1、BS ISO 11053:2009ICS 67.190; 67.200.10NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDVegetable fats and oils Determination of cocoa butter equivalents in milk chocolateThis British Standard was published under the authority of the Standards Policy and Strategy
2、Committee on 31 December2009 BSI 2009ISBN 978 0 580 63235 8Amendments/corrigenda issued since publicationDate CommentsBS ISO 11053:2009National forewordThis British Standard is the UK implementation of ISO 11053:2009. The UK participation in its preparation was entrusted to TechnicalCommittee AW/307
3、, Oil seeds, animal and vegetable fats and oils andtheir by products.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for its correct applicat
4、ion. Compliance with a British Standard cannot confer immunityfrom legal obligations.BS ISO 11053:2009Reference numberISO 11053:2009(E)ISO 2009INTERNATIONAL STANDARD ISO11053First edition2009-09-01Vegetable fats and oils Determination of cocoa butter equivalents in milk chocolate Corps gras dorigine
5、 vgtale Dtermination des quivalents au beurre de cacao dans le chocolat au lait BS ISO 11053:2009ISO 11053:2009(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces
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10、so.org Published in Switzerland ii ISO 2009 All rights reservedBS ISO 11053:2009ISO 11053:2009(E) ISO 2009 All rights reserved iiiContents Page Foreword iv 1 Scope . 1 2 Terms and definitions. 1 3 Principle. 1 4 Reagents, solutions and standards 2 5 Apparatus and equipment . 3 6 Sampling 4 7 Sample
11、preparation 4 7.1 Preparation of IRMM-801 for calibration purposes and system suitability tests. 4 7.2 Preparation of pure milk fat for system suitability tests 4 7.3 Preparation of chocolate sample 5 8 Procedure 6 8.1 Construction of calibration curve for determination of PSB content . 6 8.2 Separa
12、tion of individual TAGs of IRMM-801 by HR-GLC 7 8.3 Separation of individual TAGs of pure MF by HR-GLC. 7 8.4 Separation of individual TAGs of chocolate fat by HR-GLC 7 8.5 Identification 8 9 Calculation. 8 9.1 PSB and MF quantification in chocolate fat and chocolate . 8 9.2 CBE detection in chocola
13、te fat 9 9.3 CBE quantification in chocolate fat and chocolate. 11 10 Procedural requirements . 13 10.1 General considerations 13 10.2 System suitability . 13 11 Precision 13 11.1 Interlaboratory test . 13 11.2 Repeatability 13 11.3 Reproducibility 14 12 Test report . 14 Annex A (informative) Result
14、s of interlaboratory test 15 Annex B (informative) Example chromatograms 19 Bibliography . 22 BS ISO 11053:2009ISO 11053:2009(E) iv ISO 2009 All rights reservedForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies)
15、. The 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
16、-governmental, 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
17、 2. 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
18、a vote. 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 11053 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 11, A
19、nimal and vegetable fats and oils. BS ISO 11053:2009INTERNATIONAL STANDARD ISO 11053:2009(E) ISO 2009 All rights reserved 1Vegetable fats and oils Determination of cocoa butter equivalents in milk chocolate 1 Scope This International Standard specifies a procedure for the detection and quantificatio
20、n of cocoa butter equivalents (CBEs) and milk fat (MF) in milk chocolate by triacylglycerol (TAG) profiling using high-resolution capillary gas-liquid chromatography (HR-GLC), and subsequent data evaluation by simple and partial least-squares regression analysis. CBE admixtures can be detected at a
21、minimum level of 0,5 g CBE/100 g milk chocolate and quantified at a level of 5 % mass fraction CBE addition to milk chocolate with a predicted error of 0,7 g CBE/100 g milk chocolate. 2 Terms and definitions For the purposes of this document, the following terms and definitions apply. 2.1 milk fat c
22、ontent of milk chocolate mass fraction of milk fat in milk chocolate determined by the procedure specified in this International Standard NOTE The mass fraction is expressed in grams per 100 g of milk chocolate. 2.2 cocoa butter equivalents non-cocoa vegetable oils and fats detected in milk chocolat
23、e in accordance with the procedure prescribed in this International Standard NOTE The result is expressed qualitatively, i.e. CBEs present/CBEs not present (YES/NO). 2.3 cocoa butter equivalent content of milk chocolate mass fraction of substances determined by the procedure specified in this Intern
24、ational Standard NOTE The mass fraction is expressed in grams per 100 g of milk chocolate. 3 Principle Test samples, i.e. chocolate fats obtained from milk chocolate using a rapid fat extraction procedure, are separated by HR-GLC into TAG fractions according to their relative molecular mass and degr
25、ee of unsaturation. Individual TAG fractions, i.e. 1-palmitoyl-2-stearoyl-3-butyroyl-glycerol (PSB), 1,3-dipalmitoyl-2-oleoyl-glycerol (POP), 1-palmitoyl-2-oleoyl-3-stearoyl-glycerol (POS), 1-palmitoyl-2,3-dioleoyl-glycerol (POO), 1,3-distearoyl-2-oleoyl-glycerol (SOS), and 1-stearoyl-2,3-dioleoyl-g
26、lycerol (SOO) are used: a) to calculate the MF content in the chocolate fat (grams of MF per 100 g chocolate fat); b) to determine the presence/absence of CBEs in chocolate fat using a simple linear regression model based on the three TAGs, POP, POS, and SOS, corrected for the TAG contribution origi
27、nating from MF, and if this procedure indicates that the sample is not pure cocoa butter (CB); BS ISO 11053:2009ISO 11053:2009(E) 2 ISO 2009 All rights reservedc) to quantify the amount of the CBE admixture in chocolate fat (grams of CBE per 100 g chocolate fat) using a partial least-squares (PLS) r
28、egression model with six input variables, i.e. the five TAGs, POP, POS, POO, SOS, and SOO, normalized to 100 % and the determined MF content of the chocolate fat. To ensure the correct labelling of milk chocolate, the results obtained relating to chocolate fat are converted into grams of MF per 100
29、g chocolate and grams of CBE per 100 g chocolate, necessitating the accurate determination of the total fat content of the chocolate using a Soxhlet extraction procedure (based on AOAC Official Method 963.155). When the detection procedure proves the absence of CBEs in the chocolate fat, the quantif
30、ication and total fat content are not necessary. 4 Reagents, solutions and standards NOTE Use only reagents of recognized analytical grade, unless otherwise specified. WARNING Attention is drawn to the regulations which specify the handling of dangerous matter. Technical, organizational and personal
31、 safety measures should be followed. 4.1 Cocoa butter Certified Reference Material (IRMM-801)1)(see Reference 6), for calibration purposes and system suitability tests. 4.2 Pure milk fat, for system suitability tests. 4.3 1-Palmitoyl-2-stearoyl-3-butyroyl-glycerol (PSB)2). 4.3.1 General For calibrat
32、ion purposes, dissolve 40 mg of PSB in a 50 ml volumetric flask (5.9) with isooctane resulting in a stock solution of = 0,8 mg/ml. Mix thoroughly until complete dissolution. From this PSB stock solution prepare a series of five calibration solutions in matrix (IRMM-801) by weighing on an analytical
33、balance (5.1) IRMM-801 (4.1) into 25 ml volumetric flasks (5.9) and adding the respective volumes of the PSB stock solution as given in Table 1. Make up to the mark with isooctane. Table 1 Masses of IRMM-801 and volumes of PSB stock solution for preparation of series of PSB calibration solutions in
34、matrix Calibration solution IRMM-801 (4.1) weighed into 25 ml volumetric flask Volume taken from PSB stock solution and added to 25 ml volumetric flask Concentration of PSB in calibration solution PSBi Final IRMM-PSB concentration of solution IRMM-PSBmg ml mg/ml mg/ml 1 250 4 0,128 10 2 250 3 0,096
35、10 3 250 2 0,064 10 4 250 1 0,032 10 5 250 0,5 0,016 10 1) Commercially available from the Institute for Reference Materials and Measurements (http:/irmm.jrc.ec.europa.eu/), Belgium. This information is given for the convenience of users of this International Standard and does not constitute an endo
36、rsement by ISO of this product. 2) Commercially available from Larodan (http:/www.larodan.se/), Sweden. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of the product named. Equivalent products may be used if they ca
37、n be shown to lead to the same results. BS ISO 11053:2009ISO 11053:2009(E) ISO 2009 All rights reserved 34.3.2 Cold on-column (OCI) injection Dilute each calibration solution with isooctane, = 1 ml/5 ml, to obtain a final IRMM-PSB concentration (IRMM-PSB) of 2 mg/ml in each solution and PSB concentr
38、ations (PSB) ranging from 0,025 6 mg/ml (calibration solution 1) to 0,003 2 mg/ml (calibration solution 5). 4.3.3 Split injection (e.g. split ratio of 1:10) Dilute each calibration solution with isooctane, = 1 ml/2 ml, to obtain a final IRMM-PSB concentration (IRMM-PSB) of 5 mg/ml in each solution a
39、nd PSB concentrations (PSB) ranging from 0,064 mg/ml (calibration solution 1) to 0,008 mg/ml (calibration solution 5). NOTE The final PSB concentrations shall be calculated using the actual mass in the stock standard solution. 4.4 -Cholestane3), = 100 mg/100 ml, used as internal standard. Dissolve 5
40、0 mg -cholestane in 50 ml of isooctane. For cold on-column injection: Dilute 1:250 ( = 0,004 mg/ml). For split injection (e.g. split ratio of 1:10): Dilute 1:100 ( = 0,01 mg/ml). 4.5 Fat solvent, non-chlorinated solvents (e.g. petroleum ether, n-hexane, n-heptane, isooctane). 4.6 Hydrochloric acid,
41、c(HCl) = 4 mol/l. 5 Apparatus and equipment 5.1 Analytical balance, readable to the nearest 0,1 mg. 5.2 Drying oven. A dry heater block may be used. 5.3 Filter paper, diameter 15 cm e.g. S 30 C/min to 270 C held for 1 min;2,5 C/min to 340 C held for 7 min Oven programme for split: 200 C held for at
42、least 1 min; 14 C/min to 270 C held for 1 min;2,5 C/min to 340 C held for 10 min Detector (FID): 360 C Carrier gas for OCI: H2(purity W 99,999 %) with a constant flow rate of 3,5 ml/min (anothersuitable carrier gas is helium) Carrier gas for split: H2(purity W 99,999 %) with a constant flow rate of
43、2,5 ml/min (anothersuitable carrier gas is helium) NOTE 2 Columns and alternative experimental conditions, used in an international collaborative study (see Reference 7), are listed in Table A 1. Operating conditions may be changed to obtain optimum separation. 5.13 Chromatographic data system. 6 Sa
44、mpling Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 55553. A representative sample should have been sent to the laboratory. It should not have been damaged or changed during transport or storage. 7 Sample preparation 7.1 P
45、reparation of IRMM-801 for calibration purposes and system suitability tests Before opening and using the IRMM-801 (4.1), warm the ampoule in an oven (5.2) until the contents have melted. When a clear solution is obtained, mix the contents by repeated inversion for not less than 20 s. Then open and
46、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 pure milk fat for system suitability tests If no pure MF is available, it can be obtained from a butter sample by melting and passing the fat layer through a folded f
47、ilter paper (5.3) at 50 C in an oven (5.2). BS ISO 11053:2009ISO 11053:2009(E) ISO 2009 All rights reserved 57.3 Preparation of chocolate sample 7.3.1 General Chill approximately 200 g of chocolate until hard, and grate to a fine granular condition using a food grater (5.4). Mix thoroughly and prese
48、rve in a tightly stoppered bottle in a cool place. 7.3.2 Rapid fat extraction The chocolate fat is separated from 5 g grated chocolate (7.3.1) by extracting with two to three 10 ml portions of a suitable fat solvent (4.5). Centrifuge and decant. Combine the extracts and evaporate (5.5) most of the f
49、at solvent and finally dry it under a stream of nitrogen (5.6). The chocolate fat obtained by rapid fat extraction is used for the final TAG analysis by HR-GLC. For the detection of CBEs in chocolate, the accurate amount of total fat in chocolate is not needed. When no CBEs are detected, the second part of the standard, i.e. quantification of CBEs around the statutory limit of 5 %, is not necessary. When CBEs are detected, the quantification part should be performed using the
