1、BS EN ISO 11702:2016Animal and vegetablefats and oils Enzymaticdetermination of total sterolscontent (ISO 11702:2016)BSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06BS EN ISO 11702:2016 BRITISH STANDARDNational forewordThis British Standard is the UK implementation o
2、f EN ISO 11702:2016. It supersedes BS EN ISO 11702:2009 which iswithdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/307, Oilseeds, animal and vegetable fats and oils and their by-products.A list of organizations represented on this committee can be obtained on r
3、equest to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2016.Published by BSI Standards Limited 2016ISBN 978 0 580 92382 1 ICS 67.200.10 Compliance with a Bri
4、tish Standard cannot confer immunity from legal obligations.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 August 2016.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE
5、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 sterols content (ISO 11702:2016) Corps gras dorigines animale et vgtale - Dtermination enzymatique de la teneur en strols totaux (ISO 11702:20
6、16) Tierische und pflanzliche Fette und le - Enzymatische Bestimmung des Gesamtsterin-Gehaltes (ISO 11702:2016) This European Standard was approved by CEN on 9 August 2016. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this Europe
7、an 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 CEN-CENELEC Management Centre or to any CEN member. This European Standard exists in three official versions
8、(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-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belg
9、ium, Bulgaria, 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, Switze
10、rland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2016 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national
11、 Members. Ref. No. EN ISO 11702:2016 EBS EN ISO 11702:2016EN ISO 11702:2016 (E) 3 European foreword This document (EN ISO 11702:2016) has been prepared by Technical Committee ISO/TC 34 “Food products“ in collaboration with Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats and oils
12、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 of an identical text or by endorsement, at the latest by February 2017, and conflicting national standar
13、ds 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 not be held responsible for identifying any or all such patent rights. This document supersedes EN ISO
14、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, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,
15、 France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO 11702:2016 has been approved by CEN as E
16、N ISO 11702:2016 without any modification. BS EN ISO 11702:2016ISO 11702:2016(E)Foreword iv1 Scope . 12 Normative references 13 Terms and definitions . 14 Principle 15 Reagents 26 Apparatus . 27 Sampling 38 Preparation of the test sample . 39 Procedure. 310 Result of the determination 411 Precision
17、of the method 511.1 Interlaboratory test. 511.2 Repeatability limit 511.3 Reproducibility limit 512 Test report . 6Annex A (informative) Results of an interlaboratory test 7Bibliography 8 ISO 2016 All rights reserved iiiContents PageBS EN ISO 11702:2016ISO 11702:2016(E)ForewordISO (the International
18、 Organization for Standardization) is a worldwide federation of national standards bodies (ISO member 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 est
19、ablished has the right to be represented on that committee. International organizations, governmental 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 standardiz
20、ation.The procedures used to develop this document and those intended for its further maintenance 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 wi
21、th the editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).Attention is 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
22、patent rights identified during the development of the document will be in the Introduction and/or 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.F
23、or an explanation on the meaning of ISO specific terms and expressions related to conformity assessment, 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 comm
24、ittee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 11, Animal and vegetable 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
25、applicable to milk and milk fat products.iv ISO 2016 All rights reservedBS EN ISO 11702:2016INTERNATIONAL STANDARD ISO 11702:2016(E)Animal and vegetable fats and oils Enzymatic determination of total sterols content1 ScopeThis International Standard specifies a method for the quantitative determinat
26、ion 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 applicable to sample quantities of 1 g to 2 g of fat.The method is not applic
27、able 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 products (or fat coming from milk and milk products) are excluded from the scop
28、e 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 referenced in this document and are indispensable for its application. For dated
29、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 3696, Water for analytical laboratory use Specification and test methods3 Ter
30、ms 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 to entry: For vegetable fats and oils, the sterols content is expressed as -
31、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 determined enzymatically. Cholesterol is oxidized by cholesterol oxidase to cho
32、lestenone. 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 (3,5-diacetyl-1,4-dihydrolutidine). The latter is determined spectrophotome
33、trically 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, phytosterols like stigmasterol and sitosterol are also determined. ISO 201
34、6 All rights reserved 1BS EN ISO 11702:2016ISO 11702:2016(E)5 ReagentsWARNING Attention is drawn to the regulations which specify the handling of hazardous substances. Technical, organizational and personal safety measures shall be followed.Unless otherwise stated, use only reagents of recognized an
35、alytical 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 peroxide oxido-reductase)1)(EC 1.11.1.6) from bovine liver
36、.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 buffer solution, adjusted to pH 7.5.10 Solution 1.Add 19,1 ml
37、 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 water (5.1) in a 50 ml one-mark volumetric flask (6.4),
38、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 sterile conditions.6 Apparatus6.1 Test tubes, of diameter
39、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 and does not constitute an endorsement by ISO of this pr
40、oduct. Equivalent products may be used if they can be shown to lead to the same results.2 ISO 2016 All rights reservedBS EN ISO 11702:2016ISO 11702:2016(E)6.3 Fluted filter, suitable for the filter funnel (6.2).6.4 One-mark volumetric flasks, of capacities 25 ml, 50 ml, and 100 ml, ISO 10422class A.
41、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 nm.6.10 Glass cuvettes, pathlength 1
42、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 labora
43、tory. It should not have been damaged or changed during transport or storage.Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 5555.38 Preparation of the test samplePrepare the test sample in accordance with ISO 661. Specific t
44、reatment of the test sample (filtration, melting, etc.) shall be mentioned in the test report.9 Procedure9.1 Saponification9.1.1 Weigh 1 g to 2 g of the sample accurately to within 0,001 g into a 50 ml round bottomed flask (6.7). The sterol concentration in the test solution shall be between 0,02 g/
45、l and 0,4 g/l. This requirement shall be taken into account during the weighing and diluting steps. In the case of saturated fats, the amount weighed shall be reduced, as otherwise, free fatty acids formed after saponification and acidification are not completely removed during filtration and affect
46、 the determination. Ensure at all times that the solution obtained is clear.9.1.2 Add 10 ml of methanolic potassium hydroxide solution (5.8) and some glass beads (6.13). Heat the mixture and, when boiling, reflux for 25 min. ISO 2016 All rights reserved 3BS EN ISO 11702:2016ISO 11702:2016(E)9.1.3 Tr
47、ansfer the still warm soap solution quantitatively into a 25 ml one-mark volumetric flask (6.4) and wash out the round bottomed flask (6.7) with a few millilitres of isopropanol (5.2).9.1.4 Pipette (6.5) 1 ml of hydrochloric acid (5.7) into the 25 ml one-mark volumetric flask (6.4), make up to the m
48、ark with isopropanol (5.2) and shake vigorously. Ensure at all times that the solution obtained is clear.9.1.5 Place the flask with the mixture (9.1.4) in the refrigerator (6.12) and maintain it at 4 C for 20 min.9.1.6 Next, filter the (turbid) solution as rapidly as possible through a fluted filter
49、 (6.3) and immediately use the filtrate for the enzymatic determination.9.2 Enzymatic determination of the sterols content9.2.1 Pipette (6.6) 5 ml of solution 3 (5.12) into a test tube (6.1) and add 0,4 ml of the filtrate (9.1.6). Mix thoroughly.9.2.2 Transfer 2,5 ml of this mixture into a stoppered test tube (6.8) and add by pipette (6.5) 0,02 ml of the cholesterol oxidase suspension (5.5). Mix thoroughly.9.2.3 Transfer the rest of the solution from 9.2.1 into another stoppered test tube (6.8) for use as the blank test.9.2.4
