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1、 Reference number ISO 23275-2:2006(E) ISO 2006 INTERNATIONAL STANDARD ISO 23275-2 First edition 2006-11-01 Animal and vegetable fats and oils Cocoa butter equivalents in cocoa butter and plain chocolate Part 2: Quantification of cocoa butter equivalents Corps gras dorigines animale et vgtale quivale
2、nts au beurre de cacao dans le beurre de cacao et dans le chocolat de mnage Partie 2: Quantification des quivalents au beurre de cacao ISO 23275-2:2006(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licensing policy, this file may be printed or viewed but s
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5、y ISO member bodies. In the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below. ISO 2006 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electr
6、onic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISOs member body in the country of the requester. ISO copyright office Case postale 56 CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.or
7、g Web www.iso.org Published in Switzerland ii ISO 2006 All rights reserved -,-,- ISO 23275-2:2006(E) ISO 2006 All rights reserved iii Contents Page Foreword iv Introduction v 1 Scope 1 2 Terms and definitions .1 3 Principle1 4 Reagents and materials 2 5 Apparatus .2 6 Sampling.3 7 Preparation of tes
8、t sample3 7.1 Preparation of cocoa butter CRM for calibration purposes and system suitability check3 7.2 Preparation of chocolate sample.3 8 Procedure .3 8.1 Fat extraction .3 8.2 Separation of individual triacylglycerols by HR-GC 4 8.3 Identification.4 9 Calculation4 9.1 Determination of response f
9、actors4 9.2 Calculation of percentages of triacylglycerols.5 9.3 Calculation of CBE content in cocoa butter .5 9.4 Calculation of CBE content in chocolate 6 10 Procedural requirements 6 10.1 General6 10.2 System suitability 6 11 Precision.6 11.1 Interlaboratory test6 11.2 Repeatability.7 11.3 Reprod
10、ucibility.7 12 Test report7 Annex A (informative) Results of interlaboratory test.8 Bibliography13 -,-,- ISO 23275-2:2006(E) iv ISO 2006 All rights reserved Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). T
11、he 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 established has the right to be represented on that committee. International organizations, governmental and non-go
12、vernmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
13、 The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a v
14、ote. 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. ISO 23275-2 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 11, An
15、imal and vegetable fats and oils. ISO 23275 consists of the following parts, under the general title Animal and vegetable fats and oils Cocoa butter equivalents in cocoa butter and plain chocolate: Part 1: Determination of the presence of cocoa butter equivalents Part 2: Quantification of cocoa butt
16、er equivalents ISO 23275-2:2006(E) ISO 2006 All rights reserved v Introduction “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 extreme
17、ly difficult to quantify and even in some cases to detect. In principle, 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-distear
18、oyl-2- oleoylglycerol, miscible with cocoa butter, and obtained only 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
19、 (Shorea spp.), palm oil (Elaeis guineensis, Elaeis olifera), sal (Shorea 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 sample
20、s) in cocoa butter and plain chocolate. This part of ISO 23275 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 1 of the European Parliament and the Council. To facilitate the usage of bot
21、h parts of ISO 23275, an analytical toolbox named “CoCal-1” has 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 instrume
22、nts and an electronic evaluation sheet for Microsoft Excel to calculate 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
23、electronic evaluation sheet for subsequent data treatment to detect and quantify CBEs. -,-,- -,-,- INTERNATIONAL STANDARD ISO 23275-2:2006(E) ISO 2006 All rights reserved 1 Animal and vegetable fats and oils Cocoa butter equivalents in cocoa butter and plain chocolate Part 2: Quantification of cocoa
24、 butter equivalents 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 subsequent data evaluation by partial least-squ
25、ares 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 two methods exist in the number of individual tr
26、iacylglycerols 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 analysed. The amount of the CBE admixture is esti
27、mated 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 CBEs fats detected in cocoa butter and plain ch
28、ocolate 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 substances in chocolate determined by the procedu
29、re 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 degree of unsaturation. The added amount of CBEs
30、 is estimated by partial least squares regression analysis applied to individual triacylglycerol fractions of the fat analysed. -,-,- ISO 23275-2:2006(E) 2 ISO 2006 All rights reserved 4 Reagents and materials Use only reagents of recognized analytical grade, unless otherwise stated. WARNING Attenti
31、on 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-801 2, for calibration purposes and system suitability check. 4.2 Fat solvent, non-chlorin
32、ated 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 used. 5.3 Food grater, a kitchen blender wi
33、th 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 Gas chromatograph (GC), fitted with a cold
34、 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 separation and quantification hav
35、e 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 programme: 100 C (initial temperature), progra
36、mme 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 supercritical carbon dioxide or by using microw
37、aves) 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 drying oven (5.2) until completely melt
38、ed. 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 drying oven (5.2) in order to avoid partial f
39、at 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 the mark with the same solvent. Inject
40、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 suitability criteria (10.2) are met. 8
41、.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 comp
42、arison 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 the triacylglycerols of the cocoa but
43、ter 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 conditions identical to those used for the
44、 samples. Calculate the percentage of each of the five triacylglycerol fractions by the following equations: , , , ref ref ref 100 % i i i A P A = (1) -,-,- ISO 23275-2:2006(E) ISO 2006 All rights reserved 5 , , ref ref i i i w F P = (2) where Pref,i is the percentage of triacylglycerol i in the coc
45、oa butter CRM (from peak areas); Aref,i is the peak area of the triacylglycerol i in the cocoa butter CRM; Aref,i is the sum of the peak areas attributed to POP, POS, POO, SOS, SOO in the cocoa butter CRM; Fi is the detector response factor of triacylglycerol i in the cocoa butter CRM; wref,i is the
46、 mass fraction, in percent, of triacylglycerol i in the cocoa butter CRM as given in the certificate 2. 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 = (
47、) test, test, test, 100 % ii i ii FA w FA (3) where wtest,i is the mass fraction, in percent, of triacylglycerol i in the test samples; Atest,i is the peak area corresponding to the triacylglycerol i in the test sample; Fi is the response factor as determined in 9.1. Report the results to two decima
48、l places. 9.3 Calculation of CBE content in cocoa butter The CBE content of the cocoa butter, cCBE,CB, expressed in grams of CBE per 100 g of cocoa butter, is calculated by using a partial least squares regression analysis Equation (4) of the relative proportions of the five main triacylglycerols; i.e. %POP + %POS + %POO + %SOS + %SOO = 100 % as determined in Equation (3): cCBE,CB = 37,439 + 1,175 POP 1,939 POS 0,121 POO + 0,982 SOS 0,097 SOO (4) Report the results to two decimal places i