CEN/TS 17697:2023
(Main)Animal feeding stuffs - Methods of sampling and analysis - PFGE typing of Lactobacilli, Pediococci, Enterococci and Bacilli in animal feeds
Animal feeding stuffs - Methods of sampling and analysis - PFGE typing of Lactobacilli, Pediococci, Enterococci and Bacilli in animal feeds
This document specifies a Pulsed Field Gel Electrophoresis (PFGE) methodology for the identification of authorized probiotic strains of Lactobacillus, Pediococcus, Enterococcus and Bacillus strains. The method can be applied to purified colonies obtained from cultured premixtures and feeds. The method can be used, even in the presence of a significant microbiological background, to verify the presence of microorganisms (strains and declared concentrations) used as feed additives in animal feeding stuffs.
Futtermittel: Probenahme- und Untersuchungsverfahren - PFGE Typisierung von Laktobazillen, Pediokokken, Enterokokken und Bazillen in Futtermitteln
Dieses Dokument legt eine Methodik der Pulsfeld-Gelelektrophorese (PFGE) zur Identifizierung zugelassener probiotischer Lactobacillus-, Pediococcus-, Enterococcus- und Bacillus-Stämme fest. Das Verfahren kann auf gereinigte Kolonien angewendet werden, die aus kultivierten Vormischungen und Futtermitteln gewonnen werden. Das Verfahren kann sogar vor einem signifikanten mikrobiologischen Hintergrund angewendet werden, um das Vorhandensein von Mikroorganismen (Stämme und deklarierte Konzentrationen), die als Futtermittelzusatzstoffe verwendet werden, zu verifizieren.
Aliments des animaux : Méthodes d’analyse - Typage EGCP des lactobacilles, pédiocoques, entérocoques et bacilles dans les aliments des animaux
Le présent document définit une technique d’électrophorèse en champ pulsé (EGCP) pour identifier les souches probiotiques autorisées de lactobacilles, pédiocoques, entérocoques et bacilles. La méthode peut être appliquée aux colonies purifiées obtenues à partir de prémélanges et aliments cultivés, pour vérifier la présence de souches utilisées comme additifs alimentaires dans des concentrations déclarées, même contre l’éventuel microbiote annexe résultant de matrices non stériles.
Krma - Metode vzorčenja in analize - Tipizacija laktobacilov, pediokokov, enterokokov in bacilov z metodo PFGE v krmi
Ta dokument določa metodologijo PFGE za identifikacijo odobrenih probiotičnih sevov laktobacilov, pediokokov, enterokokov in bacilov. To metodo je mogoče uporabljati za prečiščene kolonije, pridobljene iz kultiviranih premiksov in krme, da se potrdi prisotnost sevov, ki se uporabljajo kot krmni dodatki v prijavljenih koncentracijah, tudi glede na morebitno mikrobno ozadje, ki je posledica nesterilnih matric.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-september-2023
Krma - Metode vzorčenja in analize - Tipizacija laktobacilov, pediokokov,
enterokokov in bacilov z metodo PFGE v krmi
Animal feeding stuffs - Methods of sampling and analysis - PFGE typing of Lactobacilli,
Pediococci, Enterococci and Bacilli in animal feeds
Futtermittel: Probenahme- und Untersuchungsverfahren - PFGE Typisierung von
Laktobazillen, Pediokokken, Enterokokken und Bazillen in Futtermitteln
Aliments des animaux : Méthodes d’analyse - Typage EGCP des lactobacilles,
pédiocoques, entérocoques et bacilles dans les aliments des animaux
Ta slovenski standard je istoveten z: CEN/TS 17697:2023
ICS:
65.120 Krmila Animal feeding stuffs
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
CEN/TS 17697
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
June 2023
TECHNISCHE SPEZIFIKATION
ICS 65.120
English Version
Animal feeding stuffs - Methods of sampling and analysis -
PFGE typing of Lactobacilli, Pediococci, Enterococci and
Bacilli in animal feeds
Aliments des animaux : Méthodes d'analyse - Typage Futtermittel: Probenahme- und
EGCP des lactobacilles, pédiocoques, entérocoques et Untersuchungsverfahren - PFGE Typisierung von
bacilles dans les aliments des animaux Laktobazillen, Pediokokken, Enterokokken und
Bazillen in Futtermitteln
This Technical Specification (CEN/TS) was approved by CEN on 10 March 2023 for provisional application.
The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to
submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard.
CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS
available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in
parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 17697:2023 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 6
5 Reagents . 6
6 Media . 8
7 Apparatus . 9
8 Procedure . 10
9 Test report . 13
Annex A (informative) Summary of the ring trial . 14
A.1 General . 14
A.2 Ring trial . 14
Bibliography. 16
European foreword
This document (CEN/TS 17697:2023) has been prepared by Technical Committee CEN/TC 327 “Animal
feeding stuffs: Methods of sampling and analysis”, the secretariat of which is held by NEN.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document has been prepared under a Standardization Request given to CEN by the European
Commission and the European Free Trade Association.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the
United Kingdom.
Introduction
DNA fingerprinting by pulsed field gel electrophoresis (PFGE) allows the comparison of large restriction
fragments greater than 50 kbp. This technique combined with restriction of the DNA molecule by rare
cutting endonucleases (which recognize 6 or 8 base pair sequences) has been successfully applied to
strain typing of various lactic acid bacteria including Lactobacilli and Pediococci.
This protocol describes the preparation of genomic DNA for Pulsed Field Gel Electrophoresis and further
details of the PFGE typing procedure.
1 Scope
This document specifies a Pulsed Field Gel Electrophoresis (PFGE) methodology for the identification of
authorized probiotic strains of Lactobacillus, Pediococcus, Enterococcus and Bacillus. The method can be
applied to purified colonies obtained from cultured premixtures and feeds. The method can be used, even
in the presence of a significant microbiological background, to verify the presence of microorganisms
(strains and declared concentrations) used as feed additives in animal feeding stuffs.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp/
— IEC Electropedia: available at https://www.electropedia.org/
3.1
Bacilli
any of various rodlike spore-producing bacteria constituting the family Bacillaceae
3.2
Enterococci
gram-positive, catalase negative cocci, which usually occurs in pairs or short chains
Note 1 to entry: This description is based on their characteristics as used for this document.
Note 2 to entry: Enterococci classified as aerotolerant anaerobes with the ability to reduce 2,3,5-triphenyl
tetrazolium chloride to formazan and capable of hydrolyzing aesculin at 44 °C ± 0,5 °C. They form colonies fitting
the description of this species on the specified culture media after incubation at a temperature of 37 °C under
aerobic conditions for 24 h resp. 48 h.
Note 3 to entry: See EN 15788:2021, paragraph 9.6 for the characteristics of the colonies after incubation of 24 h
to 48 h at a temperature of 37 °C under aerobic conditions with an appropriate media.
[SOURCE: EN 15788:2021, 3.1, modified – Added Note 3 to entry.]
3.3
Lactobacilli
gram-positive, catalase negative, rod-shaped bacteria in chains
Note 1 to entry: This description is based on their characteristics as used for this document.
Note 2 to entry: Lactobacilli form colonies fitting the description of these species on the specified selective media
after incubation of 48 h to 72 h at a temperature of 37 °C under anaerobic conditions.
Note 3 to entry: See EN 15787:2021, paragraph 9.7 for the characteristics of the colonies after incubation of 48 h
to 72 h at a temperature of 37 °C under anaerobic conditions with an appropriate media.
[SOURCE: EN 15787:2021, 3.1, modified – Added Note 3 to entry.]
3.4
Pediococci
gram-positive catalase negative immobile cocci that grow under aerobic as well as under anaerobic
conditions
Note 1 to entry: This description is based on their characteristics as used for this document.
Note 2 to entry: Pediococci usually occur in pairs or tetrads, rarely in chains or singly, and divide along two planes
of symmetry. They form colonies fitting the description of these species on the specified selective media after
incubation of 48 h to 72 h at a temperature of 37 °C under aerobic or anaerobic conditions.
Note 3 to entry: See EN 15786:2021, paragraph 9.7 for the characteristics of the colonies after incubation of 48 h
to 72 h at a temperature of 37 °C under anaerobic and aerobic conditions with an appropriate media.
[SOURCE: EN 15786:2021, 3.1, modified – Added Note 3 to entry.]
4 Principle
Single colonies are selected after culture on the appropriate media (see the enumeration method
described in CEN methods 7, 8, 9, 10). These colonies are cultured overnight in 10 ml of liquid cultures at
37 °C. After incubation, the liquid culture is centrifuged. The bacterial pellet is re-suspended (TE), the
cells are lysed and the chromosomal DNA are placed in agar plugs.
The chromosomal DNA is the digested to specific fragments by the action of restriction enzymes and the
obtained fragments are separated and visualized on gel after PFGE.
Genomic DNA was prepared for PFGE by modifying and adapting well known procedures described
previously for Lactobacilli and Pediococci (1, 2, 3, 5) and for Enterococci and Bacilli (4,6).
The method has been developed and assessed in the course of the EU research Contract N°
SMT4-CT98-2235 (“Methods for the Official Control of Probiotics Used as Feed Additives”).
The method has been further optimized and fully validated during the collaborative study. A summary of
the collaborative study can be found in Annex A "Results of the collaborative ring trial".
5 Reagents
5.1 Tris (hydroxymethyl)aminomethane hydrochloride (Tris-HCl)
5.2 Ethylenediaminetetraacetic acid (EDTA)
5.3 Tris-HCl TE 10 mmol Tris-Hcl, 1 mmol EDTA, adjust to pH 8,0 and autoclave
5.4 Lysozyme (≥20 000 U/mg)
5.4.1 Lysozyme stock solution
3 000000 U lysozyme (approximately 150 mg/ml – 200 mg/ml with most commercial lysozyme
preparations) per ml TE-buffer (5.3). Distribute in aliquots (100 µl to 200 µl), store at - 20 °C, thaw at
ambient temperature, use immediately after thawing.
5.5 Mutanolysin (≥4 000 U/mg)
5.5.1 Mutanolysin stock solution
2 500 U mutanolysin per ml TE-buffer (5,3). Distribute in aliquotes (100 µl to 200 µl), store at - 20 °C,
thaw at ambient temperature, use immediately after thawing.
5.6 Proteinase K (≥2,5 U/mg)
5.6.1 Proteinase K stock solution
50 U Proteinase K per mL TE-buffer (5.3) (50 U/ml). Distribute in aliquotes (100 µl to 200 µl), store at
- 20 °C, thaw at ambient temperature, use immediately after thawing.
5.7 N-Lauroylsarcosine sodium salt (Sarcosyl)
5.7.1 Sarcosyl solution: Sarcosyl 1g/100 ml in 100 mmol Tris-HCl, 100 mmol EDTA, pH8.
5.8 Proteinase K- inhibitor solution
Phenylmethylsulfonyl fluoride (PMSF) 17,5 mg/ml in isopropanol.
5.9 10 x TBE
1 M Tris-HCl, 0,9 M Boric acid, 0,01 mol EDTA, pH 8,4.
5.10 Low melting point agarose
2 % in 0,5 × TBE buffer (mix and melt in a microwave oven, keep at 45 °C to 47 °C before use).
5.11 Pulsed field certified agarose
1 % in 0,5 × TBE buffer (mix and melt in a microwave oven, keep at 45 °C to 47 °C before use).
5.12 Restriction enzymes (and respective restriction buffers)
5.12.1 SfiI (Lactobacilli)
5.12.2 SmaI (Lactobacilli, Pediococci and Enterococci)
5.12.3 SpeI (Bacilli)
5.13 Molecular weight markers (e.g. 10 kpb to 250 kbp)
5.14 Ethidium bromide
6 Media
6.1 MRS (Lactobacilli, Pedicocci)
Dipotassium hydrogen phosphate 2 g/l
Magnesium sulphate heptahydrate 0,2 g/l
Manganous sulphate tetrahydrate 0,05 g/l
Meat extract 8 g/l
Peptone 10 g/l
Sodium acetate trihydrate 5 g/l
Yeast extract, 4 g/l
Distilled water up to 1 l
pH 6,2 ± 0,2
Dissolve in boiling water, distribute into suitable portions and sterilize by autoclaving at 121 °C for
15 min. Store refrigerated (4 °C to 6 °C) for up to six months
6.2 Tryptic soy broth (Enterococci, Bacilli)
Casein peptone 17 g/l
Dipossaium hydrogen phosphate 2,5 g/l
Glu
...
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