1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS ISO 15174:2012Milk and milk products Microbial coagulants Determination of total milk-clotting activityBS ISO 15174:2012 BRITISH STANDARDNational forewordThis British Standard
2、 is the UK implementation of ISO 15174:2012.The UK participation in its preparation was entrusted to TechnicalCommittee AW/5, Chemical analysis of milk and milk products.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not purport
3、 to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2012. Published by BSI StandardsLimited 2012ISBN 978 0 580 70879 4ICS 07.100.30; 67.100.10Compliance with a British Standard cannot confer immunity fromlegal obl
4、igations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 31 May 2012.Amendments issued since publicationDate Text affectedBS ISO 15174:2012Reference numbersISO 15174:2012(E)IDF 176:2012(E)ISO and IDF 2012INTERNATIONAL STANDARD ISO15174IDF176Se
5、cond edition2012-05-15Milk and milk products Microbial coagulants Determination of total milk-clotting activity Lait et produits laitiers Coagulants microbiens Dtermination de lactivit totale de coagulation du lait BS ISO 15174:2012ISO 15174:2012(E) IDF 176:2012(E) COPYRIGHT PROTECTED DOCUMENT ISO a
6、nd IDF 2012 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, without permission in writing from either ISO or IDF at the respective address below. IS
7、O copyright office International Dairy Federation Case postale 56 CH-1211 Geneva 20 Silver Building Boulevard Auguste Reyers 70/B 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
8、 www.fil-idf.org Published in Switzerland ii ISO and IDF 2012 All rights reservedBS ISO 15174:2012ISO 15174:2012(E) IDF 176:2012(E) ISO and IDF 2012 All rights reserved iiiContents Page Foreword iv Foreword . v Introduction vi 1 Scope 1 2 Principle . 1 3 Reagents and materials 1 4 Apparatus . 2 5 Sa
9、mpling 3 6 Preparation of test sample . 3 6.1 Liquid microbial coagulant . 3 6.2 Powdered microbial coagulant 3 7 Procedure . 4 7.1 Preparation of substrate . 4 7.2 Preparation of the microbial coagulant reference solution 4 7.3 Preparation of microbial coagulant test solution 5 7.4 Clotting . 5 8 C
10、alculation and expression of results 5 8.1 Calculation . 5 8.2 Expression of results 6 9 Precision 6 9.1 Interlaboratory test 6 9.2 Repeatability 7 9.3 Reproducibility 7 10 Test report 7 Annex A (informative) Interlaboratory trial . 8 Bibliography 9 BS ISO 15174:2012ISO 15174:2012(E) IDF 176:2012(E)
11、 iv ISO and IDF 2012 All rights reservedForeword 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 bo
12、dy interested in a subject 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 Electrot
13、echnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are 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 b
14、y the technical committees 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 International Standard may be the subjec
15、t of patent rights. IDF shall not be held responsible for identifying any or all such patent rights. ISO 15174IDF 176 was prepared 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
16、 ISO and IDF. This second edition of ISO 15174IDF 176 cancels and replaces the first edition (ISO 15174IDF 176:2002), which has been technically revised. BS ISO 15174:2012ISO 15174:2012(E) IDF 176:2012(E) ISO and IDF 2012 All rights reserved vForeword IDF (the International Dairy Federation) is a no
17、n-profit organization representing the dairy sector worldwide. IDF membership comprises National Committees in every member country as well 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
18、 Committees carrying out the technical work. IDF collaborates with ISO in the development of standard methods of analysis and sampling for milk and milk products. The main task of Standing Committees is to prepare International Standards. Draft International Standards adopted by Standing Committees
19、are circulated to the National Committees for endorsement prior to publication as International Standards. Publication as an International Standard requires approval by at least 50 % of IDF National Committees casting a vote. Attention is drawn to the possibility that some of the elements of this In
20、ternational Standard may be the subject of patent rights. IDF shall not be held responsible for identifying any or all such patent rights. ISO 15174IDF 176 was prepared by the International Dairy Federation (IDF) and Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk prod
21、ucts. It is being published jointly by IDF and ISO. All work was carried out by the Joint ISO/IDF Project Group on Microbial coagulants of the Standing Committee on Analytical methods for processing aids and indicators under the aegis of its project leader, Mrs. M. Harboe (DK) and Prof. A. Andrn (SE
22、). This second edition of ISO 15174IDF 176 cancels and replaces the first edition (ISO 15174IDF 176:2002), which has been technically revised. BS ISO 15174:2012ISO 15174:2012(E) IDF 176:2012(E) vi ISO and IDF 2012 All rights reservedIntroduction Microbial coagulants are derived from various microbia
23、l sources, the most common sources being Rhizomucor miehei (EC 3.4.23.23), R. pusillus (EC 3.4.23.23) and Cryphonectria parasitica, formerly named Endothia parasitica (EC 3.4.23.22). Each of these enzymes has its own characteristics as far as milk-clotting activity and cheese-making properties are c
24、oncerned. These are differences in temperature sensitivity, pH sensitivity, sensitivity to calcium ions, and effect on the rheology of the milk-gel formed. For practical and economic reasons, therefore, it is very important to have an international method for determination of the total milk-clotting
25、 activity of microbial coagulants relative to an international recognized reference standard. Also for practical reasons, it was decided to use the R. miehei enzyme as a microbial coagulant reference standard for all types of microbial coagulants. The method is in accordance with the relative milk-c
26、lotting activity test for bovine rennets described in ISO 11815IDF 157. A qualitative determination of the microbial coagulants in a sample can be performed according to ISO 15163IDF 110:7, Annex A. For mixtures of different clotting enzymes, no correct determination of the total milk-clotting activ
27、ity for the sample can be obtained. BS ISO 15174:2012INTERNATIONAL STANDARD ISO 15174:2012(E)IDF 176:2012(E) ISO and IDF 2012 All rights reserved 1Milk and milk products Microbial coagulants Determination of total milk-clotting activity 1 Scope This International Standard specifies a method for comp
28、arison of the total milk-clotting activity of a microbial coagulant sample with the milk-clotting activity of an international microbial coagulant reference standard on a standard milk substrate prepared with a calcium chloride solution of concentration 0,5 g/l (pH 6,5). 2 Principle The time needed
29、for a visible flocculation of a standard milk substrate prepared with a 0,5 g/l calcium chloride solution (pH 6,5) is determined. The clotting time of a microbial coagulant sample is compared under identical chemical and physical conditions to that of the microbial coagulant reference standard with
30、known milk-clotting activity. 3 Reagents and materials Unless otherwise specified, use only reagents of recognized analytical grade and distilled or demineralized water or water of equivalent purity. 3.1 Buffer solution, pH 5,5. Add, using a pipette (4.1), 10,0 ml of 1 mol/l acetic acid (CH3COOH) to
31、 10,0 g of sodium acetate trihydrate (CH3COONa3H2O) and mix. Dilute with water to 1 000 ml. If necessary, adjust the pH to 5,5 with 1 mol/l acetic acid or with 1 mol/l sodium acetate solution. 3.2 Calcium chloride stock solution, (CaCl2) 500 g/l. Calcium chloride solutions with the required accurate
32、 calcium chloride concentration of 500 g/l and the actual density stated are commercially available.1)Store the solution according to the manufacturers instructions. Prior to use, bring the calcium chloride stock solution to room temperature (18 C to 22 C). Check the concentration of the calcium chl
33、oride solution by titration with EDTA (ethylenediaminetetraacetic acid) every year. 3.3 Calcium chloride working solution, (CaCl2) 0,5 g/l. Use the density of the calcium chloride stock solution (3.2) to calculate the mass needed to get a final calcium chloride concentration of 0,5 g/l in the workin
34、g solution. The mass of the solution should be equivalent to the addition of 2,00 ml of the stock solution with exact concentration of (CaCl2) 500 g/l, in which case the solution mass is 2,70 g. Weighing of the calcium chloride stock solution (3.2) is recommended to be able to prepare the calcium ch
35、loride working solution, as the viscous solution is difficult to pipette. 1) Chr. Hansens A/S, Hvidovre, Denmark is an example of a suitable supplier. This information is given for the convenience of users of this document and does not constitute an endorsement by ISO or IDF of this supplier. BS ISO
36、 15174:2012ISO 15174:2012(E) IDF 176:2012(E) 2 ISO and IDF 2012 All rights reservedWeigh, to the nearest 0,01 g, about 2,70 g of the calcium chloride stock solution (3.2) of exact known concentration at room temperature (18 C to 22 C) in a 2 000 ml one-mark volumetric flask. Dilute with water to the
37、 2 000 ml mark and mix. The calcium chloride solution shall be freshly prepared on the day of its use. NOTE Alternatively, an intermediate calcium chloride solution of 50 g/l can be prepared and further diluted before use. 3.4 Low-heat, low-fat spray-dried milk powders, of good renneting and bacteri
38、ological quality. NOTE Low-heat, low-fat spray-dried milk powders meeting the requirements are commercially available.1),2)3.5 Microbial coagulant reference standard powder (Rhizomucor miehei), in glass ampoules. The exact total milk-clotting activity is labelled on the ampoules. Store the microbial
39、 coagulant reference standard powder in the dark at 18 C, protected against moisture. For short periods, e.g. during transport, the powder may be kept at ambient temperatures. The microbial coagulant reference standard powder is a primary reference standard; a secondary liquid standard may be made a
40、nd used if it is assured that the same result is obtained. The total milk-clotting activity of the international microbial coagulant reference standard powder (R. miehei) is the amount of activity set relative to the first batch of international calf rennet reference standard powder, which was defin
41、ed to contain 1 000 IMCU/g (see ISO 11815IDF 1576). NOTE 1 The total milk-clotting activity is expressed as a percentage relative to the arithmetic mean of test results. The total milk clotting activity of the microbial coagulant reference standard powder is labelled on the glass ampoules and/or sta
42、ted on the certificate provided. There is a requirement for future preparations of microbial reference standards to be set relative to the previous batch of the microbial reference standard. NOTE 2 The total proteolytic (milk-clotting) activity of the microbial coagulant reference standard powder is
43、 checked every second year by an alternative method, e.g. on a synthetic hexapeptide substrate of NIZO3). The international microbial coagulant reference standard powder is commercially available from DSM Food Specialties4). 4 Apparatus Usual laboratory equipment and, in particular, the following. 4
44、.1 Micropipette or any other pipette, capable of delivering 0,5 ml in less than 1 s with a repeatability of 0,2 % or better. 4.2 One-mark pipettes, for delivering appropriate volumes, ISO 6481, class A. Alternatively a dilutor (e.g. a Hamilton diluter) with the same precision may be used for dilutin
45、g the coagulants. For measuring substrate, a syringe or a dispenser delivering the appropriate amount with repeatability of 0,4 % may also be used. 2) Cecalait, Poligny, France, is an example of a suitable supplier. This information is given for the convenience of users of this document Standard and
46、 does not constitute an endorsement by ISO or IDF of this supplier. 3) NIZO Food Research BV, Ede, Netherlands is an example of a suitable supplier. This information is given for the convenience of the user of this document and does not constitute an endorsement by ISO or IDF of this supplier. 4) DS
47、M Food Specialities, Dairy Ingredients Group, Delft, Netherlands is an example of a suitable supplier. This information is given for the convenience of users of this document and does not constitute an endorsement by ISO or IDF of this supplier. BS ISO 15174:2012ISO 15174:2012(E) IDF 176:2012(E) ISO
48、 and IDF 2012 All rights reserved 34.3 One-mark volumetric flasks, of required capacities, ISO 10423, class A. 4.4 Thermometer, calibrated, graduated between 20 C and 45 C, with a precision 0,1 C. 4.5 pH meter, capable of being read to the nearest 0,01 pH unit. 4.6 Analytical balance, capable of bei
49、ng read to the nearest 1 mg. 4.7 Stopwatch, capable of being read to the nearest second. 4.8 Flasks or test-tubes, for milk-clotting testing, with suitable capacity (see 7.4). 4.9 Water bath, capable of maintaining a temperature of 32 C 1 C, but also capable of maintaining the temperature constant to within 0,2 C throughout the bath. The water bath should be equipped with the following attachments. 4.9.1 Electric motor, provided with a rotating spindle to which the flask or test tub
