1、 Reference numbers ISO 29981:2010(E) IDF 220:2010(E) ISO and IDF 2010INTERNATIONAL STANDARD ISO 29981 IDF 220 First edition 2010-02-01 Milk products Enumeration of presumptive bifidobacteria Colony count technique at 37 C Produits laitiers Dnombrement des bifidobacteria prsums Technique par comptage
2、 des colonies 37 C ISO 29981:2010(E) IDF 220:2010(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces which are embedded are licensed to and installed on the comput
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6、anical, including photocopying 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 Diamant Building Boulevard Auguste Reyers 80 B-1030 Brussels Tel. + 41 22 749 01 1
7、1 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 www.fil-idf.org Published in Switzerland ii ISO and IDF 2010 All rights reservedISO 29981:2010(E) IDF 220:2010(E) ISO and IDF 2010 All rights reserved iiiContents Pag
8、e Foreword iv Foreword .v 1 Scope1 2 Normative references1 3 Terms and definitions .2 4 Principle2 5 Culture media, diluents and reagents .3 5.1 Basic materials 3 5.2 Diluent(s) 3 5.3 Culture medium (TOS-MUP medium) 3 6 Apparatus.5 7 Sampling.5 8 Procedure.6 8.1 General .6 8.2 Preparation of the tes
9、t portion and primary dilution .6 8.3 Microscopic examination7 8.4 Preparation of decimal dilutions7 8.5 Inoculation .7 8.6 Duration of the procedure 8 8.7 Incubation.8 8.8 Counting of the colonies 8 8.9 Reading of the Petri dishes confirmation.8 9 Calculation and expression of results 8 9.1 Calcula
10、tion .8 9.2 Expression of results9 10 Precision.10 10.1 Interlaboratory test10 10.2 Repeatability 10 10.3 Reproducibility 10 10.4 Precision data collectively defined for dairy products11 11 Knowledge of use of the method.13 12 Test report13 Annex A (informative) Interlaboratory trial A bifido ring t
11、rial 14 Bibliography16 ISO 29981:2010(E) IDF 220:2010(E) iv ISO and IDF 2010 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 carr
12、ied 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, governmental and non-governmental, in liaison with ISO, also take part in the work.
13、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/IEC Directives, Part 2. The main task of technical committees is to prepare Internat
14、ional 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 member bodies casting a vote. Attention is drawn to the possibility that some of the e
15、lements 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 29981|IDF 220 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federa
16、tion (IDF). It is being published jointly by ISO and IDF. ISO 29981:2010(E) IDF 220:2010(E) ISO and IDF 2010 All rights reserved vForeword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every member country. Every National Committe
17、e has 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 to prepare International Standards. Draft In
18、ternational 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 National Committees casting a vote. Attention is dr
19、awn to the possibility that some of the elements of this International Standard may be the subject of patent rights. IDF shall not be held responsible for identifying any or all such patent rights. ISO 29981|IDF 220 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Mil
20、k and milk products and the International Dairy Federation (IDF). It is being published jointly by ISO and IDF. All work was carried out by the Joint ISO-IDF Action Team on Lactic acid bacteria and starters of the Standing Committee on Microbiology methods of analysis under the aegis of its project
21、leaders, Prof. W. Kneifel (AT) and Dr. U. Zitz (AT). INTERNATIONAL STANDARD ISO 29981:2010(E) IDF 220:2010(E) ISO and IDF 2010 All rights reserved 1Milk products Enumeration of presumptive bifidobacteria Colony count technique at 37 C 1 Scope This International Standard specifies a method for the se
22、lective enumeration of presumptive bifidobacteria in milk products by using a colony count technique at 37 C under anaerobic conditions. The method is applicable to milk products such as fermented and non-fermented milks, milk powders, infant formulae, and starter cultures where these microorganisms
23、 are present and viable, and in combination with other lactic acid bacteria. (For proposed quality criteria of dairy products, see, for example, Codex Stan 243:20036 .) Bifidobacteria used in milk products usually belong to the species (e.g. see References 7816): a) Bifidobacterium adolescentis; b)
24、B. animalis subsp. animalis; c) B. animalis subsp. lactis; d) B. bifidum; e) B. breve; f) B. infantis; g) B. longum. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cites applies. For undated ref
25、erences, the latest edition of the referenced documents (including any amendments) applies. ISO 6887-1, 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 th
26、e initial suspension and decimal dilutions ISO 6887-5, 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 products ISO 7218, Microbiology of f
27、ood and animal feeding stuffs General requirements and guidance for microbiological examinations ISO 7889|IDF 117, Yogurt Enumeration of characteristic microorganisms Colony-count technique at 37 C ISO 29981:2010(E) IDF 220:2010(E) 2 ISO and IDF 2010 All rights reservedISO/TS 11133-1, Microbiology o
28、f food and animal feeding stuffs Guidelines on preparation and production of culture media Part 1: General guidelines on quality assurance for the preparation of culture media in the laboratory ISO 14461-1|IDF 169-1, Milk and milk products Quality control in microbiological laboratories Part 1: Anal
29、yst performance assessment for colony counts ISO 14461-2|IDF 169-2, Milk and milk products Quality control in microbiological laboratories Part 2: Determination of the reliability of colony counts of parallel plates and subsequent dilution steps 3 Terms and definitions For the purposes of this docum
30、ent, the following terms and definitions apply. 3.1 bifidobacteria anaerobic microorganisms that form lenticular or round whitish colonies, partially star shaped or trilobate of diameter 1 mm to 4 mm on transgalactosylated oligosaccharides-mupirocin lithium salt (TOS-MUP) medium under the conditions
31、 specified in this International Standard 4 Principle 4.1 The antibiotic, mupirocin lithium salt (MUP), inhibits the growth of most lactic acid bacteria commonly used in fermented and non-fermented dairy products. Owing to the proven selectivity of the MUP antibiotic when added to the medium, usuall
32、y there is no growth of typical yogurt bacteria (Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus), mesophilic cultures (e.g. Lactococcus lactis), Lactobacillus acidophilus, Lactobacillus casei and Lactobacillus rhamnosus on the medium specified. This property has been tested
33、with a representative number of reference strains and isolates. Additionally, TOS-agar enhances the growth of bifidobacteria used in dairy products (see Reference 17). NOTE 1 Examination under a microscope at a magnification of 100 times and oil immersion in contrast phase illumination shows rods of
34、 very varied shapes, usually curved and clubbed, often branched, arranged singly, in pairs, in V- shaped arrangements, in chains, in palisades of parallel cells, or in rosettes occasionally exhibiting swollen coccoid forms. NOTE 2 Bifidobacteria are non-acid-fast, non-spore-forming, gram-positive, n
35、on-motile and catalase-negative chemoorganotrophs, which produce acetic acid and lactic acid. Glucose is degraded exclusively and characteristically by the fructose-6-phosphate shunt in which fructose-6-phosphate phosphoketolase (F6PPK, EC 4.1.2.22) cleaves fructose-6- phosphate into acetyl phosphat
36、e and erythrose-4-phosphate. NOTE 3 The optimum growth temperature is between 37 C and 41 C. For further details, see Reference 9. 4.2 Inoculation of appropriate decimal dilutions of the homogenized sample into TOS-agar containing MUP using the pour plate technique, is followed by anaerobic incubati
37、on at 37 C for 72 h. 4.3 The colonies are counted. NOTE Optionally, selected isolates from the plates can be confirmed by means of appropriate tests (e.g. F6PPK assay, see References 1415). 4.4 The number of bifidobacteria per gram of sample is calculated from the number of colonies obtained on plat
38、es at dilution levels so as to give a significant result. ISO 29981:2010(E) IDF 220:2010(E) ISO and IDF 2010 All rights reserved 35 Culture media, diluents and reagents Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralized water or water of equi
39、valent purity. 5.1 Basic materials See ISO 6887-5 and ISO/TS 11133-1 for basic materials. 5.2 Diluent(s) See ISO 6887-5 for the preparation of diluents. To ensure comparability of the specified colony-forming unit (CFU) results, observe the following requirements. a) Use quarter-strength Ringers sol
40、ution, or any other suitable diluent which is specified in ISO 6887-5 and proven to be equivalent. b) Sterilize in bulk and use an adequate sterile dispenser unit. c) Adjust the diluent to room temperature. Transfer the diluent by dripping, without incorporating air. d) The uncertainty of measuremen
41、t of volumes used shall be in accordance with the requirements of ISO 6887-1. 5.3 Culture medium (TOS-MUP medium) Use freshly prepared transgalactosylated oligosaccharides-mupirocin lithium salt (TOS-MUP) culture medium, which has not been exposed to direct sunlight. 5.3.1 Basic medium (TOS-propiona
42、te agar medium, see Reference 10) 5.3.1.1 Composition Trypticase peptone 10,0 g Yeast extract 1,0 g KH 2 PO 4 3,0 g K 2 HPO 44,8 g (NH 4 ) 2 SO 43,0 g MgSO 4 7H 2 O 0,2 g (R)-CysteineHClH 2 O 0,5 g Sodium propionate 15,0 g TOS (see 5.3.1.2) 10,0 g Agar 15,0 gWater 950 ml ISO 29981:2010(E) IDF 220:20
43、10(E) 4 ISO and IDF 2010 All rights reserved5.3.1.2 Transgalactosylated oligosaccharide mixture A TOS mixture is obtained by enzymatic hydrolysis of lactose using Aspergillus oryceae -galactosidase. The TOS mixture contains galactose (Gal) and glucose (Glc) units in accordance with the formula () Ga
44、l Gal Glc n y x where n = 1 4; x = -1,6 -1,4 and -1,3; y = -1,4 -1,3 and -1,6. The TOS mixture is purified by chromatography under defined conditions (see References 1819). The total sugar content ( 97 % mass fraction) includes a certain proportion of tri-, tetra-, penta- and hexasaccharides. (Modif
45、ication of the ratio of oligosaccharides has no significant effect on the potential of the medium.) 5.3.1.3 Preparation Suspend the ingredients in 950 ml water while heating carefully (e.g. using a hotplate or a water bath) with frequent agitation until completely dissolved. Distribute in portions o
46、f 190 ml into bottles of 250 ml capacity. Adjust the pH (6.6), if necessary, so that after autoclaving a final pH of 6,3 0,2 pH-units is obtained at 25 C. Autoclave the basic medium at 115 C for 15 min. If not used immediately, cool the prepared basic medium, unless otherwise specified. Store the me
47、dium between 2 C and 4 C for a maximum of 1 week under conditions not producing any change in its composition. TOS-medium is sensitive to heat, thus excessive heat treatment can negatively influence the properties of the medium. Complete TOS-propionate media are commercially available and have a com
48、position in accordance with this International Standard. However, if the medium is made up in the laboratory, the results can differ significantly from one preparation to another. Therefore media should be validated to ensure that growth performance of bifidobacteria, indicated by CFU results, are o
49、n a comparable level (see also ISO/TS 11133-1). 5.3.2 MUP supplement solution (see Reference 11) Immediately before use, dissolve, for example, 50 mg MUP in 50 ml of water, or other amounts in the same proportion. Sterilize the solution obtained by filtration through a membrane (pore size 0,22 m) as specified in 5.3.3. 5.3.3 Complete medium Immediately before use, melt 190 ml portions of the prepared basic medium (5.3.1) under steam or equivale
