1、 Reference numbers ISO 26323:2009(E) IDF 213:2009(E) ISO and IDF 2009INTERNATIONAL STANDARD ISO 26323 IDF 213 First edition 2009-07-01 Milk products Determination of the acidification activity of dairy cultures by continuous pH measurement (CpH) Produits laitiers Dtermination de lactivit acidifiante
2、 des cultures laitires par mesurage continu de pH (CpH) ISO 26323:2009(E) IDF 213:2009(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 li
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7、0 B-1030 Brussels Tel. + 41 22 749 01 11 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 2009 All rights reservedISO 26323:2009(E) IDF 213:2009(E) ISO and IDF 2
8、009 All rights reserved iiiContents Page Foreword iv Foreword .v 1 Scope1 2 Normative references1 3 Terms and definitions .1 4 Principle2 5 Diluents, culture media and reagents .2 6 Apparatus.4 7 Sampling.5 8 Preparation.5 8.1 Milk preparation.5 8.2 Cleaning and calibration of pH electrodes .5 8.3 P
9、rotein and fat cleaning of the electrode6 8.4 Stabilization and storage of the pH electrode 6 8.5 Calibration of the pH electrode 6 8.6 Disinfection of the pH electrode with ethanol 6 8.7 Decontamination of the pH electrode by heat treatment 6 9 Procedure.6 9.1 Frozen cultures6 9.2 Freeze-dried prod
10、ucts.8 9.3 Termination of the analysis9 10 Precision.10 10.1 Interlaboratory test10 10.2 Repeatability 10 10.3 Reproducibility 10 11 Test report11 Annex A (informative) Interlaboratory test A CpH ring trial.12 Bibliography13 ISO 26323:2009(E) IDF 213:2009(E) iv ISO and IDF 2009 All rights reservedFo
11、reword 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 member body interested in a subject for which a tec
12、hnical 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 International Electrotechnical Commission (IEC) on all matters o
13、f 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 adopted by the technical committees are circulated
14、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 patent rights. ISO shall not be held responsible fo
15、r identifying any or all such patent rights. ISO 26323 IDF 213 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF). It is being published jointly by ISO and IDF. ISO 26323:2009(E) IDF 213:2009(E) ISO an
16、d IDF 2009 All rights reserved vForeword IDF (the International Dairy Federation) is a non-profit organization representing the dairy sector worldwide. IDF membership comprises National Committees in every member country as well as regional dairy associations having signed a formal agreement on coop
17、eration with IDF. All members of IDF have 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. Draft International Standards adopted by the Act
18、ion Teams and Standing Committees are circulated to the National Committees for voting. Publication as an International Standard requires approval by at least 50 % of the IDF National Committees casting a vote. Attention is drawn to the possibility that some of the elements of this document may be t
19、he subject of patent rights. IDF shall not be held responsible for identifying any or all such patent rights. ISO 26323 IDF 213 was prepared by the International Dairy Federation (IDF) and Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published
20、jointly by IDF and ISO. All work was carried out by the ISO-IDF Joint Action Team on Lactic acid bacteria and starters of the Standing Committee on Microbiological methods of analysis under the aegis of its project leader, Mr. L. V. Jrgensen (DK). INTERNATIONAL STANDARD ISO 26323:2009(E) IDF 213:200
21、9(E) ISO and IDF 2009 All rights reserved 1Milk products Determination of the acidification activity of dairy cultures by continuous pH measurement (CpH) 1 Scope This International Standard specifies a method for the measurement of the acidification activity of lactic acid bacteria by continuous mea
22、surement of pH. NOTE The method is based on Reference 9. The method is applicable to dairy starter cultures where these characteristic microorganisms are present. Two types of standardized milk are specified in the procedure: boiled milk with 9,5 % mass fraction dry matter (B-milk 9,5); and autoclav
23、ed milk with 9,5 % mass fraction dry matter (A-milk 9,5). It is possible that heat treatment of B-milk 9,5 does not inactivate all enzymes that are present, which can affect the activity of some cultures. In that case, cultures are tested with A-milk 9,5 in which all enzymes have been inactivated. 2
24、 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 6887-5, Microbiology of
25、 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 1)3 Terms and definitions For the purposes of this document, the following terms and definitions
26、apply. 3.1 acidification activity ability of a dairy starter culture to acidify standardized milk determined according to the procedure described in this International Standard NOTE The acidification activity can be quantified by the following parameters. a) t a The time it takes to start acidifying
27、 the standardized milk, i.e. time for pH to drop 0,08 pH-units from initial pH (after 15 min). Time t ais measured in minutes from the inoculation time, t = 0. If the software collects data every 4 min, t ais determined by interpolation. b) pH t h The pH after t h (e.g. 4 h, 6 h, 12 h or 16 h) of ac
28、idification in the standardized milk at 30 C, 37 C, 40 C or 43 C. The actual time for the parameter depends on the characteristics of the starter culture. c) t pH x The time it takes to acidify the standardized milk to a certain pH, e.g. pH 4,50. The actual time for the parameter depends on the char
29、acteristics of the starter culture and the application for which it is used. 1) Supersedes ISO 8261 IDF 122. ISO 26323:2009(E) IDF 213:2009(E) 2 ISO and IDF 2009 All rights reserved4 Principle A specified quantity of starter culture is diluted and inoculated in a specified amount of standardized mil
30、k. The inoculated culture is incubated at a specified constant temperature of 30 C, 37 C, 40 C or 43 C for a given time depending on the characteristics of the starter culture. During incubation, the acidification activity is measured by continuous pH measurements using a pH electrode and a data log
31、ger. When the fermentation curve is obtained, a number of curve parameters can be calculated or extracted. 5 Diluents, culture media and reagents Use only reagents of recognized analytical grade, unless otherwise specified, and only distilled or demineralized water or water of equivalent purity. The
32、 water shall be free of substances likely to inhibit or influence the growth of microorganisms in the reconstituted milk. If chlorinated water is used, neutralize the chlorine prior to use. Observe the following rules for the water quality: the cell count shall be below 50 cell/ml and conductivity b
33、elow 5 S/cm. Tests to determine the suitability of water for microbiological applications appear in ISO/TS 11133-1 5 . 5.1 Basic materials SAFETY PRECAUTIONS Take the required safety precautions when using the electrode cleaner (5.1.2), potassium chloride solution (5.1.3) or diaphragm cleaner (5.1.4
34、) as these can irritate skin and eyes. 5.1.1 Buffer solutions 5.1.1.1 Buffer solution of pH 4,00, capable of buffering at 20 C; e.g. Merck/VWR CertiPUR (Order No. 1.09475) 2)or equivalent. 5.1.1.2 Buffer solution of pH 7,00, capable of buffering at 20 C; e.g. Merck/VWR CertiPUR (Order No. 1.09477) 2
35、)or equivalent. 5.1.2 Electrode cleaner, capable of cleaning the electrode; e.g. pepsin/HCl solution from Mettler Toledo (Order no. 9891) 2)or equivalent. 5.1.3 Potassium chloride solution, of concentration c(KCl) = 3 mol/l; e.g. Mettler Toledo (Order no. 9823) 2)or equivalent. 5.1.4 Diaphragm clean
36、er, capable of cleaning the diaphragm; e.g. thiourea/HCl solution from Mettler Toledo (Order no. 9892) 2)or equivalent. 5.1.5 Ethanol solution, (C 2 H 5 OH) = 70 % volume fraction in water, used for disinfection. 5.1.6 Medium-heat, low fat, spray-dried milk powder. Use medium-heat, low fat, spray-dr
37、ied milk powder produced from milk of good quality and with no detectable antibiotics residues. The spray-dried milk powder shall be of good microbiological quality (see Reference 8) having a natural, pleasant taste and flavour of fresh skimmed milk and shall consist of the components specified in T
38、able 1. 2) Example of a suitable product available commercially. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by either ISO or IDF of this product. Other products may be used if they can be shown to give similar results.
39、 ISO 26323:2009(E) IDF 213:2009(E) ISO and IDF 2009 All rights reserved 3Table 1 Medium-heat, low fat, spray-dried milk powder compound a , e.g. from Chr. Hansen 3)Component Mass fraction % Milk protein 34 to 38 Lactose 48 to 56 Milk fat 1,25 Ash 7 to 9 Moisture 4 Titratable acidity, lactic acid 0,1
40、5 aThe pH of a 10 % mass fraction solution should be in the range 6,5 to 6,8. 5.2 Substrates 5.2.1 QC-Milk 9,5 5.2.1.1 Composition. Use only standardized milk with a specified dry matter content prepared from spray-dried skimmed milk powder according to the composition in Table 2. The prepared amoun
41、t may be smaller than that specified in Table 2. However, do not prepare less than 1,1 kg of milk. If using a small amount, use the analytical balance (6.1) to weigh the milk powder to the nearest 1 mg. The aim is to prepare milk with a dry matter content of 9,5 % 0,2 % mass fraction, similar to tha
42、t of bottled and sterilized milk. Table 2 Composition of milk made up from spray-dried milk powder Component Mass kg Medium-heat milk powder a10,9 to 11,2 Water 100,0 aThe quantity varies due to water content of milk powder and water loss during the preparation and heat treatment. 5.2.1.2 Preparatio
43、n. Dissolve the medium-heat milk powder in water for not more than 30 min. If needed, warm the water up to 40 C to completely dissolve the powder. Dispense the milk obtained into cylindrical bottles to reach a volume of 200 ml after heat treatment. Check the volume by weighing the bottle until it re
44、aches a net mass of 207 g 2 g (total mass minus that of the bottle). The density of QC-milk 9,5 is 1,033 g/ml. Alternatively, dispense the milk obtained into cylindrical bottles to reach a mass of 200 g 2 g (net mass). Compensate for the mass difference in the inoculation procedure. 3) Example of a
45、suitable product available commercially. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by either ISO or IDF of this product. Other products may be used if they can be shown to give similar results. ISO 26323:2009(E) IDF 2
46、13:2009(E) 4 ISO and IDF 2009 All rights reserved5.2.2 Heat treatment of B-milk 9,5. Heat treat the prepared milk (5.2.1.2) according to the temperature programme in Table 3. Table 3 Temperature programme for heat treating B-milk 9,5 Procedure Temperature C Time min Heating 99 1 20 Holding 99 1 30 1
47、 Cooling 99 1 to 40 5 40 Then, store the B-milk 9,5 obtained at below 7 C for at least 16 h. Record the evaporation during heat treatment and the tolerance of mass after the heat treatment. 5.2.3 Shelf life of B-milk 9,5. Before use, store the B-milk 9,5 (5.2.2) for a minimum of 16 h and a maximum o
48、f 12 d. Enzymes such as proteases may not have been inactivated and can affect acidification activity of some cultures. 5.2.4 Heat treatment of A-milk 9,5. Use A-milk 9,5 for cultures where B-milk 9,5 does not give the required repeatability, e.g. due to residual protease activity. Sterilize the pre
49、pared milk (5.2.1.2) according to the temperature programme in Table 4. Table 4 Temperature programme for heat treating A-milk 9,5 Procedure Temperature C Time min Heating 115 1 20 Holding 115 1 15 1 Cooling 115 1 to 40 5 40 Then, store the A-milk 9,5 obtained at below 7 C for at least 16 h. Record the evaporation during heat treatment and the tolerance of mass after that treatment. 5.2.5 Shelf life of A-milk 9,5. Before use, store the A-milk 9,5 (5.2.4) for a minimu
