6.9).6.11 Water bath, thermostatically controlled at 37 C to 40 C.6.12 Refrigerator, capable of maintaining a temperature of 4 C.6.13 Glass beads.6.14 Reflux condenser, standard ground joint.7 SamplingA representative sample should have been sent to the laboratory. It should not have been damaged or changed during transport or storage.Sampli
