1、BRITISH STANDARDBS ISO 13875:2005Liquid milk Determination of acid-soluble -lactoglobulin content Reverse-phase HPLC methodICS 67.100.10g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g3
2、8g50g51g60g53g44g42g43g55g3g47g36g58BS ISO 13875:2005This British Standard was published under the authority of the Standards Policy and Strategy Committee on 18 November 2005 BSI 18 November 2005ISBN 0 580 46215 3National forewordThis British Standard reproduces verbatim ISO 13875:2005 and implemen
3、ts 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: A list of organizations represented on this committee can be obtained on request to its secretary.Cross-referencesThe British
4、Standards which 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 publica
5、tion does not 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. aid enquirers to understand the text; present to the responsible international
6、/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.Summary of pagesThis document comprises a front cover, an inside front cover, the ISO title page, pa
7、ges ii to v, a blank page, pages 1 to 14, an inside back cover 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 CommentsReference numbersISO 13875:2005(E)IDF 178:2005(E)INTERNATIONALSTAND
8、ARDISO13875IDF178First edition2005-02-01Liquid milk Determination of acid-soluble E-lactoglobulin content Reverse-phase HPLC method Lait liquide Dtermination de la teneur en E-lactoglobuline soluble dans lacide Mthode par chromatographie liquide haute performance en phase inverse BS ISO 13875:2005ii
9、iiiContents PageForeword iv1 Scope 12 Normative references . 13 Terms and definitions. 14 Principle . 15 Reagents 16 Apparatus. 27 Sampling 38 Procedure. 38.1 Preparation of test portion. 38.2 Preparation of test solution . 48.3 Preparation of reference portion . 48.4 Preparation of reference soluti
10、ons for the multi-point calibration 48.5 Preparation of the reference solution for the “single-point” calibration procedure . 58.6 HPLC determination 58.7 Integration mode . 69 Calculation and expression of results 89.1 Multi-point calibration. 89.2 Single-point calibration 99.3 Expression of result
11、s 910 Standardization of the reference sample 1010.1 General . 1010.2 Preparation of the standard sample 1010.3 Determination of protein content 1010.4 Determination of E-LG content in the reference sample. 1010.5 Calculation of the E-LG content. 1010.6 Expression of results 1111 Precision 1111.1 In
12、terlaboratory test . 1111.2 Repeatability 1111.3 Reproducibility 1112 Test report 12Annex A (informative) Results of interlaboratory trials 13Bibliography . 14BS ISO 13875:2005ivForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (
13、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 that committee. International organizations, go
14、vernmental 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 in accordance with the rules given in the ISO/IE
15、C 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 requires approval by at least 75 % of the memb
16、er 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 13875IDF 178 was prepared by Technical Committee ISO/TC 34, Food produc
17、ts, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC International. It is being published jointly by ISO and IDF and separately by AOAC International. BS ISO 13875:2005vForeword IDF (the International Dairy Federation) is a worldwide
18、 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 Committees carrying out the technical work. IDF collaborates with ISO and AOAC International in the development of standard methods of analy
19、sis 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. Publication as an International Standard requires approval by at least 50 % of the National Committees casting a vote.
20、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 such patent rights. ISO 13875IDF 178 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Mil
21、k and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC International. It is being published jointly by ISO and IDF and separately by AOAC International. All work was carried out by the Joint ISO/IDF/AOAC Action Team, Characterization of heat treatment, of the S
22、tanding Committee on Minor components and characterization of physical properties, under the aegis of its project leader, Prof. L. Pellegrino (IT). BS ISO 13875:2005blank1Liquid milk Determination of acid-soluble E-lactoglobulin content Reverse-phase HPLC method 1 Scope This International Standard s
23、pecifies a method for the quantitative determination of the E-lactoglobulin content, soluble at pH 4,6, in liquid milk. The method has been tested over a range between 0 mg and 3 500 mg of E-lactoglobulin per litre of milk. It is suitable for distinguishing different categories of heat-treated liqui
24、d milk. 2 Normative references The following 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 8968-1IDF 20-1
25、, Milk Determination of nitrogen content Part 1: Kjeldahl methodISO 8968-2IDF 20-2, Milk Determination of nitrogen content Part 2: Block-digestion method (Macro method)3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1E-lactoglobulin content E-L
26、G content mass fraction of substance determined by the procedure specified in this International Standard NOTE It is expressed in milligrams per litre of test sample. 4 Principle Casein and denatured whey protein are precipitated isoelectrically from milk at pH 4,6. The acid whey is separated by cen
27、trifuging and filtering. The acid-soluble E-LG content in the acid whey is determined by reverse-phase HPLC. The soluble E-LG content in the test sample is quantified by single-point or a multi-point calibration using a reference sample. 5 Reagents Use only reagents of recognized analytical grade an
28、d distilled water or water of at least equivalent purity, unless otherwise specified. 5.1 Standard sample, pure E-lactoglobulin (A+B genetic variants). BS ISO 13875:20052Test the chromatographic purity of the E-LG standard sample by the HPLC procedure described in Clause 10. Determine its precise co
29、ncentration as described in 10.2. 5.2 Reference sampleThe reference sample is reconstituted from a freeze-dried raw bulk milk sample which was originally prepared from a skimmed raw bulk milk sample. It contains a known amount of soluble E-LG (A+B), which is determined by the HPLC procedure describe
30、d in Clause 10. The freeze-dried reference sample may be stored at 4 C for 6 months, preventing hydration. 5.3 Reagents for sample preparation5.3.1 Hydrochloric acid, dilute, c(HCl) = 2 mol/l. 5.3.2 Phosphate buffer solution, of pH 6,7 (final concentration 0,1 mol/l). Add 57 ml of 0,2 mol/l sodium d
31、ihydrogen orthophosphate (NaH2PO4) solution to a 200 ml volumetric flask (6.10). Add 43 ml of 0,2 mol/l disodium hydrogen orthophosphate (Na2HPO4) solution and mix the phosphate solutions. Dilute to the mark with water and mix again. 5.4 HPLC elution solventsUse elution solvents prepared from reagen
32、ts of recognized HPLC-grade. SAFETY PRECAUTIONS Take appropriate safety precautions when handling the elution solvents as the chemicals may be carcinogenic. 5.4.1 Water, of HPLC-grade. Laboratory-prepared water may be not sufficiently pure. Impure water produces column contamination and loss of reso
33、lution. If impure or improperly stored trifluoracetic acid is used, peaks can be unresolved or absent from the chromatogram. 5.4.2 Acetonitrile (CH3CN).5.4.3 Trifluoracetic acid (CF3COOH), of the highest purity. 6 ApparatusUsual laboratory equipment and, in particular, the following. 6.1 pH-meter, c
34、alibrated over the pH range 4,0 to 7,0, and accurate to 0,1 pH units. 6.2 Centrifuge, capable of operating at 2 000 g.6.3 Centrifuge glass tubes, of capacity about 30 ml. 6.4 Glass vials, of capacity about 5 ml. 6.5 Glass funnels, of diameter about 7 cm. 6.6 Filter paper, fast grade, of diameter abo
35、ut 11 cm. 6.7 Glass test tubes, of capacity about 30 ml. 6.8 One-mark pipettes, capable of delivering 1 ml, 2 ml and 5 ml. BS ISO 13875:200536.9 Beakers, of capacities 50 ml and 100 ml. 6.10 One-mark volumetric flasks, of capacities 10 ml, 20 ml, 25 ml, 50 ml and 200 ml. 6.11 Microfiltration tools.6
36、.11.1 Glass syringe, of capacity 5 ml. 6.11.2 Disposable syringe filter units, of pore size 0,22 m, used with aqueous solutions. 6.12 Analytical balance, capable of weighing to the nearest 1 mg, with a readability of 0,1 mg. 6.13 Magnetic stirrer.6.14 HPLC equipment.6.14.1 Elution gradient pumping s
37、ystem, capable of operating at 1,0 ml/min at 200 bar. 6.14.2 Manual or automatic injector, capable of injecting 20 l.6.14.3 Column heater, capable of maintaining the column at 40 C r 1C. 6.14.4 UV detector, capable of operating at 205 nm or at 280 nm wavelength and 0,1 AUFS. 6.14.5 Integrator or dat
38、a-reprocessing software, capable of measuring peak areas. 6.14.6 PLRP-S column1), of length 150 mm and internal diameter 4,6 mm, of particle size 5 m or 8 m, and pore size 30 nm; or an equivalent column packed with underivatized polystyrene divinyl benzene, giving an equivalent chromatographic patte
39、rn. 7 SamplingA representative 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. 8 Procedure8.1 Prepar
40、ation of test portion 8.1.1 Check that the reported expiry date of the test sample has not been passed. Bring the closed package of the test sample to 20 C r 2 C. Just before opening, shake the package and its contents carefully by inversion. Open the package and transfer about 50 ml of test sample
41、to a 100 ml beaker (6.9). The test sample package should not be opened until just before starting the preparation. 1) PLRP-S column is the trade name of a product supplied by Polymer Laboratories Ltd, Church Stretton, United Kingdom. This information is given for the convenience of users of this Int
42、ernational Standard and does not constitute an endorsement by ISO or IDF of this product. Equivalent products may be used if they can be shown to lead to the same results. BS ISO 13875:200548.1.2 Adjust the pH of the test portion to 4,6 by dropwise addition of the dilute hydrochloric acid (5.3.1) wh
43、ile stirring continuously. Allow the test portion to stand for 20 min at room temperature. Transfer the prepared test portion to a centrifuge tube (6.3) and centrifuge at 2 000 g for 20 min. Filter the supernatant through filter paper (6.6), collecting the casein-free acid whey in a test tube (6.7).
44、 The undiluted acid whey test portion may be stored for 24 h at 4 C or for 2 weeks at 18 C. Once defrosted, the acid whey test portion shall not be refrozen. 8.2 Preparation of test solution After defrosting, carefully mix the acid whey test portion. Using a pipette (6.8), transfer suitable amounts
45、of the acid whey test portion, depending on the type of test sample, to a 10 ml volumetric flask (6.10): a) 1 ml, if from a test sample of raw or pasteurized or high-temperature pasteurized milk (final dilution 1:10); b) 2 ml, if prepared from a test sample of UHT milk (final dilution 1:5); c) 5 ml,
46、 if prepared from a test sample of bottle-sterilized milk (final dilution 1:2). Dilute the test portion to the mark with the phosphate buffer solution (5.3.2). Mix carefully by inversion and allow to stand for 1 h. Mix again and filter using the microfiltration tools (6.11). Discard the first few mi
47、llilitres of filtrate. Collect the rest of the filtrate in a glass vial (6.4). The diluted acid whey solution may be stored at 4 C but shall be analysed within 24 h. 8.3 Preparation of reference portion Weigh, to the nearest 0,01 g, 2,50 g of reference sample (5.2) into a 30 ml beaker (6.9). Add 10
48、ml of distilled water at 40 C. Stir using a stirring rod (6.13) in order to dissolve any lumps. Quantitatively transfer the reconstituted reference sample to a 25 ml volumetric flask (6.10). Dilute to the mark with distilled water and mix thoroughly. Quantitatively transfer the 25 ml of reference so
49、lution to a 50 ml beaker (6.9). Prepare the acid whey reference portion as described in 8.1.2 for the test portion. Standardize the reference sample periodically as described in Clause 10. 8.4 Preparation of reference solutions for the multi-point calibration After defrosting, carefully mix the acid whey reference portion. Pipette 2 ml of the reference portion into a 20 ml volumetric flask (6.10) (marked D). Dilute to the mark with phosphate buffer solution (5.3.2) and mix. Immediately pipette 1 ml, 2 ml and 5 ml
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