1、BRITISH STANDARDBS EN ISO 23275-2:2008Animal and vegetable fats and oils Cocoa butter equivalents in cocoa butter and plain chocolate Part 2: Quantification of cocoa butter equivalentsICS 67.190; 67.200.10nullnull nullnullnullnullnullnullnull nullnullnullnullnullnullnull nullnullnull nullnullnullnul
2、lnullnullnullnullnullnull nullnullnullnullnullnull nullnull nullnullnullnullnullnullnullnullnull nullnull nullnullnullnullnullnullnullnullnull nullnullnullNational forewordThis British Standard is the UK implementation of EN ISO 23275-2: 2008. It is identical with ISO 23275-2:2006. It supersedes BS
3、ISO 23275-2:2006, which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/307, Oil seeds, animal and vegetable fats and oils and their by products.A list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 secretary. This public
4、ation 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 23275-2:2008This British Standard was published under the authority of the Stand
5、ards Policy and Strategy Committee on 30 November 2006 BSI 2009ISBN 978 0 580 63600 4Amendments/corrigenda issued since publicationDate Comments 30 September 2009 This corrigendum renumbers BS ISO 23275-2:2006 as BS EN ISO 23275-2:2008EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 23275-2Nove
6、mber 2008ICS 67.200.10; 67.190English VersionAnimal and vegetable fats and oils - Cocoa butter equivalents incocoa butter and plain chocolate - Part 2: Quantification ofcocoa butter equivalents (ISO 23275-2:2006)Corps gras dorigines animale et vgtale - quivalents aubeurre de cacao dans le beurre de
7、cacao et dans lechocolat de mnage - Partie 2: Quantification desquivalents au beurre de cacao (ISO 23275-2:2006)Tierische und pflanzliche Fette und le - Kakaobutter-quivalente in Kakaobutter und Zartbitterschokolade - Teil2: Mengenbestimmung von Kakaobutter-quivalenten (ISO23275-2:2006)This European
8、 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 references concern
9、ing 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 member into its own lan
10、guage 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, Latvia, Lithuania, Lux
11、embourg, 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 2008 CEN All right
12、s of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 23275-2:2008: EForeword 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) a
13、nd has been taken over as EN ISO 23275-2:2008 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 European Standard shall be given the status of a national standard,
14、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 this document may be the subject of patent rights. CEN and/or C
15、ENELEC 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 Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, De
16、nmark, 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 Kingdom. Endorsement notice The text of ISO 23275-2:2006 has been ap
17、proved by CEN as a EN ISO 23275-2:2008 without any modification. BS EN ISO 23275-2:2008EN ISO 23275-2:2008 (E)iiContents Page1Scp.12Trm adfio3Pnle4Rgts 25Au6 37p7.1 Preparation of cocoa butter CRM for calibration purposes and system suitability check37.2 Preparation of chocolate sample.38d8.1 Fat ex
18、traction .38.2 Separation of individual triacylglycerols by HR-GC 48.3 Identification.49C9.1 Determination of response factors49.2 Calculation of percentages of triacylglycerols.59.3 Calculation of CBE content in cocoa butter .59.4 Calculation of CBE content in chocolate610 Procedural requirements 6
19、10.1 General610.2 System suitability 611 Precision.611.1 Interlaboratory test611.2 Repeatability.711.3 Reproducibility.712 Test report7An n e x A (informative) Results of interlaboratory test.8Bibliography13BS EN ISO 23275-2:2008EN ISO 23275-2:2008 (E)iiiIntroduction.ivIntroduction “Cocoa butter equ
20、ivalents” is the general term for fats used to replace cocoa butter in chocolate. Theyresemble the chemical composition and physical properties of cocoa butter very closely, making themtherefore extremely difficult to quantify and even in some cases to detect. In principle, cocoa butterequivalents m
21、ust by definition be fats low in lauric acid, rich in symmetrical mono-unsaturated triacylglycerolsof 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 onlyby refining and fractionation. Wi
22、thin 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 tallow or tengkawang (Shorea spp. ), palm oil (Elaeis guineensis, Elaeis olifera), sShorea robusta), ea (Butyrospermum parki
23、i), 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 ISO23275 specifies a procedure allowing a reliablequanti
24、fication 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 ISO23275, an analytical toolbox named “CoCal-1” has beenestablished. “CoCal-1” contains the validated
25、 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 theanalysts instruments and an electronic evaluation sheet for Microsoft Excel to calculate the final result. Ananalyst working on CBE d
26、etection and quantification has only to calibrate the gas chromatographic separation system using IRMM-801, separate the triglyceride fractionsof the sample in question, and use the electronicevaluation sheet for subsequent data treatment to detect and quantify CBEs. BS EN ISO 23275-2:2008EN ISO 232
27、75-2:2008 (E)iv1A n i m a l a n d v e g e t a b l e f a t s a n d o i l s C o c o a b u t t e r e q u i v a l e n t s in cocoa butter and plain chocolate Part 2: Quantification of cocoa butter equivalents 1 Scope This part of ISO23275 specifies a procedure for the quantification of cocoa butter equi
28、valents (CBEs) incocoa butter (CB) and plain chocolate by high-resolution capillary gas chromatography (HR-GC) oftriacylglycerols, and 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
29、 chocolate assumedto be 30 %) can be determined by using the procedure explained in ISO23275-1. Differences in the procedure of the twomethods exist in the number of individual triacylglycerols used for data treatment and in the mathematical evaluationprinciple of the data. The presence of CBEs isde
30、tected by linear regression analysis applied to the relativeproportionsofthe three main triacylglycerol fractions of the fat analysed. The amount of the CBE admixture is estimated by partial leastsquares regression analysis applied to the relative proportions of the five main triacylglycerols. 2 Ter
31、ms 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 2.2 CBE content of cocoa butter massfraction of substances in cocoa butter determined by the procedure specified in this
32、part of ISO 23275 NOTE It is expressed in grams per 100 g of cocoa butter. 2.2 CBE content of chocolatemassfraction of substances in chocolate determined bythe procedure specified in this part of ISO23275 NOTE It is expressed in grams per 100 g of chocolate. 3 Principle Cocoa butter, or the fat obta
33、ined from plain chocolate, is separated by HR-GC into triacylglycerol fractionsaccording to their molecular mass and degree of unsaturation. The added amount of CBEs is estimated bypartial least squares regression analysis applied to individual triacylglycerol fractions of the fat analysed. BS EN IS
34、O 23275-2:2008EN ISO 23275-2:2008 (E)2 4 Reagents and 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, organizationaland personal safety measuresshall be follow
35、ed. 4.1 Cocoa butter Certified Reference Material (CRM) IRMM-8012, for calibration purposes and systemsuitability 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 Analytic
36、al balance,with areadbilty of 0,1 mg. 5.2 Drying oven, maintained at 55 C. A dry heater block maybe used. 5.3 Food grater, a kitchen blenderwith a design featuring the motor above the receiving container to avoidmelting the samples2). 5.4 Volumetric flasks, of capacity 20 ml. 5.5 Pipettes, of capaci
37、ty 1 ml. 5.6 Microsyringe, with maximum volume 10 l, graduated to 0,1 l, or automatic sampler. 5.7 Gas 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)
38、 or a moving-needle injector may be used provided the same results are obtained as indicated in 10.2. The separation and quantification have proven to besatisfactory 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 the
39、rmostable 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: helium or hydrogen (purityW 99,999 %). 1) Sm is the mass of the test portion (chocolate), in grams.Alt
40、ernative extraction procedures may be used (e.g. by accelerated solvent extraction, by supercritical 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 sampl
41、es cocoa butter, fat extracted from chocolate, and cocoa butterCRM (4.1) shall be warmed in a 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 th
42、e 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 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).P
43、ipette (5.5)1 ml of the resulting solution in another20 ml volumetric flask and dilute to the markwith 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
44、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-palmitoy
45、l-2,3-dioleoylglycerol (POO), 1,3-distearoyl-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 of increasing number ofcarbon atoms and
46、 of increasing unsaturation for the same number of carbon atoms.The elution order of thetriacylglycerols 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
47、, SOS and SOO by injection of the cocoa butter CRM solution using experimental conditions identical to those used for the samples. Calculatethe percentage of each of the five triacylglycerol fractions by the following equations: ,refrefref100 %iiiAPA=(1) BS EN ISO 23275-2:2008EN ISO 23275-2:2008 (E)
48、5,refrefiiiwFP= (2)where Pref,iis the percentage of triacylglycerol i in the cocoa butterCRM (from peak areas);Aref,iis 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; Fiis the detector
49、response factor of triacylglycerol i in the cocoa butter CRM; wref,iis the mass fraction, in percent, of triacylglycerol i in the cocoa butter CRM as given in the certificate2. Report the results to two decimal places. 9.2 Calculation of percentages of triacylglycerols Calculate the percentages of the triacylglycerols POP, POS, POO, SOS and SOO in the test sample by=( )test,test,test,100 %iFAw(3) where wtest,iis the mass fraction, in percent, of triacylglycerol i in the test samples;Atest,iis the peak area
