EN 15786:2009
(Main)Animal feeding stuffs - Isolation and enumeration of Pediococcus spp.
Animal feeding stuffs - Isolation and enumeration of Pediococcus spp.
This European Standard defines general rules for the enumeration of probiotic pediococci in feed samples (additives, premixtures and feeding stuffs) that contain pediococci as a single bacterial component or in a mixture with other microorganisms. This standard is not applicable for 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) [3].
There are different categories of feed samples:
a) Additives containing about 1010 (colony forming units) CFU/g
b) Premixtures containing about 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 Pediococcus spp.
Diese Europäische Norm legt allgemeine Regeln für die Zählung von probiotischen Pediokokken in Futter-mittelproben (Zusatzstoffe, Vormischungen und Futtermittel) fest, die Pediokokken 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 du Pediococcus spp.
La présente norme internationale définit les règles générales de dénombrement des pédiocoques probiotiques dans les échantillons d’aliments (additif, prémélange et aliments des animaux) qui contiennent des pédiocoques comme seul composant bactérien 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).
Il existe différentes catégories d’échantillons d’aliments :
les additifs contenant environ 1010 UFC/g ;
les prémélanges contenant environ 108 UFC/g ;
les aliments, farines ou granulés qui contiennent environ 106 UFC/g et comprennent des aliments complets et des succédanés du lait.
Krma - Izolacija in štetje domnevno prisotnih Pediococcus spp
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Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Krma - Izolacija in štetje domnevno prisotnih Pediococcus sppFuttermittel - Keimzählung von Pediococcus sppAliments des animaux - Isolement et dénombrement du Pediococcus sppAnimal feeding stuffs - Isolation and enumeration of Pediococcus spp65.120KrmilaAnimal feeding stuffsICS:Ta slovenski standard je istoveten z:EN 15786:2009SIST EN 15786:2009en,fr,de01-december-2009SIST EN 15786:2009SLOVENSKI
STANDARD
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 15786
September 2009 ICS 65.120 English Version
Animal feeding stuffs - Isolation and enumeration of Pediococcus spp.
Aliments des animaux - Isolement et dénombrement du Pediococcus spp.
Futtermittel - Keimzählung von Pediococcus spp. 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 15786:2009: ESIST EN 15786:2009
Introduction This methodology has been developed to enumerate probiotic pediococci 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 feed additives”) [1]. The described methodology was validated in an interlaboratory study [2]. SIST EN 15786:2009
1 Scope This European Standard defines general rules for the enumeration of probiotic pediococci in feed samples (additives, premixtures and feeding stuffs) that contain pediococci as a single bacterial component or in a mixture with other microorganisms. This standard is not applicable for 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) [3]. There are different categories of feed samples: a) Additives containing about 1010 (colony forming units) CFU/g b) Premixtures containing
about 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
pediococci (described by their characteristics as used for this standard) pediococci are characterised as bacteria which form colonies fitting the description of these species on the specified media after incubation of 24 h to 48 h for MRSA, AMRSA and MRSA+TTC and 24 h for Pediococcus selective medium at a temperature of 37 °C under anaerobic conditions [4].
Morphology of colonies on agar plates: a) circular; b) convex; c) entire; SIST EN 15786:2009
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. SIST EN 15786:2009
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 Media 5.2.1 General Four different media are proposed: a) MRS agar b) MRS agar supplemented with Triphenyl Tetrazolium Chloride (TTC)
c) AMRSA: Acidified MRS agar d) Selective media: MRS medium supplemented with cysteine hydrochloride, vancomycin, novobiocin and nystatin. For routine enumeration of pediococci the use of MRS agar will be sufficient assuming that the probiotic strain is present in far higher numbers than any other microorganism. The medium is designed to encourage the growth of 'lactic acid bacteria' such as pediococci, enterococci and lactobacilli. Selection can be made by pH adjustment, as pediococci and lactobacilli will tolerate a lower pH than enterococci (pH 5,0 to 6,5). When enterococci are expected to be present in similar concentrations as pediococci, acidified MRS agar (AMRSA) should be used. When pediococci in combination with lactobacilli are expected, MRS agar supplemented with TTC allows differentiation of colonies by different colouration after anaerobic incubation. The MRS medium supplemented with three antibiotics is selective for pediococci and should be used when the probiotic lactobacilli colony count exceeds the pediococci colony count by a factor of 50 or more. 5.2.2 Composition 5.2.2.1 MRS agar
The composition of the MRS agar per l of distilled water is as follows [5]: 20,0 g dextrose, 10,0 g polypeptone, 10,0 g meat extract, 5,0 g yeast extract, 5,0 g sodium acetate 3xH20, 2,0 g sodium phosphate, 2,0 g tri-ammonium citrate, 1,0 g Tween 80, 0,2 g magnesium sulphate 7xH2O, 0,05 g manganese sulphate 4xH2O, agar 15,0 g, pH 6,2 ± 0,2. 5.2.2.2 MRS agar supplemented with TTC Sterilise MRS agar (5.2.2.1) by autoclaving at 121 °C ± 1 °C for 15 min. Supplement with 1 ml of a filter sterilised 1 g/100 ml water solution of Triphenyl Tetrazolium Chloride (TTC) per 100 ml MRS agar. SIST EN 15786:2009
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