SANS 11285-2005 Milk - Determination of lactulose content - Enzymatic method《牛奶 乳果糖含量测定 酶法》.pdf

1、 Collection of SANS standards in electronic format (PDF) 1. Copyright This standard is available to staff members of companies that have subscribed to the complete collection of SANS standards in accordance with a formal copyright agreement. This document may reside on a CENTRAL FILE SERVER or INTRA

2、NET SYSTEM only. Unless specific permission has been granted, this document MAY NOT be sent or given to staff members from other companies or organizations. Doing so would constitute a VIOLATION of SABS copyright rules. 2. Indemnity The South African Bureau of Standards accepts no liability for any

3、damage whatsoever than may result from the use of this material or the information contain therein, irrespective of the cause and quantum thereof. ICS 67.100.10 ISBN 0-626-17131-8 SANS 11285:2005Edition 1ISO 11285:2004Edition 1SOUTH AFRICAN NATIONAL STANDARD Milk Determination of lactulose content E

4、nzymatic method This national standard is the identical implementation of ISO 11285:2004 and is adopted with the permission of the International Organization for Standardization. Published by Standards South Africa 1 dr lategan road groenkloof private bag x191 pretoria 0001 tel: 012 428 7911 fax: 01

5、2 344 1568 international code + 27 12 www.stansa.co.za Standards South Africa SANS 11285:2005 Edition 1 ISO 11285:2004 Edition 1 Table of changes Change No. Date Scope Abstract Provides an enzymatic method for the determination of the lactulose content of milk. Keywords agricultural products, chemic

6、al analysis and testing, dairy products, definitions, determination of content, enzymatic methods, lactose, lactulose, milk. National foreword This South African standard was approved by National Committee StanSA TC 5140.34, Dairy products, in accordance with procedures of Standards South Africa, in

7、 compliance with annex 3 of the WTO/TBT agreement. Reference numbersISO 11285:2004(E)IDF 175:2004(E)ISO and IDF 2004INTERNATIONAL STANDARD ISO11285IDF175First edition2004-09-01Milk Determination of lactulose content Enzymatic method Lait Dtermination de la teneur en lactulose Mthode enzymatique ISO

8、11285:2004(E) IDF 175:2004(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 typefaces which are embedded are licensed to and installed on the computer performing the editi

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11、found, please inform the ISO Central Secretariat at the address given below. ISO and IDF 2004 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, withou

12、t 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 Reyers 80 B-1030 Brussels Tel. + 41 22 749 01 11 Tel. + 32 2 733 98 88 Fax + 41 22 749 09 47 Fax +

13、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 2004 All rights reservedISO 11285:2004(E) IDF 175:2004(E) ISO and IDF 2004 All rights reserved iiiForeword ISO (the International Organization for Standardization

14、) 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 established has the right to be repr

15、esented 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 standardization. International Standards ar

16、e drafted in accordance with the rules given in the ISO/IEC 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 Internation

17、al 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 document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 11285IDF 175

18、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 separately by AOAC International. ISO 11285:2004(E) IDF 17

19、5:2004(E) iv ISO and IDF 2004 All rights reservedForeword IDF (the International Dairy Federation) is a worldwide 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

20、 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 the Action Teams and Standing Committees are circulated to the National Committees for voting. Public

21、ation as an International Standard requires approval by at least 50 % of IDF National Committees casting a vote. ISO 11285IDF 175 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 wi

22、th 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 milk and milk products according to heat treatment, of the Standing Committee on Minor components and characte

23、rization of physical properties, under the aegis of its project leader, Mrs E. Lechner (DE). This edition of ISO 11285IDF 175 cancels and replaces IDF 175:1995, which has been technically revised. INTERNATIONAL STANDARD ISO 11285:2004(E)IDF 175:2004(E) ISO and IDF 2004 All rights reserved 1Milk Dete

24、rmination of lactulose content Enzymatic method 1 Scope This International Standard specifies an enzymatic method for the determination of the lactulose content of milk. 2 Terms and definitions For the purposes of this document, the following terms and definitions apply. 2.1 lactulose content mass o

25、f substances determined by the procedure specified in this International Standard NOTE The lactulose content is expressed as milligrams per kilogram. 3 Principle Fat and protein are precipitated by the addition of zinc sulfate and potassium hexacyanoferrate(II) solution and are then removed by filtr

26、ation. Lactose and lactulose are hydrolysed to galactose and glucose, or galactose and fructose, respectively, in the presence of the enzyme -D-galactosidase (-gal). The amount of liberated fructose is stoichiometric with the amount of lactulose: -gal lactose + H2O galactose + glucose (1) -gal lactu

27、lose + H2O galactose + fructose (2) Lactose is present in milk in significantly higher amounts than lactulose. Thus, the glucose present after hydrolysis would interfere with the determination of fructose. Therefore, the glucose is mostly oxidized by glucose oxidase (GOD) to gluconic acid in the pre

28、sence of oxygen: GOD glucose + H2O + O2gluconic acid + H2O2 (3)By this means it is possible to determine small amounts of lactulose in the presence of excess lactose. The hydrogen peroxide formed in reaction (3) is destroyed by catalase. This reaction is used to provide the oxygen which is required

29、for the oxidation of the glucose: catalase 2 H2O22 H2O + O2 (4)The remaining unoxidized glucose and the fructose produced by the hydrolysis of the lactulose are phosphorylated by means of adenosine triphosphate (ATP) in the presence of hexokinase (HK) to yield glucose-6-phosphate and fructose-6-phos

30、phate, respectively. ISO 11285:2004(E) IDF 175:2004(E) 2 ISO and IDF 2004 All rights reservedHK glucose + ATP glucose-6-phosphate + ADP (5) HK fructose + ATP fructose-6-phosphate + ADP (6) Glucose-6-phosphate is oxidized by means of NADP+in the presence of glucose-6-phosphate dehydrogenase with cons

31、equent production of 6-phosphogluconate and NADPH. After completion of the reaction, the NADPH formed is measured by means of its absorbance at 340 nm. G-6-P-DH glucose-6-phosphate + NADP+6-phosphogluconate + NADPH + H+(7) Fructose-6-phosphate is isomerized to glucose-6-phosphate in the presence of

32、the enzyme phosphoglucose-isomerase (PGI). The glucose-6-phosphate is oxidized as given in reaction (7). The increase in NADPH is measured by means of its absorbance at 340 nm and is proportional to the fructose and lactulose content. PGI fructose-6-phosphate glucose-6-phosphate (8) In order to comp

33、ensate for any free fructose which may originally be present, a blank determination is performed with the addition of -galactosidase omitted. This blank value is taken into account in the final calculation of the lactulose content of the sample. 4 Reagents Use only reagents of recognized analytical

34、grade. The reagents for the fructose determination (4.15 to 4.18) are commercially available as test combinations. Take due account of manufacturers instructions. 4.1 Water used in the preparation of the enzyme solutions and buffer solutions shall be fresh and of at least double glass-distilled puri

35、ty. Water used for other purposes shall be glass-distilled or of at least equivalent purity. 4.2 Zinc sulfate solution, (ZnSO4) = 300 g/l. Dissolve 30,0 g of zinc sulfate heptahydrate (ZnSO47H2O) in water in a 100 ml one-mark volumetric flask (5.2). Dilute to the mark with water and mix. 4.3 Potassi

36、um hexacyanoferrate(II) solution, (K4Fe(CN)6) = 150 g/l. Dissolve 15,0 g of potassium hexacyanoferrate(II) trihydrate (K4Fe(CN)63H2O) in water in a 100 ml one-mark volumetric flask (5.2). Dilute to the mark with water and mix. 4.4 Buffer solution A, pH 7,5. Dissolve 4,80 g of disodium hydrogenphosph

37、ate (Na2HPO4), 0,86 g of sodium dihydrogenphosphate monohydrate (NaH2PO4H2O) and 0,10 g of magnesium sulfate heptahydrate (MgSO47H2O) in 80 ml of water (4.1). Adjust the pH to 7,5 0,1 at 20 C, if necessary, with 1 mol/l sodium hydroxide solution (4.10). Dilute with water to 100 ml and mix (sufficien

38、t for approximately 15 analyses). 4.5 Buffer solution B, pH 7,6. Dissolve 14,00 g of triethanolamine hydrochloride N(CH2CH2OH)3HCl and 0,25 g of magnesium sulfate heptahydrate (MgSO47H2O) in 80 ml of water (4.1). ISO 11285:2004(E) IDF 175:2004(E) ISO and IDF 2004 All rights reserved 3Adjust the pH t

39、o 7,6 0,1 at 20 C, if necessary, with 1 mol/l sodium hydroxide solution (4.10). Dilute with water to 100 ml and mix (sufficient for approximately 60 analyses). 4.6 Buffer solution C. Dilute 40,0 ml of buffer solution B (4.5) to 100 ml with water and mix (sufficient for approximately 50 analyses). 4.

40、7 Sodium hydrogen carbonate (NaHCO3). 4.8 Hydrogen peroxide (H2O2), 30 % (mass fraction). 4.9 Octan-1-ol (C8H18O). 4.10 Sodium hydroxide solutions, c(NaOH) = 0,33 mol/l and 1 mol/l respectively. 4.11 -D-Galactosidase (-gal), from E. coli (-gal EC 3.2.1.23), 5 mg/ml suspension in 3,2 mol/l ammonium s

41、ulfate (NH4)2SO4 solution, 30 units/mg (at 25 C, with lactose as substrate); 300 units/mg (at 37 C, with 2-nitrophenyl-D-galactoside (o-nitrophenyl-D-galactopyranoside) as substrate), respectively. 4.12 Glucose oxidase (GOD), from Aspergillus niger (EC 1.1.3.4), degree of purity II, lyophilizate, 20

42、0 units/mg (at 25 C) or 230 units/mg (at 37 C, both with glucose as substrate), respectively. 4.13 Oxidation solution. Dissolve 20 mg of glucose oxidase (4.12) in 1 ml of water. Prepare this solution freshly just before use. 4.14 Catalase, from beef liver (EC 1.11.1.6), suspension of 20 mg/ml in wat

43、er (stabilized), 65 000 units/mg (at 25 C; with H2O2as substrate). 4.15 Hexokinase/glucose-6-phosphate-dehydrogenase (HK/G6P-DH), mixture of enzymes from yeast (EC 2.7.1.1 and EC 1.1.1.49), suspension in 3,2 mol/l ammonium sulfate (NH42SO4) solution, HK and G6P-DH: HK: 2 mg/ml; 140 units/mg (at 25 C

44、; with glucose and ATP as substrate); G6P-DH: 1 mg/ml, 140 units/mg (at 25 C; with glucose-6-phosphate as substrate). 4.16 Phosphoglucose isomerase (PGI), from yeast (EC 5.3.1.9). Suspension in 3,2 mol/l ammonium sulfate (NH42SO4) solution: PGI: 2 mg/ml 350 units/mg (at 25 C; with fructose-6-phospha

45、te as substrate). NOTE 1 The EC numbers in 4.11, 4.12, 4.14 to 4.16 refer to the Enzymatic Classification number given by the Nomenclature Committee of the International Union of Biochemistry (see 4). NOTE 2 The unit in mentioned 4.11, 4.12, 4.14 to 4.16 (often called the International Unit or Stand

46、ard Unit) is defined as the amount of enzyme which will catalyse the transformation of 1 mol of substrate per minute under standard conditions. 4.17 ATP solution Dissolve 50 mg of adenosine-5-triphosphate disodium salt (5-ATP-Na2) and 50 mg of sodium hydrogen carbonate (NaHCO3)in 1 ml of water. 4.18

47、 NADP solution Dissolve 10 mg of -nicotinamide adenine dinucleotide phosphate disodium salt (-NADP-Na2) in 1 ml of water. ISO 11285:2004(E) IDF 175:2004(E) 4 ISO and IDF 2004 All rights reserved5 Apparatus Usual laboratory equipment and, in particular, the following. 5.1 Analytical balance, capable

48、of weighing to the nearest 0,001 g. 5.2 One-mark volumetric flasks, of capacity 10 ml, 20 ml and 100 ml. 5.3 Conical flask, with ground glass stopper, of capacity 50 ml. 5.4 Funnel, of diameter 50 mm. 5.5 Fluted filter, medium flow rate, of diameter 125 mm and 70 mm. 5.6 Water bath or drying oven, c

49、apable of maintaining a temperature of 40 C 2 C. 5.7 Spectrometer, suitable for making measurements at 340 nm. Spectral line filter photometers suitable for making measurements at 365 nm and 334 nm (mercury lamps) may also be used. The molar absorption coefficients of NADPH (see 9.1) are then 3,4 (365 nm) or 6,18 (334 nm) (lmmol1cm1). 5.8 Cuvettes, made of glass or plastic, with optical path length 10,0 mm. In the case of using plastic cuvettes, check the path