EN 15789:2021

SLOVENSKI STANDARD
01-februar-2022
Nadomešča:
SIST EN 15789:2009
Krma: metode vzorčenja in analize - Določanje in štetje Saccharomyces
cerevisiae, uporabljenih kot krmni dodatek
Animal feeding stuffs: Methods of sampling and analysis - Detection and enumeration of
Saccharomyces cerevisiae used as feed additive
Futtermittel: Probenahme- und Untersuchungs-verfahren - Nachweis und Zählung von
Saccharomyces cerevisiae als Futtermittelzusatzstoff
Aliments des animaux: Méthodes d’échantillonnage et d’analyse - Détection et
dénombrement des souches de Saccharomyces cerevisiae utilisées comme additifs pour
l’alimentation animale
Ta slovenski standard je istoveten z: EN 15789:2021
ICS:
65.120 Krmila Animal feeding stuffs
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 15789
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2021
EUROPÄISCHE NORM
ICS 65.120 Supersedes EN 15789:2009
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Detection and enumeration of Saccharomyces cerevisiae
used as feed additive
Aliments des animaux: Méthodes d'échantillonnage et Futtermittel: Probenahme- und Untersuchungs-
d'analyse - Détection et dénombrement des souches de verfahren - Nachweis und Zählung von Saccharomyces
Saccharomyces cerevisiae utilisées comme additifs cerevisiae als Futtermittelzusatzstoff
pour l'alimentation animale
This European Standard was approved by CEN on 2 August 2021.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 15789:2021 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 6
5 Diluents and culture medium . 6
5.1 Diluents . 6
5.2 Culture medium . 7
6 Apparatus . 8
7 Sampling . 9
8 Preparation of test sample . 9
9 Procedure. 9
9.1 Preparation of poured agar plates for spread plate method . 9
9.2 Preparation of YGC agar for pour plate method . 9
9.3 Preparation of the initial suspension and decimal dilutions . 9
9.4 Inoculation and incubation of plates . 11
9.5 Enumeration of colonies . 12
9.6 Confirmation . 12
10 Expression of results . 12
11 Precision . 13
11.1 General . 13
11.2 Interlaboratory study . 13
11.3 Repeatability . 13
11.4 Reproducibility . 13
12 Test report . 13
Annex A (informative)  Critical copper concentrations. 14
Annex B (informative)  Results of the interlaboratory study . 15
Bibliography . 16

European foreword
This document (EN 15789:2021) has been prepared by Technical Committee CEN/TC 327 “Animal
feeding stuffs - Methods of sampling and analysis”, the secretariat of which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by May 2022, and conflicting national standards shall be
withdrawn at the latest by May 2022.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN 15789:2009.
The main changes compared to the previous edition are as follows:
— Amendment of the title;
— Extension of the scope of application to all Saccharomyces cerevisiae strains used as feed additive;
— Updating of normative cross references; ®
— Supplement of phosphate buffered saline with Tween 80; ®
— Addition of the option to use Tween 80 supplemented phosphate buffered saline for the
preparation of the initial suspension as well as diluent for serial dilutions;
— Removal of the chromogenic culture medium for the enumeration of Saccharomyces cerevisiae;
— Addition of the option to use spread plates as well as a spiral plater for enumeration;
— Preparation of initial suspensions generally conducted with tempered tPBS;
— Unification of the homogenization time for the preparation of initial suspensions to one minute for
all feed matrices;
— Adjustment of the cultivation time and temperature to 48 h to 72 h at (30 ± 1) °C;
— Addition of a procedure for the investigation of feeding stuffs containing high amounts of copper in
the informative Annex A;
— Adjustment of the range of accepted colony numbers for counting from '≥ 30 to ≤ 350' to '≥ 10
to ≤ 200' colonies per plate.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland,
Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of North
Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United
Kingdom.
Introduction
This methodology has been developed to enumerate yeasts (Saccharomyces cerevisiae) used as feed
additives to enable the European Commission to control proper labelling of animal feeding products. It
was compiled first during the EU project SMT4-CT98-2235 “Methods for the official control of probiotics
used as feed additives” [1].
The procedure has been validated for one commercially used Saccharomyces cerevisiae strain [2]. As the
method is not selective for this particular Saccharomyces cerevisiae strain, it can be assumed, that it can
also be applied to enumerate other Saccharomyces cerevisiae strains in their respective dosage form in
feed provided that the added yeast is present in far higher numbers than any other yeast.
The method has not been validated for other yeast species (e.g. Kluyveromyces marxianus).
This method is not applicable for the detection of any ubiquitous or pathogenic yeasts in food and animal
feeding stuffs.
1 Scope
This document specifies general rules for the enumeration of Saccharomyces cerevisiae in feeding stuffs
(additives, premixtures and compound feeds excluding mineral feeds) that contain Saccharomyces
cerevisiae as a single microorganism component or in a mixture with other microorganisms. Applying the
method to premixtures and compound feeds with critical amounts of copper demands a special
procedure (see Annex A). The document is not applicable to mineral feeds, which are defined as
complementary feeding stuffs composed mainly of minerals and containing at least 40 % crude ash
(Regulation (EC) 767/2009) [3].
There are different categories of feed samples:
a) Additives containing about 10 colony forming units (CFU)/g;
b) Premixtures containing about 10 CFU/kg;
c) Compound feeds, meal or pellets which contain about 10 CFU/kg.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements 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.
EN ISO 6498, Animal feeding stuffs - Guidelines for sample preparation (ISO 6498)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
3.1
Saccharomyces cerevisiae
unicellular fungus which mostly reproduces vegetatively by budding
Note 1 to entry: This description is based on their characteristics as used for this document.
Note 2 to entry: Budding cells are broadly ellipsoidal with multilateral bud formation. They show no or simple
pseudohyphae.
Note 3 to entry: S. cerevisiae forms colonies on the specified selective medium after incubation for 48 h to 72 h at
30 °C under aerobic conditions fitting the description in 9.6.
4 Principle
— Preparation of sterile and dry poured agar plates or preparation of sterile liquid culture medium
tempered at 44 °C to 47 °C;
— Drawing a representative test sample under aseptic conditions;
— Preparation of the initial suspension with a tempered diluent to obtain a homogeneous distribution
of bacterial cells from the test portion;
— Preparation of further decimal dilutions of the initial suspension in order to reduce the number of
microorganisms per unit volume to allow, after incubation, the counting of colonies;
— Inoculation of prepared poured plates with an aliquot of the optimum dilutions and dispersion of the
inoculum using a sterile spreader or inoculation of blank plates with an aliquot of the optimum
dilutions and pouring of the molten agar medium into each plate, mixing and solidification;
— Incubation of inverted plates for 48 h to 72 h at 30 °C ± 1 °C under aerobic conditions;
— Counting of typical colonies, considering the specific properties of Saccharomyces cerevisiae;
—
...