EN 15784:2009
(Main)Animal feeding stuffs - Isolation and enumeration of presumptive Bacillus spp.
Animal feeding stuffs - Isolation and enumeration of presumptive Bacillus spp.
This European Standard defines general rules for the enumeration of probiotic bacilli in feeds containing bacilli (Bacillus species) as a single microorganism, component or mixed with other microorganisms. This method 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) [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 limits are 500 (5 x 102) colony forming units per gram (CFU/g). The limits of determination are 2 x 104 CFU/g.
Futtermittel - Keimzählung von Bacillus spp.
Diese Europäische Norm legt allgemeine Regeln für die Zählung von probiotischen Bacillus-Arten (Bacillus spp.) in Futtermitteln fest, die Bacillus-Arten als einzelnen probiotischen Bestandteil oder in einem Gemisch mit anderen probiotischen Mikroorganismen enthalten. Dieses Verfahren ist nicht anwendbar auf mineralische Futtermittel, die als Ergänzungsfuttermittel definiert sind, welche hauptsächlich aus Mineralien zusammen-gesetzt 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 Nachweisgrenzen liegen bei 500 (5 102) koloniebildenden Einheiten je Gramm (KbE/g). Die Bestim-mungsgrenzen liegen bei 2 104 KbE/g.
Aliments des animaux - Isolement et dénombrement des souches de Bacillus spp. présumées
La présente Norme européenne définit des règles générales pour le dénombrement des bacilles probiotiques dans les aliments contenant des bacilles (espèces Bacillus) sous forme de micro-organisme unique, de composant ou mélangés à d’autres micro-organismes. La présente méthode 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 (Directive 79/373/CEE du Conseil) [3].
Il existe différentes catégories d’échantillons d’aliments :
a) les additifs contenant environ 1010 unités formant colonie (UFC)/g ;
b) les prémélanges contenant environ 108 UFC/g ;
c) les aliments, farines ou granulés qui contiennent environ 106 UFC/g et qui se composent d’aliments complets et d’aliments d’allaitement.
Les limites de détection sont de 500 (5 102) unités formant colonie par gramme (UFC/g). Les seuils de détermination sont de 2 104 UFC/g.
Krma - Izolacija in štetje domnevno prisotnih Bacillus spp
General Information
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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 Bacillus sppFuttermittel - Keimzählung von vermutlichen Bacillus sppAliments des animaux - Isolement et dénombrement des souches probiotiques de BacillusAnimal feeding stuffs - Isolation and enumeration of presumptive Bacillus spp65.120KrmilaAnimal feeding stuffsICS:Ta slovenski standard je istoveten z:EN 15784:2009SIST EN 15784:2009en,fr,de01-december-2009SIST EN 15784:2009SLOVENSKI
STANDARD
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 15784
September 2009 ICS 65.120 English Version
Animal feeding stuffs -Isolation and enumeration of presumptive Bacillus spp.
Aliments des animaux - Isolement et dénombrement des souches probiotiques de Bacillus spp.
Futtermittel - Keimzählung von Bacillus 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 15784:2009: ESIST EN 15784:2009
Introduction This methodology has been developed to enumerate and differentiate probiotic bacilli spores capable of germinating, 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 in animals feeds“) [1]. Vegetative cells are not taken into account in this method, as all approved Bacillus species products at present are spores. Spores of Bacillus species survive a heat-treatment at 80 °C for 10 minutes and the Bacillus species characteristic colony morphology of the individually authorised strains is examined using the proposed method [2]. This method is not selective for probiotic bacilli but can be applied to enumerate bacilli in additives, premixtures and feeding stuffs assuming that the probiotic bacilli are present in far higher numbers than any other bacilli.
If the feeding stuffs are “contaminated” with a high level of non-probiotic Bacillus species it can be recommended to use a procedure based on antibiotics for more specific selective counting, taking the antibiotic resistance profile of the different Bacillus strains into account. SIST EN 15784:2009
1 Scope This European Standard defines general rules for the enumeration of probiotic bacilli in feeds containing bacilli (Bacillus species) as a single microorganism, component or mixed with other microorganisms. This method 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) [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 limits are 500 (5 x 102) colony forming units per gram (CFU/g). The limits of determination are 2 x 104 CFU/g. 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
bacilli (described by their characteristics as used for this standard)
bacteria of the genus Bacillus which form colonies fitting the descriptions of these species, on the surface of Tryptone Soy Agar (TSA) after heat treatment and incubation at 37°C for 16 h to 24 h under aerobic conditions
Morphology of colonies on TSA of four Bacillus species: a) Bacillus subtilis: 3 mm to 8 mm in diameter, round, surface dull, opaque, wrinkled and cream or brown coloured; b) Bacillus cereus and Bacillus coagulans: 3 mm to 8 mm in diameter, dull or of frosted glass appearance and undulate shaped; SIST EN 15784:2009
colony count of presumptive probiotic Bacillus species number of colony-forming units (CFU) which is counted and calculated according to the procedure outlined in this standard 4 Principle An initial suspension of the sample is prepared in a diluent using a suitable homogeniser. From this one new dilution is prepared and heat-treated at 80 °C for 10 min. Decimal dilutions are prepared from the heat treated sample and are spread plated on TSA agar and incubated at 37 °C for 16 h to 24 h aerobically. Colonies of Bacillus species are counted and the number of colony forming units per g or kg is calculated. 5 Diluents and selective medium 5.1 Diluents 5.1.1 Diluent for initial suspension This diluent is used to decimally dilute the sample to prepare an initial sample suspension (10-1) in appropriate containers (e.g. universals, bottles or flasks).
5.1.1.1 Initial diluent for additives
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. 5.1.1.2 Initial diluent for feeding stuffs and premixes 0,2% sodium hydroxide solution: Dissolve 2 g sodium hydroxide in one litre of distilled water, Dispense aliquots of this solution appropriate to the initial dilution of feed pellets into capped flasks. Autoclave all capped flasks containing the diluent at 121 °C ± 1 °C for 15 min. Bring the diluent to room temperature before use. 5.1.2 Diluent for serial dilutions, polysorbate peptone salt solution This diluent is used to decimally dilute the initial sample suspension and subsequent dilutions.
Peptone salt solution: SIST EN 15784:2009
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 Medium Use Tryptone Soy Agar1) as a culture medium. Composition in g/l: a) tryptone 15,0 g b) sodium chloride 5,0 g c) soya peptone 5,0 g d) agar 15,0 g e) water 1 000 ml Final pH 7,3 ± 0,2 6 Apparatus and glassware Usual microbiological laboratory equipment and, in particular, the following is applied: 6.1 Equipment for autoclaving According to EN ISO 7218. 6.2 Incubator Incubator capable of maintaining an incubation temperature 37 °C ± 1 °C. 6.3 Water bath Water bath capable of keeping a temperature of 48 °C ± 1 °C and 80 °C ± 1 °C. 6.4 Blending equipment A two-speed or a variable adjustable blender (18 000 rotations per minute (rpm) and 22 000 rpm), with a one litre bowl that has been sterilised in an oven for 1 h at 170 °C to 180 °C. 6.5 Mechanical stirrer A mechanical stirrer e.g. Vortex Mixer (see EN ISO 7218), or equivalent
1) The medium is commercially ready made available from various suppliers
6.6 Balance A balance capable of weighing to two decimal places.
6.7 Screw-cap bottles 6.8 Pipettor and tips Total-delivery graduated pipettes or automatic pipettes and sterile tips to dispense 100 µl and 1 ml. Wide bore tips to pipette homogenised feed stuff for dilution. 6.9 Spreaders Sterile L- or triangular-shaped spreaders from glass or metal or sterile disposable plastic spreaders. 6.10 Sterile Petri dishes, triple vent, 90 mm in diameter 6.11 Laminar flow cabinet 6.12 Microscope for use with 100x oil immersion lens 6.13 pH meter 7 Sampling Carry out the sampling procedure in accordance with the specific standard appropriate to the product concerned. If such a specific standard is not available, it is recommended that agreement be reached on this subject among the parties concerned. Apply community rules [1] for official control sampling of animal feeds. NOTE Sampling can be done according to ISO 6497 [4]. Although ISO 6497 is not applicable for microorganisms, due to the lack of other reference, it seems it is the most suitable protocol to be taken into account WARNING — Take precautions to avoid potential cross-contamination of samples with bacilli. Particularly after sampling additives and premixtures supplemented with bacil
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