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1、BRITISH STANDARD BS ISO 17129:2006 Milk powder Determination of soy and pea proteins using capillary electrophoresis in the presence of sodium dodecyl sulfate (SDS-CE) Screening method ICS 67.100.01 ? Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:46:18 GMT+00:00 2006, Uncontrolled Copy,
2、(c) BSI BS ISO 17129:2006 This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 July 2006 BSI 2006 ISBN 0 580 49025 4 National foreword This British Standard reproduces verbatim ISO 17129:2006 and implements it as the UK national standard. The U
3、K participation in its preparation was entrusted to Technical Committee AW/5, Milk and milk products, which has the responsibility to: A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement internation
4、al 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 purport to include all
5、 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. aid enquirers to understand the text; present to the responsible international/European committee any enquiries on
6、 the interpretation, or proposals for change, and keep UK interests informed; monitor related international and European developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the ISO title page, pages ii to vi, pages 1 to 13 and a
7、back cover. The BSI copyright notice displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. DateComments Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:46:18 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Reference numbers ISO 1712
8、9:2006(E) IDF 206:2006(E) INTERNATIONAL STANDARD ISO 17129 IDF 206 First edition 2006-07-01 Milk 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 poi
9、s par lectrophorse capillaire en prsence de dodcyl sulfate de sodium (SDS-CE) Mthode de criblage BS ISO 17129:2006 Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:46:18 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ii Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:46:18 GMT+00:00 2
10、006, Uncontrolled Copy, (c) BSI iii Contents 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 sample 3 8.2 Preparation of test portion. 3 9
11、 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 Interlaboratory test. 7 11.2 Repeatability 7 11.
12、3 Reproducibility 7 12 Test report . 8 Annex A (informative) Examples of electropherograms 9 Annex B (informative) Interlaboratory trials 11 Bibliography. 13 BS ISO 17129:2006 Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:46:18 GMT+00:00 2006, Uncontrolled Copy, (c) BSI iv Foreword ISO (
13、the International Organization for Standardization) is a worldwide 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 commi
14、ttee has been established has the right to be represented on 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 electrotec
15、hnical standardization. International Standards are drafted in 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 membe
16、r bodies for voting. Publication as an International Standard 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 identifyin
17、g any or all such patent rights. ISO 17129|IDF 206 was prepared 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 17129:2006 Licensed Copy: sheffieldun sheffie
18、ldun, na, Mon Nov 27 03:46:18 GMT+00:00 2006, Uncontrolled Copy, (c) BSI v Foreword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every member country. Every National Committee has the right to be represented on the IDF Standing C
19、ommittees 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 International Standards adopted by the Action Teams and Standing Committees are circulated to the National Committees for voting. Pub
20、lication 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 the elements of this document may be the subject of patent rights. IDF shall not be held responsible for identifying any or all s
21、uch patent rights. BS ISO 17129:2006 Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:46:18 GMT+00:00 2006, Uncontrolled Copy, (c) BSI vi Introduction The compositional standards on many milk products require that they contain no other proteins than milk proteins. However, despite the fact
22、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 the following two object
23、ives: 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 17129:2006 Licensed Copy: s
24、heffieldun sheffieldun, na, Mon Nov 27 03:46:18 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 1 Milk 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 fo
25、r the determination of the soy and pea protein isolates in low-heat milk powder, using 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
26、 referenced documents are indispensable for the application of this document. For dated 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 nit
27、rogen content Routine method using combustion according to the Dumas principle ISO 8968|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 p
28、ea proteins determined by the procedure specified in this International Standard NOTE The 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 Pr
29、inciple The milk proteins present in a test portion are removed selectively by using tetraborate 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 ord
30、er to dissociate proteins and disrupt any protein aggregates formed by S-S bonds. The proteins 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
31、specified, and double-distilled or demineralized water or water of equivalent purity. BS ISO 17129:2006 Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:46:18 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 2 5.1 Extraction buffer Dissolve 1,14 g of disodium tetraborate decahydrate (Na2B4O710H2O
32、) 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. Check whether the pH of the extraction buffer is 8,3 0,1. If the final pH value is outside that range, repea
33、t 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)aminomethane (C4H11NO3), 1,00 g of dodecyl sulfate sodium salt, (SDS; C12H25O4SNa) and 37 mg of EDTA (C10H14N2Na2
34、O82H2O) 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 ml and mix. Check whether the pH of the sample buffer is 8,7 0,1. If the final pH value is outside this
35、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 14-200 gel buffer1) or equivalent. 5.4 Sodium hydroxide solution, c(NaOH) = 0,1 mol/l. 5.5 Reference test m
36、ix, 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 percentage of plant protein, supplied by NIZO (Ede, NL)2). The protein content of the calibrant will a
37、lso 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 cap, of capacity 1,5 ml. 6.4 Analytical balance, capable of weighing to the nearest 0,1 mg. 6.5 Centrifu
38、ge, 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 compensation. 1) Beckman eCAPSDS14-200 gel buffer is an example of a suitable product available commerciall
39、y. 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 endorsement by ISO or IDF of the mentioned products. BS ISO 17129:2006 Licensed Copy: sheffieldun sheff
40、ieldun, na, Mon Nov 27 03:46:18 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 3 6.7 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
41、 20 cm (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 repr
42、esentative 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
43、of test 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
44、40 %, weigh, 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 s
45、olution 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 b
46、uffer (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
47、 min while 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
48、. 3) Eppendorf 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 17129:2006 Licensed Copy: sheffieldu
49、n sheffieldun, na, Mon Nov 27 03:46:18 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 4 After heating, the sample in the 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 this analytical procedure only. If new, flush the column wi
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