1、 g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58using capillary electrophoresis in the presence of sodium dodecyl sulfate (SDS-CE) Screening method
2、ICS 67.100.01Milk powder Determination of soy and pea proteins BRITISH STANDARDBS ISO 17129:2006BS ISO 17129:2006This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 July 2006 BSI 2006ISBN 0 580 49025 4Cross-referencesThe British Standards whic
3、h implement international publications referred to in this document may be found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Standards Online.This publication does not
4、purport to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations.Summary of pagesThis document comprises a front cover, an inside front cover, the ISO title p
5、age, pages ii to vi, pages 1 to 13 and a back cover.The BSI copyright notice displayed in this document indicates when the document was last issued.Amendments issued since publicationAmd. No. Date CommentsA list of organizations represented on this committee can be obtained on request to its secreta
6、ry. present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep UK interests informed; monitor related international and European developments and promulgate them in the UK.National forewordThis British Standard reproduces verbat
7、im ISO 17129:2006 and implements it as the UK national standard. The UK participation in its preparation was entrusted to Technical Committee AW/5, Milk and milk products, which has the responsibility to: aid enquirers to understand the text;Reference numbersISO 17129:2006(E)IDF 206:2006(E)INTERNATI
8、ONAL STANDARD ISO17129IDF206First edition2006-07-01Milk powder Determination of soy and pea proteins using capillary electrophoresis in the presence of sodium dodecyl sulfate (SDS-CE) Screening method Lait en poudre Dtermination des protines de soja et de pois par lectrophorse capillaire en prsence
9、de dodcyl sulfate de sodium (SDS-CE) Mthode de criblage BS ISO 17129:2006ii iiiContents Page Foreword iv Foreword. v Introduction . vi 1 Scope . 1 2 Normative references . 1 3 Terms and definitions. 1 4 Principle. 1 5 Reagents 1 6 Apparatus 2 7 Sampling 3 8 Preparation 3 8.1 Protein content of test
10、sample 3 8.2 Preparation of test portion. 3 9 Procedure 4 9.1 Operating conditions 4 9.2 Testing instrument suitability 4 9.3 Qualitative analysis 5 9.4 Quantitative analysis 5 10 Calculation and expression of results 6 10.1 Qualitative analysis 6 10.2 Quantitative analysis 7 11 Precision 7 11.1 Int
11、erlaboratory test . 7 11.2 Repeatability 7 11.3 Reproducibility 7 12 Test report . 8 Annex A (informative) Examples of electropherograms 9 Annex B (informative) Interlaboratory trials 11 Bibliography . 13 BS ISO 17129:2006iv Foreword ISO (the International Organization for Standardization) is a worl
12、dwide federation of national standards bodies (ISO member bodies). 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
13、that committee. International organizations, governmental and non-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 i
14、n accordance with the rules given in the ISO/IEC Directives, Part 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
15、 requires approval by at least 75 % of the member bodies casting 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 17129|IDF 206 was prepar
16、ed by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF). It is being published jointly by ISO and IDF. BS ISO 17129:2006vForeword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector
17、 with a National Committee in every member country. Every National Committee has the right to be represented on the IDF Standing Committees carrying out the technical work. IDF collaborates with ISO in the development of standard methods of analysis and sampling for milk and milk products. Draft Int
18、ernational Standards adopted by the Action Teams and Standing Committees are circulated to the National Committees for voting. Publication as an International Standard requires approval by at least 50 % of the IDF National Committees casting a vote. Attention is drawn to the possibility that some of
19、 the elements of this document may be the subject of patent rights. IDF shall not be held responsible for identifying any or all such patent rights. BS ISO 17129:2006vi Introduction The compositional standards on many milk products require that they contain no other proteins than milk proteins. Howe
20、ver, despite the fact that the low prices of some non-milk proteins could make them attractive as potential adulterants, there are at present no standardized methods for their determination in milk powders and other dairy products. The method in this International Standard has been developed with th
21、e following two objectives: a) to cover the need to control the authenticity of products; b) to assist, through its application, in controlling and restraining fraud. The choice and the first assessment of the method were carried out within the EU project SMT4-CT97-2205 (IV Frame Work). BS ISO 17129
22、:20061Milk powder Determination of soy and pea proteins using capillary electrophoresis in the presence of sodium dodecyl sulfate (SDS-CE) Screening method 1 Scope This International Standard describes a method for the determination of the soy and pea protein isolates in low-heat milk powder, using
23、capillary electrophoresis in the presence of sodium dodecyl sulfate (SDS-CE). The method is not suitable for detecting the presence of hydrolysed plant proteins in milk powder. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated
24、 references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 14891|IDF 185, Milk and milk products Determination of nitrogen content Routine method using combustion according to the Dumas principle ISO 8968
25、|IDF 20 (all parts), Milk Determination of nitrogen content 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 soy and pea proteins mass fraction of soy and pea proteins determined by the procedure specified in this International Standard NOTE T
26、he soy and pea proteins are expressed as a mass fraction of the total protein content of the sample. The factor for calculation of total proteins of a test sample is 6,38 N %, where N is the nitrogen content. 4 Principle The milk proteins present in a test portion are removed selectively by using te
27、traborate EDTA buffer to enhance the detection of small amounts of added plant protein. A tris-HCl buffer is added in the presence of sodium dodecyl sulfate and a reducing agent to dissolve the precipitate, in order to dissociate proteins and disrupt any protein aggregates formed by S-S bonds. The p
28、roteins are separated and determined by capillary electrophoresis. The amount of plant proteins is quantified by previous calibration. 5 Reagents Use only reagents of recognized analytical grade, unless otherwise specified, and double-distilled or demineralized water or water of equivalent purity. B
29、S ISO 17129:20062 5.1 Extraction buffer Dissolve 1,14 g of disodium tetraborate decahydrate (Na2B4O710H2O) and 1,49 g of ethylene-diaminetetraacetic acid disodium salt dihydrate (EDTA; C10H14N2Na2O82H2O) in a measuring cylinder (6.1) in about 80 ml of water. Dilute with water to 100 ml and mix. Chec
30、k whether the pH of the extraction buffer is 8,3 0,1. If the final pH value is outside that range, repeat the preparation of the extraction buffer while changing all reagents. The adjustment of pH by chemical agents is not acceptable. 5.2 Sample buffer Dissolve 606 mg of tris(hydroxymethyl)aminometh
31、ane (C4H11NO3), 1,00 g of dodecyl sulfate sodium salt, (SDS; C12H25O4SNa) and 37 mg of EDTA (C10H14N2Na2O82H2O) in a measuring cylinder (6.1) in about 80 ml water and mix. Add 14,7 ml of hydrochloric acid with concentration c(HCl) = 0,1 mol/l, and 2 ml of 2-mercaptoethanol. Dilute with water to 100
32、ml and mix. Check whether the pH of the sample buffer is 8,7 0,1. If the final pH value is outside this range, repeat the preparation of the sample buffer while changing all reagents. The adjustment of pH by chemical agents is not feasible. 5.3 Electrophoresis buffer, for example Beckman eCAPSDS 14-
33、200 gel buffer1)or equivalent. 5.4 Sodium hydroxide solution, c(NaOH) = 0,1 mol/l. 5.5 Reference test mix, containing proteins with molecular mass between 10 kDa and 200 kDa. NOTE The reference test mix can be obtained as a commercially available mixture. 5.6 Reference sample calibrant, with a known
34、 percentage of plant protein, supplied by NIZO (Ede, NL)2). The protein content of the calibrant will also be known. 6 Apparatus Usual laboratory apparatus and, in particular, the following. 6.1 Graduated measuring cylinder, of capacity up to 100 ml. 6.2 Pasteur pipette. 6.3 Micro-vial, with screw c
35、ap, of capacity 1,5 ml. 6.4 Analytical balance, capable of weighing to the nearest 0,1 mg. 6.5 Centrifuge, capable of spinning with radial acceleration of up to 6 500 g. 6.6 pH meter, of minimum sensitivity 0,1 pH unit, with a glass electrode and a suitable reference electrode, with temperature comp
36、ensation. 1) Beckman eCAPSDS14-200 gel buffer is an example of a suitable product available commercially. 2) Address: NIZO Food Research B.V., P.O. Box 20, 6710 BA, Ede, The Netherlands. This information is given for the convenience of users of this International Standard and does not constitute an
37、endorsement by ISO or IDF of the mentioned products. BS ISO 17129:200636.7 Vortex mixer. 6.8 Thermomixer, Eppendorf 54363)or similar. 6.9 Capillary electrophoresis instrument, with linear voltage gradient. 6.9.1 Columns, hydrophilically coated fused-silica capillary, of effective length about 20 cm
38、(injector to detector) and internal diameter of 75 m. 6.9.2 UV detector, capable of measuring at approximately 214 nm. 6.9.3 Software, capable of subtracting the blank value from the sample run. 6.10 Data system, capable of producing information required in Clauses 9 and 10. 7 Sampling A representat
39、ive sample should have been sent to the laboratory. It should not have been damaged or changed during transport or storage. Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 707|IDF 50. 8 Preparation 8.1 Protein content of test
40、 sample Determine the total protein content of the test samples by using the method specified in ISO 14891|IDF 185 or the method specified in the relevant part of ISO 8968|IDF 20. 8.2 Preparation of test portion If the protein content of the test sample is between a mass fraction of 30 % and 40 %, w
41、eigh, to the nearest 0,1 mg, 126 mg of test sample in a micro-vial with screw cap (6.3). If the protein content of the test sample is outside this range, change the mass of the sample proportionally. Add 1 ml of extraction buffer (5.1) to the test portion in the micro-vial. Mix the obtained solution
42、 with the vortex mixer (6.7) at about 2 500 r/min for 1,5 min. Allow to stand for 5 min then mix the solution again for 1,5 min. Centrifuge the mixture at 6 500 g for 30 min. Use a Pasteur pipette to carefully remove the supernatant from the residue. Wash the residue with 1 ml of extraction buffer (
43、5.1). Centrifuge the washed residue at 6 500 g for 20 min. Use a Pasteur pipette to carefully remove the supernatant. Wash the remaining residue once more by the same procedure. Add 250 l of sample buffer (5.2) to the washed residue in the micro-vial. Close the vial and heat it at 95 C for 10 min wh
44、ile stirring with the thermomixer (6.8) set at about 1 000 r/min. Cool the vial in cold (ice) water or on ice. After cooling, centrifuge the vial and its contents again at 3 000 g for 5 min. Transfer about 200 l of the clear supernatant to a suitable injection vial to use as the test solution. 3) Ep
45、pendorf Thermomixer 5436 is an example of a suitable product available commercially. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO or IDF of the mentioned product. BS ISO 17129:20064 After heating, the sample in th
46、e micro-vial may also be stored in a freezer. The above centrifuging step may be omitted if the contents of the micro-vial are already visually clear. 9 Procedure 9.1 Operating conditions 9.1.1 Running conditions for capillary electrophoresis (CE) Reserve a capillary column (6.9.1) to be used for th
47、is analytical procedure only. If new, flush the column with water. Before each series of analyses, flush the used capillary with sodium hydroxide solution (5.4) for 0,5 min, followed by flushing with electrophoresis buffer (5.3) for 5 min. Before each separation, flush the capillary again, preferabl
48、y in the reversed direction, with electrophoresis buffer (5.3) at 241 325 Pa (35 psi) for 1 min. Migrations are run at 25 C. Start the electrophoresis with the capillary electrophoresis instrument (6.9) set at a voltage of 2 kV followed by a linear voltage gradient from 2 kV to 7 kV over 1,7 min. Th
49、en keep the voltage constant at 7 kV up to a total electrophoresis time period of 16 min. The current should be about 20 A and the instrument shall be grounded at the injector side. Set the detector at 214 nm, the data collection at 2 Hz and the rise time at 0,5 s. On the basis of instrumental characteristics, operators should adapt the operating conditions in order to obtain comparable qualitative separations. 9.1.2 Injection Inject the test solution (8.2) at 3 447,5 Pa (0,5 psi) for 60 s. Then dip the in
