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 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 International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.

The procedures used to develop this 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 document should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).

ISO draws attention to the possibility that the implementation of this document may involve the use of (a) patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a) patent(s) which may be required to implement this document. However, implementers are cautioned that this may not represent the latest information, which may be obtained from the patent database available at www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.

Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about ISO’s adherence to the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.

This document 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.

This second edition cancels and replaces the first edition (ISO 7889 | IDF 117:2003), which has been technically revised.

The main changes are as follows:

The culture medium M17 has been amended: lactose is replaced by sucrose to (also) enumerate lactose-negative strains of S. thermophilus.
The concentration ß-glycerophosphate, the main selective agent of M17 culture medium and a powerful buffer,^[14^][15^] is lowered from 1,9 % to 1 %. This reduction improved the size and morphology of S. thermophilus colonies during pilot studies and can improve the productivity.^[10^]
For better performance, microaerobic incubation of M17-S has been changed to mandatory.
Spread plates have been added to the method as alternative for pour plates.
Performance characteristics, with the results of an interlaboratory study, which are based on the method of this second edition have been included as Annex C.

Any feedback or questions on this document should be directed to the user’s national standards body. A complete listing of these bodies can be found at www.iso.org/members.html.

IDF (the International Dairy Federation) is a non-profit private 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 industry, 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 and reference numbers of both organizations.

IDF draws attention to the possibility that the implementation of this document may involve the use of (a) patent(s). IDF takes no position concerning the evidence, validity or applicability of any claimed patent rights in respect thereof. As of the date of publication of this document, IDF had not received notice of (a) patent(s) which may be required to implement this document. However, implementers are cautioned that this may not represent the latest information, which may be obtained from the patent database available at www.iso.org/patents. IDF shall not be held responsible for identifying any or all such patent rights.

Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement.

This document was prepared by IDF Standing Committee on Methods for Dairy Microbiology and ISO Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published jointly by ISO and IDF.

The work was carried out by the IDF/ISO Action Team (D14) of the Standing Committee on Methods for Dairy Microbiology under the aegis of its project leader Arnold F. Dijkstra (NL).

Introduction

Lactobacillus delbrueckii subsp. bulgaricus (in short: L. bulgaricus) and Streptococcus thermophilus (in short: S. thermophilus) are used to produce yoghurt as defined in CXS 243‑2003^[7^].

S. thermophilus ferments lactose in milk, producing lactic acid, formic acid and carbon dioxide. These compounds lower the pH of the milk, which stimulates the growth of L. bulgaricus. Some strains of S. thermophilus also produce folic acid and fatty acids, which are beneficial for L. bulgaricus. L. bulgaricus possesses proteolytic enzymes (proteases) that break down milk proteins (casein) into amino acids. These amino acids are essential for the growth of both L. bulgaricus and S. thermophilus. For well protocooperation and the physiological and sensory characteristics of the yoghurt, the ratio of S. thermophilus and L. bulgaricus is very important.^[8^] Proper protocooperation also controls the post acidification when yoghurt is stored until the shelf life.^[17^]

An accurate enumeration method for L. bulgaricus and S. thermophilus by means of the colony-count technique, such as the one given in this document, is therefore important to guarantee the quality of yoghurt.

The main technical changes listed in the Foreword, introduced in this document compared to ISO 7889 | IDF 117:2003, are considered as major (see ISO 17468).

These technical changes have a major impact on the performance characteristics of the method.

1 Scope

This document specifies a method for the enumeration of the characteristic microorganisms Lactobacillus delbrueckii subsp. bulgaricus (in short: L. bulgaricus) and Streptococcus thermophilus (in short: S. thermophilus) by means of the colony-count technique.

The method is applicable to yoghurts (for the definition see CXS 243‑2003^[7^]).

The colony-count technique (pour plates) is suitable for, but not limited to, the enumeration of L. bulgaricus and S. thermophilus in test samples with a minimum of 10 colonies counted on a plate. This corresponds to a level of the characteristic microorganisms L. bulgaricus and S. thermophilus that is expected to be higher than 100 cfu/g.

The colony-count technique (spread plates) is suitable for, but not limited to, the enumeration of L. bulgaricus and S. thermophilus in test samples with a minimum of 10 colonies counted on a plate. This corresponds to a level of the characteristic microorganisms L. bulgaricus and S. thermophilus that is expected to be higher than 1 000 cfu/g.

2 Normative references

3 Terms and definitions

4 Principle

4.1 General

4.2 Preparation of dilutions

4.3 Inoculation

4.4 Incubation

4.5 Enumeration and confirmation

5 Culture media and reagents

6 Equipment and consumables

6.1

6.2

6.3

6.4

6.5

6.6

6.7

6.8

6.9

6.10

6.11

6.12

6.13

7 Sampling

8 Preparation of test sample

9 Procedure

9.1 Test portion, initial suspension and dilutions

9.2 Pour plates: Inoculation and incubation

9.3 Spread plates: Inoculation and incubation

9.4 Counting of colonies

9.5 Confirmation (optional)

10 Expression of results

11 Validation of the method

11.1 Validation in accordance with ISO 17468

11.2 Performance characteristics

12 Test report

13 Quality assurance

Bibliography