1、December 2009DEUTSCHE NORM English price group 11No part of this standard may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 65.120!$“1562558www.d
2、in.deDDIN EN 15789Animal feeding stuffs Isolation and enumeration of yeast probiotic strainsEnglish version of DIN EN 15789:2009-12Futtermittel Keimzhlung von HefestmmenEnglische Fassung DIN EN 15789:2009-12www.beuth.deDocument comprises pages17DIN EN 15789:2009-12 National foreword This standard ha
3、s been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs Methods of sampling and analysis” (Secretariat: NEN, Netherlands). The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products St
4、andards Committee), Technical Committee NA 057-03-03 AA Futtermittel. The DIN Standard corresponding to the International Standard referred to in this document is as follows: EN ISO 6497 DIN EN ISO 6497 National Annex NA (informative) Bibliography DIN EN ISO 6497, Animal feeding stuffs Sampling 2 EU
5、ROPEAN 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 cerevisiae) Futtermittel -
6、 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 alteration. Up-to-date list
7、s 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 resp
8、onsibility 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, I
9、celand, 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 NORMUNG Management Centre: Ave
10、nue 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: EEN 15789:2009 (E) 2 Contents Page Foreword 3 Introduction .4 1 Scope 5 2 Normative references 5 3 Terms and definitions .5 4 Principle
11、 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 Bi
12、bliography . 15 DIN EN 15789:2009-12 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 pub
13、lication 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 sh
14、all 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 f
15、ollowing 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,
16、Slovenia, Spain, Sweden, Switzerland and the United Kingdom. DIN EN 15789:2009-12 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 feedin
17、g 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
18、 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 an elective enumeration of the p
19、robiotic 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 co
20、mmercially 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 product. Equivalent products may be
21、used if they can be shown to lead to the same results. DIN EN 15789:2009-12 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 compon
22、ent 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) Addit
23、ives 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 EN ISO 7218. 2 Normative refe
24、rences 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 an
25、imal 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 - Gene
26、ral 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
27、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.
28、 b) Drawing a representative test sample under sterile conditions. DIN EN 15789:2009-12 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 i
29、n 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 i
30、nverted 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 colo
31、ny 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 samp
32、le 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
33、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 dilu
34、ent 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 diges
35、t 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
36、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. DIN EN 15789:2009-12 EN 15789:2009 (E) 7 5.2 Media 5.2.1 Yeast extract dextrose chloramphenicol (oxytetracycline) agar (CGYE) Yeast
37、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
38、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 0,85% sterile saline
39、 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
40、) 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 commercially. This info
41、rmation 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. DIN EN 15789:2009-12 EN 15789:2009 (E) 8 i) Opaque; j) Vary in size (1 mm and 3 mm in diameter). 5.4 Biochemical characterisation Presumptive yeast col
42、onies 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 Equip
43、ment 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
44、 (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
45、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
46、, 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. DIN EN 15789:2009-12 EN 15789:2009 (E) 9 6.10 Sterile spreaders Sterile L- or triangu
47、lar-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 procedure
48、 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
49、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, particularly 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 av
