1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationMilk and milk products Calf rennet and adult bovine rennet Determination by chromatography of chymosin and bovine pepsin contentsBS ISO 15163:2012National forewordThis British St
2、andard is the UK implementation of ISO 15163:2012.The UK participation in its preparation was entrusted to Technical CommitteeAW/5, Chemical analysis of milk and milk products.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not
3、purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2012Published by BSI Standards Limited 2012 ISBN 978 0 580 70430 7 ICS 67.100.01Compliance with a British Standard cannot confer immunity from legal obl
4、igations.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 July 2012.Amendments issued since publicationDate Text affectedBRITISH STANDARDBS ISO 15163:2012Reference numbersISO 15163:2012(E)IDF 110:2012(E)ISO and IDF 2012INTERNATIONAL STANDAR
5、D ISO15163IDF110First edition2012-05-15Milk and milk products Calf rennet and adult bovine rennet Determination by chromatography of chymosin and bovine pepsin contents Lait et produits laitiers Prsure de veau et coagulant issu de bovin adulte Dtermination des teneurs en chymosine et en pepsine bovi
6、ne par chromatographie BS ISO 15163:2012ISO 15163:2012(E) IDF 110:2012(E) COPYRIGHT PROTECTED DOCUMENT ISO and 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 photocop
7、ying and microfilm, without 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 Silver Building Boulevard Auguste Reyers 70/B B-1030 Brussels Tel. + 41 22 749 01 11 Tel. + 32 2 733 98 88 F
8、ax + 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 www.fil-idf.org Published in Switzerland ii ISO and IDF 2012 All rights reservedBS ISO 15163:2012ISO 15163:2012(E) IDF 110:2012(E) ISO and IDF 2012 All rights reserved iiiContents Page Forewo
9、rd iv Foreword . v Introduction vi 1 Scope 1 2 Normative references 1 3 Principle . 1 4 Reagents 2 5 Apparatus . 3 6 Sampling 4 7 Procedure . 4 7.1 Check 4 7.2 Preparation of a fresh column with Fractogel 4 7.3 Regeneration and equilibration of the Fractogel resin in the column . 4 7.4 Storage of th
10、e column with Fractogel . 5 7.5 Preparation of test sample . 5 7.6 Analysis of the desalted rennet . 6 7.7 Determination of the clotting times . 7 8 Calculation and expression of results 8 8.1 Calculation of the activity of chymosin and pepsin, expressed as a percentage 8 8.2 Calculation of active c
11、hymosin and active bovine pepsin, in milligrams per litre . 9 8.3 Expression of results 10 9 Precision 10 9.1 Interlaboratory test 10 9.2 Repeatability 10 9.3 Reproducibility 11 10 Test report 11 Annex A (informative) Qualitative determination by double immunodiffusion of milk-coagulating enzymes in
12、 commercial coagulants 12 Annex B (informative) Interlaboratory test . 17 Bibliography 19 BS ISO 15163:2012ISO 15163:2012(E) IDF 110:2012(E) iv ISO and IDF 2012 All rights reservedForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
13、 (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 on that committee. International organizations,
14、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 drafted in accordance with the rules given in the ISO/
15、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 Standard requires approval by at least 75 % of the me
16、mber 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 15163IDF 110 was prepared by Technical Committee ISO/TC 34, Food prod
17、ucts, Subcommittee SC 5, Milk and milk products and the International Dairy Federation (IDF). It is being published jointly by IDF and ISO. This first edition of ISO 15163IDF 110 cancels and replaces IDF 110B:1997, which has been technically revised. BS ISO 15163:2012ISO 15163:2012(E) IDF 110:2012(E
18、) ISO and IDF 2012 All rights reserved vForeword 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 well as regional dairy associations having signed a formal agreement
19、 on cooperation with IDF. All members of IDF have the right to be represented on the IDF Standing 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
20、to prepare International Standards. Draft International Standards adopted by the Standing Committees are circulated to the National Committees for endorsement prior to publication as an International Standard. Publication as an International Standard requires approval by at least 50 % of IDF Nationa
21、l Committees casting a vote. Attention is drawn 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 15163IDF 110 was prepared by the International Dairy Federation (IDF
22、) and Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published jointly by IDF and ISO. All work was carried out by an ISO-IDF Project Group on Chymosin and bovine pepsin determination, of the Standing Committee on Analytical methods for processin
23、g aids and indicators, under the aegis of its project leader, Prof. A. Andrn (SE). This first edition of ISO 15163IDF 110 cancels and replaces IDF 110B:1997, which has been technically revised. BS ISO 15163:2012ISO 15163:2012(E) IDF 110:2012(E) vi ISO and IDF 2012 All rights reservedIntroduction Cal
24、f rennet and adult bovine rennet preparations contain both chymosin and bovine pepsin in various amounts as main clotting enzymes. The proportion of chymosin decreases relative to pepsin in the abomasum (the fourth “true” stomach) with age and at weaning of the calf. The ratio of abomasa from young
25、cattle to that of old cattle in the raw material for rennet production thus highly influences the composition of chymosin and pepsin in the final rennet. The higher the abomasa from young milk-fed calves, the higher the proportion of chymosin and vice versa56. Both chymosin and pepsin have special c
26、haracteristics relevant to milk-clotting activity and suitability for cheese making. The milk-clotting activity of pepsin is, for example, much more pH-dependent than chymosin and pepsin also has a more general proteolytic activity than chymosin. Therefore, it is very important to analyse the conten
27、t of chymosin and pepsin in addition to the strength (total milk-clotting activity) of the rennet67. BS ISO 15163:2012INTERNATIONAL STANDARD ISO 15163:2012(E)IDF 110:2012(E) ISO and IDF 2012 All rights reserved 1Milk and milk products Calf rennet and adult bovine rennet Determination by chromatograp
28、hy of chymosin and bovine pepsin contents 1 Scope This International Standard specifies a reference method for the determination of the amounts of chymosin and bovine pepsin present in a test sample of calf rennet and adult bovine rennet. In addition, it can be used for mixtures of calf/bovine renne
29、t with fermentation-produced bovine chymosin (FPC). 2 Normative references The following referenced document is indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including a
30、ny amendments) applies. ISO 11815IDF 157:2007, Milk Determination of total milk-clotting activity of bovine rennets 3 Principle As a first step, the rennet sample is desalted and the enzymes chymosin and bovine pepsin separated on an anion exchange column89. In a second step, the milk-clotting activ
31、ity of each of the separated two enzymes is determined by ISO 11815IDF 157 (reconstituted milk with pH 6,5). The enzymatic composition of the rennet sample is expressed in percentage chymosin activity and percentage pepsin activity of the sum of the activities in International Milk-Clotting Units (I
32、MCU) of both components, or the results are expressed in milligrams per litre of active chymosin and milligrams per litre of active pepsin. The total milk-clotting activity of the first batch of calf rennet reference standard powder and the first batch of adult bovine rennet reference standard powde
33、r has once and for all been set at 1 000 IMCU/g. Future preparations of reference standards shall be set relative to the previous reference standards (see ISO 11815IDF 157). This International Standard specifies both a manual set-up of the anion exchange chromatography and an alternative automated s
34、et-up. This is a reference method and changes may therefore only be made if confirmed to give the same result, and repeatability and reproducibility at least as high as the original standard method. Any change to what is stated in this International Standard method shall also be mentioned in the tes
35、t report (see Clause 10). BS ISO 15163:2012ISO 15163:2012(E) IDF 110:2012(E) 2 ISO and IDF 2012 All rights reserved4 Reagents Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralized water or water of equivalent purity. 4.1 Resin, FractogelEMD DEAE
36、 (M) (Merck cat. no. 1.16883)1)or Mono Q1 ml prepacked column (HR 5/5 or 5/50 GL from GE Healthcare)2)or equivalent resin. NOTE 1 FractogelEMD DEAE (M) is a suitable resin for manual chromatography and Mono Qis suitable for the automated chromatography. NOTE 2 If the Fractogelor Mono Q resins are su
37、bstituted by another resin, it will most likely be necessary to change the buffers in 4.12, resulting in a need to re-evaluate the method. 4.2 Piperazine hexahydrate (C4H10N26H2O). 4.3 Sodium chloride (NaCl). 4.4 Thymol, optional preservative. 4.5 Sodium hydroxide (NaOH). 4.6 Hydrochloric acid solut
38、ion, c(HCl) 1 mol/l. 4.7 Ethanol (C2H5OH), with a volume fraction of at least 96 %. 4.8 Ethanol (C2H5OH), with an approximate volume fraction of at least 20 %. Add 105 ml ethanol 96 % (4.7) to 400 ml water and mix. If a sterile filtration is desired, filter the water before mixing it with the ethano
39、l. 4.9 Urea, c(N2H4CO) 8 mol/l. Dissolve 48 g urea in water and fill to a total volume of 100 ml. 4.10 Dialysis tubing, of diameter approximately 1 cm (Union Carbide)3)or equivalent (optional). NOTE The quality of the dialysis tubing is not critical. 4.11 Desalting columns, Bio-Rad Econopac 10DG (ca
40、t. no. 732-2010)4)or equivalent (optional). Use either the dialysis tubing (4.10) or the desalting columns for desalting the rennet. 4.12 Buffer solutions 4.12.1 Buffer solution I, piperazine (CH2)4(NH)2, c(CH2)4(NH)2 = 0,025 mol/l. 1) FractogelEMD DEAE (M) is an example of a suitable product availa
41、ble commercially. This information is given for the convenience of the users of this document and does not constitute an endorsement of the product by ISO or IDF. 2) Mono Q1 ml prepacked column is an example of a suitable product available commercially. This information is given for the convenience
42、of the users of this document and does not constitute an endorsement of the product by ISO or IDF. 3) Union Carbide is an example of a suitable product available commercially. This information is given for the convenience of users of this document and does not constitute an endorsement of the produc
43、t by ISO and IDF. 4) BioRAD - Econopac 10DG is an example of a suitable product available commercially. This information is given for the convenience of users of this document and does not constitute an endorsement of the product by ISO or IDF. BS ISO 15163:2012ISO 15163:2012(E) IDF 110:2012(E) ISO
44、and IDF 2012 All rights reserved 3Weigh 4,85 g of piperazine (4.2) and 42,8 g of hydrochloric acid solution (4.6) in a beaker and mix. Transfer quantitatively the contents of beaker to a 1 000 ml one-mark volumetric flask (5.5), dilute with water to the 1 000 ml mark, and mix. The pH shall be 5,30 0
45、,05. If not, adjust with piperazine or hydrochloric acid. Before use, degas and preserve the buffer solution as described in 4.12.5. 4.12.2 Buffer solution II, c(NaCl) 0,25 mol/l. Weigh 14,6 g of NaCl into a 1 000 ml one-mark volumetric flask (5.5). Add the buffer solution I (4.12.1) to the 1 000 ml
46、 mark and mix. Do not adjust the pH. Buffer solution II is used only for the manual method. Before use, degas and preserve the buffer solution as described in 4.12.5. 4.12.3 Buffer solution III, c(NaCl) 0,50 mol/l. Weigh 29,2 g of NaCl into a 1 000 ml one-mark volumetric flask (5.5). Add buffer solu
47、tion I (4.12.1) to the 1 000 ml mark and mix. Do not adjust the pH. Buffer solution III is used only for the manual version. Before use, degas and preserve the buffer solution as described in 4.12.5. 4.12.4 Buffer solution IV, c(NaCl) 1,0 mol/l. Weigh 58,4 g of NaCl into a 1 000 ml one-mark volumetr
48、ic flask (5.5). Add buffer solution I (4.12.1) to the 1 000 ml mark and mix. Do not adjust the pH. Before use, degas and preserve the buffer solution as described in 4.12.5. 4.12.5 Degassing and preservation Before use, degas the buffer solutions I to IV (4.12.1 to 4.12.4) under vacuum or by use of
49、an ultrasound water bath. Preserve buffer solutions I to IV for use in the manual method by adding a few thymol crystals and in the automated method by sterile filtering using a filter of 0,2 m. Buffer solutions I to IV can be kept for at least 5 days at room temperature or for 2 months in a refrigerator. 5 Apparatus Usual laboratory apparatus and, in particular, the following. 5.1 Multiway peristaltic pump or other suitable pump (for manual set-up only). 5.2 pH-meter, of
