ISO 7889-2003 Yogurt - Enumeration of characteristic microorganisms - Colony-count technique at 37C《酸奶 特性微生物的计数 37℃时的菌落计数法》.pdf

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1、 Reference numbers ISO 7889:2003(E) IDF 117:2003(E) ISO and IDF 2003INTERNATIONAL STANDARD ISO 7889 IDF 117 First edition 2003-02-01 Yogurt Enumeration of characteristic microorganisms Colony-count technique at 37 C Yaourt Dnombrement des micro-organismes caractristiques Technique de comptage des co

2、lonies 37 C ISO 7889:2003(E) IDF 117:2003(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 computer perfo

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5、ating to it is found, please inform the ISO Central Secretariat at the address given below. ISO and IDF 2003 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 mi

6、crofilm, 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 11 Tel. + 32 2 733 98 88 Fax + 41 22 7

7、49 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 2003 All rights reservedISO 7889:2003(E) IDF 117:2003(E) ISO and IDF 2003 All rights reserved iiiContents Page Foreword iv Foreword. v 1 Scope 1 2

8、Normative references . 1 3 Terms and definitions. 1 4 Principle . 2 5 Culture media, diluents and reagents. 2 6 Apparatus and glassware. 4 7 Sampling 5 8 Preparation of test sample. 5 9 Procedure. 5 9.1 Preparation of test portion. 5 9.2 Microscopic examination . 6 9.3 Preparation of primary dilutio

9、n . 6 9.4 Preparation of decimal dilutions . 6 9.5 Duration of the procedure 6 9.6 Inoculation and incubation 6 9.7 Counting of colonies 6 9.8 Confirmation 7 10 Calculation and expression of results 7 10.1 Calculation. 7 10.2 Expression of results 7 10.3 Examples of calculation . 8 11 Precision 9 11

10、.1 General. 9 11.2 Repeatability 9 12 Test report 9 Annex A (informative) Notes on procedure . 10 Bibliography . 11 ISO 7889:2003(E) IDF 117:2003(E) iv ISO and IDF 2003 All rights reservedForeword ISO (the International Organization for Standardization) is a worldwide federation of national standard

11、s 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 committee. International organiz

12、ations, 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

13、the ISO/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 % o

14、f the member 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 7889IDF 117 was prepared by Technical Committee ISO/TC 34, Fo

15、od products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC International. It is being published jointly by ISO and IDF and separately by AOAC International. ISO 7889:2003(E) IDF 117:2003(E) ISO and IDF 2003 All rights reserved vFo

16、reword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every member country. Every National Committee has the right to be represented on the IDF Standing Committees carrying out the technical work. IDF collaborates with ISO and AOAC

17、 International in the development of standard methods of analysis and sampling for milk and milk products. Draft International Standards adopted by the Action Teams and Standing Committees are circulated to the National Committees for voting. Publication as an International Standard requires approva

18、l by at least 50 % of the National Committees casting a vote. ISO 7889IDF 117 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC International. It is being published jointly

19、 by ISO and IDF and separately by AOAC International. All work was carried out by the Joint ISO/IDF/AOAC Action Team, Lactic acid bacteria and starters, of the Standing Committee on Microbiological methods of analysis, under the aegis of its project leader, Prof. B. Bianchi Salvadori (IT). This edit

20、ion cancels and replaces the first edition of IDF 117A:1988, which has been technically revised. INTERNATIONAL STANDARD ISO 7889:2003(E) IDF 117:2003(E) ISO and IDF 2003 All rights reserved 1Yogurt Enumeration of characteristic microorganisms Colony-count technique at 37 C 1 Scope This International

21、 Standard specifies a method for the enumeration of characteristic microorganisms in yogurt by means of the colony-count technique at 37 C. The method is applicable to yogurts in which both characteristic microorganisms (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus) are

22、 present and viable. 2 Normative references The following referenced documents are 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 any amendments) applies. ISO 6

23、887-1, 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 the initial suspension and decimal dilutions ISO 7218, Microbiology of food and animal feeding stuf

24、fs General rules for microbiological examinations ISO 8261IDF 122, Milk and milk products General guidance for the preparation of test samples, initial suspensions and decimal dilutions for microbiological examination 3 Terms and definitions For the purposes of this document, the following terms and

25、 definitions apply. 3.1 characteristic microorganisms in yogurt Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus as detected under the conditions specified in this International Standard 3.2 Lactobacillus delbrueckii subsp. bulgaricus thermophilic microorganism which forms

26、lenticular, often sharp-shaped, colonies of diameter 1 mm to 3 mm on acidified MRS medium under the conditions specified in this International Standard NOTE Under a microscope, these microorganisms appear as rods, generally short, but sometimes in longer forms. They are non-spore forming, Gram-posit

27、ive, non-motile and catalase-negative. 3.3 Streptococcus thermophilus thermophilic microorganism which forms lenticular colonies of diameter 1 mm to 2 mm on M17 medium under the conditions specified in this International Standard ISO 7889:2003(E) IDF 117:2003(E) 2 ISO and IDF 2003 All rights reserve

28、dNOTE Under a microscope, these microorganisms appear as spherical or ovoid cells (of diameter 0,7 m to 0,9 m) in pairs or in long chains. They are Gram-positive and catalase-negative. 4 Principle 4.1 Decimal dilutions of the test sample are inoculated into: a) acidified MRS medium, followed by anae

29、robic incubation at 37 C 1 C for 72 h, for the count of Lactobacillus delbrueckii subsp. bulgaricus; b) complete medium (M17), followed by aerobic incubation at 37 C 1 C for 48 h, for the count of Streptococcus thermophilus. 4.2 The colonies are counted and confirmed by means of appropriate tests. 4

30、.3 The number of characteristic microorganisms per gram of sample is calculated from the number of colonies obtained on plates at dilution levels so as to give a significant result. 5 Culture media, diluents and reagents Use only reagents of recognized analytical grade, unless otherwise specified, a

31、nd distilled or demineralized water or water of equivalent purity. See also ISO 6887-1 and ISO 8261IDF 122. 5.1 Diluent See ISO 6887-1 and ISO 8261IDF 122. 5.2 Culture media Use freshly prepared culture media (MRS and M17) which shall not be exposed to direct sunlight. If the prepared culture media

32、are not used immediately, they shall, unless otherwise specified, be cooled and stored at between 2 C and 4 C for no longer than 1 week and under conditions which do not produce any change in their composition. As for reagents, see storage conditions specified in ISO 7218. 5.2.1 Acidified MRS medium

33、 (see reference 7) 5.2.1.1 Composition Peptone 1 (tryptic digest of casein) 10,0 g Meat extract 10,0 g Yeast extract (dried) 5,0 g Glucose (C 6 H 12 O 6 ) 20,0 g Tween 80 (sorbitan mono-oleate) 1,0 ml Dipotassium hydrogen orthophosphate (K 2 HPO 4 ) 2,0 g Sodium acetate trihydrate (CH 3 CO 2 Na3H 2

34、O) 5,0 g Diammonium citrate C 6 H 6 O 7 (NH 4 )2 2,0 g Magnesium sulfate heptahydrate (MgSO 4 7H 2 O) 0,2 g Manganese sulfate tetrahydrate (MnSO 4 4H 2 O) 0,05 g Agar 9,0 g to 18,0 g 1)Water up to 1 000 ml1) Depending on the gel strength of the agar or according to manufacturers instructions. ISO 78

35、89:2003(E) IDF 117:2003(E) ISO and IDF 2003 All rights reserved 35.2.1.2 Preparation Separately dissolve each component in a water bath (6.7) set at boiling. Cool in another water bath (6.7) to 50 C. Adjust the pH so that after sterilization it is 5,4 0,1 at 25 C 1 C by adding acetic acid (5.3.3) an

36、d checking with the pH-meter (6.8). Transfer the medium in 100 ml portions into 150 ml bottles (6.10) or in 200 ml portions into 250 ml bottles (6.10). Sterilize for 15 min in an autoclave at 121 C 1 C. NOTE 1 MRS medium is highly sensitive to heat treatment which may cause differences according to

37、the autoclave used. NOTE 2 Comparative tests have shown that commercially available MRS media may give counts that are lower than those given by the MRS medium prepared in accordance with this International Standard. Therefore, if used, the former should be checked against the medium prepared accord

38、ing to this International Standard. This may cause problems for yogurt producers and the authorities investigating the requested minimum cell count in yogurt products. Before beginning the bacteriological examination, completely melt the required amount of medium in a water bath (6.7) set at boiling

39、, or by steaming in a partially closed container. Then cool it in another water bath (6.7). 5.2.2 M17 medium (see reference 8) 5.2.2.1 Basic medium 5.2.2.1.1 Composition Peptone 1 (tryptic digest of casein) 2,5 g Peptone 2 (peptic digest of meat) 2,5 g Peptone 3 (papain digest of soya) 5,0 g Yeast e

40、xtract (dried) 2,5 g Meat extract 5,0 g -Glycerophosphate (disodium salt) (C 3 H 7 O 6 PNa 2 ) 19,0 g Magnesium sulfate heptahydrate (MgSO 4 7H 2 O) 0,25 g Ascorbic acid (C 6 H 8 O 6 ) 0,50 g Agar 9,0 g to 18,0 g 1)Water up to 950 ml 5.2.2.1.2 Preparation Separately, dissolve each component in a wat

41、er bath (6.7) set at boiling. Cool in another water bath (6.7) to 50 C. Adjust the pH so that, after sterilization, it is 6,8 0,1 at 25 C 1 C by using a reagent (5.3) and checking with the pH-meter (6.8). Transfer the medium in 95 ml portions into 150 ml bottles (6.10). Sterilize for 15 min in an au

42、toclave at 121 C 1 C. 5.2.2.2 Lactose solution 5.2.2.2.1 Composition Lactose (C 12 H 22 O 11 ) 10,0 g Water up to 100 ml 5.2.2.2.2 Preparation Dissolve the lactose in the water. Dilute with water to 100 ml. Sterilize for 15 min in an autoclave at 121 C 1 C. ISO 7889:2003(E) IDF 117:2003(E) 4 ISO and

43、 IDF 2003 All rights reserved5.2.2.3 Complete medium (M17) 5.2.2.3.1 Composition Basic medium (5.2.2.1) 95,0 ml Lactose solution (5.2.2.2) 5,0 ml 5.2.2.3.2 Preparation Immediately before use, melt the basic medium in a water bath (6.7) set at boiling. Cool in another water bath (6.7) to 50 C. Prehea

44、t the lactose solution in a water bath (6.7) set at 50 C. Add the lactose solution to the basic medium and mix by swirling. Cool the medium in the water bath to between 44 C and 47 C. NOTE Complete M17 media are commercially available but, as in the case of commercially available MRS media, the resu

45、lts obtained may differ significantly from one supplier to the other. Therefore they should be checked against M17 medium prepared according to this International Standard. This may cause problems for yogurt producers and the authorities investigating the requested minimum bacterial count in yogurt

46、products. 5.3 Reagents for pH adjustment 5.3.1 Sodium hydroxide solution, c(NaOH) = 0,1 mol/l approximately. 5.3.2 Dilute hydrochloric acid, c(HCl) = 0,1 mol/l approximately. 5.3.3 Acetic acid (CH 3 COOH), 100 % (glacial). 5.4 Reagent for staining, ethanolic solution of methylene blue, 6 g/l. 5.5 Re

47、agent for cleaning the container surface, ethanol 70 % (volume fraction). 6 Apparatus and glassware Sterilization of equipment that will come into contact with the test sample, the diluent, the dilutions or the culture medium shall be carried out in accordance with the requirements of ISO 8261IDF 12

48、2. The glassware shall be resistant to repeated sterilization. Usual microbiological laboratory equipment (see ISO 7218) for the preparation of test samples and dilutions, as specified in ISO 8261IDF 122 and, in particular, the following. 6.1 Incubator, capable of operating at 37 C 1 C. 6.2 Anaerobi

49、c incubation cabinet or anaerobic jars, capable of being maintained at 37 C 1 C, providing an atmosphere of 90 % nitrogen and 10 % carbon dioxide. 6.3 Blender, either a peristaltic-type blender (stomacher) with sterile plastic bags, or a rotary blender, capable of operating at a minimum rotational frequency of 20 000 min 1 , with sterile 200 ml round-bottom centrifuge tubes made of strengthened glass, or metal containers of appropriate capacity. 6.4 Te