1、BSI Standards PublicationBS ISO 19344:2015Milk and milk products Starter cultures, probioticsand fermented products Quantification of lactic acidbacteria by flow cytometryBS ISO 19344:2015 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of ISO 19344:2015.The UK partic
2、ipation 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 necessaryprovisions of a contract. Us
3、ers are responsible for its correctapplication. The British Standards Institution 2015.Published by BSI Standards Limited 2015ISBN 978 0 580 85108 7ICS 67.100.10Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of
4、theStandards Policy and Strategy Committee on 31 December 2015.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS ISO 19344:2015Milk and milk products Starter cultures, probiotics and fermented products Quantification of lactic acid bacteria by flow cytometryLait et produit
5、s laitiers Cultures, probiotiques et produits ferments Quantification de bactries lactiques par cytomtrie en fluxINTERNATIONAL STANDARDFirst edition2015-12-15Reference numbersISO 19344:2015(E)IDF 232:2015(E)ISO19344IDF232 ISO and IDF 2015BS ISO 19344:2015ISO 19344:2015(E)IDF 232:2015(E)ii ISO and ID
6、F 2015 All rights reservedCOPYRIGHT PROTECTED DOCUMENT ISO and IDF 2015, 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 post
7、ing on the internet 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 7
8、0/B B-1030 BrusselsCH-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 19344:2015ISO 19344:2015(E)IDF 232:2015(E)Forewords .ivIntroduction vi1 Scope . 12 Normative re
9、ferences 13 Terms and definitions . 14 Principle 25 Diluents and reagents . 35.1 General . 35.2 Peptone-salt solution . 35.3 Diluents and reagents for staining protocols 35.3.1 Protocol A . 45.3.2 Protocol B . 45.3.3 Protocol C . 56 Apparatus . 67 Sampling 78 Preparation of test sample . 78.1 Genera
10、l . 78.2 Freeze-dried cultures 78.3 Frozen cultures . 88.4 Fermented milk products 89 Procedure. 89.1 General . 89.2 Staining 89.2.1 Protocol A . 99.2.2 Protocol B . 99.2.3 Protocol C . 99.3 Flow cytometry analysis 109.3.1 General. 109.3.2 Instruments and settings . 109.4 Gating . 119.4.1 General. 1
11、19.4.2 Protocol A 119.4.3 Protocol B 139.4.4 Protocol C 1410 Calculation and expression of results 1511 Critical factors affecting results .1612 Precision 1712.1 Interlaboratory test1712.2 Repeatability 1812.3 Reproducibility 1813 Test report 18Annex A (informative) Diagram of staining protocols .19
12、Annex B (informative) Calculation example of the appropriate sample dilution .21Annex C (informative) Interlaboratory test22Bibliography .25 ISO and IDF 2015 All rights reserved iiiContents PageBS ISO 19344:2015ISO 19344:2015(E)ForewordsISO (the International Organization for Standardization) is a w
13、orldwide 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
14、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.The procedures used to develop this
15、 document 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 Dir
16、ectives, 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 devel
17、opment 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 for the convenience of users and does not constitute an endorsement.For an explanation on the meaning of ISO s
18、pecific terms 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 product
19、s, Subcommittee SC 5, Milk and milk products and the International Dairy Federation (IDF). This document is being published jointly by ISO and IDF.IDF 232:2015(E)iv ISO and IDF 2015 All rights reservedBS ISO 19344:2015ISO 19344:2015(E)IDF (the International Dairy Federation) is a non-profit private
20、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 composed of representatives of dairy-related national interest groups including dairy farmers, dairy processing in
21、dustry, 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 milk and milk products. Since 2001, ISO and IDF jointly publish their International Standards using the logos
22、 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 responsible for identifying any or all such patent rights. Details of any patent rights identified during the devel
23、opment 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 for the convenience of users and does not constitute an endorsement.ISO 19344 | IDF 232 was prepared by the ID
24、F Standing Committee on Analytical Methods for Dairy Microorganisms and the ISO Technical Committee ISO/TC 34 on Food products, Subcommittee SC 5 on Milk and milk products.The work was carried out by the IDF/ISO Project Group on Quantification of Lactic Acid Bacteria by Flow Cytometry of the Standin
25、g Committee on Analytical Methods for Dairy Microorganisms under the aegis of its project leader, Sandra Casani (DK), Ph.D.IDF 232:2015(E) ISO and IDF 2015 All rights reserved vBS ISO 19344:2015ISO 19344:2015(E)IntroductionQuantification of lactic acid bacteria is an important factor in assessing th
26、e quality of starter cultures, probiotics and fermented milk products. Examination of lactic acid bacteria in these products can be done following different method principles, with plate count techniques being the most traditional and widely used. Newer techniques include flow cytometry, which is ab
27、le to determine cells as active and/or total units. Advantages of the use of flow cytometry include low variation, differentiation between active and total cells, and possibility of high analysis throughout. Furthermore, the quantification and use of the fraction of active cells per total cells is a
28、 key feature and an important flow cytometry tool to evaluate the fitness of a given cell population. This is of special relevance for certain applications such as optimization of production process and stability assessment during shelf-life.The International Organization for Standardization (ISO) a
29、nd the International Dairy Federation (IDF) draw attention to the fact that compliance with this document may involve the use of patents concerning the staining of protocol C as described in this document.Neither ISO nor IDF take position concerning the evidence, validity and scope of these patent r
30、ights.The holder of these patent rights has ensured ISO and IDF that he/she is willing to negotiate licences either free of charge or under reasonable and non-discriminatory terms and conditions with applicants throughout the patented territory. In this respect, the statement of the holder of these
31、patent rights is registered with ISO. Information may be obtained from:Chr. Hansen A/S Boege Alle 10-12 2970 Hoersholm DenmarkAttention is drawn to the possibility that some of the elements of this document may be the subject of patent rights other than those identified above. Neither ISO nor IDF sh
32、all be held responsible for identifying any or all such patent rights.ISO (www.iso.org/patents) and IEC (http:/patents.iec.ch) maintain online databases of patents relevant to their standards. Users are encouraged to consult the databases for the most up-to-date information concerning patents.IDF 23
33、2:2015(E)vi ISO and IDF 2015 All rights reservedBS ISO 19344:2015INTERNATIONAL STANDARDISO 19344:2015(E)IDF 232:2015(E)Milk and milk products Starter cultures, probiotics and fermented products Quantification of lactic acid bacteria by flow cytometryWARNING The use of this International Standard may
34、 involve hazardous materials and operations. This International Standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this International Standard to establish safety and health practices and to determine the applicability of reg
35、ulatory limitations prior to use.1 ScopeThis International Standard specifies a standardized method for the quantification of active and/or total lactic acid bacteria and probiotic strains in starter cultures used in dairy products by means of flow cytometry. The method is also applicable to probiot
36、ics used in dairy products and to fermented milk products such as yogurts containing primarily lactic acid bacteria.This International Standard does not apply to taxonomical differentiation of bacteria. Due to its non-specificity, the method may quantify other bacteria than those within the scope of
37、 this International Standard, when present in the sample. This may lead to overestimation of the counts.The minimum bacterial cell concentration in the sample before applying this standardized method depends on the dilution rates used in the individual protocols. Typically 106cells per gram or ml ar
38、e considered within the minimum range.2 Normative referencesThe following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the ref
39、erenced document (including any amendments) applies.ISO 6887-1:1999, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 1: General rules for the preparation of the initial suspension and decimal di
40、lutionsISO 6887-5:2010, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 5: Specific rules for the preparation of milk and milk productsISO 7218, Microbiology of food and animal feeding stuffs Ge
41、neral requirements and guidance for microbiological examinationsISO 7889 | IDF 117, Yogurt Enumeration of characteristic microorganisms Colony-count technique at 37 CISO 15214, Microbiology of food and animal feeding stuffs Horizontal method for the enumeration of mesophilic lactic acid bacteria Col
42、ony-count technique at 30 C3 Terms and definitionsFor the purposes of this document, the following terms and definitions apply. ISO and IDF 2015 All rights reserved 1BS ISO 19344:2015ISO 19344:2015(E)3.1lactic acid bacteriagram-positive, non-motile, non-spore forming, catalase-negative, nitrate-redu
43、ctase-negative and cytochrome oxidase-negative bacterium that does not liquefy gelatine or produce indoleNote 1 to entry: Lactic acid bacteria have a fermentative metabolism which is mainly saccharolytic. Lactic acid is the major end product from carbohydrate utilization.EXAMPLE Lactic acid bacteria
44、 of importance for the dairy industry are: Streptococcus thermophilus, Lactococcus lactis, Pediococcus, Enterococcus, Leuconostoc and Lactobacillus.3.2probiotic strainsprobiotic strains are live microorganisms which, when administered in adequate amounts, are intended to confer a health benefit to t
45、he hostEXAMPLE Probiotic strains of importance are: Bifidobacterium animalis, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus plantarum and Propionibacterium freudenreichii.Note 1 to entry: See Reference 1.3.3acti
46、ve fluorescent unitsAFUevents counted in a gate specific for scatter/fluorescence characteristics of presumed live cells, i.e. cells stained for the specific activity indicator used in the protocol3.4non-active fluorescent unitsn-AFUevents counted in a gate specific for scatter/fluorescence characte
47、ristics of presumed dead cells, i.e. cells damaged to an extend that they do not stain for the specific activity indicator used in the protocol3.5total fluorescent unitsTFUsum of AFU and n-AFU3.6% active fluorescent units% AFUpercentage ratio of AFU to TFU4 Principle4.1 A test portion or sample is p
48、repared, and diluted if necessary.4.2 Initial suspensions, and/or dilutions if needed, are stained according to one of the following three protocols, differing on the target of fluorescent cell staining, in order to discriminate active and total fluorescent units:a) dual staining targeting nucleic a
49、cid with the non-permeant red-fluorescent dye propidium iodide (PI) and intracelullar enzyme activity based on cleavage of 5(6)-carboxyfluorescein diacetate (cFDA) mixed isomers to green-fluorescent carboxyfluorescein by intracellular esterases;IDF 232:2015(E)2 ISO and IDF 2015 All rights reservedBS ISO 19344:2015ISO 19344:2015(E)b) dual nucleic acid staining with PI and a cell-permeant green fluorescent dye, i.e. SYTO1)24 green fluorescent cell-permeant nucleic acid stain;c) single staining with the membrane-pote
