1、BS EN 15789:2009ICS 65.120NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDAnimal feeding stuffs Isolation andenumeration of yeastprobiotic strainsThis British Standardwas published underthe authority of theStandards Policy andStrategy Committee on 30September 20
2、09 BSI 2009ISBN 978 0 580 61805 5Amendments/corrigenda issued since publicationDate CommentsBS EN 15789:2009National forewordThis British Standard is the UK implementation of EN 15789:2009.The UK participation in its preparation was entrusted to TechnicalCommittee AW/10, Animal feeding stuffs.A list
3、 of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for its correct application.Compliance with a British Standard cannot confer immunityfrom legal obl
4、igations.BS EN 15789:2009EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE 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 dnombrement de souches probiotiques de levures (saccharomyces
5、 cerevisiae) Futtermittel - Keimzhlung von Hefestmmen 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
6、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
7、 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,
8、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 COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NOR
9、MUNG 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: EBS EN 15789:2009EN 15789:2009 (E) 2 Contents Page Foreword 3 Introduction .4 1 Scope 5 2 Normative referenc
10、es 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) R
11、esults of the interlaboratory study . 14 Bibliography . 15 BS EN 15789:2009EN 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
12、 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 o
13、f 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 nat
14、ional 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,
15、 Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. BS EN 15789:2009EN 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
16、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 selecti
17、ve 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 chromogenic1agar can be used allowing
18、 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 bee
19、n validated for four probiotic commercially used yeast strains, belonging to Saccharomyces cerevisiae, in premixtures and feeding stuffs 3. 1e.g. CHROMagar Candida from CHROMagar This is a suitable product available commercially. This information does not constitute an endorsement by CEN of this pro
20、duct. Equivalent products may be used if they can be shown to lead to the same results. BS EN 15789:2009EN 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
21、 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 categori
22、es of feed samples: a) Additives which contain about 109CFU/g to 1010CFU/g (CFU = colony forming units): b) Premixtures which contain about 108CFU/g, c) Feeds, meal or pellets, which contain about 106CFU/g and include complete feedingstuffs, and milk replacers. The detection limit is as defined in E
23、N 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,
24、 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
25、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
26、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
27、 48 C 1 C for poured plates. b) Drawing a representative test sample under sterile conditions. BS EN 15789:2009EN 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 t
28、he 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)
29、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)
30、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
31、 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 distille
32、d 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. Mea
33、sure 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 solu
34、tion 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
35、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. BS EN 15789:2009EN 15789:2009 (E) 7 5.2 Media 5.2.1 Yeast extract dextrose chloramphenicol (oxytetracycline)
36、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 b
37、e added or it can be purchased as a complete medium NOTE Chloramphenicol may be replaced by oxytetracycline (C22H24N2O9) at a final concentration of 100 g/ml of medium. 5.2.2 Chromogenic agar2)5.3 Phenotypic characterisation Selected colonies are checked microscopically by suspension in a drop of 0,
38、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 (oxytet
39、racycline) 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; 2e.g. CHROMagar Candida from CHROMagar This is a suitable product available comme
40、rcially. 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. BS EN 15789:2009EN 15789:2009 (E) 8 i) Opaque; j) Vary in size (1 mm and 3 mm in diameter). 5.4 Biochemical characterisation Presumpt
41、ive 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 appli
42、ed: 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 adjus
43、table 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 a
44、utoclaving 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, bottle
45、s, 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 l and 1 ml. Wide bore tips to pipette homogenised feed stuff for dilution. BS EN 15789:2009EN 15789:2009 (E) 9 6.10 Sterile spreaders Sterile L- or
46、 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-imaging (400x) and for use with oil immersion (1 000x) 7 Sampling Carry out the sampling p
47、rocedure 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 S
48、ampling can be done according to ISO 6497 5. Although ISO 6497 is not applicable for micro organisms, 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 yeast, particul
49、arly after sampling additives and premixtures supplemented with yeast. 8 Preparation of test samples See 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. ISO 6498 gives general guidelines on test sample preparation and test sample preparation is recommended to be done in accordance with this ISO 6498. 9 Procedure 9.1 Preparation of agar plates 9.1.1 Preparat
