1、BS EN 15787:2009ICS 65.120NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDAnimal feedingstuffs Isolationand enumeration ofLactobacillus spp.This British Standardwas published underthe authority of theStandards Policy andStrategy Committee on 30September 2009 BSI
2、 2009ISBN 978 0 580 61803 1Amendments/corrigenda issued since publicationDate CommentsBS EN 15787:2009National forewordThis British Standard is the UK implementation of EN 15787:2009.The UK participation in its preparation was entrusted to TechnicalCommittee AW/10, Animal feeding stuffs.A list of or
3、ganizations 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 obligatio
4、ns.BS EN 15787:2009EUROPEAN 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 Lactobac
5、illus 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 bibliograp
6、hical 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
7、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,
8、 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: Avenue Marnix 17, B
9、-1000 Brussels 2009 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 15787:2009: EBS EN 15787:2009EN 15787:2009 (E) 2 Contents Page Foreword 3 Introduction .4 1 Scope 5 2 Normative references 5 3 Terms and definitions .5 4 Principle
10、 6 5 Diluent, selective media and 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
11、the interlaboratory study . 15 Bibliography . 17 BS EN 15787:2009EN 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 nati
12、onal 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 r
13、ights. 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 stan
14、dards 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, P
15、ortugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. BS EN 15787:2009EN 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 (
16、EU project SMT4-CT98-2235 “Methods for the official control of probiotics (microorganisms) used as feed additives”) 1. The described methodology was validated in an interlaboratory study 2. BS EN 15787:2009EN 15787:2009 (E) 5 1 Scope This European Standard defines general rules for the enumeration o
17、f probiotic lactobacilli 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 mainl
18、y 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 1010colony forming units (CFU)/g; b) Premixtures containing about 108CFU/g; c) Feeds, meal or pellets, which contain about 106CFU/g and
19、include complete feeding 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 la
20、test 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
21、 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
22、 this document, the 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 te
23、mperature of 37 C under anaerobic conditions 4: Morpholoy of colonies: a) circular; b) regular or irregular (starry) surrounding; c) convex or conical; d) dull or glistening surface; BS EN 15787:2009EN 15787:2009 (E) 6 e) translucent, white, pale green, dark green. Colony size varies between 0,5 mm
24、and 3 mm in diameter. 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) Preparati
25、on of sterile 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
26、 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. f) Incubation of inverted plates for 48 h to
27、72 h at 37 C 1 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
28、 of feed sample. 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 conta
29、iners (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.
30、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 capacit
31、y. BS EN 15787:2009EN 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
32、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 us
33、e 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 Media 5.2.1 General Four different media are proposed: a) MRS medium; b) MRS supplemented with Triphenyl Tetrazolium Chloride (TTC);
34、c) AMRSA: 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 d
35、esigned to 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 expecte
36、d to be present 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 selec
37、tive medium 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
38、Tween 80, 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 Tetrazoli
39、um Chloride (TTC) per 100 ml MRS agar. BS EN 15787:2009EN 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 bro
40、th 104,4 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 sterili
41、se by 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 autoc
42、laving. 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. S
43、olution 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
44、 C aseptically. 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 iden
45、tifies 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
46、 Usual microbiological laboratory equipment and, in particular, the following: BS EN 15787:2009EN 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 equipme
47、nt 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 nea
48、rest 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 Equip
49、ment 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 Microscope Capable of phase-contrast microscopy at 600x to 1 000x magnification. 6.15 Bacterial Cell spreaders Sterile L- or triangular-shaped spreaders from glass or metal or sterile disposable plastic spreaders. BS EN 15787:2009EN 15787:2009 (E) 10 7 Sampling Carry out the sampling procedure in accordance with the
