1、BRITISH STANDARD BS ISO 9232:2003 Yogurt Identification of characteristic microorganisms (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus) ICS 07.100.30 BS ISO 9232:2003 This British Standard was published under the authority of the Standards Policy and Strategy Committee
2、on 7 February 2003 BSI 7 February 2003 ISBN 0 580 41190 7 National foreword This British Standard reproduces verbatim ISO 9232:2003 and implements it as the UK national standard. The UK participation in its preparation was entrusted to Technical Committee AW/5, Milk and milk products, which has the
3、responsibility to: A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international publications referred to in this document may be found in the BSI Catalogue under the section entitled “Internati
4、onal Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Standards Online. This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a Britis
5、h Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and
6、European developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the ISO title page, pages ii to v, a blank page, pages 1 to 17 and a back cover. The BSI copyright date displayed in this document indicates when the document was last
7、issued. Amendments issued since publication Amd. No. Date Comments Reference numbers ISO 9232:2003(E) IDF 146:2003(E)INTERNATIONAL STANDARD ISO 9232 IDF 146 First edition 2003-02-01 Yogurt Identification of characteristic microorganisms (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus
8、thermophilus) Yaourt Identification des micro-organismes caractristiques (Lactobacillus delbrueckii subsp. bulgaricus et Streptococcus thermophilus) BSISO9232:2003IS:2329 O3002(E) ID:641 F3002(E) DPlcsid Fremia ihTs PDF file mya ctnoian emdebt dedyfepcaes. In ccacnadrow eitA hebods licnesilop gnic,y
9、 tihs file mirp eb yatnde iv roweb detu slahl ton ide ebtlnu deess teh typfecaes wihce era hml era deddebicsnede ti dna onstlalde t noeh comuptfrep reromign tide ehtin.g In wodlnidaot gnihs fil,e raptitpecca se tierehn ter ehssnopiiblito yf ton ifnirigngn Aebods licnesign lopic.y ieNtt reheh ISeC Ot
10、nrla eScretarain tor hte DIa Fccepts any ailbytili ni htsi area. Ai ebods a tedarmfo kra Aebod SystemI snctaropro.de teDials fo teh sfotwcudorp erats sut deo crtaee tihs PDF file cna f ebi dnuon tlareneG eh Ifnler oatit evt oeh file; tP ehDc-Frtaeino marapteres wtpo ereimizf dero irptni.gn Evyre cne
11、eb sah era tkane to sneeru ttah teh file is siutlbae fsu roe yb ISO memdob rebeis dna IDF antilano ocmmittees. In the unlikely veent that a problem relating to it is f,dnuo lpsaei efnmro tI ehStneC Olar Scerteirata ta teh serddas igleb nevow. ISO dna ID3002 F All irthgs erse.devr lnUeto sswrehise sp
12、ecified, on trap fo this lbupictaion maeb y cudorperro de tuilizi den yna form ro na ybm ynae,s lecetrinoc ro mceinahcla, incliduntohp gcoiypodna gn micrfoilm, wittuoh repmissii now nritign from eitI rehSro O IDF ta ter ehstcepiev serddas lebwo. ISO cirypothg fofice Itntanreilano iaDtaredeF yrino sa
13、Ce tsopale 65 eneG 1121-HC 02 av Dimanat Builid gn BoulA draveugust 08 sreyeR e B 0301-Bssurels leT. 14 + 20 947 2111 eTl. 23 + 29 337 888 aFx0 947 22 14 + 974 aFx0 337 2 23 + 431 -Email copyrightisoo.rg -Emailni off-lidif.org We bwww.is.o gro We bwww.fil-ifd. groii BSISO9232:2003 iiiContents Page F
14、oreword iv Foreword. v 1 Scope 1 2 Normative references . 1 3 Terms and definitions. 1 4 Principle . 1 5 Culture media, diluents and reagents. 2 6 Apparatus and glassware. 5 7 Procedure. 6 7.1 Isolation of colonies . 6 7.2 Phenotypic characteristics required for identification of L. delbrueckii subs
15、p. bulgaricus. 6 7.3 Phenotypic characteristics required for identification of S. thermophilus. 7 8 Expression of results 8 9 Test report 8 Annex A (normative) Main attributes tables 9 Annex B (normative) Milk cultures of lactic acid bacteria Determination of the contents of lactic acid and lactate
16、enantiomers 11 Bibliography . 17 BSISO9232:2003iv Foreword 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 carried out through ISO technical committees. Each
17、 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. ISO collaborates closely with the Internationa
18、l 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 International Standards. Draft International Standards
19、 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 elements of this document may be the subject of
20、 patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 9232 IDF 146 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 Internat
21、ional. It is being published jointly by ISO and IDF and separately by AOAC International. BSISO9232:2003 vForeword 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 re
22、presented on the IDF Standing Committees carrying out the technical work. IDF collaborates with ISO and AOAC 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
23、 circulated to the National Committees for voting. Publication as an International Standard requires approval by at least 50 % of the National Committees casting a vote. ISO 9232 IDF 146 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the
24、International Dairy Federation (IDF), in collaboration with AOAC International. It is being published jointly 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 Microbiol
25、ogical methods of analysis, under the aegis of its project leader, Prof. B. Bianchi Salvadori (IT). BSISO9232:2003NITERNATNOIAL STANDARD IS:2329 O3002(E) ID:641 F002(3)E1Yogurt Identification of characteristic microorganisms (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus
26、) 1 Scope This International Standard specifies tests for the identification of the characteristic microorganisms in yogurt on the basis of their morphological, cultural and physiological properties. It is applicable to strains isolated from yogurts in which both characteristic microorganisms are pr
27、esent 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 6887
28、-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 stuffs
29、General rules for microbiological examinations ISO 7889 IDF 117:2002, Yogurt Enumeration of characteristic microorganisms Colony-count technique at 37 C ISO 8261 IDF 122, Milk and milk products General guidance for the preparation of test samples, initial suspensions and decimal dilutions for microb
30、iological examination 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 characteristic microorganisms in yogurt Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus 4 Principle 4.1 The morphological, cultural and biochemic
31、al characteristics of L. delbrueckii subsp. bulgaricus are determined. 4.2 The morphological, cultural and biochemical characteristics of S. thermophilus are determined. BSISO9232:20032 5 Culture media, diluents and reagents Use only reagents of recognized analytical grade, unless otherwise specifie
32、d, and glass-distilled or demineralized water or water of equivalent purity. The water used for the preparation of the enzyme solutions should be at least double glass-distilled. See also ISO 6887-1 and ISO 8261 IDF 122. For other materials, see ISO 7889 IDF 117. 5.1 Culture media Use only freshly p
33、repared culture media which shall not be exposed to direct sunlight. If the prepared culture media 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 com
34、position. As for reagents, see storage conditions in ISO 7218. 5.1.1 Skimmed milk 5.1.1.1 Composition Low-heat-treated, spray-dried skimmed milk, free from growth inhibitors 100 g Water up to 1 000 ml 5.1.1.2 Preparation Dissolve the dried milk in the water. Distribute 10 ml portions of the obtained
35、 solution in test tubes of 16 mm 160 mm (6.5). Sterilize in an autoclave at 110 C 1 C for 30 min or at 115 C 1 C for 20 min. After sterilization and before use, check the sterility by incubating the test tubes in the incubator (6.1) set at 37 C for 3 days. 5.1.2 MRS broth 5.1.2.1 Composition Peptone
36、 1(tryptic digest of casein) 10,00 g Meat extract 10,00 g Yeast extract (dried) 5,00 g Glucose (C 6 H 12 O 6 ) 20,00 g Tween 80 (sorbitan mono-oleate) 1,00 ml Dipotassium hydrogen phosphate (K 2 HPO 4 ) 2,00 g Sodium acetate trihydrate (CH 3 CO 2 Na3H 2 O) 5,00 g Diammonium citrate C 6 H 6 O 7 (NH 4
37、 ) 2 2,00 g Magnesium sulfate heptahydrate (MgSO 4 7H 2 O) 0,20 g Manganese sulfate tetrahydrate (MnSO 4 4H 2 O) 0,05 g Water up to 1 000 ml 5.1.2.2 Preparation Separately dissolve each component in the already boiling water. Cool in a water bath (6.8) to 50 C. Adjust the pH so that after sterilizat
38、ion it is 6,5 0,2 at 25 C 1 C, by using a reagent (5.2) and checking with the pH-meter (6.4). BSISO9232:2003 3Distribute 20 ml portions of the obtained medium in test tubes of 20 mm 200 mm (6.5). Sterilize in an autoclave at 121 C 1 C for 15 min. NOTE When using commercially available MRS media, the
39、 obtained results may differ significantly from one supplier to the other. Therefore, always check commercially MRS medium against the medium prepared as described above. 5.1.3 Basic medium for fermentation tests 5.1.3.1 Composition Use the composition as described in 5.1.2.1 for the MRS broth, but
40、omitting the meat extract and the glucose component. 5.1.3.2 Preparation Prepare the basic medium as described in 5.1.2.2 for the MRS broth, using the components described in 5.1.3.1 and adjusting the pH so that after sterilization it is 6,95 0,05 instead of pH 6,5 at 25 C 1 C. 5.1.4 Culture medium
41、for production of CO 25.1.4.1 Composition Use the composition as described in 5.1.2.1 for the MRS broth, but omitting the meat extract component and replacing the 20 g of glucose with 50 g of glucose. 5.1.4.2 Preparation Prepare the culture medium as described in 5.1.2.2 for the MRS broth using the
42、components described in 5.1.4.1 and adjusting the pH so that after sterilization it is 6,95 0,05 instead of pH 6,5 at 25 C 1 C. Distribute 10 ml portions instead of 20 ml (as described in 5.1.2.2) of the obtained medium in the test tubes of 16 mm 160 mm (6.5). Sterilize in an autoclave at 121 C 1 C
43、for 15 min. 5.1.5 Overlay agar 5.1.5.1 Composition Bacteriological agar 20 g Water up to 1 000 ml 5.1.5.2 Preparation Dissolve the agar in the water. Distribute 10 ml portions in tubes of 16 mm 160 mm (6.5). Sterilize in an autoclave at 121 C 1 C for 15 min. 5.1.6 Litmus milk 5.1.6.1 Composition Lit
44、mus powder 0,70 g Skimmed milk (5.1.1) up to 1 000 ml BSISO9232:20034 5.1.6.2 Preparation Prepare the litmus milk as described in 5.1.1.2 for the skimmed milk, using the components as described in 5.1.6.1. NOTE Litmus powder or skimmed milk with litmus is commercially available. 5.1.7 M17 broth 5.1.
45、7.1 Basic medium 5.1.7.1.1 Composition Peptone 1 (tryptic digest of casein) 2,50 g Peptone 2 (peptic digest of meat) 2,50 g Peptone 3 (papain digest of soya) 5,00 g Yeast extract (dried) 2,50 g Meat extract 5,00 g -Glycerophosphate (disodium salt) (C 3 H 7 O 6 PNa 2 ) 19,00 g Magnesium sulfate hepta
46、hydrate (MgSO 4 7H 2 O) 0,25 g Ascorbic acid (C 6 H 8 O 6 ) 0,50 g Water up to 950 ml 5.1.7.1.2 Preparation Separately dissolve the components in the boiling water. Cool on a water bath (6.8) to 50 C. Adjust the pH so that after sterilization it is 6,8 0,2 at 25 C 1 C by using a reagents (5.2) and c
47、hecking with the pH-meter (6.4). Distribute 19 ml portions of the obtained medium in test tubes of 20 mm 200 mm (6.5). Sterilize for 15 min in an autoclave at 121 C 1 C. 5.1.7.2 Lactose solution 5.1.7.2.1 Composition Lactose (C 12 H 22 O 11 ) 10 g Water up to 100 ml 5.1.7.2.2 Preparation Dissolve th
48、e lactose in the water. Sterilize for 15 min in an autoclave at 121 C 1 C. 5.1.7.3 Complete medium 5.1.7.3.1 Composition Lactose solution (5.1.7.2) 1 ml Basic medium (5.1.7.1) 19 ml BSISO9232:2003 55.1.7.3.2 Preparation Immediately before use, add the lactose solution to the test tubes with the basi
49、c medium (5.1.7.1). Mix by swirling. NOTE When using commercially available M17 media, the obtained results may differ significantly from one supplier to the other. Therefore, always check commercially M17 medium against the medium prepared as described above. 5.1.8 Culture medium for growth in presence of 6,5 % NaCl 5.1.8.1 Composition Use the composition as described in 5.1.7.1.1 for
