1、BRITISH STANDAR BS EN ISO 23275-1:2008Animal and vegetable fats and oils Cocoa butter equivalents in cocoa butter and plain chocolate Part 1: Determination of the presence of cocoa butter equivalentsICS 67.190; 67.200.10nullnull nullnullnullnullnullnullnull nullnullnullnullnullnullnull nullnullnull
2、nullnullnullnullnullnullnullnullnullnull nullnullnullnullnullnull nullnull nullnullnullnullnullnullnullnullnull nullnull nullnullnullnullnullnullnullnullnull nullnullnullNational forewordThis British Standard is the UK implementation of EN ISO 23275-1:2008. It is identical with ISO 23275-1:2006. It
3、supersedes BS ISO 23275-1:2006, which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/307, Oil seeds, animal and vegetable fats and oils and their by products.A list of organizations represented on this committee can be obtained on request to its secretar
4、y.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application.Compliance with a British Standard cannot confer immunity from legal obligations.BS EN ISO 23275-1:2008This British Standard was published under the authority
5、of the Standards Policy and Strategy Committee on 30 November 2006 BSI 2009ISBN 978 0 580 63597 7Amendments/corrigenda issued since publicationDate Comments 30 June 2009 This corrigendum renumbers BS ISO 23275-1:2006 as BS EN ISO 23275-1:2008EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 2327
6、5-1November 2008ICS 67.200.10; 67.190English VersionAnimal and vegetable fats and oils - Cocoa butter equivalents incocoa butter and plain chocolate - Part 1: Determination of thepresence of cocoa butter equivalents (ISO 23275-1:2006)Corps gras dorigines animale et vgtale - quivalents aubeurre de ca
7、cao dans le beurre de cacao et dans lechocolat de mnage - Partie 1: Dtermination de laprsence dquivalents au beurre de cacao (ISO 23275-1:2006)Tierische und pflanzliche Fette und le - Kakaobutter-quivalente in Kakaobutter und Zartbitterschokolade - Teil1: Bestimmung der Prsenz von Kakaobutter-quival
8、enten(ISO 23275-1:2006)This European Standard was approved by CEN on 23 October 2008.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists an
9、d bibliographical references concerning such nationalstandards may be obtained on application to the CEN 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 translationunder the responsibil
10、ity of a CEN member into its own language and notified to the CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ir
11、eland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 3
12、6 B-1050 Brussels 2008 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 23275-1:2008: EEN ISO 23275-1:2008 (E) ii Foreword The text of ISO 23275-1:2006 has been prepared by Technical Committee ISO/TC 34 “Agricultural food products
13、” of the International Organization for Standardization (ISO) and has been taken over as EN ISO 23275-1:2008 by 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 Europ
14、ean Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by May 2009, and conflicting national standards shall be withdrawn at the latest by May 2009. Attention is drawn to the possibility that some of the elements of
15、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. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European S
16、tandard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingd
17、om. Endorsement notice The text of ISO 23275-1:2006 has been approved by CEN as a EN ISO 23275-1:2008 without any modification. BS EN ISO 23275-1:2008iiiContentsPageIntroduction v1Scope12Terms and definitions .13Principle14Reagents and materials15Apparatus.26Sampling.27Preparation of test sample37.1
18、 Preparation of cocoa butter CRM for calibration purposes and system suitability check37.2 Preparation of chocolate sample.38Procedure.38.1Fat extraction .38.2 Separation of individual triacylglycerols by HR-GC38.3Identification.39Calculation49.1Determination of response factors49.2 Calculation of p
19、ercentages of triacylglycerols.49.3 Decision if sample is pure cocoa butter410 Procedural requirements510.1 General considerations.510.2 System suitability511 Precision.511.1 Interlaboratory test511.2 Repeatability.611.3 Reproducibility.612 Test report6Annex A (informative) Results of interlaborator
20、y test.7Bibliography12BS ISO 23275-1:2006iEN ISO 23275-1:2008 (E) BS EN ISO 23275-1:2008Introduction “Cocoa butter equivalents” is the general term forfats used to replace cocoa butter in chocolate. Theyresemble the chemical composition and physic al properties of cocoa butter very close ly, making
21、themtherefore extremelydifficult to quantify and even in some cases to detect. In principle, cocoa butterequivalents must by definition be fats low in lauric acid, rich in symmetrical mono-unsaturated triacylglycerolsof the type 1,3-dipalmitoyl-2-oleoylglycerol, 1-palmitoyl-2-oleo yl-3-stearoylglyce
22、rol and 1,3-distearoyl-2-oleoylglycerol, miscible with cocoa butter, and obtained onlyby refining and fractionation. Within the European Union, the following vegetable fats, obtained from the plants listed below, maybe used singlyor in blends, according to Directive 2000/36/EC 1: illip, Borneo tallo
23、w or tengkawang ( Shorea spp . ), palm oil ( Elaeis guineensis, Elaeis olifera ), sal( Shorea robusta), shea ( Butyrospermum parkii), kokum gurgi ( Garcinia indica ), and mango kernel ( Mangifera indica ). This part of ISO23275 specifies a procedure for the detection of these fats (restrictions are
24、only made forpure illip fat samples) in cocoa butter and plain chocolate. ISO 23275-2 specifies a procedure allowing areliable quantification of these fats at the level of 5 %, complying with the statutorylimit laid down in Directive2000/36/EC 1 of the European Parliament and the Council. To facilit
25、ate the usage of both parts of ISO23275, an analytical toolbox named “CoCal-1” has beenestablished. “CoCal-1” contains the validated methods for detection (part 1) and quantification (part 2) ofCBEs in plain chocolate, and also a certified cocoa butter reference material(IRMM-801) to calibrate thean
26、alysts instruments and an electronic evaluation sheet for Microsoft Excel to calculate the final result. Ananalyst working on CBE detection and quantification has only to calibrate the gas chromatographic separation system using IRMM-801, separate the triglyceride fractions of the sample in question
27、, and use the electronicevaluation sheet for subsequent data treatment to detect and quantify CBEs. Information on “CoCal-1” isavailable on thewebsite of the Institute for Reference Materials and Measurements: http:/www.irmm.jrc.be. EN ISO 23275-1:2008 (E) ivBS EN ISO 23275-1:20081Animal and vegetab
28、le fats and oils Cocoa butter equivalents in cocoa butter and plain chocolate Part 1: Determination of the presence of cocoa butter equivalents 1 Scope This part of ISO23275 specifies a procedure for the detection of cocoa butter equivalents (CBEs) in cocoa butter (CB)and plain chocolate by high-res
29、olution capillarygas liquid chromatography(HR-GC)of triacylglycerols and subsequent data evaluation by regression analysis. The method is applicable for the detection of2 % CBE admixture to cocoa butter,corresponding to about 0,6 % CBE in chocolate (i.e. the assumed fat content of chocolate is 30 %)
30、. 2 Terms and definitions For the purposes of this document, the following terms and definitions apply.2.1 cocoa butter equivalentsCBEs fats detected in cocoa butter and plain chocolate according the procedure specified in this part of ISO 23275 NOTE The result is expressed as a qualitative result,
31、i.e. CBEs present/CBEs not present (YES/NO). 3 Principle Cocoa butter, or the fat obtained by solvent extraction from plain chocolate, is separated by HR-GC into triacylglycerol fractions according to their molecular mass and degree of unsaturation. The presence of CBEsis detected bylinear regressio
32、n analysis applied to individual triacylglycerol fractions of the fat analysed. 4 Reagentsand materials Use only reagents of recognized analytical grade, unless otherwise stated. WARNING Attention is drawn to the regulations which specify the handling of dangerous matter.Technical, organizational an
33、d personal safety measures shall be followed. 4.1 Cocoa butter Certified Reference Material (CRM) IRMM-801 2, for calibration purposes and systemsuitability check.4.2 Fat solvent , non-chlorinated solvents (e.g. diethyl ether, n -heptane, iso-octane). EN ISO 23275-1:2008 (E) BS EN ISO 23275-1:20082
34、5 Apparatus 5.1 Analyticalbalance,with a readability of 0,1 mg. 5.2 Drying oven, maintained at 55 C. A dry heater blockmaybe used. 5.3 Food grater, i.e. a kitchen blender with a design featuring the motor above the receiving container toavoid melting the samples1). 5.4 Rotary evaporator. Alternative
35、 evaporation procedures maybe used. 5.5 Pipettes , of capacity 1 ml.5.6 Volumetric flasks , ofcapacity 20 ml. 5.7 Microsyringe, with maximum volume 10 l, graduated to 0,1 l, or automatic sampler. 5.8 Gas chromatograph (GC), fitted with a cold on-column injection system and a flame ionization detecto
36、r(FID). Alternative injection systems e.g. a split injector, a programmed-temperature vaporizer (PTV) ora moving-needle injector may be used provided the same results are obtained as indicated in 1 0.2. The separation and detection have been found to be satisfactory if the following experimental con
37、ditions are followed: GC column: 25 m to 30 m length, with 0,25 mm i.d., fused silica coated with thermostable 50 %phenylmethylpolysiloxane to a film thickness of 0,1 m to 0,15 m. temperature programme: 100 C (initial temperature), programme rate 30 C/min to 340 C (final temperature). carrier gas: h
38、elium or hydrogen (purity W 99,999 %). NOTE Suitable columns and alternative experimental conditions, used in an international collaborative study, arelisted in Annex A. Operating conditions may be changed to obtain optimumseparation of cocoa butter triacylglycerols. 5.9 Chromatographic datasystem.
39、6 Sampling A representative sample should have been sent to the laboratory. It should not have been damaged orchanged during transport or storage. Sampling is not part of the method specified in this part of ISO23275. A recommended sampling method isgiven in ISO5555. 1) Philips HR2833 is an example
40、of suitable equipment available commercially. This information is given for the convenience of the users of this part of ISO23275 and does not constitute an endorsement by ISO of this product. EN ISO 23275-1:2008 (E) BS EN ISO 23275-1:200837 Preparation of test sample 7.1Preparation of cocoa butter
41、CRM for calibration purposes and system suitability check Before opening and using the cocoa butter CRM (4.1), the ampoule shall be warmed in a drying oven (5.2)until the contents have melted. When a clear so lutionis obtained, mix the contents by repeated inversion fornot less than 20 s. Then open
42、and transfer the contents to a clean vial, which can be tightly sealed and preserved in a cool place for future usage. 7.2Preparation of chocolate sample Chill approx. 200 g of chocolate until hard, and grate to fine granular condition using a mechanica l device (5.3).Mix thoroughly and preserve in
43、tightly stoppered bottle in a cool place. 8 Procedure 8.1 Fat extraction Separate the fat from 10 g to 40 g of grated chocolate (as described in 7.2) byextracting with two or three 100 mlportionsofafat solvent (4.2). Centrifuge and decant. Combine the extracts and evaporate most of the fat solvent (
44、5.4) then finallydry it under a stream of nitrogen. Alternative extraction procedures may be used (e.g. by Soxhlet, by supercritical carbon dioxide or by using microwaves) provided that the same results are obtained. 8.2Separation of individual triacylglycerols by HR-GC The test samples cocoa butter
45、, fat extracted from chocolate, cocoa butter CRM (4.1) shall be warmed in adrying oven (5.2) until completely melted. If the liquid sample contains sediment, filter the sample inside theoven to obtain a clear filtrate. Pipettes (or similar equipment) used for transferring the sample during weighingo
46、perations should be brought to a temperature of ca. 55 C in a drying oven (5.2) in order to a void partial fat fractionation. Weigh ca. 0,2 g of test sample in a 20 ml volumetric flask (5.6) and dilute to the mark with a suitable fat solvent (4.2). Pipette (5.5) 1 ml of the resulting solution into a
47、nother 20 ml volumetric flask and dilute to the mark with the same solvent.Inject 0,5 l to 1,0 l of the final test solution ( fat = 0,5 mg/ml) into the HR-GC system using the cold on-column injection system. Alternative sample amounts and injectors may be used provided that the detection system empl
48、oyed gives a linear response and the system suitability criteria (10.2) are met. 8.3 Identification Identification of the five major triacylglycerol fractions 1,3-dipalmitoyl-2-oleoylglycerol (POP), 1-palmitoyl-2-oleoyl-3-stearoylglycerol (POS), 1-palmitoyl-2,3-dioleoylglycerol (POO), 1,3-distearoyl
49、-2-oleoylglycerol (SOS),and 1-stearoyl-2,3-dioleoylglycerol (SOO) is made by comparison of the retention times of the test sampleswith those of the cocoa butter CRM (4.1). In general, triacylglycerols appear in order ofincreasing number ofcarbon atoms and of increasing unsaturation for the same number ofcarbon atoms. The elution order of thetriacylglycerols of the cocoa butter CRM is given in the example chromatogram (Figure A.1). EN ISO 23275-1:2008 (E) BS EN ISO 23275-1:20084 9 Calculation 9.1Determination of r
