1、December 2009DEUTSCHE NORM English price group 10No 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!$c“1562564www.
2、din.deDDIN EN 15784Animal feeding stuffs Isolation and enumeration of presumptive Bacillus spp.English version of DIN EN 15784:2009-12Futtermittel Keimzhlung von Bacillus spp.Englische Fassung DIN EN 15784:2009-12www.beuth.deDocument comprises pages16DIN EN 15784:2009-12 National foreword This stand
3、ard 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 Produ
4、cts 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 Sampling 2
5、 EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 15784 September 2009 ICS 65.120 English Version Animal feeding stuffs - Isolation and enumeration of presumptive Bacillus spp. Aliments des animaux - Isolement et dnombrement des souches probiotiques de Bacillus spp. Futtermittel - Keimzhlung von
6、 Bacillus 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 biblio
7、graphical 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 o
8、f 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, Iceland, Irel
9、and, 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 1
10、7, 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 15784:2009: EEN 15784:2009 (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
11、s and selective medium 6 6 Apparatus and glassware .7 7 Sampling .8 8 Preparation of test sample 8 9 Procedure .8 10 Expression of results . 10 11 Precision 11 12 Test report . 11 Annex A (informative) Notes on procedure 12 Annex B (informative) Results of the interlaboratory study . 13 Bibliography
12、 . 14 DIN EN 15784:2009-12 EN 15784:2009 (E) 3 Foreword This document (EN 15784:2009) 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 o
13、f 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 shall not be
14、 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 following c
15、ountries 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,
16、Spain, Sweden, Switzerland and the United Kingdom. DIN EN 15784:2009-12 EN 15784:2009 (E) 4 Introduction This methodology has been developed to enumerate and differentiate probiotic bacilli spores capable of germinating, to enable the European Commission to control proper labelling of animal feeding
17、 products (EU project SMT4-CT98-2235 - “Methods for the official control of probiotics (microorganisms) used in animals feeds“) 1. Vegetative cells are not taken into account in this method, as all approved Bacillus species products at present are spores. Spores of Bacillus species survive a heat-tr
18、eatment at 80 C for 10 minutes and the Bacillus species characteristic colony morphology of the individually authorised strains is examined using the proposed method 2. This method is not selective for probiotic bacilli but can be applied to enumerate bacilli in additives, premixtures and feeding st
19、uffs assuming that the probiotic bacilli are present in far higher numbers than any other bacilli. If the feeding stuffs are “contaminated” with a high level of non-probiotic Bacillus species it can be recommended to use a procedure based on antibiotics for more specific selective counting, taking t
20、he antibiotic resistance profile of the different Bacillus strains into account. DIN EN 15784:2009-12 EN 15784:2009 (E) 5 1 Scope This European Standard defines general rules for the enumeration of probiotic bacilli in feeds containing bacilli (Bacillus species) as a single microorganism, component
21、or mixed with other microorganisms. This method is not applicable to mineral feeds which are defined as complementary feeding stuffs composed mainly of minerals and containing at least 40% crude ash (Council Directive 79/373/EEC) 3. There are different categories of feed samples: a) Additives contai
22、ning 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. The detection limits are 500 (5 x 102) colony forming units per gram (CFU/g). The limits of determin
23、ation are 2 x 104CFU/g. 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 any amendments) applies. EN
24、 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) EN ISO 7218, Microbiology
25、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 apply. 3.1 bacilli (descri
26、bed by their characteristics as used for this standard) bacteria of the genus Bacillus which form colonies fitting the descriptions of these species, on the surface of Tryptone Soy Agar (TSA) after heat treatment and incubation at 37C for 16 h to 24 h under aerobic conditions Morphology of colonies
27、on TSA of four Bacillus species: a) Bacillus subtilis: 3 mm to 8 mm in diameter, round, surface dull, opaque, wrinkled and cream or brown coloured; b) Bacillus cereus and Bacillus coagulans: 3 mm to 8 mm in diameter, dull or of frosted glass appearance and undulate shaped; DIN EN 15784:2009-12 EN 15
28、784:2009 (E) 6 c) Bacillus licheniformis: 4 mm to 8 mm diameter, convex, from matt, chondroid, to very mucous, lobbulated, shape surrounded by smaller sub-colonies. The central mucous part of a colony may dry up and become flat, white and opaque while the colonies are still surrounded by small, muco
29、us sub-colonies. Some parts of a colony will adhere more strongly to the substrate than others. 3.2 colony count of presumptive probiotic Bacillus species number of colony-forming units (CFU) which is counted and calculated according to the procedure outlined in this standard 4 Principle An initial
30、suspension of the sample is prepared in a diluent using a suitable homogeniser. From this one new dilution is prepared and heat-treated at 80 C for 10 min. Decimal dilutions are prepared from the heat treated sample and are spread plated on TSA agar and incubated at 37 C for 16 h to 24 h aerobically
31、. Colonies of Bacillus species are counted and the number of colony forming units per g or kg is calculated. 5 Diluents and selective medium 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 (10-1) in appropr
32、iate containers (e.g. universals, bottles or flasks). 5.1.1.1 Initial diluent for additives 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. Aliquot
33、e 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 the diluent to room temperature before use. Measure the pH of t
34、he diluent to ensure the suitable buffer capacity. 5.1.1.2 Initial diluent for feeding stuffs and premixes 0,2% sodium hydroxide solution: Dissolve 2 g sodium hydroxide in one litre of distilled water, Dispense aliquots of this solution appropriate to the initial dilution of feed pellets into capped
35、 flasks. Autoclave all capped flasks containing the diluent at 121 C 1 C for 15 min. Bring the diluent to room temperature before use. 5.1.2 Diluent for serial dilutions, polysorbate peptone salt solution This diluent is used to decimally dilute the initial sample suspension and subsequent dilutions
36、. Peptone salt solution: DIN EN 15784:2009-12 EN 15784:2009 (E) 7 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
37、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 bottles to avoid weight loss during autoclaving. Sterilise in the autoclave for 15 min at 121 C 1 C. Bring the dilue
38、nt to room temperature before use. 5.2 Medium Use Tryptone Soy Agar1)as a culture medium. Composition in g/l: a) tryptone 15,0 g b) sodium chloride 5,0 g c) soya peptone 5,0 g d) agar 15,0 g e) water 1 000 ml Final pH 7,3 0,2 6 Apparatus and glassware Usual microbiological laboratory equipment and,
39、in particular, the following is applied: 6.1 Equipment for autoclaving According to EN ISO 7218. 6.2 Incubator Incubator capable of maintaining an incubation temperature 37 C 1 C. 6.3 Water bath Water bath capable of keeping a temperature of 48 C 1 C and 80 C 1 C. 6.4 Blending equipment A two-speed
40、or a variable adjustable blender (18 000 rotations per minute (rpm) and 22 000 rpm), with a one litre bowl that has been 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 1) The medium is commercially ready
41、 made available from various suppliers DIN EN 15784:2009-12 EN 15784:2009 (E) 8 6.6 Balance A balance capable of weighing to two decimal places. 6.7 Screw-cap bottles 6.8 Pipettor and tips Total-delivery graduated pipettes or automatic pipettes and sterile tips to dispense 100 l and 1 ml. Wide bore
42、tips to pipette homogenised feed stuff for dilution. 6.9 Spreaders Sterile L- or triangular-shaped spreaders from glass or metal or sterile disposable plastic spreaders. 6.10 Sterile Petri dishes, triple vent, 90 mm in diameter 6.11 Laminar flow cabinet 6.12 Microscope for use with 100x oil immersio
43、n lens 6.13 pH meter 7 Sampling Carry out the sampling procedure 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
44、 1 for official control sampling of animal feeds. NOTE Sampling can be done according to ISO 6497 4. Although ISO 6497 is not applicable for microorganisms, 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 potent
45、ial cross-contamination of samples with bacilli. Particularly after sampling additives and premixtures supplemented with bacilli. If needed, clean and disinfect sampling equipment between each sample, particularly after sampling additives and premixtures containing bacilli. Put the sample in a steri
46、le container. 8 Preparation of test sample The test sample preparation shall be done in accordance with ISO 6498 and the congruent product standard. If such a specific standard is not available, it is recommended that agreement be reached on this subject among the parties concerned. ISO 6498 gives g
47、eneral guidelines on test sample preparation. 9 Procedure 9.1 Preparation of poured agar plates Prepare the medium according to the manufacturers directions. Autoclave the medium for 15 min at 121 C 1C and then cool in a water bath to a temperature of 48 C 1 C, subsequently pour portions of 12 ml to
48、 15 ml medium into each Petri dish (6.10) under sterile conditions and spread to give a homogeneous layer. DIN EN 15784:2009-12 EN 15784:2009 (E) 9 When the medium has solidified, pile quantities of four plates reversed on each other and dry at room temperature or in an incubator at 37C 1C for appro
49、ximately 12 h or over night. Alternatively spread the plates out in a lamina flow cabinet and dry the agar surface with the lids partially removed for about 30 min. Check the dried plates for sterility. WARNING If the surface of the agar plates is not dry or the portions of media are more than 15 ml per Petri dish, the Bacillus species strains will very often make swarming colonies on the entire plate and the detection of viable cell count will not be possible. Dried plates, if correctly protected f
