EN 15789:2021

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

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 1010 colony forming units (CFU)/g;
b)   Premixtures containing about 1011 CFU/kg;
c)   Compound feeds, meal or pellets which contain about 109 CFU/kg.

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 Vormischungen und Mischfuttermittel mit kritischen Kupfermengen erfordert ein besonderes Vorgehen (siehe Anhang A). Dieses Dokument ist nicht anwendbar auf mineralische Futtermittel, die als Ergänzungsfuttermittel definiert sind, die hauptsächlich aus Mineralstoffen zusammengesetzt sind und mindestens 40 % Rohasche enthalten (Verordnung (EG) Nr. 767/2009) [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, Mehle oder Pellets, die etwa 109 KbE/kg enthalten.

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 spécifie 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 prémélanges et aux aliments composés pour animaux présentant une teneur critique en cuivre nécessite de mettre en œuvre 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 (CE) 767/2009) [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 contenant environ 109 UFC/kg.

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
• Publication Date: 23-Nov-2021
• Withdrawal Date: 30-May-2022
• ICS: 65.120 - Animal feeding stuffs
• Technical Committee: CEN/TC 327 - Animal feeding stuffs - Methods of sampling and analysis
• Drafting Committee: CEN/TC 327/WG 3 - Feed additives and drugs
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 24-Nov-2021
• Due Date: 06-Jul-2020
• Completion Date: 24-Nov-2021
• Directive: 882/2004 - Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 onofficial controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules
• Ref Project: SIST EN 15789:2022 - Animal feeding stuffs: Methods of sampling and analysis - Detection and enumeration of Saccharomyces cerevisiae used as feed additive

Relations

• Replaces: EN 15789:2009 - Animal feeding stuffs - Isolation and enumeration of yeast probiotic strains
• Effective Date: 01-Dec-2021

Overview

EN 15789:2021 (CEN) - Animal feeding stuffs: Methods of sampling and analysis - Detection and enumeration of Saccharomyces cerevisiae used as feed additive - defines a validated laboratory method for detecting and enumerating Saccharomyces cerevisiae in animal feed matrices (additives, premixtures and compound feeds, excluding mineral feeds). The standard supports official control and quality assurance by specifying sampling rules, diluents, culture media, incubation conditions and counting procedures for yeast used as a feed additive.

Key topics and technical requirements

• Scope & applicability
  • Applies to feeds containing S. cerevisiae as a single microorganism or in mixtures; not applicable to mineral feeds (≥ 40% crude ash).
  • Not validated for other yeast species or for detection of ubiquitous/pathogenic yeasts.
• Target sample categories and typical concentrations
  • Additives: about 10^10 CFU/g
  • Premixtures: about 10^11 CFU/kg
  • Compound feeds (meal/pellets): about 10^9 CFU/kg
• Sampling and sample preparation
  • Follow representative aseptic sampling and prepare initial suspensions with tempered diluent; homogenization time unified to one minute for all feed matrices.
• Diluents and media
  • tPBS (phosphate buffered saline + Tween 80) recommended for initial suspension and serial dilutions; alternative: peptone salt solution (PSS) per EN ISO 6887-1.
  • YGC agar (yeast extract dextrose agar + chloramphenicol) is the specified culture medium; chloramphenicol may be replaced by oxytetracycline.
• Enumeration methods
  • Options include spread plate, pour plate or spiral plater techniques.
  • Incubation: 48–72 h at (30 ± 1) °C under aerobic conditions.
  • Acceptable colony counts for calculation: 10 to 200 colonies per plate.
• Special procedures
  • Annex A provides an informative procedure for premixtures and compound feeds with critical/high copper concentrations.
• Quality & reporting
  • Includes morphologic confirmation, calculation of CFU/g or CFU/kg, precision data from interlaboratory studies and required test-report elements.

Applications

• Quality control in feed additive and compound feed manufacturing
• Official control and regulatory verification of product labelling (EU)
• Microbiological testing laboratories performing enumeration of probiotic yeasts in feed
• Product development and stability testing for feed probiotics

Who should use this standard

• Feed manufacturers and additive producers performing in-house QC
• Accredited microbiology laboratories and contract testing services
• National food/feed control authorities and inspectors
• R&D and formulation scientists working with yeast-based feed additives

Related standards

• EN ISO 6498 - Guidelines for sample preparation (animal feeding stuffs)
• EN ISO 6887-1 - Preparation of test samples, dilution methods (PSS)
• EN ISO 7218 - General requirements for microbiology laboratories

Keywords: EN 15789:2021, Saccharomyces cerevisiae, animal feeding stuffs, feed additive, enumeration, detection, CFU, YGC agar, phosphate buffered saline, Tween 80, premixtures, compound feeds.

Standards Content (Sample)

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;
— 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.
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
a
Agar
12 g to 15 g
Water, distilled or deionized 1 000 ml
a
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).
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
-1
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
...