1、BRITISH STANDARD BS ISO 5765-1:2002 Dried milk, dried ice-mixes and processed cheese Determination of lactose content Part 1: Enzymatic method utilizing the glucose moiety of the lactose ICS 67.100.10; 67.100.30; 67.100.40 BS ISO 5765-1:2002 This British Standard, having been prepared under the dire
2、ction of the Consumer Products and Services Sector Policy and Strategy Committee, was published under the authority of the Standards Policy and Strategy Committee on 8 October 2002 BSI 8 October 2002 ISBN 0 580 40506 2 National foreword This British Standard reproduces verbatim ISO 5765-1:2002 and i
3、mplements 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-references Th
4、e British 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. T
5、his publication 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 in
6、ternational/European committee any enquiries on the interpretation, or proposals for change, and keep the 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 I
7、SO title page, pages ii to vi, pages 1 to 14, an inside back cover and a back cover. The BSI copyright date displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date Comments Reference numbers ISO 5765-1:2002(E) IDF 79-1:2002(E)INTERNAT
8、IONAL 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, mlanges secs pour crmes glaces et fromages fondus Dtermination de la teneur en
9、lactose Partie 1: Mthode enzymatique par la voie glucose BSISO57651:2002BSISO57651:2002iiIS-5675 O1:(2002)E ID-97 F1:(2002E) I SO 2002 All irthgs ersedevr iiiContents Foreword iv Foreword . v Introduction vi 1 Scope 1 2 Term and definition. 1 3 Principle . 1 4 Reagents 2 5 Apparatus. 3 6 Sampling 4
10、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 Calculation and expression of results 7 8.1 Calculation . 7 8.2 Expression of results 8 9 Precision 8 9.1 Interlaboratory trial
11、 . 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 BSISO57651:2002iiiIS-5675 O1:(2002)E ID-97 F1:(2002E) vi I SO 2002 All irthgs ersedevrForeword ISO (the International
12、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 committee has been esta
13、blished 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 electrotechnical standardiza
14、tion. 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 committees are circulated to the member bodies for votin
15、g. 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. ISO shall not be held responsible for identifying an
16、y 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 being published jointly by ISO and IDF separately by
17、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 2: Enzymatic method utilizing the galactose moiety
18、of the lactose Annex A forms a normative part of this part of ISO 5765IDF 79. BSISO57651:2002ivIS-5675 O1:(2002)E ID-97 F1:(2002E) I SO 2002 All irthgs ersedevr vForeword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every member
19、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 analysis and sampling for milk and milk products. Draft International Standards
20、 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 IDF National Committees casting a vote. ISO 5765-1IDF 79-1 was prepared by Technical Committee ISO/TC 34, Food pro
21、ducts, 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. ISO 5765IDF 79 consists of the following parts, under the general title Dried m
22、ilk, dried ice-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 d
23、etermination, of the Standing Committee on Main components of milk, under the aegis of its project leader, Mr J. Labrijn (NL). BSISO57651:2002vIS-5675 O1:(2002)E ID-97 F1:(2002E) iv I SO 2002 All irthgs ersedevrIntroduction This part of ISO 5765IDF 79 describes the enzymatic method for the determina
24、tion of lactose utilizing the glucose moiety of the lactose. It is complementary 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
25、the sample to be analysed. If the glucose content of a sample is considerably 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
26、 the method described in this part of ISO 5765IDF 79. For samples with a low 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
27、methods. In heat-treated milk and milk products, a proportion of lactose may 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 det
28、ermined as lactose. Moreover, in intensively heat- treated milk (e.g. sterilized 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. Onl
29、y when the good laboratory practice (GLP) rules for enzymatic analyses have been applied strictly, will reliable results be obtained. The GLP rules are stated in annex A. BSISO57651:2002viNITERNATNOIAL STANDARD IS-5675 O1:(2002)E ID-97 F1:(2002E)I SO 2002 All irthgs ersedevr 1Dried milk, dried ice-m
30、ixes and processed cheese Determination of lactose content Part 1: Enzymatic method utilizing the glucose moiety of the lactose 1 Scope This 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 p
31、resence of other carbohydrates and reducing substances, and of processed cheese. 2 Term and definition For the purposes of this part of ISO 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
32、 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 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) -galact
33、osidase, to split the lactose into glucose and galactose; b) hexokinase and adenosine triphosphate (ATP), to phosphorylate the glucose (both that originally present and that liberated by the -galactosidase) to glucose 6-phosphate (G6P); c) glucose 6-phosphate dehydrogenase (G6P-DH) in the presence o
34、f nicotinamide adenine dinucleotide phosphate (NADP + ), to catalyse the oxidation of G6P to 6-phosphogluconate, the NADP +being reduced to 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
35、 the amount of NADPH if a correction is made for the glucose present in the test sample at the start of the analysis. BSISO57651:20021IS-5675 O1:(2002)E ID-97 F1:(2002E) 2 I SO 2002 All irthgs ersedevr4 Reagents Use only reagents of recognized analytical grade unless otherwise specified. The water u
36、sed 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 of the production and expiry dates of the reagents given by the manufacturer. If an enzyme suspen
37、sion 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 described in 4.3 and in 4.5 to 4.7 inclusive may be obtained commercially as a test combination, for exam
38、ple, Boehringer test kit. 1)4.1 Potassium hexacyanoferrate(II) solution, K 4 Fe(CN) 6 Dissolve 3,6 g of potassium hexacyanoferrate(II) trihydrate in water. Dilute with water to 100 ml and mix. 4.2 Zinc sulfate solution, ZnSO 4Dissolve 7,2 g of zinc sulfate heptahydrate in water. Dilute with water to
39、 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 of trisodium citrate dihydrate (C 6 H 5 O 7 Na 3 2H 2 O), 0,042 g of citric acid monohydrate
40、(C 6 H 8 O 7 H 2 O), and 0,625 g of magnesium sulfate heptahydrate (MgSO 4 7H 2 O) 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 water to 50 ml and mix. This solution may be kept for 3 months if stored in
41、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 (C 6 H 15 NO 3 HCl) and 0,25 g of magnesium sulfate heptahydrate (MgSO 4 7H 2 O) in about 80 ml of water. Adjust the pH to 7,6 0,1 at 20 C with about 5 ml
42、 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 adenine dinucleotide phosphate disodium salt C 21 H 26 N 7 O
43、17 P 3 Na 2 , (98 to 99) % purity and 170 mg of adenosine 5-triphosphate disodium salt C 10 H 14 N 5 O 13 P 3 Na 2 , (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 between 0 C and 5 C. 1) Boehringer tes
44、t 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. BSISO57651:20022IS-5675 O1:(2002)E ID-97 F1:(2002E) I SO 2002 All irthgs ersed
45、evr 34.7 -Galactosidase suspension (from Escherichia coli), suspension in 3,2 mol/l ammonium sulfate solution, of pH approximately 6. The activity of the suspension of -galactosidase (EC 3.2.1.23) 5shall be at least 60 units/ml (lactose as substrate at 25 C). The suspension may be kept for about 12
46、months if stored in a refrigerator set at between 0 C and 5 C. When using the suspension, the vessel containing the suspension shall be kept immersed in crushed ice. NOTE A -galactosidase suspension which contains not more than 0,001 % each of -fructosidase, -galactosidase, glucose dehydrogenase, -g
47、lucosidase and NADH-oxidase, calculated in terms of the specific activity of -galactosidase, has been found to be suitable. 4.8 HK/G6P-DH suspension (from yeast), suspension in 3,2 mol/l ammonium sulfate solution, of pH approximately 6. The activity of the suspension of hexokinase (EC 2.7.1.1) 5shal
48、l be at least 280 units/ml at 25 C. That of glucose- 6-phosphate dehydrogenase (EC 1.1.1.49) 5shall be at least 140 units/ml at 25 C. The suspension may be kept for at least 1 year if stored in a refrigerator set at between 0 C and 5 C. When using the suspension, the vessel containing the suspension
49、 shall be kept immersed in crushed ice. NOTE An HK/G6P-DH suspension which contains not more than 0,01 % each of 6-phosphogluconate dehydrogenase and phosphoglucomutase, not more than 0,002 % of phosphogluco-isomerase and not more than 0,02 % of glutathion reductase, calculated in terms of the specific activity of HK/G6P-DH, has been found to be suitable. 4.9 L
