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!$“1562557www.d
2、in.deDDIN EN 15787Animal feeding stuffs Isolation and enumeration of Lactobacillus spp.English version of DIN EN 15787:2009-12Futtermittel Keimzhlung von Lactobacillus spp.Englische Fassung DIN EN 15787:2009-12www.beuth.deDocument comprises pages19DIN EN 15787:2009-12 National foreword This standard
3、 has 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
4、 Standards 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
5、 EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 15787 September 2009 ICS 65.120 English Version Animal feeding stuffs - Isolation and enumeration of Lactobacillus spp. Aliments des animaux - Isolement et dnombrement du Lactobacillus spp. Futtermittel - Keimzhlung von Lactobacillus spp. This Eu
6、ropean 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 c
7、oncerning 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 it
8、s 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, Li
9、thuania, 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: Avenue Marnix 17, B-1000 Brussels 200
10、9 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 15787:2009: EEN 15787:2009 (E) 2 Contents Page Foreword 3 Introduction .4 1 Scope 5 2 Normative references 5 3 Terms and definitions .5 4 Principle 6 5 Diluent, selective media and
11、phenotypic characterisation .6 6 Apparatus and glassware .8 7 Sampling 10 8 Preparation of test sample . 10 9 Procedure 10 10 Expression of results . 12 11 Precision 12 12 Test report . 13 Annex A (informative) Notes on procedure 14 Annex B (informative) Results of the interlaboratory study . 15 Bib
12、liography . 17 DIN EN 15787:2009-12 EN 15787:2009 (E) 3 Foreword This document (EN 15787:2009) 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 publ
13、ication 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 sha
14、ll 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 fo
15、llowing 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, S
16、lovenia, Spain, Sweden, Switzerland and the United Kingdom. DIN EN 15787:2009-12 EN 15787:2009 (E) 4 Introduction This methodology has been developed to enumerate probiotic lactobacilli to enable the European Commission to control proper labelling of animal feeding products (EU project SMT4-CT98-223
17、5 “Methods for the official control of probiotics (microorganisms) used as feed additives”) 1. The described methodology was validated in an interlaboratory study 2. DIN EN 15787:2009-12 EN 15787:2009 (E) 5 1 Scope This European Standard defines general rules for the enumeration of probiotic lactoba
18、cilli in feed samples (additives, premixtures and feeding stuffs) that contain lactobacilli as a single bacterial component or in a mixture with other microorganisms. This standard is not applicable to mineral feeds, which are defined as complementary feeding stuffs composed mainly of minerals and c
19、ontaining at least 40% crude ash (Council Directive 79/373/EEC 3). There are different categories of feed samples: a) Additives containing about 1010colony forming units (CFU)/g; b) Premixtures containing about 108CFU/g; c) Feeds, meal or pellets, which contain about 106CFU/g and include complete fe
20、eding 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
21、 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 dec
22、imal 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
23、 following terms and definitions apply. 3.1 lactobacilli (described by their characteristics as used for this standard) lactobacilli are bacteria which form colonies fitting the description of these species on the specified selective medium after incubation of 48 h to 72 h at a temperature of 37 C u
24、nder anaerobic conditions 4: Morpholoy of colonies: a) circular; b) regular or irregular (starry) surrounding; c) convex or conical; d) dull or glistening surface; DIN EN 15787:2009-12 EN 15787:2009 (E) 6 e) translucent, white, pale green, dark green. Colony size varies between 0,5 mm and 3 mm in di
25、ameter. Phase contrast microscopic a examination of selected colonies shows that cells are varying from long and slender sometimes bent rods, to short, often coryneform coccobacilli and chain formation is common. NOTE For a detailed account of morphology see 4. 4 Principle a) Preparation of sterile
26、and dry poured agar plates. b) Drawing a representative test sample under sterile conditions. c) Preparation of the initial suspension to obtain a homogeneous distribution of bacterial cells from the test portion. d) Preparation of further decimal dilutions of the initial suspension in order to redu
27、ce 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. f) Incubation of inverted plates for 48 h to 72 h at 37 C 1
28、 C, under anaerobic conditions. g) Counting of typical colonies, considering the specific properties of lactobacilli. h) Morphological verification of isolates within the Lactobacillus genus through the use of microscope analysis. i) Calculation of the colony count per gram or kilogram of feed sampl
29、e. 5 Diluent, selective media and 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 sample suspension (10-1) in appropriate containers (e.g. un
30、iversals, 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, bo
31、ttles 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. DIN EN 1578
32、7:2009-12 EN 15787:2009 (E) 7 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 casei
33、n 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 c
34、ap 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 medium; b) MRS supplemented with Triphenyl Tetrazolium Chloride (TTC); c) AMRSA:
35、 Acidified MRS agar; d) LAMVAB: Lactobacillus Anaerobic MRS with vancomycin and bromocresol green. For routine enumeration of lactobacilli 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 t
36、o encourage the growth of the lactic acid bacteria such as lactobacilli, enterococci and pediococci. Selection can be made by pH adjustment, as lactobacilli will tolerate a lower pH than enterococci (pH 5,0 to pH 6,5), with pediococci growing best in this range. When enterococci are expected to be p
37、resent in similar concentrations as lactobacilli, acidified MRS agar (AMRSA) should be used. When lactobacilli in combination with pediococci are expected, MRS agar supplemented with TTC allows differentiation of colonies by different coloration after anaerobic incubation. LAMVAB is a selective medi
38、um for lactobacilli. 5.2.2 Composition 5.2.2.1 MRS agar The composition of the 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,
39、 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 Chlori
40、de (TTC) per 100 ml MRS agar. DIN EN 15787:2009-12 EN 15787:2009 (E) 8 5.2.2.3 AMRSA Acidified MRS agar can be obtained by adjusting the pH of MRS agar (see 5.2.2.1) to 5,4 0,1 with HCl prior to autoclaving. 5.2.2.4 LAMVAB The medium consists of three different components: a) Solution A: MRS broth 1
41、04,4 g/l with cysteine hydrochloride (0,5 g/l) and bromocresol green (0,05 g/l). b) Solution B: Agar 40 g/l. c) Solution C: 2 mg/ml of vancomycin hydrochloride (potency about 1 000 g/mg) in distilled water. 5.2.3 Preparation 5.2.3.1 MRS agar Suspend all ingredients in distilled water and sterilise b
42、y autoclaving at 121 C 1 C for 15 min. 5.2.3.2 MRS agar supplemented with TTC Prepare 1 g Triphenyl Tetrazolium Chloride (TTC) in 100 ml water and filter sterilise. Add 1 ml per 100 ml MRS agar medium (see 5.2.3.1) which is temperated at 48 C 1 C after autoclaving. NOTE TTC is destroyed by autoclavi
43、ng. 5.2.3.3 AMRSA Adjust the pH of MRS agar with HCl to 5,4 0,1 prior to autoclaving. Sterilise at 121 C 1 C for 15 min. 5.2.3.4 LAMVAB Adjust the pH of solution A with HCl to 5,0 0,1. Sterilise solutions A and B at 121 C 1 C for 15 min. Sterilise solution C by filtration using a 0,2 m filter. Solut
44、ion C is stable for at least three months in a fridge. Preparation of the medium involves sterilisation of equal volumes of solutions A and B. Cool solution B down to 50 C in an incubator or water bath. Cool solution A down to room temperature. Add to 500 ml of this solution A, 10 ml of solution C a
45、septically. Finally, add solution B to the MRS-vancomycin (A+C) mixture. Pour plates immediately after mixing. This procedure results in a final vancomycin concentration of 20 mg/l. 5.3 Phenotypic characterisation Microscopical observation of the Gram stained microorganism and catalase test identifi
46、es the bacteria. A test for catalase is done with a drop of hydrogen peroxide (H2O2) on a colony. The formation of bubbles is a positive reaction. Only bacteria that are regular, nonsporing Gram-positive rods, catalase negative, and non-obligatory aerobe are considered. 6 Apparatus and glassware Usu
47、al microbiological laboratory equipment and, in particular, the following: DIN EN 15787:2009-12 EN 15787:2009 (E) 9 6.1 Equipment for dry sterilisation (oven) and wet sterilisation (autoclave) According to EN ISO 7218. 6.2 Incubator Capable of maintaining a temperature of 37C 1C. 6.3 Blending equipm
48、ent Two-speed or a variable adjustable blender (18 000 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. 6.4 Mechanical stirrer A mechanical stirrer e.g. Vortex Mixer (see EN ISO 7218), or equivalent 6.5 Balance Balance capable of weighing to the ne
49、arest 0,01 g. 6.6 Flasks or screw-cap bottles of appropriate capacities 6.7 Test tubes of appropriate capacities 6.8 Pipettor and sterile tips to dispense 100 l and 1 ml Wide bore tips to pipette homogenised feed stuff for dilution. 6.9 pH meter 6.10 Sterile Petri dishes, 90 mm in diameter 6.11 Equipment for anaerobic incubation Appropriate anaerobic jars or chambers 6.12 Laminar flow cabinet 6.13 Water bath Capable of maintaining temperatures of 48 C 1 C resp. 50 C 1 C. 6.14 Microsco
