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!$Z“1562555www.
2、din.deDDIN EN 15786Animal feeding stuffs Isolation and enumeration of Pediococcus spp.English version of DIN EN 15786:2009-12Futtermittel Keimzhlung von Pediococcus spp.Englische Fassung DIN EN 15786:2009-12www.beuth.deDocument comprises pages19DIN EN 15786:2009-12 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: ISO 6497 DIN EN ISO 6497 National Annex NA (informative) Bibliography DIN EN ISO 6497, Animal feeding stuffs Sampling 2 EUROP
5、EAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 15786 September 2009 ICS 65.120 English Version Animal feeding stuffs - Isolation and enumeration of Pediococcus spp. Aliments des animaux - Isolement et dnombrement du Pediococcus spp. Futtermittel - Keimzhlung von Pediococcus spp. This European Stand
6、ard 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 su
7、ch 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 langua
8、ge 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, Lux
9、embourg, 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 2009 CEN All ri
10、ghts of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 15786:2009: EEN 15786:2009 (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 phenotypic c
11、haracterisation .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 Bibliography .
12、17 DIN EN 15786:2009-12 EN 15786:2009 (E) 3 Foreword This document (EN 15786:2009) 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 a
13、n 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 he
14、ld 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 coun
15、tries 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, Spa
16、in, Sweden, Switzerland and the United Kingdom. DIN EN 15786:2009-12 EN 15786:2009 (E) 4 Introduction This methodology has been developed to enumerate probiotic pediococci to enable the European Commission to control proper labelling of animal feeding products (EU project SMT4-CT98-2235 “Methods for
17、 the official control of probiotics (microorganisms) used as feed additives”) 1. The described methodology was validated in an interlaboratory study 2. DIN EN 15786:2009-12 EN 15786:2009 (E) 5 1 Scope This European Standard defines general rules for the enumeration of probiotic pediococci in feed sa
18、mples (additives, premixtures and feeding stuffs) that contain pediococci as a single bacterial component or in a mixture with other microorganisms. This standard is not applicable for mineral feeds which are defined as complementary feeding stuffs composed mainly of minerals and containing at least
19、 40% crude ash (Council Directive 79/373/EEC) 3. There are different categories of feed samples: a) Additives containing about 1010(colony forming units) CFU/g b) Premixtures containing about 108CFU/g c) Feeds, meal or pellets, which contain about 106CFU/g and include complete feeding stuffs, and mi
20、lk 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 documen
21、t (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
22、 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 an
23、d definitions apply. 3.1 pediococci (described by their characteristics as used for this standard) pediococci are characterised as bacteria which form colonies fitting the description of these species on the specified media after incubation of 24 h to 48 h for MRSA, AMRSA and MRSA+TTC and 24 h for P
24、ediococcus selective medium at a temperature of 37 C under anaerobic conditions 4. Morphology of colonies on agar plates: a) circular; b) convex; c) entire; DIN EN 15786:2009-12 EN 15786:2009 (E) 6 d) white; e) glistening surface; f) opaque. Colony size varies between 1 mm and 3 mm in diameter. Phas
25、e contrast microscopical examination of selected colonies shows that cells are spherical arranged in pairs or tetrads. 4 Principle a) Preparation of sterile and dry poured agar plates. b) Drawing a representative test sample under sterile conditions. c) Preparation of the initial suspension to obtai
26、n a homogeneous distribution of bacterial 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 wi
27、th an aliquot of the optimum dilutions and dispersion of the inoculum by using a sterile spreader. f) Incubation of inverted plates for 24 h to 48 h at 37 C 1 C, under anaerobic conditions. g) Counting of typical colonies, considering the specific properties of pediococci as listed above. h) Morphol
28、ogical verification of isolates within the Pediococcus genus through the use of microscope analysis. i) Calculation of the colony count per g or kg of feed sample. 5 Diluent, selective media and phenotypic characterisation 5.1 Diluents 5.1.1 Diluent for initial suspension This diluent is used to dec
29、imally 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 dihyd
30、rogen 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 t
31、he diluent to room temperature before use. Measure the pH of the diluent to ensure the suitable buffer capacity. DIN EN 15786:2009-12 EN 15786:2009 (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 s
32、olution: 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 t
33、o 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. 5.2 Media 5.2.1 Genera
34、l Four different media are proposed: a) MRS agar b) MRS agar supplemented with Triphenyl Tetrazolium Chloride (TTC) c) AMRSA: Acidified MRS agar d) Selective media: MRS medium supplemented with cysteine hydrochloride, vancomycin, novobiocin and nystatin. For routine enumeration of pediococci the use
35、 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 to encourage the growth of lactic acid bacteria such as pediococci, enterococci and lactobacilli. Selection can be made by pH adjustment, as pedioc
36、occi and lactobacilli will tolerate a lower pH than enterococci (pH 5,0 to 6,5). When enterococci are expected to be present in similar concentrations as pediococci, acidified MRS agar (AMRSA) should be used. When pediococci in combination with lactobacilli are expected, MRS agar supplemented with T
37、TC allows differentiation of colonies by different colouration after anaerobic incubation. The MRS medium supplemented with three antibiotics is selective for pediococci and should be used when the probiotic lactobacilli colony count exceeds the pediococci colony count by a factor of 50 or more. 5.2
38、.2 Composition 5.2.2.1 MRS agar The composition of the MRS 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, 0,2 g magnesium sul
39、phate 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 Chloride (TTC) per 100 ml
40、MRS agar. DIN EN 15786:2009-12 EN 15786:2009 (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 Selective medium MRS agar supplemented with 0,05% cysteine hydrochloride. The medium is supplemented wit
41、h 10 g/ml vancomycin, 0,1 g/ml novobiocin and 50 U/ml nystatin. 5.2.3 Preparation 5.2.3.1 MRS agar Suspend all ingredients in distilled water and sterilise 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
42、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 autoclaving. 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 Selective medi
43、um Prepare MRS medium with cysteine hydrochloride according to the manufacturers directions. Autoclave the medium for 15 min at 121 C 1 C. Melt the medium the day before use and keep at 48 C 1 C. Add the selective agents from filter-sterilised stocks to the medium immediately before pouring. Pour ap
44、proximately 15 ml portions of medium into each Petri dish (6.12) under sterile conditions and spread to give a homogeneous layer. Add filter sterilised (0,45 m pore-size) selective agents, vancomycin (10 g/ml), novobiocin (0,1 g/ml), and nystatin (50 U/ml), to sterile molten media held at 48C, prior
45、 to plate pouring and drying. Warning Care is needed in preparing the novobiocin stock as levels above 0,1 g/ml may be inhibitory to the probiotic pediococci component. At a concentration lower than 0,1 g/ml the probiotic lactobacilli component may not be inhibited. A stock solution of 10 g/ml shoul
46、d be made through a series of ten-fold dilutions from an initial 1 mg/ml stock. Stack plates when the medium has solidified, and dryd in an incubator at 37 C for approximately 12 h. Check the dried plates for possible loss of sterility. 5.3 Phenotypic characterisation Characteristic appearance of th
47、e pediococci under phase contrast microscopy is spherical cells arranged in pairs or tetrads. 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 catalase negative colonies are considered as Pediococcus. 6 Apparatus a
48、nd glassware Usual microbiological laboratory equipment and, in particular, the following: DIN EN 15786:2009-12 EN 15786:2009 (E) 9 6.1 Equipment for dry sterilisation (oven) and wet sterilization (autoclave) According to EN ISO 7218. 6.2 Incubator Capable of maintaining a temperature of 37 C 1 C. 6
49、.3 Water bath Capable of maintaining a temperature of 48 C 1 C. 6.4 Blending equipment Two-speed or a 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 Balance Balance capable of weighing to the nearest 0,01 g. 6.7 Microscope Capable of phase-contrast microscopy at 600x to 1
