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1、 Reference numbers ISO 5765-1:2002(E) IDF 79-1:2002(E) ISO and IDF 2002 INTERNATIONAL STANDARD ISO 5765-1 IDF 79-1 First edition 2002-09-01 Dried milk, dried ice-mixes and processed cheese Determination of lactose content Part 1: Enzymatic method utilizing the glucose moiety of the lactose Lait sec,
2、 mlanges secs pour crmes glaces et fromages fondus Dtermination de la teneur en lactose Partie 1: Mthode enzymatique par la voie glucose Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/1
3、9/2007 03:30:05 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 5765-1:2002(E) IDF 79-1:2002(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 edited unless the
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6、mber bodies and IDF national committees. In the unlikely event that a problem relating to it is found, please inform the ISO Central Secretariat at the address given below. ISO and IDF 2002 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in
7、any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO or IDF at the respective address below. ISO copyright office International Dairy Federation Case postale 56 CH-1211 Geneva 20 Diamant Building Boulevard Auguste Rey
8、ers 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 Printed in Switzerland ii ISO and IDF 2002 All rights reserved Copyright International Organization for St
9、andardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/19/2007 03:30:05 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 5765-1:2002(E) IDF 79-1:2002(E) ISO 2002 All rights reserved iii Contents Foreword iv
10、Foreword. v Introduction vi 1 Scope 1 2 Term and definition. 1 3 Principle . 1 4 Reagents 2 5 Apparatus. 3 6 Sampling 4 7 Procedure. 4 7.1 Test to check the procedure 4 7.2 Preparation of test sample. 5 7.3 Test portion 5 7.4 Reagent blank test 5 7.5 Deproteination. 5 7.6 Determination 6 8 Calculati
11、on and expression of results 7 8.1 Calculation. 7 8.2 Expression of results 8 9 Precision 8 9.1 Interlaboratory trial . 8 9.2 Repeatability 8 9.3 Reproducibility 9 10 Test report 9 Annex A (normative) Good Laboratory Practice (GLP) rules for the performance of enzymatic analyses 10 Bibliography 14 C
12、opyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/19/2007 03:30:05 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 5765-1:2002(E) IDF 79-1:2002(E) iv ISO 2002
13、All rights reserved Foreword 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 out through ISO technical committees. Each member body interested in a s
14、ubject 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 collaborates closely with the International Electrotechnical Commission
15、(IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical comm
16、ittees 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 elements of this part of ISO 5765IDF 79 may be the subject of patent rights.
17、ISO shall not be held responsible for identifying any or all such patent rights. ISO 5765-1IDF 79-1 was prepared by Technical Committee ISO/TC 34, Food Products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC International. It is b
18、eing published jointly by ISO and IDF separately by AOAC International. ISO 5765IDF 79 consists of the following parts, under the general title Dried milk, dried ice-mixes and processed cheese Determination of lactose content: Part 1: Enzymatic method utilizing the glucose moiety of the lactose Part
19、 2: Enzymatic method utilizing the galactose moiety of the lactose Annex A forms a normative part of this part of ISO 5765IDF 79. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/19/2007
20、03:30:05 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 5765-1:2002(E) IDF 79-1:2002(E) ISO 2002 All rights reserved v Foreword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every member country. Eve
21、ry 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 analysis and sampling for milk and milk products. Draft International Standards adopted by
22、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 IDF National Committees casting a vote. ISO 5765-1IDF 79-1 was prepared by Technical Committee ISO/TC 34, Food products, Subco
23、mmittee 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. ISO 5765IDF 79 consists of the following parts, under the general title Dried milk, dried i
24、ce-mixes and processed cheese Determination of lactose content: Part 1: Enzymatic method utilizing the glucose moiety of the lactose Part 2: Enzymatic method utilizing the galactose moiety of the lactose All work was carried out by the Joint ISO/IDF/AOAC Action Team Lactose and lactate determination
25、, of the Standing Committee on Main components of milk, under the aegis of its project leader, Mr J. Labrijn (NL). Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/19/2007 03:30:05 MDTNo
26、reproduction or networking permitted without license from IHS -,-,- ISO 5765-1:2002(E) IDF 79-1:2002(E) vi ISO 2002 All rights reserved Introduction This part of ISO 5765IDF 79 describes the enzymatic method for the determination of lactose utilizing the glucose moiety of the lactose. It is compleme
27、ntary to ISO 5765-2IDF 79-2 which utilizes the galactose moiety of the lactose. The choice of whether to use the method described in part 1 or part 2 of ISO 5765IDF 79 depends on the amount of glucose or galactose present in the sample to be analysed. If the glucose content of a sample is considerab
28、ly higher than its lactose content, it is recommended to use the method described in ISO 5765-2IDF 79-2. Conversely, for a sample with a considerably higher galactose content than its lactose content, it is recommended to use the method described in this part of ISO 5765IDF 79. For samples with a lo
29、w content of both glucose and galactose, either method may be used without preference. For samples with a high content of both glucose and galactose, the accuracy of the lactose determination is considerably reduced for both methods. In heat-treated milk and milk products, a proportion of lactose ma
30、y have been converted to lactulose. Lactulose cannot be determined by applying the method described in this part of ISO 5765IDF 79. If, however, the method in ISO 5765-2IDF 79-2 is applied, the lactulose will partially be determined as lactose. Moreover, in intensively heat- treated milk (e.g. steri
31、lized milk) or milk products, a proportion of the lactose may be bound to protein because of a Maillard reaction. In such cases the bound lactose cannot be determined by the method described either part of ISO 5765IDF 79. Only when the good laboratory practice (GLP) rules for enzymatic analyses have
32、 been applied strictly, will reliable results be obtained. The GLP rules are stated in annex A. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/19/2007 03:30:05 MDTNo reproduction or net
33、working permitted without license from IHS -,-,- INTERNATIONAL STANDARD ISO 5765-1:2002(E) IDF 79-1:2002(E) ISO 2002 All rights reserved 1 Dried milk, dried ice-mixes and processed cheese Determination of lactose content Part 1: Enzymatic method utilizing the glucose moiety of the lactose 1 Scope Th
34、is part of ISO 5765IDF 79 specifies an enzymatic method for the determination of the lactose content of all types of dried milk, of ice-mixes in dry form in the presence of other carbohydrates and reducing substances, and of processed cheese. 2 Term and definition For the purposes of this part of IS
35、O 5765IDF 79, the following term and definition applies. 2.1 lactose content mass fraction of substances determined by the procedure specified in this part of ISO 5765IDF 79 NOTE The lactose content is expressed as a percentage by mass. 3 Principle 3.1 A solution or suspension of the test portion is
36、 deproteinated to obtain a pure extract. 3.2 The purified extract of the test portion is reacted with the following enzymes and biochemical substances: a) -galactosidase, to split the lactose into glucose and galactose; b) hexokinase and adenosine triphosphate (ATP), to phosphorylate the glucose (bo
37、th that originally present and that liberated by the -galactosidase) to glucose 6-phosphate (G6P); c) glucose 6-phosphate dehydrogenase (G6P-DH) in the presence of nicotinamide adenine dinucleotide phosphate (NADP+), to catalyse the oxidation of G6P to 6-phosphogluconate, the NADP+ being reduced to
38、NADPH. 3.3 The amount of NADPH is determined from the absorbance of the test solution at 340 nm. 3.4 The lactose content is calculated, which is proportional to the amount of NADPH if a correction is made for the glucose present in the test sample at the start of the analysis. Copyright Internationa
39、l Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/19/2007 03:30:05 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 5765-1:2002(E) IDF 79-1:2002(E) 2 ISO 2002 All rights reserved 4
40、Reagents Use only reagents of recognized analytical grade unless otherwise specified. The water used for the preparation of the enzyme solutions shall be of at least doubly glass-distilled purity. The water used for other purposes shall be glass-distilled or of at least equivalent purity. Take note
41、of the production and expiry dates of the reagents given by the manufacturer. If an enzyme suspension is applied with other than the prescribed activity, the volume of the suspension as stated in the pipetting scheme (7.6.1) should be increased or decreased proportionally. NOTE The reagents describe
42、d in 4.3 and in 4.5 to 4.7 inclusive may be obtained commercially as a test combination, for example, Boehringer test kit.1) 4.1 Potassium hexacyanoferrate(II) solution, K4Fe(CN)6 Dissolve 3,6 g of potassium hexacyanoferrate(II) trihydrate in water. Dilute with water to 100 ml and mix. 4.2 Zinc sulf
43、ate solution, ZnSO4 Dissolve 7,2 g of zinc sulfate heptahydrate in water. Dilute with water to 100 ml and mix. 4.3 Sodium hydroxide solution, c(NaOH) = 0,1 mol/l Dissolve 4,0 g of sodium hydroxide in water. Dilute with water to 1 000 ml and mix. 4.4 Citrate buffer solution, pH 6,6 0,1 Dissolve 2,8 g
44、 of trisodium citrate dihydrate (C6H5O7Na32H2O), 0,042 g of citric acid monohydrate (C6H8O7H2O), and 0,625 g of magnesium sulfate heptahydrate (MgSO47H2O) in about 40 ml of water. Adjust the pH to 6,6 0,1 at 20 C with sulfuric acid (2 mol/l) or sodium hydroxide solution (0,1 mol/l). Dilute with wate
45、r to 50 ml and mix. This solution may be kept for 3 months if stored in a refrigerator set at between 0 C and 5 C. 4.5 Triethanolamine (TEA) buffer solution, pH 7,6 0,1 Dissolve 14,0 g of triethanolamine hydrochloride (C6H15NO3HCl) and 0,25 g of magnesium sulfate heptahydrate (MgSO47H2O) in about 80
46、 ml of water. Adjust the pH to 7,6 0,1 at 20 C with about 5 ml of sodium hydroxide solution (5 mol/l). Dilute with water to 100 ml and mix. This solution may be kept for 8 weeks if stored in a refrigerator set at between 0 C and 5 C. 4.6 NADP+/ATP/TEA buffer solution Dissolve 65 mg of nicotinamide a
47、denine dinucleotide phosphate disodium salt C21H26N7O17P3Na2, (98 to 99) % purity and 170 mg of adenosine 5-triphosphate disodium salt C10H14N5O13P3Na2, (99 to 100) % purity in 30 ml of triethanolamine buffer solution (4.6). This solution may be kept for 2 weeks if stored in a refrigerator set at be
48、tween 0 C and 5 C. 1) Boehringer test kit is an example of a suitable product available commercially. This information is given for the convenience of users of this part of ISO 5765IDF 79 and does not constitute an endorsement by ISO or IDF of this product. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/19/2007 03:30:05 MDTNo reproduction