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!$7x“1562085www
2、.din.deDDIN EN 15785Animal feeding stuffs Isolation and enumeration of Bifidobacterium spp.English version of DIN EN 15785:2009-12Futtermittel Keimzhlung von Bifidobacterium spp.Englische Fassung DIN EN 15785:2009-12www.beuth.deDocument comprises pages18DIN EN 15785:2009-12 National foreword This st
3、andard 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 Pr
4、oducts 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: ISO 6497 DIN EN ISO 6497 National Annex NA (informative) Bibliography DIN EN ISO 6497, Animal feeding stuffs Samplin
5、g 2 EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 15785 September 2009 ICS 65.120 English Version Animal feeding stuffs - Isolation and enumeration of Bifidobacterium spp. Aliments des animaux - Isolement et dnombrement du Bifidobacterium spp. Futtermittel - Keimzhlung von Bifidobacterium spp
6、. 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 bibliographical ref
7、erences 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 CEN membe
8、r 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, Italy, L
9、atvia, 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-1000 Bru
10、ssels 2009 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 15785:2009: EEN 15785:2009 (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, media and p
11、henotypic characterisation 6 6 Apparatus and glassware .8 7 Sampling .9 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 procedures 14 Annex B (informative) Results of the interlaboratory study . 15 Bibl
12、iography . 16 DIN EN 15785:2009-12 EN 15785:2009 (E) 3 Foreword This document (EN 15785:2009) 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 publi
13、cation 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 shal
14、l 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 fol
15、lowing 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, Sl
16、ovenia, Spain, Sweden, Switzerland and the United Kingdom. DIN EN 15785:2009-12 EN 15785:2009 (E) 4 Introduction This methodology has been developed to enumerate and differentiate probiotic bifidobacteria, to enable the European Commission to control proper labelling of animal feed stuffs (EU projec
17、t SMT4-CT98-2235 - “Methods for the official control of probiotics (microorganisms) used in animals feeds“) 1. The proposed enumeration method for probiotic bifidobacteris was validated in an interlaboratory study 2. The method is not selective for probiotic bifidobacteria but can be applied to enum
18、erate them in additives, premixtures and feeding stuffs assuming that they are present in higher numbers than any other bacteria. DIN EN 15785:2009-12 EN 15785:2009 (E) 5 1 Scope This European Standard defines general rules for the enumeration of probiotic bifidobacteria in feed samples (additives,
19、premixtures and feeding stuffs) that contain bifidobacteria 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 40% crude ash
20、 (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 feeding stuffs, and milk replacers.
21、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 document (including a
22、ny 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 6887-1:1999)
23、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 and definitions
24、apply. 3.1 bifidobacteria (described by their characteristics as used for this standard) bacteria which form colonies fitting the descriptions of the species, on the specified media of 36 h to 48 h at a temperature of 37C under incubation; anaerobic conditions) Morphology of colonies: a) circular; b
25、) convex; c) entire; d) cream; DIN EN 15785:2009-12 EN 15785:2009 (E) 6 e) shiny surface; f) opaque. Colony size varies between 0,5 mm and 1 mm in diameter. Phase contrast microscopical examination of selected colonies shows that cells are of various rod shapes. 4 Principle a) Preparation of sterile
26、 and dry poured agar plates. b) Drawing of 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
27、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 36 h to 48 h at 3
28、7 C 1 C, under anaerobic conditions. g) Counting of typical colonies, considering the specific properties of bifidobacteria as listed above. h) Morphological verification of isolates within the Bifidobacterium genus using microscope analysis. i) Calculation of the colony count per g or kg of feed sa
29、mple. 5 Diluent, media and phenotypic characterisation 5.1 Diluents 5.1.1 Diluent for initial suspension This diluent is used to decimally dilute the sample to prepare an initial sample suspension of 10-1in appropriate containers (e.g. universals, bottles or flasks). 5.1.1.1 Initial diluent for addi
30、tives 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 is dissolved in 1 l of distilled water. Aliquote this saline is aliquoted into appropriate containers (e.g. universals,
31、bottles or flasks). Autoclave all capped containers with the initial diluent are autoclaved 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 capa
32、city. DIN EN 15785:2009-12 EN 15785:2009 (E) 7 5.1.2 Diluent for serial dilutions This diluent is used to decimally dilute the initial sample suspension and subsequent dilutions. A peptone salt solution is made complying with EN ISO 6887-1. Compose the solution of enzymatic digest of 1 g casein such
33、 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 cap bot
34、tles 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 agar; b) MRS agar supplemented with Triphenyl Tetrazolium Chloride (TTC); c) AMRSA: Ac
35、idified MRS agar; d) Selective medium: MRS medium supplemented with cysteine hydrochloride and mupirocin. For routine enumeration of bifidobacteria 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
36、esigned to encourage the growth of lactic acid bacteria such as pediococci, enterococci and lactobacilli. Selection can be made by pH adjustment, as pediococci, lactobacilli and bifidobacteria will tolerate a lower pH than enterococci (pH 5.0 to pH 6,5) and will grow on acidified MRS agar. When pedi
37、ococci and lactobacilli are expected, MRS agar supplemented with TTC allows differentiation of bifidobacteria colonies by different translucent brown-red coloration after anaerobic incubation. The MRS medium supplemented with mupirocin is selective for bifidobacteria and should be used when other pr
38、obiotic lactic acid bacteria are present in higher numbers than bifidobacteria. 5.2.2 Composition 5.2.2.1 MRS agar The composition of the MRS agar per l of distilled water is as follows: 20,0 g dextrose, 10,0 g polypeptone, 10,0 g meat extract, 5,0 g yeast extract, 5,0 g sodium acetate 3 x H20, 2,0
39、g sodium phosphate, 2,0 g tri-ammonium citrate, 1,0 g Tween 80, 0,2 g magnesium sulphate 7 x H2O, 0,05 g manganese sulphate 4 x H2O, agar 15,0 g, pH 6,5 0,2. NOTE BactoTMLactobacilli MRS agar from Difco Laboratories or from any other supplier producing a medium of same composition may be used. 5.2.2
40、.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. DIN EN 15785:2009-12 EN 15785:2009 (E) 8 5.2.2.3 AMRSA Acidified MRS agar can
41、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 (see 5.2.2.1) supplemented with 0,05 % cysteine hydrochloride (e.g. Sigma C-1276, or equivalent) and with a final concentration of 50 g mupirocin (e.g. Oxoid CT0523B,
42、 or equivalent) per ml. 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 and filter sterilise. Add 1 ml per 100 m
43、l 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 121C 1C for 15 min. 5.2.3.4 Selective medium Selective medium (see 5.2.2.4) suppleme
44、nted with 0,05% (wt/vol) cysteine hydrochloride (e.g. Sigma C-1276, or equivalent). In order to prepare a mupirocin solution add 50 disks (e.g. Oxoid CT0523B or equivalent with 200 g per disk) to 10 ml of 37 C warm sterile distilled water and shake for 30 min. Decant the solution from disks and add
45、to 190 ml molten medium held at about 48 C prior to plate pouring and drying. Final mupirocin concentration shall be 50 g/ml. 5.3 Phenotypic characterisation Characteristic appearance of the bifidobacteria under phase contrast microscopy is rod shaped. Cell morphology can be used to distinguish bifi
46、dobacteria from lactobacilli, enterococci, pediococci and bacilli. 6 Apparatus and glassware Usual microbiological laboratory equipment and, in particular, the following is applied: 6.1 Equipment for dry sterilisation (oven) and wet sterilisation (autoclave) According to EN ISO 7218. 6.2 Incubator C
47、apable of maintaining a temperature of 37 C 1 C. DIN EN 15785:2009-12 EN 15785:2009 (E) 9 6.3 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 that
48、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 Capable of weighing to the nearest 0,01 g. 6.7 Microscope Capable of phase-contrast microscopy at 600x to 1 000x magnification. 6.8 Flasks or
49、 screw-cap bottles of appropriate capacities 6.9 Test tubes of appropriate capacities 6.10 Pipettor and sterile tips to dispense 100 l and 1 ml Wide bore tips to pipette homogenised feed stuff for dilution 6.11 pH meter 6.12 Sterile Petri dishes, 90 mm in diameter 6.13 Bacterial Cell Spreaders Sterile L- or triangular-shaped spreaders from glass or metal or sterile disposable plastic spreaders 6.14 Equipment for anaerobic incubation Appropriate anaerobic jars or chambers 7 Sampling Carry out the sampli
