Animal feeding stuffs - Isolation and enumeration of Enterococcus (E. faecium) spp.

This European Standard defines general rules for the enumeration of enterococci in feed samples (additives, premixtures and feeding stuffs) that contain enterococci (E. faecium) as a single microorganism component or in a mixture with other microorganisms. This standard is not applicable to mineral feeds which are defined as complementary feedingstuffs composed mainly of minerals and containing at least 40% crude ash (Council Directive 79/373/EEC) [3].
There are different categories of feed samples:
a)   Additives containing about 1010 colony forming units (CFU)/g;
b)   Premixtures containing 108 CFU/g;
c)   Feeds, meal or pellets which contain about 106 CFU/g and include complete feeding stuffs,and milk replacers.
The detection limit is as defined in ISO 7218.

Futtermittel - Keimzählung von Enterococcus spp. (E. faecium)

Diese Europäische Norm legt allgemeine Regeln für die Zählung von Enterokokken in Futtermittelproben (Zusatzstoffe, Vormischungen und Futtermittel) fest, die Enterokokken (E. faecium) als einzelnen probiotischen Bestandteil oder in einem Gemisch mit anderen probiotischen Mikroorganismen enthalten. Diese Norm ist nicht anwendbar auf mineralische Futtermittel, die als Ergänzungsfuttermittel definiert sind, hauptsächlich aus Mineralien zusammengesetzt sind und mindestens 40 % Rohasche enthalten (Richtlinie 79/373/EWG) [3].
Es gibt unterschiedliche Kategorien von Futtermittelproben:
a) Zusatzstoffe, die etwa 1010 koloniebildende Einheiten (KbE/g) enthalten;
b) Vormischungen, die etwa 108 KbE/g enthalten;
c) Futtermittel, Mehl oder Pellets, die etwa 106 KbE/g enthalten, einschließlich Fertigfuttermittel und Milch-austauscher.
Die Nachweisgrenze entspricht der in EN ISO 7218 festgelegten.

Aliments des animaux - Isolement et dénombrement de l'entérocoque (E. faecium) spp.

La présente Norme européenne définit les règles générales de dénombrement des entérocoques dans les échantillons alimentaires (additif, prémélange, aliments des animaux) qui contiennent des entérocoques (E. faecium) comme seul composant de type micro-organisme ou dans un mélange comportant d’autres micro-organismes. La présente norme ne s’applique pas aux aliments minéraux définis comme aliments complémentaires constitués principalement de minéraux et contenant au moins 40 % de cendre brute (Directive 79/373/CEE du Conseil) 3.
Il existe différentes catégories d’échantillons d’aliments :
les additifs contenant environ 1010 UFC/g ;
les prémélanges contenant 108 UFC/g ;
les aliments, farines ou granulés qui contiennent environ 106 UFC/g et comprennent des aliments complets et des aliments d’allaitement.
Les limites de détection sont telles que définies dans la norme EN ISO 7218.

Krma - Izolacija in štetje domnevno prisotnih Enterococcus (E. faecium) spp

General Information

Status
Withdrawn
Publication Date
01-Sep-2009
Withdrawal Date
23-Nov-2021
Current Stage
9960 - Withdrawal effective - Withdrawal
Completion Date
24-Nov-2021

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Krma - Izolacija in štetje domnevno prisotnih Enterococcus (E. faecium) sppFuttermittel - Keimzählung von Enterococcus (E. faecium) sppAliments des animaux - Isolement et dénombrement de l'Entérocoque (E. faecium) sppAnimal feeding stuffs - Isolation and enumeration of Enterococcus (E. faecium) spp65.120KrmilaAnimal feeding stuffsICS:Ta slovenski standard je istoveten z:EN 15788:2009SIST EN 15788:2009en,fr,de01-december-2009SIST EN 15788:2009SLOVENSKI
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SIST EN 15788:2009



EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 15788
September 2009 ICS 65.120 English Version
Animal feeding stuffs - Isolation and enumeration of Enterococcus (E. faecium) spp.
Aliments des animaux - Isolement et dénombrement de l'Entérocoque (E. faecium) spp.
Futtermittel - Keimzählung von Enterococcus spp. (E. faecium) This European Standard was approved by CEN on 1 August 2009.
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 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 Management Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre:
Avenue Marnix 17,
B-1000 Brussels © 2009 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 15788:2009: ESIST EN 15788:2009



EN 15788:2009 (E) 2 Contents Page Foreword .3 Introduction .4 1 Scope .5 2 Normative references .5 3 Terms and definitions .5 4 Principle .6 5 Diluents, selective medium and test kit for phenotypic characterisation .6 6 Apparatus and glassware .7 7 Sampling .8 8 Preparation of test sample .9 9 Procedure .9 10 Expression of results . 10 11 Precision . 11 12 Test report . 11 Annex A (informative) Notes on procedure . 12 Annex B (informative) Results of the interlaboratory study . 13 Bibliography . 14
SIST EN 15788:2009



EN 15788:2009 (E) 3 Foreword This document (EN 15788:2009) has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs”, 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 March 2010, and conflicting national standards shall be withdrawn at the latest by March 2010. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights. This document has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. SIST EN 15788:2009



EN 15788:2009 (E) 4
Introduction This methodology has been developed to enumerate enterococci (E. faecium) 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 method is based on an extensive screening of 12 pre-selected, commercially available media for the detection and enumeration of enterococci. The described methodology was validated in an interlaboratory study [2]. This method is not selective for probiotic enterococci (E. faecium) but can be applied to enumerate enterococci in additives, premixtures and feeding stuffs assuming that the probiotic enterococci (E. faecium) is present in far higher numbers than any other enterococci. SIST EN 15788:2009



EN 15788:2009 (E) 5
1 Scope This European Standard defines general rules for the enumeration of enterococci in feed samples (additives, premixtures and feeding stuffs) that contain enterococci (E. faecium) as a single microorganism component or in a mixture with other microorganisms. This standard is not applicable to mineral feeds which are defined as complementary feedingstuffs composed mainly of minerals and containing at least 40% crude ash (Council Directive 79/373/EEC) [3]. There are different categories of feed samples: a) Additives containing about 1010 colony forming units (CFU)/g; b) Premixtures containing 108 CFU/g; c) Feeds, meal or pellets which contain about 106 CFU/g and include complete feeding stuffs,and milk replacers. The detection limit is as defined in EN ISO 7218. 2 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. EN ISO 6887-1, Microbiology of food and animal feeding stuffs - Preparation of test samples, initial suspension and decimal dilutions for microbiological examination - Part 1: General rules for the preparation of the initial suspension and decimal dilutions (ISO 6887-1:1999) EN ISO 7218, Microbiology of food and animal feeding stuffs - General requirements and guidance for microbiological examinations (ISO 7218:2007) ISO 6498, Animal feeding stuffs – Preparation of test samples 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1
enterococcus faecium (described by their characteristics as used for this standard) enterococcus faecium is charaterised as a bacterium which forms colonies fitting the description of the species on the specified selective medium after incubation of 24 h at a temperature of 37 °C under aerobic conditions: a) morphology of colonies on selective medium; b) circular; c) convex to dome-shaped; d) entire; SIST EN 15788:2009



EN 15788:2009 (E) 6 e) white; f) glistening surface; g) opaque. Colony size varies between 1 mm and 2 mm in diameter The medium surrounding the colonies shows a dark brown to black coloration, due to the hydrolysis of esculin Phase contrast microscopical examination of selected colonies typically shows spherical cells arranged in pairs.
4 Principle An initial suspension of the sample is prepared in a diluent with suitable buffer capacity using a suitable homogeniser. Dilutions of the initial suspension have to be immediately prepared before the suspension settles. Spread plates (Bile Esculin Azide Agar) are inoculated with the chosen dilutions. The plates are incubated aerobically for 24 h ± 2 h at 37 °C ± 1 °C. Presumptive enterococci (E. faecium) colonies are counted and the number of colony forming units per g or kg is calculated. NOTE To verify the colony count, a phenotypic characterisation and confirmation of a selection of colonies may be done by means of an identification kit.
5 Diluents, selective medium and test kit for phenotypic characterisation 5.1 Diluents 5.1.1 Diluent for initial suspension of premixtures, additives and feeding stuffs This diluent is used to decimally dilute the sample to prepare an initial decimally diluted sample suspension (10-1) in appropriate containers (e.g. universals, bottles or flasks).
Phosphate buffered saline (PBS): Dissolve 8 g sodium chloride, 0,2 g potassium chloride, 1,15 g disodium hydrogen phosphate, 0,2 g potassium dihydrogen phosphate, pH 7,3 ± 0,2 in 1 l of distilled water. Aliquote this saline into appropriate containers (e.g. universals, bottles or flasks). Autoclave all capped containers with the initial diluent at 121 °C ± 1 °C for 10 min. To avoid loss during autoclaving, screw cap bottles are recommended. Bring the diluent to room temperature before use. Measure the pH of the diluent to ensure the suitable buffer capacity. 5.1.2 Diluent for serial dilutions This diluent is used to decimally dilute the initial sample suspension and subsequent dilutions.
Peptone salt solution: A peptone salt solution is made complying with EN ISO 6887-1. SIST EN 15788:2009



EN 15788:2009 (E) 7 Compose the solution of enzymatic digest of 1 g casein such as pancreatic peptone of casein (or peptone of same quality)
and 8,5 g sodium chloride) per liter (l)
distilled water. Dissolve the ingredients in water. Adjust the pH to 7,0 ± 0,2 at 25 °C ± 1 °C. For decimal dilutions, prepare test tubes containing 9,0 ml ±
0,1 ml after sterilisation or use screw cap bottles to avoid weight loss during autoclaving. Sterilise in the autoclave for 15 min at 121 °C ± 1 °C. Bring the diluent to room temperature before use. 5.2 Selective medium Bile Esculin Azide Agar1) is used as a selective medium. Composition in g/l final medium: a) Peptone 1 (pancreatic digest of casein) 17,0 g b) Peptone 2 (peptic digest of meat) 3,0 g c) Yeast extract 5,0 g d) Ox bile (dehydrated) 10,0 g e) Sodium chloride 5,0 g f) Esculin 1,0 g g) Ferric ammonium citrate 0,5 g h) Sodium azide 0,25 g i) Agar 13,5 g Final pH 7,1 ± 0,2. 5.3 Phenotypic characterization and confirmation Check selected colonies microscopically for enterococci-like morphology.
NOTE A phenotypic kit may be used for phenotypic characterisation of isolated colonies if required.
6 Apparatus and glassware Usual microbiological laboratory equipment and, in particular, the following: 6.1 Equipment for dry sterilisation (oven) and wet sterilisation (autoclave) According to EN ISO 7218. 6.2 Incubator Capable of maintaining a temperature of 37 °C ± 1 °C.
1) th
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