1、BRITISH STANDARD BS ISO 5765-2:2002 Dried milk, dried ice-mixes and processed cheese Determination of lactose content Part 2: Enzymatic method utilizing the galactose moiety of the lactose ICS 67.100.10; 67.100.30; 67.100.40 BS ISO 5765-2:2002 This British Standard, having been prepared under the di
2、rection 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 40507 9 National foreword This British Standard reproduces verbatim ISO 5765-2:2002 and
3、 implements 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
4、The 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.
5、 This 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
6、international/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
7、 ISO 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 5765-2:2002(E) IDF 79-2:2002(E)INTERN
8、ATIONAL STANDARD ISO 5765-2 IDF 79-2 First edition 2002-09-01 Dried milk, dried ice-mixes and processed cheese Determination of lactose content Part 2: Enzymatic method utilizing the galactose moiety of the lactose Lait sec, mlanges secs pour crmes glaces et fromages fondus Dtermination de la teneur
9、 en lactose Partie 2: Mthode enzymatique par la voie galactose BSISO57652:2002BSISO57652:2002iiIS-5675 O2:(2002)E ID-97 F2:(2002E) I SO 2002 All irthgs ersedevr iiiContents Foreword iv Foreword . v Introduction vi 1 Scope 1 2 Terms and definitions. 1 3 Principle . 1 4 Reagents 1 5 Apparatus. 3 6 Sam
10、pling 4 7 Procedure. 4 7.1 Test to check the procedure 4 7.2 Preparation of test sample . 4 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 Interlaborato
11、ry trial . 8 9.2 Repeatability 8 9.3 Reproducibility 8 10 Test report 9 Annex A (normative) Good Laboratory Practice (GLP) rules for the performance of enzymatic analyses 10 Bibliography 14 BSISO57652:2002iiiIS-5675 O2:(2002)E ID-97 F2:(2002E) vi I SO 2002 All irthgs ersedevrForeword ISO (the Intern
12、ational 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 b
13、een 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 electrotechnical sta
14、ndardization. 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 f
15、or 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. ISO shall not be held responsible for identi
16、fying any or all such patent rights. ISO 5765-2IDF 79-2 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 and se
17、parately 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 2: Enzymatic method utilizing the galac
18、tose moiety of the lactose Annex A forms a normative part of this part of ISO 5765IDF 79. BSISO57652:2002ivIS-5675 O2:(2002)E ID-97 F2:(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 e
19、very 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 analysis and sampling for milk and milk products. Draft Internation
20、al 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 National Committees casting a vote. ISO 5765-2IDF 79-2 was prepared by Technical Committee ISO/TC 34,
21、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 and separately by AOAC International. ISO 5765IDF 79 consists of the following parts, under the general title
22、 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 of the lactose All work was carried out by the Joint ISO/IDF/AOAC Action Team Lactose and l
23、actate determination, of the Standing Committee on Main components of milk, under the aegis of its project leader, Mr J. Labrijn (NL). BSISO57652:2002vIS-5675 O2:(2002)E ID-97 F2:(2002E) iv I SO 2002 All irthgs ersedevrIntroduction This part of ISO 5765IDF 79 describes the enzymatic method for the d
24、etermination of lactose utilizing the galactose moiety of the lactose. It is complementary to ISO 5765-1IDF 79-1 which utilizes the glucose moiety of the lactose. The choice of whether to use the method described in part 1 or part 2 of ISO 5765IDF 79 depends from the amount of glucose or galactose p
25、resent in 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 this part of ISO 5765IDF 79. Conversely, for a sample with a considerably higher galactose content than its lactose content, it is
26、 recommended to use the method described in ISO 5765-1IDF 79-1. 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
27、 for both 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 ISO 5765-1IDF 79-1. If, however, the method in this part of ISO 5765IDF 79 is applied, the lactulose will partia
28、lly be determined 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 in part of ISO 576
29、5IDF 79. Only 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. BSISO57652:2002viNITERNATNOIAL STANDARD IS-5675 O2:(2002)E ID-97 F2:(2002E)I SO 2002 All irthgs ersedevr 1Dried milk,
30、 dried ice-mixes and processed cheese Determination of lactose content Part 2: Enzymatic method utilizing the galactose 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
31、 form in the presence of other carbohydrates and reducing substances, and of processed cheese. 2 Terms and definitions 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 in this part
32、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 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) -
33、galactosidase, to split the lactose into glucose and galactose; b) -galactosidase dehydrogenase in the presence of nicotinamide adenine dinucleotide phosphate (NAD + ) to catalyse the oxidation of galactose into galactonic acid, the NAD +being reduced to NADH. 3.3 The amount of NADH is determined fr
34、om the absorbance of the test solution at 340 nm. 3.4 The lactose content is calculated, which is proportional to the amount of NADH if a correction is made for the galactose present in the test sample at the start of the analysis. 4 Reagents Use only reagents of recognized analytical grade unless o
35、therwise 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. BSISO57652:20021IS-5675 O2:(2002)E ID-97 F2:(2002E) 2 I SO 2002 All irthgs e
36、rsedevrTake note 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
37、reagents described in 4.4 and in 4.6 to 4.8 inclusive may be obtained commercially as a test combination, for example, Boehringer test kit.1)4.1 Potassium hexacyanoferrate(II) solution, K 4Fe(CN) 6 Dissolve 3,6 g of potassium hexacyanoferrate(II) trihydrate in water. Dilute with water to 100 ml and
38、mix. 4.2 Zinc sulfate solution, ZnSO 4Dissolve 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
39、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 (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 hydroxid
40、e solution (0,1 mol/l). Dilute with water 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 Phosphate (KH 2 PO 4 ) buffer solution, pH 8,6 0,1 Dissolve 16,6 g of potassium dihydrogen phosphate in about 80 ml of water. Adjust the pH t
41、o 8,6 0,1 at 20 C with sodium hydroxide solution (1 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 NAD + + + + /citrate buffer solution Dissolve 25 mg of nicotinamide adenine dinucleotide (C 21 H 27 N 7 O
42、 17 P 2 ; approximately 98 % assay) in 5 ml of citrate buffer solution (4.4). This solution may be kept for 3 weeks if stored in a refrigerator set at between 0 C and 5 C. 4.7 -Galactosidase suspension (from Escherichia coli), suspension in 3,2 mol/l ammonium sulfate solution, of pH 6 0,1. The activ
43、ity 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 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 i
44、mmersed in crushed ice. NOTE A -galactosidase suspension which contains not more than 0,001 % each of -fructosidase, -galactosidase, glucose dehydrogenase, -glucosidase and NADH-oxidase, calculated in terms of the specific activity of -galactosidase, has been found to be suitable. 1) Boehringer test
45、 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. BSISO57652:20022IS-5675 O2:(2002)E ID-97 F2:(2002E) I SO 2002 All irthgs ersede
46、vr 34.8 -Galactose dehydrogenase suspension (from Pseudomonas fluorescens), suspension in 3,2 mol/l of ammonium sulfate solution, of pH 6 0,1. The activity of the suspension of -galactosidase dehydrogenase (EC 1.1.1.48) 5shall be at least 8 units/ml. The suspension may be kept for at least 6 months
47、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 dehydrogenase suspension which contains not more than 0,01 % each of alcohol dehydrogenase and -galactosidase, not more
48、 than 0,1 % of NADH-oxidase and glucose-reacting enzymes, and not more than 0,5 % of lactate dehydrogenase has been found to be suitable. 4.9 Lactose standard solution, c(C 12 H 22 O 11 H 2 O) = 0,8 mg/ml. Before use, dry the lactose monohydrate to a constant mass in a drying oven set at 87 C. Disso
49、lve 400 mg of dried lactose monohydrate in water. Dilute with water to 500 ml and mix. The solution may be kept for 2 days if stored in a refrigerator set at between 0 C and 5 C. Warm the solution to about 20 C just before use. 5 Apparatus Usual laboratory equipment and, in particular, the following. 5.1 Analytical balance, capable of weighing to
