1、BSI Standards PublicationBS ISO 27105:2016Milk and cheese Determination of hens eggwhite lysozyme content byhigh performance liquidchromatographyBS ISO 27105:2016 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of ISO 27105:2016.It supersedes DD ISO/TS 27105:2009 whic
2、h is withdrawn.The UK participation in its preparation was entrusted to TechnicalCommittee AW/5, Chemical analysis of milk and milk products.A list of organizations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 to include all the necessary
3、provisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2016.Published by BSI Standards Limited 2016ISBN 978 0 580 85005 9ICS 67.100.01Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was publi
4、shed under the authority of theStandards Policy and Strategy Committee on 30 April 2016.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS ISO 27105:2016Milk and cheese Determination of hens egg white lysozyme content by high performance liquid chromatographyLait et fromage
5、s Dtermination de la teneur en lysozyme de blanc doeuf par chromatographie liquide haute performanceINTERNATIONAL STANDARDFirst edition2016-04-01Reference numbersISO 27105:2016(E)IDF 216:2016(E)ISO27105IDF216 ISO and IDF 2016BS ISO 27105:2016ISO 27105:2016(E)IDF 216:2016(E)ii ISO and IDF 2016 All ri
6、ghts reservedCOPYRIGHT PROTECTED DOCUMENT ISO and IDF 2016, Published in SwitzerlandAll rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the in
7、ternet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below or ISOs member body in the country of the requester.ISO copyright office International Dairy FederationCh. de Blandonnet 8 CP 401 Silver Building Bd Auguste Reyers 70/B B-1030 Br
8、usselsCH-1214 Vernier, Geneva, SwitzerlandTel. +41 22 749 01 11 Tel. + 32 2 325 67 40Fax +41 22 749 09 47 Fax + 32 2 325 67 41copyrightiso.org infofil-idf.orgwww.iso.org www.fil-idf.orgBS ISO 27105:2016ISO 27105:2016(E)IDF 216:2016(E)Forewords .ivIntroduction vi1 Scope . 12 Terms and definitions . 1
9、3 Principle 14 Reagents and reference substances . 14.1 Reagents and materials . 14.2 Lysozyme . 25 Apparatus . 26 Sampling 37 Procedure. 37.1 Preparation of lysozyme standard solution . 37.1.1 Lysozyme standard stock solution . 37.1.2 Lysozyme standard working solution . 37.2 Test portion 37.2.1 Mi
10、lk 37.2.2 Cheese . 37.2.3 Preparation of test solution 37.3 HPLC determination . 47.3.1 Chromatographic conditions . 48 Calculation and expression of results . 58.1 Single-point calibration . 58.2 Expression of results 69 Precision . 69.1 Interlaboratory test. 69.2 Repeatability . 69.3 Reproducibili
11、ty . 610 Test report . 6Annex A (informative) Verification by LC/MS 8Annex B (informative) Interlaboratory test .10Bibliography .11 ISO and IDF 2016 All rights reserved iiiContents PageBS ISO 27105:2016ISO 27105:2016(E)ForewordsISO (the International Organization for Standardization) is a worldwide
12、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 on that c
13、ommittee. 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.The procedures used to develop this document
14、 and those intended for its further maintenance are described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives,
15、Part 2 (see www.iso.org/directives).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. Details of any patent rights identified during the development of
16、 the document will be in the Introduction and/or on the ISO list of patent declarations received (see www.iso.org/patents).Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement.For an explanation on the meaning of ISO specific t
17、erms and expressions related to conformity assessment, as well as information about ISOs adherence to the WTO principles in the Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary informationThe committee responsible for this document is ISO/TC 34, Food products, Subcom
18、mittee SC 5, Milk and milk products and the International Dairy Federation (IDF) and it is being published jointly by ISO and IDF.This first edition of ISO 27105|IDF 216 cancels and replaces ISO/TS 27105|IDF/RM 216:2009, which has been technically revised.IDF 216:2016(E)iv ISO and IDF 2016 All right
19、s reservedBS ISO 27105:2016ISO 27105:2016(E)IDF (the International Dairy Federation) is a non-profit private sector organization representing the interests of various stakeholders in dairying at the global level. IDF members are organized in National Committees, which are national associations compo
20、sed of representatives of dairy-related national interest groups including dairy farmers, dairy processing industry, dairy suppliers, academics and governments/food control authorities.ISO and IDF collaborate closely on all matters of standardization relating to methods of analysis and sampling for
21、milk and milk products. Since 2001, ISO and IDF jointly publish their International Standards using the logos and reference numbers of both organizations.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 respon
22、sible for identifying any or all such patent rights. Details of any patent rights identified during the development of the document will be in the Introduction and/or on the ISO list of patent declarations received (see www.iso.org/patents).Any trade name used in this document is information given f
23、or the convenience of users and does not constitute an endorsement.ISO 27105|IDF 216 was prepared by the IDF Standing Committee on Analytical Methods for Additives and contaminants and ISO Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products.All work was carried ou
24、t by the ISO/IDF Project Group on Determination of hens egg white lysozyme content by high performance liquid chromatography of the Standing Committee on Analytical Methods for Additives and Contaminants under the aegis of the project leaders, T. Berger (CH) and Prof. L. Pellegrino (IT).This first e
25、dition of ISO 27105|IDF 216 cancels and replaces ISO/TS 27105|IDF/RM 216:2009, which has been technically revised.IDF 216:2016(E) ISO and IDF 2016 All rights reserved vBS ISO 27105:2016ISO 27105:2016(E)IntroductionLysozyme (EC 3.2.1.17, muramidase) is an enzyme widely dispersed in nature; it appears
26、, e.g. in hens egg white (approximately 3 % to 4 %), saliva and in tear liquid. Lysozyme has a preservative effect because of the lytic activity on the cell wall of some bacteria. Hens egg white lysozyme is used in cheese making to prevent late blowing of semi-hard and hard cheeses.IDF 216:2016(E)vi
27、 ISO and IDF 2016 All rights reservedBS ISO 27105:2016INTERNATIONAL STANDARDISO 27105:2016(E)IDF 216:2016(E)Milk and cheese Determination of hens egg white lysozyme content by high performance liquid chromatography1 ScopeThis International Standard specifies a method for the quantitative determinati
28、on of hens egg white lysozyme content in milk and cheese.The method is suitable for measuring low levels of hens egg white lysozyme with a quantification limit of 10 mg/kg.2 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.2.1hens egg white lysozyme c
29、ontentmass fraction of substance determined by the procedure specifiedNote 1 to entry: The lysozyme content is expressed as milligram per kilogram.3 PrincipleCasein and denatured whey proteins from milk and cheese are precipitated isoelectrically at pH 4,3 (cheese) or at pH 2,2 (milk). Acid-soluble
30、hens egg white lysozyme is then determined by reversed-phase high-performance liquid chromatography (HPLC) and fluorescence detection. The lysozyme peak can be verified by LC/MS (see Annex A).4 Reagents and reference substancesUse only reagents of recognized analytical grade, unless otherwise specif
31、ied, and only distilled water or water of equivalent purity.4.1 Reagents and materials4.1.1 Sodium chloride solution, c(NaCl) = 1 mol/l.Dissolve 58,44 g of sodium chloride in 1 l water.4.1.2 Hydrochloric acid, c(HCl) = 1 mol/l.Dissolve 4,0 ml of hydrochloric acid with mass fraction 37 % in a 50 ml o
32、ne-mark volumetric flask. Dilute to the mark with water.4.1.3 Sodium hydroxide, c(NaOH) = 1 mol/l.Dissolve 2,6 ml of sodium hydroxide with a mass fraction of 50 % in a 50 ml one-mark volumetric flask. Dilute to the mark with water.4.1.4 Trifluoracetic acid (CF3COOH), analytical grade. ISO and IDF 20
33、16 All rights reserved 1BS ISO 27105:2016ISO 27105:2016(E)4.1.5 Acetonitrile (CH3CN), HPLC grade.4.1.6 Water, HPLC grade.4.2 LysozymePure hens egg white lysozyme1). Sufficiently pure and characterized lysozyme is difficult to obtain. A control on lot-to-lot variability is needed.5 ApparatusUsual lab
34、oratory equipment and, in particular, the following.5.1 pH-meter, accurate to 0,1 unit.5.2 Fluted filter, diameter 15 cm2).5.3 Membrane filter, pore size 0,22 m3).5.4 Balance, capable of weighing to the nearest 100 mg, with a readability of 10 mg.5.5 Analytical balance, capable of weighing to the ne
35、arest 0,1 mg, with a readability of 0,01 mg.5.6 Magnetic stirrer.5.7 Homogenizer 4), capable of spinning at a rotational frequency of 3 000 rpm to 3 500 rpm.5.8 HPLC equipment.5.8.1 Gradient pumping system, capable of operating with a speed of 1,0 ml/min.5.8.2 Manual or automatic injector, capable o
36、f injecting amounts of 50 l.5.8.3 Column heater, capable of maintaining a column temperature of 45 C 2 C.5.8.4 Column, reversed phase5), 5 m, 250 mm 4,6 mm.1) Lysozyme SIGMA L-6876 is an example of a suitable product available commercially. This information is given for the convenience of users of t
37、his document and does not constitute an endorsement by either ISO or IDF of this product.2) Fluted filter Schleicher stock solution II: 1 ml of trifluoracetic acid (4.1.4) in 1 l of acetonitrile (4.1.5).Use the following elution solvents for HPLC: elution solvent A contains: stock solution I:stock s
38、olution II = 72,25:27,75 (w/w); elution solvent B contains: stock solution II.Table 1 Suggested gradient elutionTime(min)Elution solvent Aa(%)Elution solvent Ba(%)0,0 100 020,0 100 021,0 50 5022,0 50 5023,0 100 035,0 100 0aThe gradient elution might require slight modification in order to achieve th
39、e resolution shown in Figure 1 and Figure 2. Isocratic elution in the range of the lysozyme peak, as used in this International Standard, leads to a cleaner chromatogram but to larger peak width. The use of a gradient may lead to smaller peak widths and more stable retention times but also to more p
40、eaks in the lysozyme range with a risk of misinterpretation.Set the flow rate of the gradient elution pumping system of the HPLC equipment at 1,0 ml/min.Set the temperature of the column heater at 45 C. Determine the equilibration time by monitoring the column elution. The detector response at the e
41、nd of the run (baseline) should be equal to its initial value. An isocratic flushing of 15 min is usually sufficient.Injection: Use a manual or automatic injector to inject 50 l of the solutions into the column.IDF 216:2016(E)4 ISO and IDF 2016 All rights reservedBS ISO 27105:2016ISO 27105:2016(E)Ke
42、y1 lysozymeFigure 1 HPLC fluorescence signal from the standard working solution (7.1.2)Key1 lysozymeFigure 2 HPLC fluorescence signal from a hard cheese sample8 Calculation and expression of results8.1 Single-point calibrationCalculate the lysozyme content of the test sample, w, expressed in milligr
43、ams per kilogram, by using Formula (1):wHcVHm=tstst(1)whereIDF 216:2016(E) ISO and IDF 2016 All rights reserved 5BS ISO 27105:2016ISO 27105:2016(E)csis the concentration, in milligrams per litre, of the standard working solution (see 7.1.2);Htis the numerical value of the peak height or area of the
44、test solution (see 7.2), in counts;Hsis the numerical value of the peak height or area of the standard working solution (see 7.1.2), in counts;mtis the mass, in grams, of the test portion (see 7.2.1 or 7.2.2);Vtis the volume, in millilitres, of the test solution (see 7.2.3).Check equipment linearity
45、 and reagent blank regularly. This may be needed in case of lysozyme contents of more than 100 mg/kg of cheese which can lead to a non-linear response. In such cases, the use of a multiple point calibration is necessary.8.2 Expression of resultsExpress the results to one decimal place. Express test
46、results of below 10 mg/kg as: “less than 10 mg/kg”, or equivalent expression.9 Precision9.1 Interlaboratory testThe values for repeatability and reproducibility derived from an interlaboratory test7were determined in accordance with ISO 5725-12and ISO 5725-2.3The values for the repeatability and the
47、 reproducibility limit are expressed for the 95 % probability level and may not be applicable to concentration ranges and matrices other than those given. Details of the interlaboratory test on the precision of the method are given in Annex B.9.2 RepeatabilityThe absolute difference between two indi
48、vidual single test results, obtained with the same method on identical test material in the same laboratory by the same operator using the same equipment within a short interval of time, shall in not more than 5 % of the cases be greater than 7,7 % (95 % confidence intervals 6,2 % to 8,5 %).9.3 Repr
49、oducibilityThe absolute difference between two individual single test results, obtained with the same method on identical test material in different laboratories with different operators using different equipment, shall in not more than 5 % of the cases be greater than 33,8 % (95 % confidence intervals 20,3 % to 57,5 %).Running the chromatographic system on another temperature, using a different column type, or other differing conditions do not result automatically in false or less sensitive results but n
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