FprCEN/TS 17697

Animal feeding stuffs: Methods of analysis - PFGE typing of Lactobacilli, Pediococci, Enterococci and Bacilli in animal feeds

This document defines a Pulsed Field Gel Electrophoresis (PFGE) methodology for the identification of authorized probiotic Lactobacillus, Pediococcus, Enterococcus and Bacillus strains. The method can be applied to purified colonies obtained from cultured premixtures and feeds, in order to verify the presence of strains used as feed additives in declared concentrations, even against eventual microbial background resulting from nonsterile matrices.

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, um das Vorhandensein von Stämmen, die als Futtermittelzusatzstoffe in deklarierten Konzentrationen verwendet werden, sogar vor einem möglichen mikrobiellen Hintergrund, der aus nicht sterilen Matrices resultiert.

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 analize - Tipizacija laktobacilov, pediokokov, enterokokov in bacilov z metodo PFGE v krmi

General Information

• Status: Not Published
• Publication Date: 09-Aug-2023
• ICS: 65.120 - Animal feeding stuffs
• Technical Committee: CEN/TC 327 - Animal feeding stuffs - Methods of sampling and analysis
• Drafting Committee: CEN/TC 327 - Animal feeding stuffs - Methods of sampling and analysis
• Current Stage: 5060 - Closure of Vote - Formal Approval
• Start Date: 09-Feb-2023
• Completion Date: 09-Feb-2023
• Mandate: M/521 - Mandate for standardisation addressed to CEN for methods of analysis in the field of animal nutrition - part 1
• Ref Project: kSIST FprEN 17697:2022 - Animal feeding stuffs: Methods of analysis - PFGE typing of Lactobacilli, Pediococci, Enterococci and Bacilli in animal feeds

Standards Content (Sample)

SLOVENSKI STANDARD
oSIST prEN 17697:2021
01-oktober-2021

Krma: Metode analize - Tipizacija laktobacilov, pediokokov, enterokokov in bacilov

z metodo PFGE v krmi

Animal feeding stuffs: Methods of analysis - PFGE typing of Lactobacilli, Pediococci,

Enterococci and Bacilli in animal feeds
Futtermittel: Probenahme- und Untersuchungsverfahren - PFGE Typisierung von
Laktobazillen, Pediokokken, Enterokokken und Bazillen
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: prEN 17697
ICS:
65.120 Krmila Animal feeding stuffs
oSIST prEN 17697:2021 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN 17697:2021
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oSIST prEN 17697:2021
DRAFT
EUROPEAN STANDARD
prEN 17697
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2021
ICS 65.120
English Version
Animal feeding stuffs: Methods of 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

This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee

CEN/TC 327.

If this draft becomes a European Standard, 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.

This draft European Standard was established by CEN 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 language and notified to the CEN-CENELEC

Management Centre has the same status as the official versions.

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, Turkey and

United Kingdom.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are

aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without

notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17697:2021 E

worldwide for CEN national Members.
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Contents Page

European foreword ....................................................................................................................................................... 4

Introduction .................................................................................................................................................................... 5

1 Scope .................................................................................................................................................................... 6

2 Normative references .................................................................................................................................... 6

3 Terms and definitions ................................................................................................................................... 6

4 Principle ............................................................................................................................................................. 7

5 Reagents ............................................................................................................................................................. 7

5.1 Tris (hydroxymet yl) aminomethane hydrochloride (Tris-HCl) .................................................... 7

5.2 Ethylenediaminetetraacetic acid (EDTA) ............................................................................................... 7

5.3 TE-buffer: TE 10 mM Tris-Hcl, 1 mM EDTA, adjust to pH 8,0 and autoclave .............................. 7

5.4 Lysozyme (≥20 000 U/mg)........................................................................................................................... 7

5.4.1 Lysozyme stock solution ............................................................................................................................... 7

5.5 Mutanolysin (≥4000 U/mg) ......................................................................................................................... 8

5.5.1 Mutanolysin stock solution ......................................................................................................................... 8

5.6 Proteinase K (≥2.5 U/mg) ............................................................................................................................ 8

5.6.1 Proteinase K stock solution ......................................................................................................................... 8

5.7 N-Lauroylsarcosine sodium salt (Sarcosyl) ........................................................................................... 8

5.7.1 Sarcosyl solution: Sarcosyl 1 % (w/v) in 100mM Tris-HCl, 100 mM EDTA, pH8 ..................... 8

5.8 Proteinase K-inhibitor solution ................................................................................................................. 8

5.9 10 x TBE .............................................................................................................................................................. 8

5.10 Low melting point agarose .......................................................................................................................... 8

5.11 Pulsed field certified agarose ..................................................................................................................... 8

5.12 Restriction enzymes (and respective restriction buffers) ............................................................... 8

5.12.1 Sfil (Lactobacilli) ............................................................................................................................................. 8

5.12.2 Smal (Lactobacilli, Pediococci and Enterococci).................................................................................. 8

5.12.3 Spel (Bacilli) ...................................................................................................................................................... 8

5.13 Molecular weight markers (e.g. 10 – 250 kbp) ..................................................................................... 8

5.14 Ethidium bromide ........................................................................................................................................... 8

6 Media ................................................................................................................................................................... 8

6.1 MRS (Lactobacilli, Pediococci) .................................................................................................................... 8

6.2 Tryptic soy broth (Enterococci, Bacilli) .................................................................................................. 9

7 Apparatus ........................................................................................................................................................... 9

7.1 Gel electrophoreses ........................................................................................................................................ 9

7.1.1 Pulse Field Gel Electrophoresis system with Contour-Clamped Homogeneous

Electric Fields (CHEF) .................................................................................................................................... 9

7.1.2 Microwave oven ............................................................................................................................................... 9

7.1.3 Conical flask ...................................................................................................................................................... 9

7.1.4 Water bath ......................................................................................................................................................... 9

7.1.5 Plug moulds (usually provided together with the PFGE apparatus) ............................................ 9

7.1.6 Conical centrifuge tubes (50 ml) ............................................................................................................... 9

7.1.7 Screened Plug Caps ......................................................................................................................................... 9

7.1.8 Image analysis system or Polaroid camera and transilluminator ................................................ 9

7.1.9 Thermoblock .................................................................................................................................................... 9

7.2 Turbidmeter................................................................................................................................................... 10

7.3 Refrigerator .................................................................................................................................................... 10

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7.4 Deep freezer ................................................................................................................................................... 10

7.5 Table top centrifuge ..................................................................................................................................... 10

7.6 Centrifuge tubes ............................................................................................................................................ 10

7.6.1 Conical tubes 50 ml ...................................................................................................................................... 10

7.6.2 Conical tubes 15 ml ...................................................................................................................................... 10

7.6.3 Eppendorf tubes 1,5 ml............................................................................................................................... 10

7.7 Balance ............................................................................................................................................................. 10

7.8 Automatic pipettes and pipette tips (range 2 – 1000 µL) .............................................................. 10

7.9 Transfer loops ................................................................................................................................................ 10

8 Procedure ........................................................................................................................................................ 10

8.1 Harvesting the cells ...................................................................................................................................... 10

8.2 Preparation of plugs .................................................................................................................................... 10

8.3 Lysis of the cells ............................................................................................................................................. 11

8.4 Restriction of chromosomal DNA............................................................................................................ 11

8.5 Preparation of gels and PFGE ................................................................................................................... 11

8.6 Visualization and interpretation ............................................................................................................ 11

9 Test report ...................................................................................................................................................... 13

Anhang A (informative) Summary of the Validation study ......................................................................... 14

A.1 General ............................................................................................................................................................. 14

A.2 Ring trial .......................................................................................................................................................... 14

Bibliography ................................................................................................................................................................. 16

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European foreword

This document (prEN 17697:2021) has been prepared by Technical Committee CEN/TC 327 “Animal

feeding stuffs: Methods of analysis”, the secretariat of which is held by NEN.
This document is currently submitted to the CEN Enquiry.

This document has been prepared under a standardization request given to CEN by the European

Commission.
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Introduction

DNA fingerprinting by pulsed field gel electrophoresis (PFGE) allows the comparison of large restriction

fragments greater than 50 Kb. 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.

The method described in this document defines the preparation of genomic DNA for Pulsed Field Gel

Electrophoresis and further details of the PFGE typing procedure.
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1 Scope

This document defines a Pulsed Field Gel Electrophoresis (PFGE) methodology for the identification of

authorized probiotic Lactobacillus, Pediococcus, Enterococcus and Bacillus strains. The method can be

applied to purified colonies obtained from cultured premixtures and feeds, in order to verify the presence

of strains used as feed additives in declared concentrations, even against eventual microbial background

resulting from nonsterile matrices.
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 terminological 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
[SOURCE: EN 15788:2020, 3.1]
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 (see 9.7)

[SOURCE: EN 15787:2020, 3.1]
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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 (see 9.7).

[SOURCE: EN 15786:2020, 3.1]
4 Principle

Single colonies can be selected from the appropriate media used, obtained by using the best available

specific enumeration method, as for example described in CEN methods for the enumeration of probiotics

Lactobacilli, Pediococci and Enterococci [7, 8, 9,10]. These are cultured overnight in 10 ml liquid cultures

at 37 °C. Cells are harvested by centrifugation and included in agar plugs, where they are then submitted

to lysis, restriction of chromosomal DNA, and PFGE to separate fragments of DNA so obtained.

Genomic DNA from pediococci or lactobacilli was prepared for PFGE by modifying and adapting weIl

known procedures described previously (Tynkkynen et al. 1999; Luchansky et al., 1992, Smith and

Cantor, 1987). The methods described by Turabelidze et al. (2002) and Zhang et al. (2016) were adapted

for enterococci and bacilli, respectively.

The method has been developed and assessed in the course of the EU research Contract N° SMT4-CT98-

2235 (“Meth
...