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!$VZI“1515538ww
2、w.din.deDDIN EN ISO 23275-2Animal and vegetable fats and oils Cocoa butter equivalents in cocoa butter and plain chocolate Part 2: Quantification of cocoa butter equivalents (ISO 23275-2:2006)English version of DIN EN ISO 23275-2:2009-03Tierische und pflanzliche Fette und le Kakaobutter-quivalente i
3、n Kakaobutter und Zartbitterschokolade Teil 2: Quantifizierung von Kakaobutter-quivalenten (ISO 23275-2:2006)Englische Fassung DIN EN ISO 23275-2:2009-03www.beuth.deDocument comprises 19 pagesDIN EN ISO 23275-2:2009-03 2 National foreword This standard has been prepared by Technical Committee ISO/TC
4、 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 sampling and analysis” (Sec
5、retariat: 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 Research Society) in the
6、 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 Annex NA (informative)
7、 Bibliography DIN EN ISO 5555, Animal and vegetable fats and oils Sampling EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 23275-2November 2008ICS 67.200.10; 67.190English VersionAnimal and vegetable fats and oils - Cocoa butter equivalents incocoa butter and plain chocolate - Part 2: Quantifi
8、cation ofcocoa butter equivalents (ISO 23275-2:2006)Corps gras dorigines animale et vgtale - quivalents aubeurre de cacao dans le beurre de cacao et dans lechocolat de mnage - Partie 2: Quantification desquivalents au beurre de cacao (ISO 23275-2:2006)Tierische und pflanzliche Fette und le - Kakaobu
9、tter-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 and bibliographical
10、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 responsibility of a CEN membe
11、r 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, Ireland, Italy, Latv
12、ia, 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, 36 B-1050 Brussels
13、2008 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 23275-2:2008: Equivalente in Kakaobutter und Zartbitterschokolade - (ISO 23275-2:2006)Teil 2: Quantifizierung von Kakaobutter-quivalenten Contents Page 1 2 3 4 5 6 7 7.1 7.2 8
14、8.1 8.2 8.3 9 9.1 9.2 9.3 9.4 10 10.1 10.2 11 11.1 11.2 11.3 12 EN ISO 23275-2:2008 (E) 2DIN EN ISO 23275-2:2009-03 Introduction 4 Scope 5 Terms and definitions .5 Principle5 Reagents and materials 6 Apparatus .6 Sampling.7 Preparation of test sample7 Preparation of cocoa butter CRM for calibration
15、purposes and system suitability check7 Preparation of chocolate sample .7 Procedure .7 Fat extraction .7 Separation of individual triacylglycerols by HR-GC 8 Identification.8 Calculation8 Determination of response factors 8 Calculation of percentages of triacylglycerols.9 Calculation of CBE content
16、in cocoa butter .9 Bibliography 17 Calculation of CBE content in chocolate 10 Procedural requirements 10 General10 System suitability 10 Precision.10 Interlaboratory test 10 Repeatability.11 Reproducibility.11 Test report 11 Annex A (informative) Results of interlaboratory test.12 Foreword. 3 Forewo
17、rd The text of ISO 23275-2:2006 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 23275-2:2008 by Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats and oils a
18、nd 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 standards sha
19、ll 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 Internal Regul
20、ations, 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, Malta, Nethe
21、rlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of ISO 23275-2:2006 has been approved by CEN as a EN ISO 23275-2:2008 without any modification. EN ISO 23275-2:2008 (E) 3DIN EN ISO 23275-2:2009-03 Introducti
22、on “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 principle, coc
23、oa 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 by refin
24、ing 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 ( Shorea
25、robusta), shea ( Butyrospermum parkii), kokum gurgi ( Garcinia indica), and mango kernel ( Mangifera indica). ISO 23275-1 specifies a procedure for the detection of these fats (restrictions are only made for pure illip fat samples) in cocoa butter and plain chocolate. This part of ISO 23275 specifie
26、s 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 been est
27、ablished. “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 calculate
28、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 detect and
29、quantify CBEs. EN ISO 23275-2:2008 (E) 4DIN EN ISO 23275-2:2009-03 1 Scope This part of ISO 23275 specifies a procedure for the quantification of cocoa butter equivalents (CBEs) in cocoa butter (CB) and plain chocolate by high-resolution capillary gas chromatography (HR-GC) of triacylglycerols, and
30、subsequent data evaluation by partial least-squares regression analysis. NOTE The presence of CBEs in CB and plain chocolate down to a level of 0,6 % (fat content of chocolate assumed to be 30 %) can be determined by using the procedure explained in ISO 23275-1. Differences in the procedure of the t
31、wo methods exist in the number of individual triacylglycerols used for data treatment and in the mathematical evaluation principle of the data. The presence of CBEs is detected by linear regression analysis applied to the relative proportions of the three main triacylglycerol fractions of the fat an
32、alysed. The amount of the CBE admixture is estimated by partial least squares regression analysis applied to the relative proportions of the five main triacylglycerols. 2 Terms and definitions For the purposes of this document, the following terms and definitions apply. 2.1 cocoa butter equivalents
33、CBEs fats detected in cocoa butter and plain chocolate 2.2 CBE content of cocoa butter mass fraction of substances in cocoa butter determined by the procedure specified in this part of ISO 23275 NOTE It is expressed in grams per 100 g of cocoa butter. 2.2 CBE content of chocolate mass fraction of su
34、bstances in chocolate determined by the procedure specified in this part of ISO 23275 NOTE It is expressed in grams per 100 g of chocolate. 3 Principle Cocoa butter, or the fat obtained from plain chocolate, is separated by HR-GC into triacylglycerol fractions according to their molecular mass and d
35、egree of unsaturation. The added amount of CBEs is estimated by partial least squares regression analysis applied to individual triacylglycerol fractions of the fat analysed. EN ISO 23275-2:2008 (E) 5DIN EN ISO 23275-2:2009-03 4 Reagents and materials Use only reagents of recognized analytical grade
36、, unless otherwise stated. WARNING Attention is drawn to the regulations 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 suitab
37、ility check. 4.2 Fat solvent, non-chlorinated solvents (e.g. diethyl ether, n-heptane, iso-octane). 4.3 Hydrochloric acid, c = 8 mol/l. 4.4 Fluted filter paper1),15 cm. 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 us
38、ed. 5.3 Food grater, a kitchen blender with a design featuring the motor above the receiving container to avoid melting the samples2). 5.4 Volumetric flasks, of capacity 20 ml. 5.5 Pipettes, of capacity 1 ml. 5.6 Microsyringe, with maximum volume 10 l, graduated to 0,1 l, or automatic sampler. 5.7 G
39、as chromatograph (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.
40、2. The separation and quantification have proven 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 progra
41、mme: 100 C (initial temperature), programme rate 30 C/min to 340 C (final temperature). carrier gas: helium or hydrogen (purity W 99,999 %). 1) S m is the mass of the test portion (chocolate), in grams. Alternative extraction procedures may be used (e.g. by accelerated solvent extraction, by supercr
42、itical carbon dioxide or by using microwaves) provided that the same results are obtained. Report the fat content to two decimal places. 8.2 Separation of individual triacylglycerols by HR-GC The test samples cocoa butter, fat extracted from chocolate, and cocoa butter CRM (4.1) shall be warmed in a
43、 drying oven (5.2) until completely melted. If the liquid sample contains some sediment, filter the sample inside the 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 dryin
44、g oven (5.2) in order to avoid partial fat fractionation during handling of samples. Weigh ca. 0,2 g of test sample in a 20 ml volumetric flask (5.4) and dilute to the mark with a suitable fat solvent (4.2). Pipette (5.5) 1 ml of the resulting solution in another 20 ml volumetric flask and dilute to
45、 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 employed gives a linear response and the system
46、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-d
47、ioleoylglycerol (SOO) is made by comparison of the retention times of the test samples with those of the cocoa butter CRM (4.1). In general, triacylglycerols appear in order of increasing number of carbon atoms and of increasing unsaturation for the same number of carbon atoms. The elution order of
48、the triacylglycerols of the cocoa butter CRM is given in the example chromatogram (Figure A.1). 9 Calculation 9.1 Determination of response factors Determine the response factors of the triacylglycerols POP, POS, POO, SOS and SOO by injection of the cocoa butter CRM solution using experimental condi
49、tions identical to those used for the samples. Calculate the percentage of each of the five triacylglycerol fractions by the following equations: ,refrefref100 %iiiAPA=(1) EN ISO 23275-2:2008 (E) 8DIN EN ISO 23275-2:2009-03 ,refrefiiiwFP= (2) where Pref,iis the percentage of triacylglycerol i in the cocoa butter CRM (from peak areas); Aref,i is the peak area of the triacylglycerol i in the cocoa butter CRM; Aref,iis the sum of the peak areas attributed to POP, POS, POO, SOS, SOO in the cocoa butter CRM;
