1、BS ISO 3356:2009ICS 67.100.10NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDMilk Determinationof alkalinephosphataseThis British Standard was published under the authority of the Standards Policy and Strategy Committee on 30November 2009 BSI 2009ISBN 978 0 580
2、57345 3Amendments/corrigenda issued since publicationDate CommentsBS ISO 3356:2009National forewordThis British Standard is the UK implementation of ISO 3356:2009. The UK participation in its preparation was entrusted to TechnicalCommittee AW/5, Chemical analysis of milk and milk products.A list of
3、organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for its correct application. Compliance with a British Standard cannot confer immunityfrom legal obliga
4、tions.BS ISO 3356:2009Reference numbersISO 3356:2009(E)IDF 63:2009(E)ISO and IDF 2009INTERNATIONAL STANDARD ISO3356IDF63Second edition2009-03-01Milk Determination of alkaline phosphatase Lait Dtermination de la phosphatase alcaline BS ISO 3356:2009ISO 3356:2009(E) IDF 63:2009(E) PDF disclaimer This
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9、ng 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 + 32 2 733 04 13 E-mail
10、 copyrightiso.org E-mail infofil-idf.org Web www.iso.org Web www.fil-idf.org Published in Switzerland ii ISO and IDF 2009 All rights reservedBS ISO 3356:2009ISO 3356:2009(E) IDF 63:2009(E) ISO and IDF 2009 All rights reserved iiiForeword ISO (the International Organization for Standardization) is a
11、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 represented
12、 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 are draft
13、ed 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 International Stan
14、dard 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 3356IDF 63 was prepa
15、red by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF). It is being published jointly by ISO and IDF. This second edition of ISO 3356IDF 63 cancels and replaces the first edition of ISO 3356:1975, which has been te
16、chnically revised. BS ISO 3356:2009ISO 3356:2009(E) IDF 63:2009(E) iv ISO and IDF 2009 All rights reservedForeword IDF (the International Dairy Federation) is a non-profit organization representing the dairy sector worldwide. IDF membership comprises National Committees in every member country as we
17、ll as regional dairy associations having signed a formal agreement on cooperation with IDF. All members of IDF have the right to be represented at the IDF Standing Committees carrying out the technical work. IDF collaborates with ISO in the development of standard methods of analysis and sampling fo
18、r milk and milk products. Draft International 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 IDF National Committees casting a vote. Attention is drawn
19、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 any or all such patent rights. ISO 3356|IDF 63 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products.
20、 It is being published jointly by ISO and IDF. All work was carried out by the Joint ISO-IDF Action Team on Heat treatment of the Standing Committee on Minor components and characterization of physical properties under the aegis of its project leader, Mrs. M. Nicolas (FR). This edition of ISO 3356ID
21、F 63 cancels and replaces IDF 63:1971, which has been technically revised. BS ISO 3356:2009INTERNATIONAL STANDARD ISO 3356:2009(E)IDF 63:2009(E) ISO and IDF 2009 All rights reserved 1Milk Determination of alkaline phosphatase WARNING The use of this International Standard may involve hazardous mater
22、ials, operations and reagents. Persons using this International Standard should be familiar with normal laboratory practice. This International Standard does not purport to address all the safety problems, if any, associated with its use. It is the responsibility of the user to establish appropriate
23、 safety and health practices and to ensure compliance with any national regulatory conditions. 1 Scope This International Standard specifies a method for the determination of alkaline phosphatase activity in milk. The method applies to alkaline phosphatase activities not less than 1 g of phenol per
24、millilitre. The method is also suitable for the determination of alkaline phosphatase activity in milk powder, buttermilk and buttermilk powder, whey and whey powder. 2 Terms and definitions For the purposes of this document, the following terms and definitions apply. 2.1 alkaline phosphatase activi
25、ty ALP activity alkaline phosphatase activity in milk quantity of phenol liberated by the sample determined according to the procedure specified in this International Standard NOTE The alkaline phosphatase activity is expressed as the quantity of phenol, in micrograms, liberated by 1 ml of the sampl
26、e or of reconstituted sample, under the conditions specified in this International Standard. Other International Standards (e.g. ISO 11816-1IDF 155-16, ISO 22160IDF 2097) express alkaline phosphatase activity in milliunits per litre. The literature gives information on the equivalence of the differe
27、nt units used to express the alkaline phosphatase activity. 3 Principle The sample is diluted with a buffer at pH 10,6 and incubated at a temperature of 37 C for 1 h. Under these conditions, any alkaline phosphatase present in the sample liberates phenol from the disodium phenylphosphate added. The
28、phenol liberated reacts with a quinoneimide (dibromoquinonechlorimide) to produce dibromoindophenol (blue colour) which is measured photometrically at 610 nm. 4 Reagents Use only reagents of recognized analytical grade, unless otherwise specified, and only distilled water or water of equivalent puri
29、ty. BS ISO 3356:2009ISO 3356:2009(E) IDF 63:2009(E) 2 ISO and IDF 2009 All rights reserved4.1 Barium borate-hydroxide buffer solution Dissolve 25,0 g of barium hydroxide Ba(OH)28H2O, carbonate free, in water in a 500 ml one-mark volumetric flask (5.8). Make up to the mark with water and mix. Dissolv
30、e 11,0 g of boric acid (H3BO3) in water in another 500 ml one-mark volumetric flask (5.8). Make up to the mark with water and mix. Warm both solutions to 50 C. Add one to the other and mix by stirring. Cool the solution obtained rapidly to about 20 C. Adjust the pH of the solution, if necessary, to
31、10,6 0,1 with an additional amount of barium hydroxide solution. Filter the solution through filter paper (5.10). Store the filtered barium borate-hydroxide buffer solution in a tightly stoppered container. Before use, dilute the buffer solution with an equal volume of water. 4.2 Colour development
32、buffer solutions 4.2.1 Colour buffer solution I Dissolve 6,0 g of sodium metaborate (NaBO2) or 12,6 g of NaBO24H2O, and 20,0 g of sodium chloride (NaCl) in water in a 1 000 ml one-mark volumetric flask (5.8). Make up to the mark with water and mix. 4.2.2 Colour buffer solution II Transfer 10 ml of b
33、uffer solution I (4.2.1) to a 100 ml one-mark volumetric flask (5.8). Make up to the mark with water and mix. 4.3 Buffer substrate solution 4.3.1 Disodium phenylphosphate dihydrate (Na2C6H5PO42H2O), containing no more than 0,01 % mass fraction phenol. 4.3.2 Dissolve 0,1 g of disodium phenylphosphate
34、 dihydrate (4.3.1) in 100 ml of diluted barium borate-hydroxide buffer solution (4.1). 4.4 Zinc-copper precipitant solution Dissolve 3,0 g of zinc sulfate (ZnSO47H2O) and 0,6 g of copper sulfate (CuSO45H2O) in water in a 100 ml one-mark volumetric flask (5.8). Make up to the mark with water and mix.
35、 4.5 2,6-Dibromoquinonechloroimide (BQC) solution, Gibbs reagent. Dissolve 40 mg 1 mg of BQC (C6H2Br2ClNO) in 10 ml of ethanol 96 % volume fraction. Store the solution in a dark coloured bottle at 4 C 2 C. Reject if discoloured or more than 1 month old. 4.6 Copper sulfate solution Dissolve 0,05 g of
36、 copper sulfate (CuSO45H2O) in water in a 100 ml one-mark volumetric flask (5.8). Make up to the 100 ml mark with water and mix. BS ISO 3356:2009ISO 3356:2009(E) IDF 63:2009(E) ISO and IDF 2009 All rights reserved 34.7 Sodium hydroxide solution, c(NaOH) = 0,5 mol/l. 4.8 Phenol standard solutions 4.8
37、.1 Phenol standard stock solution Transfer a weighed amount of 200 mg 2 mg of anhydrous phenol of purity higher than 99,5 % mass fraction into a 100 ml one-mark volumetric flask (5.8). Dissolve the phenol in water. Make up to the mark with water and mix. The phenol standard stock solution remains st
38、able at 4 C 2 C for 6 weeks. 4.8.2 Phenol standard working solutions Pipette 10 ml of phenol standard stock solution (4.8.1) into a 100 ml one-mark volumetric flask (5.8). Make up to the mark with water and mix (1 ml contains 200 g of phenol). Use the diluted standard solution to prepare the appropr
39、iate phenol standard working solutions, containing 2 g, 5 g, 10 g and 20 g of phenol per millilitre respectively. 5 Apparatus Usual laboratory equipment and, in particular, the following. 5.1 Analytical balance, capable of weighing to the nearest 1 mg, with a readability of 0,1 mg. 5.2 Photometer, s
40、uitable for measuring at a wavelength of 610 nm. 5.3 Water bath, capable of being maintained at 37 C 1 C under thermostatic control. 5.4 Boiling water bath. 5.5 Vortex mixer. 5.6 Pipettes, capacities 0,1 ml, 1 ml, 5 ml and 10 ml, ISO 6481, class A. 5.7 Glass test tubes, of appropriate volumes, with
41、closures made from phenolic-free liners. 5.8 One-mark volumetric flasks, capacities 100 ml, 500 ml and 1000 ml, ISO 10423, class A. 5.9 Glass funnels, diameters about 60 mm and about 100 mm. 5.10 Filter paper, fast grade, diameters about 110 mm and about 185 mm. 6 Sampling A representative sample sh
42、ould have been sent to the laboratory. It should not have been damaged or changed during transport or storage. Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 707IDF 502. Store the test sample in such a way that deterioration
43、 and change in composition are prevented. BS ISO 3356:2009ISO 3356:2009(E) IDF 63:2009(E) 4 ISO and IDF 2009 All rights reserved7 Preparation of test sample 7.1 Preparation Carefully mix the test sample before use. Usually, it is not necessary to pre-warm the test sample for proper mixing. If pre-wa
44、rming is necessary, however, the temperature shall under no circumstances exceed 35 C. 7.2 Milk powder, buttermilk powder and whey powder Dissolve 10 g of test sample in 90 ml of water by heating, if necessary. The temperature, however, applied in dissolving the sample completely, shall under no cir
45、cumstances exceed 35 C. 7.3 Neutralization of acid test samples If the test sample is acidic (pH 7,0), adjust to neutral pH with sodium hydroxide solution (4.7) 8 Procedure 8.1 Preparation of the standard curve 8.1.1 Prepare a range of standard matching solutions in five glass test tubes (5.7) by pi
46、petting 1 ml of water for the control or blank test tube and 1 ml of each of the four phenol standard working solutions (4.8.2) into each of the remaining four test tubes respectively. The standard test tubes contain 0 g (control or blank), 2 g, 5 g, 10 g and 20 g of phenol respectively. 8.1.2 Add t
47、o each test tube (8.1.1) 1 ml of copper sulfate solution (4.6), 5 ml of colour buffer solution II (4.2.2), 3 ml of water and 0,1 ml of BQC solution (4.5) and mix. Allow the colour to develop at room temperature for 30 min. 8.1.3 Measure the optical density of the standard solutions against that of t
48、he control or blank solution at a wavelength of 610 nm. 8.1.4 Plot the optical density against the quantities of phenol in micrograms (8.1.1). Calculate the equation of the standard curve. 8.2 Determination 8.2.1 Avoid exposure to direct sunlight during the determination. Contamination with traces o
49、f saliva or perspiration can give false positive results and should be avoided. 8.2.2 Pipette into each of two test tubes (5.7) 1 ml of test sample or reconstituted sample. Use one of the tubes as control or blank test. 8.2.3 Stopper the blank test tube. Place the tube in a beaker with boiling water. Cover the beaker with aluminium foil. Heat the tube in the boiling water for 2 min while keeping the tube and the beaker covered with the foil to ensure the entire tube is heated. Then
