1、DEUTSCHE NORM10482-1Translation by DIN-Sprachendienst.In case of doubt, the German-language original should be consulted as the authoritative text. No part of this translation may be reproduced without the prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 B
2、erlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).April 2005Determination of the annatto content of cheesePart 1: Photometric method (includes DIN 10482-1:2005-05 Corrigendum 1)Document comprises 6 pages.ICS 67.100.30Bestimmung des Annattogehaltes in Kse Teil 1: Photom
3、etrisches VerfahrenAll liquid mixtures are in parts by volume.ContentsPageForeword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Scope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4、. . 22 Normative references . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Concept. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Abbreviation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5、. . . . . . . . . . . . . . . . . 25 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Reagents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Apparatus . . . . . . . . . . . . .
6、 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Sampling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7、 310 Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411 Precision. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412 Test report . . . . . . . . . . . . . . . . . . . . . . . . . . .
8、 . . . . . . . . . . . . . . . . . . . . . . 5Annex A (informative) Results of interlaboratory testing . . . . . . . . . . . . . . 6Bibliography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6ForewordThis standard has been prepared by Technical
9、Committee Chemische und physikalische Milchunter- suchung of the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Foodstuffs and Agricul-tural Products Standards Committee).NOTE: The precision of this standard is regarded as equivalent to that of the high performance liquid chro-matogr
10、aphy method specied in DIN 10482-2 and the user is free to choose which method to adopt.English price group 8 www.din.de www.beuth.de02.06 9670303Page 2 DIN 10482-1:2005-041 ScopeThis standard species a photometric method of determining annatto (E 160b) in cheese after solid phase extraction (SPE) 1
11、.2 Normative referencesThis standard incorporates, by dated or undated reference, provisions from other publications. For dated ref-erences, only the document referred to applies. For undated references, the latest edition of the publication referred to applies (including amendments).DIN EN ISO 707
12、Milk and milk products Guidance on sampling (ISO 707:1997)3 ConceptAnnatto (E 160b) is a natural yellow dye found in the ripe seedpods of Bixa orellana.NOTE: Depending on the method of extraction, the dye extracted will consist of the carotenoids bixin (C25H30O4) and/or norbixin (C24H28O4), both of
13、which may occur as cis- or trans-isomers 2.4 AbbreviationSPE: Solid Phase Extraction.5 PrincipleThe cheese sample is extracted with acetone containing hydrochloric acid. Using a solid phase extraction column, fat and b-carotin are separated with n-hexane/diethyl ether and the annatto is then eluted
14、with a (7+3) mixture of methanol/glacial acetic acid. Finally, the annatto is determined photometrically at a wavelength of 488 nm with a (7+3) mixture of methanol/glacial acetic acid as reference.6 Reagents6.1 GeneralAnalytical grade reagents shall be used, and the water used shall be double-distil
15、led or water of at least equivalent purity.The following reagents shall be used.6.2 Reagents for extracting annatto from cheese6.2.1 Sea sand, as ltration aid.6.2.2 Acetone containing hydrochloric acid, prepared by adding 2 ml of 2 mol/l hydrochloric acid, HCl, to 200 ml of 100 % ( m/m) acetone, C3H
16、6O.6.2.3 100 % ( m/m) absolute alcohol, C2H6O.6.2.4 Particle-free gaseous nitrogen, for stripping the acetone containing hydrochloric acid. Its pressure shall be nely regulated with a reducing valve.6.2.5 Acetone.6.3 Reagents used for the Solid Phase Extraction (SPE)6.3.1 n-Hexane.6.3.2 (1+1) mixtur
17、e of n-hexane/diethyl ether, prepared by mixing 10 ml of n-hexane and 10 ml of diethyl ether.6.3.3 (7+3) mixture of methanol/glacial acetic acid, prepared by mixing 70 ml of methanol and 30 ml of glacial acetic acid.Use a fresh solution for each analysis.7 ApparatusIn addition to standard laboratory
18、 equipment, the following shall be used.Page 3 DIN 10482-1:2005-047.1 Mortar and pestle.7.2 Sealable centrifuge tubes, of nominal capacity 100 ml, suitable for the centrifuge specied in sub-clause 7.3.7.3 Centrifuge, capable of being operated at an effective centrifugal acceleration of 1 000 g and m
19、ain-tained at a temperature of 4 C.7.4 Glass funnel, having a diameter of about 10 cm.7.5 Fluted lter, moistened with acetone (6.2.5) prior to ltration.7.6 Tapered ask, of nominal capacity 100 ml, suitable for a rotary evaporator as specied in sub-clause 7.7.7.7 Rotary evaporator with temperature-co
20、ntrolled water bath, capable of being maintained at a temperature of 40 C.7.8 Ultrasonic bath.7.9 Volumetric ask, of nominal capacity 10 ml.7.10 Membrane lter, having a pore size of 0,45 m, suitable for ltering the sample solution (9.1.2).7.11 Analytical balance.7.12 Reducing valve, suitable for ste
21、el nitrogen bottles.7.13 One-mark bulb pipettes, of nominal capacities 2 ml, 10 ml, 20 ml and 50 ml.7.14 Peleus ball or aspirette, suitable as a suction aid for one-mark bulb pipettes (7.13).7.15 Pasteur pipettes.7.16 Water bath or heating block, suitable for heating a 100 ml tapered ask and capable
22、 of being main-tained at 40 C.7.17 Solid phase extraction apparatus.7.18 Solid phase extraction column, e.g. IST Isolute NH22 g/6 ml column (Separtis GmbH, Grenzach-Whylen, Germany) 1).7.19 Double beam spectrophotometer, for measuring absorbance at a wavelength of 488 nm, suitable for cells having a
23、 path length of 1 cm.8 SamplingAs specied in DIN EN ISO 707.9 Procedure9.1 Preparation of sample solutionCAUTION: Inhalation or swallowing shall be avoided when working with acetone, acetone containing hydrochloric acid, diethyl ether, n-hexane, methanol and glacial acetic acid, and gloves shall be
24、worn to prevent contact with the skin. All extractions shall be performed in a fume cupboard.9.1.1 Cheese extractionStore cheese samples at 20 C and grate pieces of cheese or cheese rind prior to analysis.Grind 5 g of the cheese sample or edible 2) rind to be tested together with 15 g of sea sand in
25、 a mortar and transfer the resultant mixture quantitatively to a sealable 100 ml centrifuge tube. After adding 20 ml of acetone containing hydrochloric acid and shaking the solution vigorously for ten minutes, centrifuge for ve minutes at 1 000 g.1)Reference to the Isolute product is for the purpose
26、 of providing the user with an example of a suitable com-mercial product and does not imply any approval by DIN. Other products may be used if they are shown to yield identical results.2)Edible as dened in ZZulV 3.Page 4 DIN 10482-1:2005-04Filter the supernatant obtained through a uted lter paper mo
27、istened with acetone and collect the ltrate in a 100 ml tapered ask. Rinse the lter paper with acetone and repeat the extraction two more times.If the upper rim of the lter paper has a slight yellow coloration after ltration, use scissors to cut off a strip about 1 cm wide from it. Rinse this strip
28、with drops of acetone until the colour disappears and transfer the resultant extract to the 100 ml tapered ask.Evaporate down the extracts collected in the tapered ask on a rotary evaporator in vacuo at 40 C. Remove residual water by adding 10 ml of ethanol (6.2.3) and evaporating down again. Strip
29、off residual solvent with nitrogen for 15 minutes at 40 C. Use the extract obtained for the SPE. For the blank test proceed in the same way, but omitting the cheese sample.9.1.2 Solid phase extraction (SPE)During extraction, particularly if it is vacuum-aided, ensure that the eluates leave the colum
30、n dropwise at as constant a volumetric rate as possible.1) Condition the stationary phase by rinsing it with 15 ml of n-hexane.2) Take up the acetone extraction residue in 2 ml of acetone in the tapered ask and, after treating it for a short time in an ultrasonic bath, transfer the solution to the S
31、PE column. To ensure quantitative transfer, add a further 2 ml of acetone to the tapered ask, repeat the ultrasonic treatment and add this solution to the column.3) To separate fat and b-carotin, rinse the SPE column, rst with 10 ml of n-hexane/diethyl ether (6.3.2) and then with 2 ml of acetone.4)
32、To isolate the annatto dyes bixin and norbixin, apply 8 ml of the (7+3) mixture of methanol/glacial acetic acid to the SPE column and collect the eluate in a 10 ml volumetric ask. Make the eluate up to the mark with the same solvent and lter it through a membrane lter (7.10) prior to the determinati
33、on to obtain the sample solution for the photometric analysis. The solution is photosensitive and will be stable only for one day.5) Treat the sample for the blank test in the same way.9.2 DeterminationThe annatto content is determined by measuring the contents of the dyes bixin and norbixin photome
34、trically. Prior to the analysis, the photometer shall be adjusted to 0,000 absorbance units at a wavelength of 488 nm with air as reference (no cell in the beam path). The absorbance of the sample solution shall be determined using the (7+3) mixture of methanol/glacial acetic acid as reference.To de
35、termine a reagent blank value, perform the extraction (see 9.1.1) without a cheese sample by separating the residue obtained by SPE (see 9.1.2) in the same way as for the sample solution and measuring the reagent blank solution obtained photometrically at a wavelength of 488 nm.10 EvaluationCalculat
36、e the annatto content, wAnnatto, in mg/kg, in accordance with the Lambert-Beer law from the ratio of the absorbance to the concentration using equation (1):(1)whereAlP is the absorbance measured in the sample solution for bixin and norbixin at a wavelength of 488 nm in the (7+3) mixture of methanol/
37、glacial acetic acid;AlBwis the absorbance measured in the reagent blank solution at a wavelength of 488 nm in the (7+3) mix-ture of methanol/glacial acetic acid;E1cm1 %is the bixin and norbixin absorbance at 488 nm in a 1 % solution of the (7+3) mixture of methanol/gla -cial acetic acid for a path l
38、ength of the cell of 1 cm (the absorbance is linear in the 0 to 1,45 range, which is equivalent to 0 mg/kg to 10 mg/kg of annatto);10 000 is the factor for converting E1cm1 %from 10 g/l to mg/l of sample solution; VML is the total volume of the SPE extract, in ml (here: 10 ml);M is the initial sampl
39、e mass, in g.11 Precision11.1 GeneralThe precision data were obtained in an interlaboratory test carried out in 2003 on various types of cheese in accordance with DIN ISO 5725-2 and involving 12 laboratories. Details of this test will be found in Annex A.Page 5 DIN 10482-1:2005-04As part of its qual
40、ity assurance, any laboratory using this standard shall conform to the precision data con-tained in this clause and provide conrmation of this, for example by participating in interlaboratory tests de-signed to assess quality assurance and laboratory performance and by using reference materials (pre
41、ferably during regular in-house statistical checks). 11.2 Repeatability limit(same operator, same equipment)The absolute difference between two successive results obtained under repeatability conditions shall not ex-ceed a relative repeatability limit, rrel, of 18 % in more than 5 % of cases.If the
42、repeatability limit is exceeded, both results shall be deemed to be unreliable.11.3 Reproducibility limit(different operators, different equipment)The absolute difference between two individual results obtained under reproducibility conditions shall not ex-ceed a relative reproducibility limit, Rrel
43、, of 32 % in more than 5 % of cases.If the reproducibility limit is exceeded, both results shall be deemed to be unreliable.12 Test reportThe test report shall refer to this standard and include the following details as a minimum:a) type of sample;b) method and date of sampling;c) result;d) date of
44、analysis;e) reasons for any deviations from this standard.Page 6 DIN 10482-1:2005-04Annex A (informative)Results of interlaboratory testingThe precision data shown in table A.1 were obtained in an interlaboratory test carried out on various cheese samples by the DIN Annatto Team in 2003.Table A.1: R
45、esults of interlaboratory testSample 1aSample 2bSample 3cNumber of laboratories 12 12 12Number of laboratories after eliminating outliers (n) 10 10 11Number of test results in all the laboratories 36 36 36Mean, x (mg/kg) 1,6 0,7 6,7Repeatability standard deviation, sr(mg/kg) 0,14 0,04 0,32Repeatabil
46、ity coefcient of variation (%) 8,8 5,7 4,8Repeatability limit, r (mg/kg) 0,4 0,1 0,9Reproducibility standard deviation, sR(mg/kg) 0,19 0,09 0,57Reproducibility coefcient of variation (%) 11,9 12,9 8,5Reproducibility limit, R (mg/kg) 0,5 0,3 1,6a Naturally-ripened hard cheese.b Ready-sliced semi-hard
47、 cheese (Butterkse).c Soft cheese (cheese rind).BibliographyDIN 10482-2 *) Determination of Annatto content in cheese Part 2: High performance liquid chromato-graphic methodDIN ISO 5725-2 Accuracy (trueness and precision) of measurement methods and results Part 2: Basic method for the determination
48、of repeatability and reproducibility of a standard measurement method (ISO 5725-2:1994, including Technical Corrigendum 1:2002)1 Bareth, A., Strohmar, W. and Kitzelmann, E.: HPLC and spectrophotometric determination of annatto in cheese, Eur. Food Res. Technol., 2002: 215, 359364.2 Commission direct
49、ive 95/45/EC laying down specic purity criteria concerning colours for use in food-stuffs, as of 26 July 1995, Ofcial Journal of the European Communities 226.3 Verordnung ber die Zulassung von Zusatzstoffen zu Lebensmitteln zu technologischen Zwecken (ZZulV), Artikel 1 der Verordnung zur Neuordnung lebensmittelrechtlicher Vorschriften ber Zusatzstoffe (LMRZStoffNOV) (German Regulation on the approval of food additives for technical purposes, Article 1 of the Regulation amending the Food Act with respect to additives),
