Animal feeding stuffs - Isolation and enumeration of yeast probiotic strains

This European Standard defines general rules for the enumeration of probiotic yeasts in feed samples (additives, premixtures and feeding stuffs) that contain yeast as a single microorganism component or in a mixture with other microorganisms. The 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) [4].
There are different categories of feed samples:
a)   Additives which contain about 109 CFU/g to 1010 CFU/g (CFU = colony forming units).
b)   Premixtures which contain about 108 CFU/g
c)   Feeds, meal or pellets, which contain about 106 CFU/g and include complete feedingstuffs, and milk replacers.
The detection limit is as defined in ISO 7218.

Futtermittel - Keimzählung von probiotischen Hefestämmen

Diese Internationale Norm legt allgemeine Regeln für die Zählung von probiotischer Hefe in Futtermittelproben (Zusatzstoffe, Vormischung und Futtermittel) fest, die Hefe als einzelnen mikroorganischen Bestandteil oder in einem Gemisch mit anderen 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).
Es gibt unterschiedliche Kategorien von Futtermittelproben:
-   Zusatzstoffe, die etwa 1010 KbE/g enthalten;
-   Vormischungen, die etwa 108 KbE/g enthalten;
-               Futtermittel, Mehl oder Pellets, die etwa 106 KbE/g enthalten und fertige Futtermittel und Milchersatz berücksichtigen.

Aliments des animaux - Isolation et dénombrement de souches probiotiques de levures (saccharomyces cerevisiae)

La présente Norme internationale donne des directives générales pour le dénombrement des levures probiotiques présentes dans des échantillons d’aliments (additifs, prémélanges et aliments des animaux) qui contiennent de la levure en tant que composant sous forme d’un micro-organisme simple ou en mélange avec d’autres micro-organismes. La présente norme ne s’applique pas aux aliments minéraux qui sont définis comme étant des aliments complémentaires des animaux constitués principalement de minéraux et renfermant au moins 40 % de cendres brutes (Directive du Conseil 79/373/CEE).
Il existe différentes catégories d’échantillons d’aliments des animaux :
   les additifs qui contiennent environ 109 UFC/g à 1010 UFC/g ;
   les prémélanges qui contiennent environ 108 UFC/g ;
   les aliments des animaux, les farines ou les granulés qui contiennent environ 106 UFC/g et incluent des aliments complets et des succédanés de lait.

Krma - Izolacija in štetje domnevno prisotnih kvasovk probiotičnih sevov

General Information

Status
Withdrawn
Publication Date
15-Nov-2009
Withdrawal Date
27-Dec-2021
Current Stage
9900 - Withdrawal (Adopted Project)
Start Date
28-Dec-2021
Due Date
20-Jan-2022
Completion Date
28-Dec-2021

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Futtermittel - Keimzählung von probiotischen HefestämmenAliments des animaux - Isolation et dénombrement de souches probiotiques de levures (saccharomyces cerevisiae)Animal feeding stuffs - Isolation and enumeration of yeast probiotic strains65.120KrmilaAnimal feeding stuffsICS:Ta slovenski standard je istoveten z:EN 15789:2009SIST EN 15789:2009en,fr,de01-december-2009SIST EN 15789:2009SLOVENSKI

STANDARD
SIST EN 15789:2009
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 15789
September 2009 ICS 65.120 English Version
Animal feeding stuffs - Isolation and enumeration of yeast probiotic strains

Aliments des animaux - Isolation et dénombrement de souches probiotiques de levures (saccharomyces cerevisiae)

Futtermittel - Keimzählung von Hefestämmen 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 15789:2009: ESIST EN 15789:2009

EN 15789:2009 (E) 2 Contents Page Foreword ..............................................................................................................................................................3 Introduction .........................................................................................................................................................4 1 Scope ......................................................................................................................................................5 2 Normative references ............................................................................................................................5 3 Terms and definitions ...........................................................................................................................5 4 Principle ..................................................................................................................................................5 5 Diluent, selective media and test kit for phenotypic characterisation .............................................6 6 Apparatus and glassware .....................................................................................................................8 7 Sampling .................................................................................................................................................9 8 Preparation of test samples ..................................................................................................................9 9 Procedure ...............................................................................................................................................9 10 Expression of results ......................................................................................................................... 12 11 Precision .............................................................................................................................................. 13 12 Test report ........................................................................................................................................... 13 Annex A (informative) Results of the interlaboratory study ......................................................................... 14 Bibliography ..................................................................................................................................................... 15

SIST EN 15789:2009

EN 15789:2009 (E) 3 Foreword This document (EN 15789:2009) 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 15789:2009

EN 15789:2009 (E) 4

Introduction This method has been developed to enumerate probiotic yeasts in additives, premixtures and feeding stuffs. 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]. It is based on ISO 7954, a pour plate method using extract dextrose chloramphenicol (CGYE) (alternatively oxytetracycline) agar, a selective agar for yeasts [1]. This method is not selective for probiotic yeast but can be applied to enumerate yeast in feed assuming that the probiotic yeast is present in far higher numbers than any other yeast. In addition or alternatively a spread plate method and a chromogenic1 agar can be used allowing an elective enumeration of the probiotic yeast species for example Saccharomyces cerevisiae, which forms distinct mauve/purple colonies. The presence of other yeasts will be identified on the elective agar by different colouration. The application of both agars (CGYE and Chromagar® Candida) have been validated for four probiotic commercially used yeast strains, belonging to Saccharomyces cerevisiae, in premixtures and feeding stuffs [3].

1 e.g. CHROMagar® Candida from CHROMagar

This is a suitable product available commercially. This information does not constitute an endorsement by CEN of this product. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 15789:2009

EN 15789:2009 (E) 5

1 Scope This European Standard defines general rules for the enumeration of probiotic yeasts in feed samples (additives, premixtures and feeding stuffs) that contain yeast as a single microorganism component or in a mixture with other microorganisms. The 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) [4]. There are different categories of feed samples: a) Additives which contain about 109 CFU/g to 1010 CFU/g (CFU = colony forming units): b) Premixtures which contain about 108 CFU/g, c) Feeds, meal or pellets, which contain about 106 CFU/g and include complete feedingstuffs, 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

yeasts (described by their characteristics as used for this standard) microorganisms which form colonies on the selective yeast extract dextrose chloramphenicol (oxytetracycline) agar according to the method specified in this Standard 4 Principle a) Preparation of sterile and dry poured agar plates, and sterile molten agar at 48 °C ± 1 °C for poured plates.

b) Drawing a representative test sample under sterile conditions.
SIST EN 15789:2009

EN 15789:2009 (E) 6 c) Preparation of the initial suspension to obtain a homogeneous distribution of yeast-like cells from the test portion. d) 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. e) Inoculation of the prepared plates with an aliquot of the optimum dilutions and dispersion of the inoculum by using a sterile spreader, or poured plate. f) Aerobic incubation of inverted spread plates for 3 days at 30 °C ± 1 °C, or 35 °C ± 1 °C for 2 days for poured plates. g) Counting of typical colonies, considering the specific properties of yeast as listed above.

h) Morphological verification of isolates of yeast through the use of microscope analysis. i) Calculation of the colony count per g or kg of feed sample. 5 Diluent, selective media 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. 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. SIST EN 15789:2009

EN 15789:2009 (E) 7 5.2 Media 5.2.1 Yeast extract dextrose chloramphenicol (oxytetracycline) agar (CGYE) Yeast extract dextrose chloramphenicol agar (CGYE) is composed of:

a) yeast extract 5 g; b) dextrose 20 g; c) chloramphenicol 0,1 g; d) agar 12 g to 15 g; e) made up to 1 000 ml with distilled water. The base without antibiotic can be purchased and the chloramphenicol supplement has to be added or it can be purchased as a complete medium

NOTE Chloramphenicol may be replaced by oxytetracycline (C22H24N2O9) at a final concentration of 100 µg/ml of medium. 5.2.2 Chromogenic agar2)

5.3 Phenotypic characterisation Selected colonies are checked microscopically by suspension in a drop of 0,85% sterile saline with a coverslip and a feasible magnification (e.g. oil immersion) for morphology. Yeast obtains large cells of varying shape and size, possibly with 'budding'.

Typical microscopic and phenotypic profile for Saccharomyces cerevisiae: On yeast extract dextrose chloramphenicol (oxytetracycline) agar: a) Cream and opaque; b) Irregular shaped; c) Vary in size (1 mm to 6 mm in diameter). On chromogenic agar2: d) Circular; e) Convex to dome-shaped; f) Entire; g) Mauve/purple ; h) Matt or shiny surface;

2 e.g. CHROMagar® Candida from CHROMagar

This is a suitable product available commercially. This information does not constitute an endorsement by CEN of this product. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 15789:2009

EN 15789:2009 (E) 8 i) Opaque; j) Vary in size (1 mm and 3 mm in diameter). 5.4 Biochemical characterisation Presumptive yeast colonies can be biochemically characterised by classical or commercial test kits

(e.g. API 20 C AUX or equivalent) for confirmation according to the manufacturers specifications. 6 Apparatus and glassware Usual microbiological laboratory equipment and, in particular, the following is applied: 6.1 Equipment for dry sterilisation (oven) and wet sterilisation (autoclave) According to EN ISO 7218. 6.2 Incubators Capable of maintaining a temperature of 30 °C ± 1 °C and 35 °C ± 1 °C. 6.3 Water bath Capable of maintaining a temperature of 48 °C ± 1 °C. 6.4 Blending equipment Two-speed or variable adjustable blender (18 000 rotations per minute (rpm) and 22 000 rpm, with a one litre bowl which is 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

6.6 Steamer, or similar equipment for melting agar without autoclaving A boiling water bath (or steamer) is used for the preparation of 500 ml volumes of agar, while a media preparator and plate pourer are used for more than 1 000 ml. 6.7 Balance Capable of weighing to two decimal places. 6.8 Screw-cap bottles Appropriate capacities, 25 ml universals, bottles, test tubes, flasks and 1 000 ml Duran bottles. 6.9 Pipettors 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. SIST EN 15789:2009

EN 15789:2009 (E) 9 6.10 Sterile spreaders Sterile L- or triangular-shaped spreaders from glass or metal or sterile disposable plastic spreaders.

6.11 Sterile Petri dishes, triple vent, 90 mm in diameter
6.12 Laminar flow cabinet
6.13 Microscope With phase-contrast-ima
...

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