1、March 2009DEUTSCHE NORM English price group 11No part of this standard 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!$VZL“1515541ww
2、w.din.deDDIN EN ISO 23275-1Animal and vegetable fats and oils Cocoa butter equivalents in cocoa butter and plain chocolate Part 1: Determination of the presence of cocoa butter equivalents(ISO 23275-1:2006)English version of DIN EN ISO 23275-1:2009-03Tierische und pflanzliche Fette und le Kakaobutte
3、r-quivalente in Kakaobutter und Zartbitterschokolade Teil 1: Bestimmung der Prsenz von Kakaobutter-quivalenten (ISO 23275-1:2006)Englische Fassung DIN EN ISO 23275-1:2009-03www.beuth.deDocument comprises 18 pagesDIN EN ISO 23275-1:2009-03 2 National foreword This standard has been prepared by Techni
4、cal Committee ISO/TC 34 “Food products” (Secretariat: AFNOR, France), Subcommittee SC 11 “Animal and vegetable fats and oils” (Secretariat: BSI, United Kingdom) ) in collaboration with Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats and oils and their by-products Methods of sampl
5、ing and analysis” (Secretariat: AFNOR, France). The responsible German bodies involved in its preparation were the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee) and the Deutsche Gesellschaft fr Fettwissenschaft e. V. (German Fat Re
6、search Society) in the Joint Committee NA 057-05-05 AA Gemeinschaftsausschuss fr die Analytik von Fetten, len, Fettprodukten, verwandten Stoffen und Rohstoffen. The DIN Standard corresponding to the International Standard referred to in this document is as follows: ISO 5555 DIN EN ISO 5555 National
7、Annex NA (informative) Bibliography DIN EN ISO 5555, Animal and vegetable fats and oils Sampling EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 23275-1November 2008ICS 67.200.10; 67.190English VersionAnimal and vegetable fats and oils - Cocoa butter equivalents incocoa butter and plain chocol
8、ate - Part 1: Determination of thepresence of cocoa butter equivalents (ISO 23275-1:2006)Corps gras dorigines animale et vgtale - quivalents aubeurre de cacao dans le beurre de cacao et dans lechocolat de mnage - Partie 1: Dtermination de laprsence dquivalents au beurre de cacao (ISO 23275-1:2006)Ti
9、erische und pflanzliche Fette und le - Kakaobutter-quivalente in Kakaobutter und Zartbitterschokolade - Teil1: Bestimmung der Prsenz von Kakaobutter-quivalenten(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 Interna
10、l Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the CEN Management Centre or to any CEN member.T
11、his European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility 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 t
12、he national standards bodies of 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
13、 and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 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 232
14、75-1:2008: EContents Page 1 2 3 4 5 6 7 7.1 7.2 8 8.1 8.2 8.3 9 9.1 9.2 9.3 10 10.1 10.2 11 11.1 11.2 11.3 12 EN ISO 23275-1:2008 (E) 2DIN EN ISO 23275-1:2009-03 Introduction 4 Scope 5 Terms and definitions .5 Principle5 Reagents and materials 5 Apparatus .6 Sampling.6 Preparation of test sample7 Pr
15、eparation of cocoa butter CRM for calibration purposes and system suitability check7 Preparation of chocolate sample .7 Procedure .7 Fat extraction .7 Separation of individual triacylglycerols by HR-GC 7 Identification.7 Calculation8 Determination of response factors 8 Calculation of percentages of
16、triacylglycerols.8 Decision if sample is pure cocoa butter8 Procedural requirements 9 System suitability 9 Interlaboratory test 9 General considerations.9 Precision.9 Bibliography 16 Test report 10 Annex A (informative) Results of interlaboratory test.11 Reproducibility.10 Foreword. 3 Repeatability.
17、10 Foreword The text of ISO 23275-1:2006 has been prepared by Technical Committee ISO/TC 34 “Agricultural food products” 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 a
18、nd 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 of an identical text or by endorsement, at the latest by May 2009, and conflicting national stan
19、dards shall be withdrawn at the latest by May 2009. 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. According to the CEN/CENELEC Inter
20、nal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Mal
21、ta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of ISO 23275-1:2006 has been approved by CEN as a EN ISO 23275-1:2008 without any modification. EN ISO 23275-1:2008 (E) 3DIN EN ISO 23275-1:2009-03 I
22、ntroduction “Cocoa butter equivalents” is the general term for fats used to replace cocoa butter in chocolate. They resemble the chemical composition and physical properties of cocoa butter very closely, making them therefore extremely difficult to quantify and even in some cases to detect. In princ
23、iple, cocoa butter equivalents must by definition be fats low in lauric acid, rich in symmetrical mono-unsaturated triacylglycerols of the type 1,3-dipalmitoyl-2-oleoylglycerol, 1-palmitoyl-2-oleoyl-3-stearoylglycerol and 1,3-distearoyl-2-oleoylglycerol, miscible with cocoa butter, and obtained only
24、 by refining and fractionation. Within the European Union, the following vegetable fats, obtained from the plants listed below, may be used singly or in blends, according to Directive 2000/36/EC 1: illip, Borneo tallow or tengkawang ( Shorea spp.), palm oil ( Elaeis guineensis, Elaeis olifera), sal
25、( Shorea robusta), shea ( Butyrospermum parkii), kokum gurgi ( Garcinia indica), and mango kernel ( Mangifera indica). This part of ISO 23275 specifies a procedure for the detection of these fats (restrictions are only made for pure illip fat samples) in cocoa butter and plain chocolate. ISO 23275-2
26、 specifies a procedure allowing a reliable quantification of these fats at the level of 5 %, complying with the statutory limit laid down in Directive 2000/36/EC 1of the European Parliament and the Council. To facilitate the usage of both parts of ISO 23275, an analytical toolbox named “CoCal-1” has
27、 been established. “CoCal-1” contains the validated methods for detection (part 1) and quantification (part 2) of CBEs in plain chocolate, and also a certified cocoa butter reference material (IRMM-801) to calibrate the analysts instruments and an electronic evaluation sheet for Microsoft Excel to c
28、alculate the final result. An analyst 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, and use the electronic evaluation sheet for subsequent data treatment to de
29、tect and quantify CBEs. Information on “CoCal-1” is available on the website of the Institute for Reference Materials and Measurements: http:/www.irmm.jrc.be. EN ISO 23275-1:2008 (E) 4DIN EN ISO 23275-1:2009-03 1 Scope This part of ISO 23275 specifies a procedure for the detection of cocoa butter eq
30、uivalents (CBEs) in cocoa butter (CB) and plain chocolate by high-resolution capillary gas liquid chromatography (HR-GC) of triacylglycerols and subsequent data evaluation by regression analysis. The method is applicable for the detection of 2 % CBE admixture to cocoa butter, corresponding to about
31、0,6 % CBE in chocolate (i.e. the assumed fat content of chocolate is 30 %). 2 Terms and definitions For the purposes of this document, the following terms and definitions apply. 2.1 cocoa butter equivalents CBEs fats detected in cocoa butter and plain chocolate according the procedure specified in t
32、his part of ISO 23275 NOTE The result is expressed as a qualitative result, 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 an
33、d degree of unsaturation. The presence of CBEs is detected by linear regression analysis applied to individual triacylglycerol fractions of the fat analysed. 4 Reagents and materials Use only reagents of recognized analytical grade, unless otherwise stated. WARNING Attention is drawn to the regulati
34、ons which specify the handling of dangerous matter. Technical, organizational and personal safety measures shall be followed. 4.1 Cocoa butter Certified Reference Material (CRM) IRMM-8012, for calibration purposes and system suitability check. 4.2 Fat solvent, non-chlorinated solvents (e.g. diethyl
35、ether, n-heptane, iso-octane). EN ISO 23275-1:2008 (E) 5DIN EN ISO 23275-1:2009-03 5 Apparatus 5.1 Analytical balance, with a readability of 0,1 mg. 5.2 Drying oven, maintained at 55 C. A dry heater block may be used. 5.3 Food grater, i.e. a kitchen blender with a design featuring the motor above th
36、e receiving container to avoid melting the samples1). 5.4 Rotary evaporator. Alternative evaporation procedures may be used. 5.5 Pipettes, of capacity 1 ml. 5.6 Volumetric flasks, of capacity 20 ml. 5.7 Microsyringe, with maximum volume 10 l, graduated to 0,1 l, or automatic sampler. 5.8 Gas chromat
37、ograph (GC), fitted with a cold on-column injection system and a flame ionization detector (FID). Alternative injection systems e.g. a split injector, a programmed-temperature vaporizer (PTV) or a moving-needle injector may be used provided the same results are obtained as indicated in 10.2. The sep
38、aration and detection have been found to be satisfactory if the following experimental conditions 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
39、(initial temperature), programme rate 30 C/min to 340 C (final temperature). carrier gas: helium or hydrogen (purity W 99,999 %). NOTE Suitable columns and alternative experimental conditions, used in an international collaborative study, are listed in Annex A. Operating conditions may be changed to
40、 obtain optimum separation of cocoa butter triacylglycerols. 5.9 Chromatographic data system. 6 Sampling A representative sample should have been sent to the laboratory. It should not have been damaged or changed during transport or storage. Sampling is not part of the method specified in this part
41、of ISO 23275. A recommended sampling method is given in ISO 5555. 1) Philips HR2833 is an example of suitable equipment available commercially. This information is given for the convenience of the users of this part of ISO 23275 and does not constitute an endorsement by ISO of this product. EN ISO 2
42、3275-1:2008 (E) 6DIN EN ISO 23275-1:2009-03 7 Preparation of test sample 7.1 Preparation of cocoa butter 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. W
43、hen a clear solution is obtained, mix the contents by repeated inversion for not less than 20 s. Then open and transfer the contents to a clean vial, which can be tightly sealed and preserved in a cool place for future usage. 7.2 Preparation of chocolate sample Chill approx. 200 g of chocolate until
44、 hard, and grate to fine granular condition using a mechanical device (5.3). Mix thoroughly and preserve in 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) by extracting with two or three 100 ml por
45、tions of a fat solvent (4.2). Centrifuge and decant. Combine the extracts and evaporate most of the fat solvent (5.4) then finally dry 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
46、the same results are obtained. 8.2 Separation of individual triacylglycerols by HR-GC The test samples cocoa butter, fat extracted from chocolate, cocoa butter CRM (4.1) shall be warmed in a drying oven (5.2) until completely melted. If the liquid sample contains sediment, filter the sample inside t
47、he oven to obtain a clear filtrate. Pipettes (or similar equipment) used for transferring the sample during weighing operations should be brought to a temperature of ca. 55 C in a drying oven (5.2) in order to avoid partial fat fractionation. Weigh ca. 0,2 g of test sample in a 20 ml volumetric flas
48、k (5.6) and dilute to the mark with a suitable fat solvent (4.2). Pipette (5.5) 1 ml of the resulting solution into another 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-c
49、olumn injection system. Alternative sample amounts and injectors may be used provided that the detection system employed 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-2-oleoylglycerol (SOS), and 1-stearoyl-2,3-dioleoylglycerol (SOO) is made by comparison of t
