Animal feeding stuffs: Methods of sampling and analysis - Detection and enumeration of Saccharomyces cerevisiae used as feed additive

This European Standard defines general rules for the enumeration of probiotic yeasts (Saccharomyces cerevisiae) in feed samples (additives, premixtures and feeding stuffs) that contain yeast as a single microorganism component or in a mixture with other microorganisms. Applying the method to feeds with a high copper content (> 400 mg/kg) demands a special procedure (see Annex B).The standard is not applicable to mineral feeds which are defined as complementary feeding stuffs composed mainly of minerals and containing at least 40 % crude ash (Council Directive 79/373/EEC).
There are different categories of feed samples:
a)   Additives which contain about 10+9 CFU/g to 10+10 CFU/g (CFU = colony forming units).
b)   Premixtures which contain about 10+8 CFU/g
c)   Feeds, meal or pellets, which contain about 10+6 CFU/g and include complete feedingstuffs, and milk replacers.
The detection limit is as defined in EN ISO 7218.

Futtermittel: Probenahme- und Untersuchungs-verfahren - Nachweis und Zählung von Saccharomyces cerevisiae als Futtermittelzusatzstoff

Dieses Dokument legt allgemeine Regeln für die Zählung von Saccharomyces cerevisiae in Futtermitteln (Zusatzstoffe, Vormischungen und Mischfuttermittel mit Ausnahme von mineralischen Futtermitteln) fest, die Saccharomyces cerevisiae als einzelnen mikrobiellen Bestandteil oder in einem Gemisch mit anderen Mikroorganismen enthalten. Die Anwendung des Verfahrens auf Mischfuttermittel mit kritischen Kupfer-mengen erfordert ein besonderes Verfahren (siehe Anhang A). Dieses Dokument ist nicht anwendbar auf mineralische Futtermittel, die als Ergänzungsfuttermittel definiert sind, hauptsächlich aus Mineralstoffen zusammengesetzt sind und mindestens 40 % Rohasche enthalten (Verordnung R767/2009) [3].
Es gibt unterschiedliche Kategorien von Futtermittelproben:
a) Zusatzstoffe, die etwa 1010 koloniebildende Einheiten (KbE/g) enthalten;
b) Vormischungen, die etwa 1011 KbE/kg enthalten;
c) Mischfuttermittel, Mehl oder Pellets, die etwa 109 KbE/kg enthalten.
Die Nachweisgrenze entspricht der in EN ISO 7218 festgelegten.

Aliments des animaux: Méthodes d’échantillonnage et d’analyse - Détection et dénombrement des souches de Saccharomyces cerevisiae utilisées comme additifs pour l’alimentation animale

Le présent document définit des règles générales pour le dénombrement de Saccharomyces cerevisiae présents dans les aliments pour animaux (additifs, prémélanges et aliments composés, à l’exception des aliments minéraux) qui contiennent des Saccharomyces cerevisiae comme seul micro-organisme constitutif ou en mélange avec d’autres micro-organismes. L’application de la méthode aux aliments composés pour animaux présentant une teneur critique en cuivre nécessite de mettre en oeuvre un mode opératoire spécial (voir Annexe A). Le présent document ne s’applique pas aux aliments minéraux, qui se définissent comme des aliments complémentaires constitués principalement de minéraux et contenant au moins 40 % de cendre brute (Règlement R767/2009) [3].
Il existe différentes catégories d’échantillons d’aliments pour animaux :
a) les additifs contenant environ 1010 UFC/g (UFC : unités formant colonie) ;
b) les prémélanges contenant environ 1011 UFC/kg ;
c) les aliments composés, farines ou granulés qui contiennent environ 109 UFC/kg.
La limite de détection est définie dans l’EN ISO 7218.

Krma: metode vzorčenja in analize - Določanje in štetje Saccharomyces cerevisiae, uporabljenih kot krmni dodatek

Ta evropski standard določa splošna pravila za štetje probiotičnih kvasovk (Saccharomyces cerevisiae) v vzorcih krme (dodatki, premiksi in krma), ki vsebujejo kvasovke kot eno komponento mikroorganizma ali v kombinaciji z drugimi mikroorganizmi. Uporaba metode za krmo z visoko vsebnostjo bakra (> 400 mg/kg) zahteva poseben postopek (glej dodatek B). Ta standard se ne uporablja za mineralno krmo, ki je opredeljena kot dopolnilna krma, sestavljena predvsem iz mineralov, in vsebuje najmanj 40 % surovega pepela (Direktiva Sveta 79/373/EGS).
Obstajajo različne kategorije vzorcev krme:
a)   dodatki, ki vsebujejo približno od 10+9 CFU/g do 10+10 CFU/g (CFU = enote, ki tvorijo kolonije);
b)   premiksi, ki vsebujejo približno 10+8 CFU/g;
c)   krma, moka ali peleti, ki vsebujejo približno 10+6 CFU/g ter vključujejo popolno krmo in mlečne nadomestke.
Meja zaznavanja je opredeljena v standardu EN ISO 7218.

General Information

Status
Published
Public Enquiry End Date
19-Mar-2020
Publication Date
13-Jan-2022
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
28-Dec-2021
Due Date
04-Mar-2022
Completion Date
14-Jan-2022

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SLOVENSKI STANDARD
SIST EN 15789:2022
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
SIST EN 15789:2022 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
SIST EN 15789:2022
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SIST EN 15789:2022
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.
---------------------- Page: 3 ----------------------
SIST EN 15789:2022
EN 15789:2021 (E)
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

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SIST EN 15789:2022
EN 15789:2021 (E)
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.
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SIST EN 15789:2022
EN 15789:2021 (E)
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.
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SIST EN 15789:2022
EN 15789:2021 (E)
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.
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SIST EN 15789:2022
EN 15789:2021 (E)
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;

— Morphological verification of isolates by use of microscopy;

— Calculation of the colony forming units of Saccharomyces cerevisiae per g or kg of feed sample.

5 Diluents and culture medium
5.1 Diluents
5.1.1 Diluent for initial suspension

The diluent is used for the preparation of the initial suspension and may also be used for the preparation

of further decimal dilutions. The composition of the diluent is given in Table 1.

® 1
Table 1 — Phosphate buffered saline with Polysorbate 80 (Tween 80) (tPBS)
Sodium chloride NaCl 8,00 g
Potassium chloride KCl 0,20 g
Disodium hydrogen phosphate anhydrous Na HPO 1,15 g
2 4
Potassium dihydrogen phosphate anhydrous KH PO 0,20 g
2 4
Polyoxyethylene (20) sorbitan monooleate C H O 1 ml
64 124 26
® 1
(Tween 80)
Water, distilled or deionized H O 1 000 ml

Dissolve the components (see Table 1) in water. If necessary, adjust to a final pH of 7,3 ± 0,2 at 25 °C after

sterilization. Fill the solution into appropriate containers (e.g. bottles, flasks, or test tubes) and sterilize

at 121 °C ± 3 °C for 15 min. To avoid loss during autoclaving, screw cap bottles are recommended.

Tween 80 is an example of a suitable product available commercially. This information is given for the

convenience of users of this document and does not constitute an endorsement by CEN of this product.

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SIST EN 15789:2022
EN 15789:2021 (E)
For immediate use hold at 40 °C ± 1 °C in a water bath or incubator.

NOTE When using commercially available PBS buffer tablets, please take note that variations in composition

and pH can occur between products from different manufacturers and could therefore give results different from

the ones obtained with the buffer as specified in this document.
5.1.2 Diluents for serial dilutions

For serial dilutions, the diluent for initial suspension (5.1.1) or alternatively a peptone salt solution (PSS)

according to EN ISO 6887-1 [4] can be used.
Table 2 — Peptone salt solution (PSS) according to EN ISO 6887-1
Enzymatic digest casein 1,0 g
Sodium chloride NaCl 8,5 g
Water, distilled or deionized H O 1 000 ml

Dissolve the components (see Table 2) in water in flasks or bottles. Adjust the pH if necessary so that,

after sterilization, it is 7,0 ± 0,2 at 25 °C. For decimal dilutions, prepare test tubes containing

9,0 ml ± 0,1 ml after sterilization or use screw cap bottles to avoid volume loss during autoclaving.

Sterilize at 121 °C ± 3 °C for 15 min. Bring the diluent to room temperature before use.

NOTE Commercially available, ready-to-use PSS tubes of 9 ml are suitable.
5.2 Culture medium
5.2.1 Yeast extract dextrose chloramphenicol (YGC) agar

The composition of the yeast extract dextrose chloramphenicol (YGC) agar is given in Table 3. The

resulting pH value at 25 °C is 6,6 ± 0,2.
Table 3 — Composition of the YGC agar
Yeast extract 5 g
D(+)-glucose 20 g
Chloramphenicol 0,1 g
Agar
12 g to 15 g
Water, distilled or deionized 1 000 ml
Depending on the gel strength of the agar.

The base agar (see Table 3) without the antibiotic can be purchased and the chloramphenicol supplement

has to be added, or the YGC agar can be purchased as a complete medium.

NOTE 1 Chloramphenicol can be replaced by oxytetracycline (C H N O ) at a final concentration of 100 µg/ml

22 24 2 9
of medium.

NOTE 2 Any other medium leading to comparable results can be used (e.g. Sabouraud dextrose agar (SDA) or

Wort agar supplemented with chloramphenicol).
---------------------- Page: 9 ----------------------
SIST EN 15789:2022
EN 15789:2021 (E)
5.2.2 Preparation

Dissolve all components (see Table 2) in water under heating and fill into appropriate containers (e.g.

bottles or flasks with non-toxic metal screw-caps). If necessary, adjust to a final pH of 6,6 ± 0,2 at 25 °C

after sterilization. Sterilize at 121 °C ± 3 °C for 15 min. Excessive heating during sterilization shall be

avoided.

If chloramphenicol is replaced by oxytetracycline, the basic medium is prepared in the same way. Prepare

a 1 % mass concentration (m/m) solution of oxytetracycline hydrochloride in water and sterilize by

filtration. Just prior to use, add 10 ml of this solution aseptically to 1 000 ml of the basic medium after

sterilization (in order to obtain a final concentration of 0,1 g/l of medium), that has been maintained at

44 °C to 47 °C.
6 Apparatus
Usual microbiological laboratory equipment and, in particular, the following:

6.1 Equipment for dry sterilization (oven) and wet sterilization (autoclave), for example

according to EN ISO 7218 [5].

6.2 Incubator, capable of maintaining a temperature of 30 °C ± 1 °C. Optionally also capable of

maintaining a temperature of 40 °C ± 1 °C and/or between 44 °C and 47 °C.

6.3 Water bath, capable of maintaining a temperature of 40 °C ± 1 °C and between 44 °C and 47 °C.

6.4 Blending equipment, e.g. a rotary homogenizer (blender), with a fixed or variable speed of

minimum 22 000 min , with aseptic glass or metals bowls equipped with covers according to

EN ISO 7218 [5].
6.5 Mechanical stirrer.

A mechanical stirrer (e.g. Vortex Mixer) facilitates the homogenous mixing of decimal dilutions, as

described in e.g. EN ISO 7218 [5].

6.6 Balances, of the required range and accuracy, for example according to EN ISO 7218 [5], for the

different products to be weighed.
6.7 Flasks or screw-cap bottles, of appropriate capacities.
6.8 Test tubes, of appropriate capacities.
6.9 Pipettes or pipettor and sterile tips, to dispense 0,1 ml to 1 ml.
6.10 Sterile pipettes
...

SLOVENSKI STANDARD
oSIST prEN 15789:2020
01-marec-2020
Krma: metode vzorčenja in analize - Izolacija in štetje prisotnih kvasovk
probiotičnih sevov (Saccharomyces cerevisiae)

Animal feeding stuffs: Methods of sampling and analysis - Isolation and enumeration of

yeast probiotic strains (Saccharomyces cerevisiae)
Futtermittel: Probenahme- und Untersuchungsverfahren - Trennung und Zählung von
Hefestämmen (Saccharomyces cerevisiae)
Aliments des animaux - Méthodes d’échantillonnage et d’analyse - Isolement et
dénombrement de souches probiotiques de levures (Saccharomyces cerevisiae)
Ta slovenski standard je istoveten z: prEN 15789
ICS:
65.120 Krmila Animal feeding stuffs
oSIST prEN 15789:2020 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
oSIST prEN 15789:2020
---------------------- Page: 2 ----------------------
oSIST prEN 15789:2020
DRAFT
EUROPEAN STANDARD
prEN 15789
NORME EUROPÉENNE
EUROPÄISCHE NORM
January 2020
ICS 65.120 Will supersede EN 15789:2009
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Isolation and enumeration of yeast probiotic strains
(Saccharomyces cerevisiae)

Aliments des animaux - Méthodes d'échantillonnage et Futtermittel: Probenahme- und

d'analyse - Isolement et dénombrement de souches Untersuchungsverfahren - Trennung und Zählung von

probiotiques de levures (Saccharomyces cerevisiae) Hefestämmen (Saccharomyces cerevisiae)

This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee

CEN/TC 327.

If this draft becomes a European Standard, 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.

This draft European Standard was established by CEN 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.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are

aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without

notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 15789:2020 E

worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
oSIST prEN 15789:2020
prEN 15789:2019 (E)
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 selective media ...................................................................................................................... 6

6 Apparatus and glassware ............................................................................................................................. 8

7 Sampling ............................................................................................................................................................. 9

8 Preparation of test sample .......................................................................................................................... 9

9 Procedure........................................................................................................................................................... 9

9.1 Preparation of poured agar plates ............................................................................................................ 9

9.2 Preparation of YGC agar for pour plate method .................................................................................. 9

9.3 Preparation of the initial suspension and decimal dilutions ....................................................... 10

9.4 Inoculation and incubation of plates .................................................................................................... 11

Counting of colonies .................................................................................................................................................. 11

9.5 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) Notes on procedure ..................................................................................................... 14

Annex B (informative) Results of the interlaboratory study ..................................................................... 15

Bibliography ................................................................................................................................................................. 16

---------------------- Page: 4 ----------------------
oSIST prEN 15789:2020
prEN 15789:2019 (E)
European foreword

This document (prEN 15789:2019) 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 document is currently submitted to the CEN Enquiry.
This document will supersede EN 15789:2009.
---------------------- Page: 5 ----------------------
oSIST prEN 15789:2020
prEN 15789:2019 (E)
Introduction

This method 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 (EU project

SMT4-CT98-2235 “Methods for the official control of probiotics (microorganisms) used as animal feeds”

[1].

The procedure has been validated for one commercially used Saccharomyces cerevisiae strain [1]. 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).

---------------------- Page: 6 ----------------------
oSIST prEN 15789:2020
prEN 15789:2019 (E)
1 Scope

This document defines 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 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

R767/2009) [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.
The detection limit is defined in EN ISO 7218.
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)

EN ISO 7218, Microbiology of food and animal feeding stuffs – General requirements and guidance for

microbiological examinations (ISO 7218)
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:

• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at http://www.iso.org/obp
3.1
saccharomyces cerevisiae
unicellular fungus which mostly reproduces vegetatively by budding

Note1 to entry This description is based on their characteristics as used for this standard

Note 2 to entry: Budding cells are broadly ellipsoidal with multilateral bud formation. It shows no or simple

pseudohyphae.

Note 3 to enty: S. cerevisiae forms colonies on the specified selective media after incubation for 48 h to 72 h at

30 °C under aerobic conditions fitting the description in 9.6.
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4 Principle

a) Preparation of sterile and dry poured agar plates or preparation of sterile liquid selective medium

tempered at 44 °C to 47 °C;
b) A representative test sample is taken under aseptic conditions;

c) An initial suspension is prepared with a tempered diluent to obtain a homogeneous distribution of

yeast cells from the test portion;

d) The number of microorganisms per unit volume is reduced by the preparation of further decimal

dilutions from the initial suspension to obtain a countable number of colonies on the selective

enumeration media;

e) 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 petri dishes with an aliquot of the

optimum dilutions and pouring of the molten agar medium into each Petri dish, mixing and

solidification;

f) The inoculated plates are incubated for 48 h to 72 h at 30 °C ± 1 °C under aerobic conditions;

g) Counting of typical colonies, considering the specific properties of Saccharomyces cerevisiae as listed

in 3.1;
h) Morphological verification of isolates by use of microscopy;

i) Calculation of the colony forming units of Saccharomyces cerevisiae per g or kg of feed sample.

5 Diluents and selective media
5.1 Diluents
5.1.1 Diluent for initial suspension

This diluent is used for the preparation of the initial suspension and may also be used for the

preparation of further decimal dilutions.
Table 1 — Phosphate buffered saline supplemented with Tween® 80 (tPBS)
Sodium chloride NaCl 8,00 g
Potassium chloride KCl 0,20 g
Disodium hydrogen phosphate anhydrous Na HPO 1,15 g
2 4
Potassium dihydrogen phosphate anhydrous KH PO 0,20 g
2 4
® 1 ml
Polyoxyethylensorbitanmonooleate (Tween
80)
Water, distilled or deionized 1 000 ml

Tween® is an example of a suitable product available commercially. This information is given for the

convenience of users of this document and does not constitute an endorsement by CEN of this product

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Dissolve the components in water. If necessary, adjust to a final pH of 7,3 ± 0,2 at 25 °C after

sterilization. The solution is filled into appropriate containers (e.g. bottles or flasks, test tubes) and

sterilized at 121 °C ± 1 °C for 15 min. To avoid loss during autoclaving, screw cap bottles are

recommended.
Temper to 40 ± 1°C in a water bath or incubator immediately before usage.

NOTE The use of commercially available PBS buffer tablets is acceptable. However, please take note that

variations in composition and pH can occur between products from different manufacturers and could therefore

give results different from the ones obtained with the medium as specified in this International Standard.

5.1.2 Diluents for serial dilutions

For serial dilutions, the diluent for initial suspension (5.1.1) or alternatively peptone salt solution (PSS)

according to EN ISO 6887-1 can be used.
Table 2 — Peptone salt solution PSS according to EN ISO 6887-1
Enzymatic digest casein 1.0g
Sodium chloride NaCl 8.5g
Water, distilled or 1000 ml
deionized

Dissolve the components in the water in flasks, bottles or test tubes. Adjust the pH if necessary so that,

after sterilization, it is 7,0 ± 0,2 at 25 °C. For decimal dilutions, prepare test tubes containing

9,0 ml ± 0,1 ml after sterilization or use screw cap bottles to avoid weight loss during autoclaving.

Sterilize at 121 °C ± 1 °C for 15 min. Bring the diluent to room temperature before use.

NOTE Commercially available, ready-to-use PSS tubes of 9 ml are suitable.
5.2 Enumeration media
5.2.1 Yeast extract dextrose chloramphenicol (oxytetracycline) agar (YGC agar)
Table 3 — Composition of the YGC agar
Yeast extract 5 g
D(+)-Glucose 20 g
Chloramphenicol 0,1 g
Agar agar a
12–15 g
Water, distilled or deionized 1 000 ml
pH 6,6 ± 0,2 at 25 °C
a Depending on the gel strength of the agar.

The base agar without antibiotic can be purchased and the chloramphenicol supplement has to be

added or it can be purchased as a complete medium.

NOTE 1 Chloramphenicol can be replaced by oxytetracycline (C H N O ) at a final concentration of

22 24 2 9
100 µg/ml of medium.

NOTE 2 Any other medium leading to comparable results can be used (e.g. Sabouraud Dextrose Agar (SDA) or

Wort agar supplemented with chloramphenicol)
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5.2.2 Preparation

Dissolve all components in water under heating and fill into appropriate containers (e.g. bottles or

flasks with non-toxic metal screw-caps). If necessary, adjust to a final pH of 6,6 ± 0,2 at 25 °C after

sterilization. Sterilize at 121 °C ± 1 °C for 15 min. Excessive heating shall be avoided.

NOTE If chloramphenicol is replaced by oxytetracycline the basic medium is prepared in the same way but

without chloramphenicol. Prepare a 1 % mass concentration (m/m) solution of oxytetracycline hydrochloride in

water and sterilize by filtration. Just prior to use, add 10 ml of this solution aseptically to 1 000 ml of the basic

medium after sterilization (in order to obtain a final concentration of 0,1 g/l of medium), that has been maintained

at 44 °C – 47 °C.
6 Apparatus and glassware
Usual microbiological laboratory equipment and, in particular, the following:
6.1 Equipment for dry sterilization (oven) and wet sterilization (autoclave)
According to EN ISO 7218.
6.2 Incubator

Capable of maintaining a temperature of 30 °C ± 1 °C. Optionally also capable of maintaining a

temperature of 40 ± 1°C and/or 44 °C to 47 °C.
6.3 Water bath
Capable of maintaining a temperature of 44 °C to 47 °C and 40 °C ± 1 °C.
6.4 Blending equipment

A rotary homogenizer (blender), with a fixed or variable speed of minimum 22 000 r/min, with aseptic

glass or metals bowls equipped with covers (see EN ISO 7218).
6.5 Mechanical stirrer
A mechanical stirrer e.g. Vortex Mixer (see EN ISO 7218), or equivalent.
6.6 Balance

Balances of the required range and accuracy according ISO 7218 for the different products to be

weighed.
6.7 Flasks or screw-cap bottles of appropriate capacities
6.8 Test tubes of appropriate capacities
6.9 Pipettes or Pipettor and sterile tips to dispense 0,1 ml to 1 ml
6.10 Sterile 5 ml graduated pipettes

For full outlet with wide (approx. 3 mm) tips (e.g. serological pipette; alternatively: 5 ml-graduated

pipettes without tips).
6.11 Bacterial Cell spreaders

Sterile L- or triangular-shaped spreaders from glass or metal or sterile disposable plastic spreaders.

Alternatively a spiral plater with a sanitized dispensing system or disposable one–way microsyringes

can be used.
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6.12 Sterile petri dishes, 90 mm in diameter
6.13 Laminar flow cabinet
6.14 Microscope
Capable of phase-contrast mi
...

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