1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS ISO 26462:2010Milk Determination oflactose content Enzymaticmethod using difference in pHBS ISO 26462:2010 BRITISH STANDARDNational forewordThis British Standard is the UK imp
2、lementation of ISO 26462:2010.The UK participation in its preparation was entrusted to TechnicalCommittee AW/5, Chemical analysis of milk and milk products.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not purport to include al
3、l the necessaryprovisions of a contract. Users are responsible for its correctapplication. BSI 2010ISBN 978 0 580 56535 9ICS 67.100.10Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Str
4、ategy Committee on 31 July 2010Amendments issued since publicationDate Text affectedBS ISO 26462:2010Reference numbersISO 26462:2010(E)IDF 214:2010(E)ISO and IDF 2010INTERNATIONAL STANDARD ISO26462IDF214First edition2010-06-15Milk Determination of lactose content Enzymatic method using difference in
5、 pH Lait Dtermination de la teneur en lactose Mthode enzymatique par pH-mtrie diffrentielle BS ISO 26462:2010ISO 26462:2010(E) IDF 214:2010(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
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10、neva 20 Diamant Building Boulevard Auguste Reyers 80 B-1030 Brussels Tel. + 41 22 749 01 11 Tel. + 32 2 733 98 88 Fax + 41 22 749 09 47 Fax + 32 2 733 04 13 E-mail copyrightiso.org E-mail infofil-idf.org Web www.iso.org Web www.fil-idf.org Published in Switzerland ii ISO and IDF 2010 All rights rese
11、rvedBS ISO 26462:2010ISO 26462:2010(E) IDF 214:2010(E) ISO and IDF 2010 All rights reserved iiiContents Page Foreword iv Foreword .v 1 Scope1 2 Terms and definitions .1 3 Principle .1 4 Reagents 1 5 Apparatus.3 6 Sampling 3 7 Preparation of test sample .3 8 Procedure.3 8.1 General .3 8.2 Blank deter
12、mination4 8.3 Calibration4 8.4 Checking the calibration.5 8.5 Determination 5 8.6 Checking the stability .5 8.7 Cleaning procedure.5 9 Maintenance of the electrodes.6 9.1 Regeneration6 9.2 Strong regeneration 6 10 Calculation and expression of results 6 10.1 Calculation .6 10.2 Expression of results
13、6 11 Precision 7 11.1 Interlaboratory test7 11.2 Repeatability 7 11.3 Reproducibility 7 12 Test report7 Annex A (informative) Basic diagram of a differential pH apparatus 8 Annex B (informative) Collaborative trial .9 Annex C (informative) Comparison between the pH and HPLC methods.10 Bibliography11
14、 BS ISO 26462:2010ISO 26462:2010(E) IDF 214:2010(E) iv ISO and IDF 2010 All rights reservedForeword ISO (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 o
15、ut 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-governmental, in liaison with ISO, also take part in the work. ISO c
16、ollaborates 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 2. The main task of technical committees is to prepare International
17、 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 a vote. Attention is drawn to the possibility that some of the elemen
18、ts 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 26462IDF 214 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (
19、IDF). It is being published jointly by ISO and IDF. BS ISO 26462:2010ISO 26462:2010(E) IDF 214:2010(E) ISO and IDF 2010 All rights reserved vForeword IDF (the International Dairy Federation) is a non-profit organization representing the dairy sector worldwide. IDF membership comprises National Commi
20、ttees in every member country as well as regional dairy associations having signed a formal agreement on cooperation with IDF. All members of IDF have the right to be represented on the IDF Standing Committees carrying out the technical work. IDF collaborates with ISO in the development of standard
21、methods of analysis and sampling for milk and milk products. The main task of Standing Committees is to prepare International Standards. Draft International Standards adopted by the Standing Committees are circulated to the National Committees for endorsement prior to publication as an International
22、 Standard. Publication as an International Standard requires approval by at least 50 % of 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 an
23、y or all such patent rights. ISO 26462IDF 214 was prepared by the International Dairy Federation (IDF) and Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published jointly by IDF and ISO. All work was carried out by the ISO-IDF Joint Project Grou
24、p on Enzymatic determination of lactose of the Standing Committee on Analytical methods for composition under the aegis of its project leader, Mr. P. Trossat (FR). BS ISO 26462:2010BS ISO 26462:2010INTERNATIONAL STANDARD ISO 26462:2010(E)IDF 214:2010(E) ISO and IDF 2010 All rights reserved 1Milk Det
25、ermination of lactose content Enzymatic method using difference in pH 1 Scope This International Standard specifies an enzymatic method for the determination of the lactose content of milk and reconstituted milk by measurement of the difference in pH (differential pH measurement). 2 Terms and defini
26、tions For the purpose of this International Standard, the following terms and definitions apply. 2.1 lactose content in milk amount of substance concentration of compounds determined by the procedure specified in this International Standard NOTE The lactose content of milk is expressed in millimoles
27、 per litre. For conversion of the result into other units, see Table 1. 2.2 unit of enzyme activity international unit standard unit U amount of enzyme which catalyses the transformation of one micromole of substrate per minute under standard conditions 3 Principle -Galactosidase is added to cleave
28、lactose into glucose and galactose. At pH 7,8, glucose is phosphorylated by glucokinase, thereby releasing protons that induce a change in pH. The pH change varies as a function of the lactose content of the sample and is measured by using a differential pH analyser. 4 Reagents During the analysis,
29、unless otherwise stated, use only reagents of recognized analytical grade and distilled or demineralized water or water of equivalent purity. 4.1 Buffer solution, pH 7,8 Dissolve 0,242 g of tris(hydroxymethyl)methylamine (tris), 0,787 g of adenosine 5-triphosphate disodium salt (ATP), 0,304 g of tri
30、sodium phosphate (Na3PO412H2O), 0,009 g of sodium hydroxide (NaOH), 0,203 g of BS ISO 26462:2010ISO 26462:2010(E) IDF 214:2010(E) 2 ISO and IDF 2010 All rights reservedmagnesium chloride hexahydrate (MgCl26H2O), 2 g of octylphenoxypolyethoxyethanol e.g. Triton X1001), 0,820 g of potassium chloride (
31、KCl) and 0,010 g of 2-bromo-2-nitropropan-1,3-diol e.g. Bronopol1) in a 100 ml beaker containing 50 ml of water under continuous stirring. Adjust the final pH to 7,8 0,1, if needed. Transfer to a 100 ml one-mark volumetric flask (5.4), make up to the mark with water and mix. The buffer solution can
32、be kept for 2 months if stored at 4 C. 4.2 Enzyme solutions 4.2.1 Glucokinase enzyme solution Dissolve 2,57 mg of lyophilized glucokinase-1 (GK1; 1 mg = 350 U; EC 2.7.1.2) in 3 ml of glycerol with a volume fraction of 50 %. The activity of the glucokinase solution obtained shall be 290 U/ml 30 U/ml
33、(see 2.2). The glucokinase enzyme solution can be kept for 6 months if stored at 4 C. 4.2.2 -Galactosidase enzyme solution Dilute a concentrated -galactosidase (EC 3.2.1.23) extract purified from contaminating enzymes with glycerol with a volume fraction of 50 %. The activity of the -galactosidase s
34、olution obtained shall be 1 500 U/ml 200 U/ml. The -galactosidase enzyme solution can be kept for 6 months if stored at 4 C. 4.3 Lactose standard solution (150 mmol/l) Before use, determine the water content of lactose monohydrate powder by a Karl Fischer titration method, in order to correct for th
35、e quantity of lactose monohydrate used for the lactose standard solution. The correction should be based on the percentage of the determined water content in order to prepare a lactose standard solution containing 5,404 g lactose monohydrate per 100 ml. Dissolve 5,404 g lactose monohydrate powder, 0
36、,745 g of potassium chloride (KCl) and 0,01 g of 2-bromo-2-nitropropan-1,3-diol e.g. Bronopol1) in the buffer solution at pH 7,8 (4.1) in a 100 ml one-mark volumetric flask (5.4). Make up to the mark with water and mix. The lactose standard solution can be kept for 6 months if stored at 4 C. 4.4 Cle
37、aning solution Dissolve 1,742 g of dipotassium monohydrogenphosphate (K2HPO4), 1,361 g of potassium dihydrogenphosphate (KH2PO4), 7,455 g of potassium chloride (KCl), 1,00 g of sodium azide (NaN3), 2 g of octylphenoxypolyethoxyethanol, 2 g of polyoxyethyleneglycol dodecylether e.g. Brij 351) and 3 g
38、 of lauryl maltoside e.g. LM1) in a 1 000 ml one-mark volumetric flask (5.4). Make up to the mark with water and mix. The cleaning solution can be kept for 1 year if stored at room temperature. 4.5 Regenerating solution Use a 0,1 mol/l hydrochloric acid (HCl) solution as regenerating solution. The r
39、egenerating solution can be kept for 1 year if stored at room temperature. 1) Example of a suitable product available commercially. This information is given for the convenience of users of this document and does not constitute an endorsement by ISO or IDF of this product. BS ISO 26462:2010ISO 26462
40、:2010(E) IDF 214:2010(E) ISO and IDF 2010 All rights reserved 34.6 Strong regenerating solution DANGER The use of sodium fluoride (NaF) alone and in combination with HCl may cause health problems due to inhalation and/or skin absorption. This International Standard does not purport to address all th
41、e safety problems, if any, associated with its use. It is the responsibility of the user to establish appropriate safety and health practices and to ensure compliance with any national regulatory conditions. Dissolve 30 g of nitric acid (HNO3) with a mass fraction, w(HNO3) 69 %, 30 g of hydrochloric
42、 acid (HCl) with a mass fraction, w(HCl) 37 %, 30 g of sodium fluoride (NaF), and 1 g of octylphenoxypolyethoxyethanol in a 1 000 ml one-mark volumetric flask (5.6). Make up to the mark with water and mix. The strong regeneration solution can be kept for 1 year if stored in non-corroding material at
43、 room temperature. 5 Apparatus Usual laboratory equipment and, in particular, the following. 5.1 Analytical balance, capable of weighing to the nearest 1 mg. 5.2 Micropipettes, capacity 20 l, ISO 75505, with positive displacement. 5.3 Water bath, capable of maintaining a temperature of 38 C 1 C. 5.4
44、 One-mark volumetric flasks, capacities 100 ml and 1 000 ml, ISO 10422class A. 5.5 Differential pH apparatus, shown schematically in Figure A.1. The differential pH apparatus consists of peristaltic pumps to circulate liquids, a mixing chamber, two glass capillary flow-through electrodes (E1 and E2)
45、, and an electronic system for measurement. 5.6 One-mark volumetric flasks, capacity 1 000 ml and of material capable of storing the extremely corrosive strong regenerating solution (4.6). 6 Sampling Sampling is not part of the method specified in this International Standard. A recommended sampling
46、method is given in ISO 707IDF 501. It is important that the laboratory receive a truly representative sample which has not been damaged or changed during transport or storage. 7 Preparation of test sample Warm the test sample to 38 C in the water bath (5.3) while mixing. Cool the sample to 20 C, bef
47、ore preparing the test portion. 8 Procedure 8.1 General Since the various types of differential pH apparatus (5.5) available differ in design and handling, the operator shall carefully follow the instrument manufacturers instructions for setting up, calibration, and operation of the instrument. Swit
48、ch the instrument on and allow its operating conditions to stabilize. BS ISO 26462:2010ISO 26462:2010(E) IDF 214:2010(E) 4 ISO and IDF 2010 All rights reservedIf the time between two consecutive measurements is 5 min or more, renew the buffer solution (4.1) in the mixing chamber of the apparatus. 8.
49、2 Blank determination Using a micropipette (5.2), add 20 l of glucokinase enzyme solution (4.2.1) into the mixing chamber of the differential pH apparatus (5.5). Dilute the enzyme solution with buffer solution (4.1) to a total volume of 1 200 l and mix. Fill the flow-through electrodes, E1 and E2 (see Figure A.1), of the pH apparatus (5.5) with the buffer and glucokinase mixture obtained. Measure the offset differential pH, D1, between the two electrodes. The difference betwe
