Animal feeding stuffs: Methods of sampling and analysis - Detection and enumeration of Pediococcus spp. used as feed additive

This international standard defines general rules for the enumeration of probiotic pediococci in feed samples (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 40 % crude ash (Council Directive 79/373/EEC).
There are different categories of feed samples:
a)   Additives containing about 10+10 (colony forming units) CFU/g
b)   Premixtures containing  about 10+8 CFU/g
c)   Feeds, meal or pellets, which contain about 10+6 CFU/g and include complete feeding stuffs, and milk replacers.

Futtermittel: Probenahme- und Untersuchungs-verfahren - Nachweis und Zählung von Pediococcus spp. als Futtermittelzusatzstoff

Dieses Dokument legt allgemeine Regeln für die Zählung von Pediokokken in Futtermitteln (Zusatzstoffe, Vormischungen und Mischfuttermittel mit Ausnahme von mineralischen Futtermitteln) fest, die Pediokokken als einzelnen mikrobiellen Bestandteil oder in einem Gemisch mit anderen Mikroorganismen enthalten. Die Anwendung des Verfahrens auf Mischfuttermittel mit kritischen Kupfermengen erfordert ein besonderes Verfahren (siehe Anhang A). Dieses Dokument ist nicht anwendbar auf mineralische Futtermittel, die als Ergänzungsfuttermittel definiert sind, hauptsächlich aus Mineralstoffen zusammengesetzt sind und mindestens 40 % Rohasche enthalten (Verordnung R767/2009) [3].
Es gibt unterschiedliche Kategorien von Futtermittelproben:
a) Zusatzstoffe, die etwa 1010 koloniebildende Einheiten (KbE/g) enthalten;
b) Vormischungen, die etwa 1011 KbE/kg enthalten;
c) Mischfuttermittel, Mehl oder Pellets, die etwa 109 KbE/kg enthalten.
Die Nachweisgrenze entspricht der in EN ISO 7218 festgelegten.

Aliments des animaux: Méthodes d’échantillonnage et d’analyse - Détection et dénombrement des souches de Pediococcus spp. utilisées comme additifs pour l’alimentation animale

Le présent document définit des règles générales pour le dénombrement des pédiocoques présents dans les aliments pour animaux (additifs, prémélanges et aliments composés, à l’exception des aliments minéraux) qui contiennent des pédiocoques comme seul micro-organisme constitutif ou en mélange avec d’autres micro-organismes. L’application de la méthode aux aliments composés pour animaux présentant une teneur critique en cuivre nécessite de mettre en oeuvre un mode opératoire spécial (voir Annexe A). Le présent document ne s’applique pas aux aliments minéraux, qui se définissent comme des aliments complémentaires constitués principalement de minéraux et contenant au moins 40 % de cendre brute (Règlement R767/2009) [3].
Il existe différentes catégories d’échantillons d’aliments pour animaux :
a) les additifs contenant environ 1010 UFC/g (UFC : unités formant colonie) ;
b) les prémélanges contenant environ 1011 UFC/kg ;
c) les aliments composés, farines ou granulés qui contiennent environ 109 UFC/kg.
La limite de détection est définie dans l’EN ISO 7218.

Krma: metode vzorčenja in analize - Določanje in štetje prisotnih Pediococcus spp., uporabljenih kot krmni dodatek

Ta mednarodni standard določa splošna pravila za štetje probiotičnih pediokokov v vzorcih krme (dodatki, premiksi in krma), ki vsebujejo pediokoke kot eno bakterijsko komponento ali v kombinaciji z drugimi mikroorganizmi. Ta standard se ne uporablja za mineralno krmo, ki je opredeljena kot dopolnilna krma, sestavljena predvsem iz mineralov, in vsebuje najmanj 40 % surovega pepela (Direktiva Sveta 79/373/EGS).
Obstajajo različne kategorije vzorcev krme:
a)   dodatki, ki vsebujejo približno 10+10 enot, ki tvorijo kolonije (CFU)/g;
b)   premiksi, ki vsebujejo približno 10+8 CFU/g;
c)   krma, moka ali peleti, ki vsebujejo približno 10+6 CFU/g ter vključujejo popolno krmo in mlečne nadomestke.

General Information

Status
Published
Public Enquiry End Date
19-Mar-2020
Publication Date
13-Jan-2022
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
28-Dec-2021
Due Date
04-Mar-2022
Completion Date
14-Jan-2022

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Standards Content (sample)

SLOVENSKI STANDARD
SIST EN 15786:2022
01-februar-2022
Nadomešča:
SIST EN 15786:2009
Krma: metode vzorčenja in analize - Določanje in štetje prisotnih Pediococcus
spp., uporabljenih kot krmni dodatek

Animal feeding stuffs: Methods of sampling and analysis - Detection and enumeration of

Pediococcus spp. used as feed additive
Futtermittel: Probenahme- und Untersuchungs-verfahren - Nachweis und Zählung von
Pediococcus spp. als Futtermittelzusatzstoff
Aliments des animaux: Méthodes d’échantillonnage et d’analyse - Détection et
dénombrement des souches de Pediococcus spp. utilisées comme additifs pour
l’alimentation animale
Ta slovenski standard je istoveten z: EN 15786:2021
ICS:
65.120 Krmila Animal feeding stuffs
SIST EN 15786:2022 en,fr,de

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

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SIST EN 15786:2022
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SIST EN 15786:2022
EN 15786
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2021
EUROPÄISCHE NORM
ICS 65.120 Supersedes EN 15786:2009
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Detection and enumeration of Pediococcus spp. used as
feed additive

Aliments des animaux: Méthodes d'échantillonnage et Futtermittel: Probenahme- und Untersuchungs-

d'analyse - Détection et dénombrement des souches de verfahren - Nachweis und Zählung von Pediococcus

Pediococcus spp. utilisées comme additifs pour spp. als Futtermittelzusatzstoff
l'alimentation animale
This European Standard was approved by CEN on 2 August 2021.

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 such national standards may be obtained on application to the CEN-CENELEC 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 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.
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. EN 15786:2021 E

worldwide for CEN national Members.
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SIST EN 15786:2022
EN 15786:2021 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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

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

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

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

4 Principle ............................................................................................................................................................. 6

5 Diluents and culture media ......................................................................................................................... 7

5.1 Diluents ............................................................................................................................................................... 7

5.2 Culture media ................................................................................................................................................... 8

6 Apparatus ........................................................................................................................................................ 10

7 Sampling .......................................................................................................................................................... 11

8 Preparation of test sample ....................................................................................................................... 11

9 Procedure........................................................................................................................................................ 12

9.1 Samples with a single or multi-component added microflora .................................................... 12

9.2 Preparation of poured agar plates for spread plate method ....................................................... 12

9.3 Preparation of MRS agar for pour plate method .............................................................................. 12

9.4 Preparation of the initial suspension and decimal dilutions ....................................................... 12

9.5 Inoculation and incubation of plates .................................................................................................... 13

9.6 Enumeration of colonies ............................................................................................................................ 14

9.7 Confirmation .................................................................................................................................................. 14

10 Expression of results ................................................................................................................................... 15

11 Precision .......................................................................................................................................................... 16

11.1 General ............................................................................................................................................................. 16

11.2 Interlaboratory study ................................................................................................................................. 16

11.3 Repeatability .................................................................................................................................................. 16

11.4 Reproducibility ............................................................................................................................................. 16

12 Test report ...................................................................................................................................................... 16

Annex A (informative) Notes on the procedure .............................................................................................. 17

A.1 General ............................................................................................................................................................. 17

A.2 Critical copper concentration .................................................................................................................. 17

Annex B (informative) Results of the interlaboratory study ....................................................................... 18

Bibliography ................................................................................................................................................................. 20

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SIST EN 15786:2022
EN 15786:2021 (E)
European foreword

This document (EN 15786:2021) 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.

This European Standard shall be given the status of a national standard, either by publication of an

identical text or by endorsement, at the latest by May 2022, and conflicting national standards shall be

withdrawn at the latest by May 2022.

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 supersedes EN 15786:2009.
The main changes compared to the previous edition are as follows:
— Amendment of the title;

— Extension of the scope of application to all Pediococci spp. used as feed additive;

— Updating of normative cross references;
— Supplement of phosphate buffered saline with Tween 80;

— Addition of the option to use Tween 80 supplemented phosphate buffered saline for the

preparation of the initial suspension as well as diluent for serial dilutions;

— Adjustment of the composition of the MRS agar to commercially available formulations;

— Replacement of the required laboratory mixer with a rotation speed of 18 000 min to

22 000 min by homogenization devices, for example according to EN ISO 7218, with a maximal

requested rotation speed of 10 000 min ;

— Unification of the homogenization time for the preparation of initial suspensions to five minutes for

all feed matrices;
— Addition of the option to use a spiral plater for plating;
— Preparation of initial suspensions generally conducted with tempered tPBS;
— Addition of the pour plate method as an alternative cultivation technique;

— Addition of a procedure for the investigation of feeding stuffs containing high amounts of copper in

the informative Annex A;

— Adjustment of the range of accepted colony numbers for counting from '≥ 30 to ≤ 350' to '≥ 10

to ≤ 200' colonies per plate.

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 organisations of the

following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia,

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SIST EN 15786:2022
EN 15786:2021 (E)

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 the United

Kingdom.
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SIST EN 15786:2022
EN 15786:2021 (E)
Introduction

This methodology has been developed to enumerate pediococci used as feed additives to enable the

European Commission to control proper labelling of animal feeding products. It was compiled first during

the EU project SMT4-CT98-2235 “Methods for the official control of probiotics used as feed additives” [1].

The specified methodology was validated in an interlaboratory study [2]. The method is validated in this

project for one strain of Pediococcus acidilactici. It can be assumed that the method is suitable also for

other Pediococcus strains used as feed additives.

This method is not selective for pediococci used as feed additives, but can be applied to enumerate

Pediococci spp. in additives, premixtures and compound feeds assuming that the added pediococci are

present in far higher numbers than any other pediococci.

This method is not applicable for the detection of ubiquitous contaminants of Pediococcus spp. and any

other lactic acid bacteria in food and animal feeding stuffs.
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EN 15786:2021 (E)
1 Scope

This document specifies general rules for the enumeration of pediococci in feeding stuffs (additives,

premixtures and compound feeds excluding mineral feeds) that contain pediococci as a single

microorganism component or in a mixture with other microorganisms. Applying the method to

premixtures and compound feeds with critical amounts of copper demands a special procedure (see A.2).

The document is not applicable to mineral feeds which are defined as complementary feeding stuffs

composed mainly of minerals and containing at least 40 % crude ash (Regulation (EC) 767/2009) [3].

There are different categories of feed samples:
a) Additives containing about 10 colony forming units (CFU)/g;
b) Premixtures containing about 10 CFU/kg;
c) Compound feeds, meal or pellets which contain about 10 CFU/kg.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements 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 ISO 6498, Animal feeding stuffs - Guidelines for sample preparation (ISO 6498)

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
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).

4 Principle

a) Preparation of sterile and dry poured agar plates or preparation of sterile liquid culture medium

tempered at 44 °C to 47 °C;
b) Drawing a representative test sample under aseptic conditions;

c) Preparation of the initial suspension with a tempered diluent to obtain a homogeneous distribution

of Pediococcus cells from the test portion;
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EN 15786:2021 (E)

d) Preparation of further decimal dilutions of the initial suspension in order to reduce the number of

microorganisms per unit volume to allow the counting of colonies on the selective enumeration

media;

e) Inoculation of the prepared poured plates with an aliquot of the optimum dilutions and dispersion

of the inoculum by using a sterile spreader or inoculation of blank plates with an aliquot of the

optimum dilutions and pouring of the molten agar medium into each plate, mixing and solidification;

f) Incubation of inoculated plates for 48 h to 72 h at 37 °C ± 1 °C under aerobic or anaerobic conditions;

g) Counting of typical colonies, considering the specific properties of Pediococcus spp.;

h) Morphological verification of isolates by use of microscopy;

i) Calculation of the colony forming units of Pediococcus spp. per gram or kilogram of feed sample.

5 Diluents and culture media
5.1 Diluents
5.1.1 Diluent for initial suspension

The diluent is used for the preparation of the initial suspension and may also be used for the preparation

of further decimal dilutions. The composition of the diluent is given in Table 1.

® 1
Table 1 — Phosphate buffered saline with Polysorbate 80 (Tween 80) (tPBS)
Sodium chloride NaCl 8,00 g
Potassium chloride KCl 0,20 g
Na HPO
Disodium hydrogen phosphate anhydrous 1,15 g
2 4
KH PO
Potassium dihydrogen phosphate anhydrous 0,20 g
2 4
® 1
C H O
1 ml
Polyoxyethylen (20) sorbitan monooleate (Tween 80)
64 124 26
Water, distilled or deionized H O 1 000 ml

Dissolve the components (see Table 1) in water. If necessary, adjust to a final pH of 7,3 ± 0,2 at 25 °C after

sterilization. Fill the solution into appropriate containers (e.g. bottles, flasks, or test tubes) and sterilize

at 121 °C ± 3 °C for 15 min. To avoid loss during autoclaving, screw cap bottles are recommended.

For immediate use, hold at 40 °C ± 1 °C in a water bath or incubator.

NOTE When using commercially available PBS buffer tablets, please take note that variations in composition

and pH can occur between products from different manufacturers and could therefore give results different from

the ones obtained with the buffer as specified in this document.
5.1.2 Diluents for serial dilutions

For serial dilutions, the diluent for initial suspension (5.1.1) or alternatively a peptone salt solution (PSS)

according to EN ISO 6887-1 [4] can be used. The composition of PSS is given in Table 2.

1 ®

Tween 80 is an example of a suitable product available commercially. This information is given for the

convenience of users of this document and does not constitute an endorsement by CEN of this product.

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EN 15786:2021 (E)
Table 2 — Peptone salt solution (PSS) according to EN ISO 6887-1
Enzymatic digest casein 1,0 g
Sodium chloride NaCl 8,5 g
Water, distilled or deionized H O 1 000 ml

Dissolve the components (see Table 2) in water in flasks or bottles. Adjust the pH if necessary so that,

after sterilization, it is 7,0 ± 0,2 at 25 °C. For decimal dilutions, prepare test tubes containing

9,0 ml ± 0,1 ml after sterilization or use screw cap bottles to avoid volume loss during autoclaving.

Sterilize at 121 °C ± 3 °C for 15 min. Bring the diluent to room temperature before use.

NOTE Commercially available, ready-to-use PSS tubes of 9 ml are suitable.
5.2 Culture media
5.2.1 General
Four different culture media are proposed:
a) De Man, Rogosa and Sharpe (MRS) agar;
b) MRS agar supplemented with triphenyl tetrazolium chloride (TTC) (MRS+TTC);
c) Acidified MRS agar (AMRSA);

d) Selective media: MRS medium supplemented with cysteine hydrochloride, vancomycin and

novobiocin.

For routine enumeration of pediococci, the use of MRS agar will be sufficient assuming that the added

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 pediococci and lactobacilli will tolerate a lower pH than enterococci

(pH 5,0 to pH 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 TTC allows differentiation of colonies by different colouration

after anaerobic incubation but can exert a negative influence on the colony amount. The MRS medium

supplemented with two antibiotics is selective for pediococci and should be used when the added

lactobacilli colony count exceeds the pediococci colony count by a factor of 50 or more.

NOTE An antifungal agent like nystatin (50 IU/ml) can be added to MRS agar, AMRSA or MRS+TTC to inhibit

moulds and yeasts.
5.2.2 Composition
5.2.2.1 MRS agar

The composition of the MRS agar is given in Table 3. The resulting pH value at 25 °C is 6,2 ± 0,2.

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EN 15786:2021 (E)
Table 3 — Composition of the MRS agar
Glucose 20 g
Peptone 10 g
Meat extract 8 g
Yeast extract 4 g
Sodium acetate trihydrate 5 g
Dipotassium hydrogen phosphate 2 g
Triammonium citrate 2 g
1 ml
Polyoxyethylen (20) sorbitan monooleate (Tween 80)
Magnesium sulphate heptahydrate 0,2 g
Manganese sulphate tetrahydrate 0,05 g
Agar
10 g to 15 g
Water, distilled or deionized 1 000 ml
Depending on the gel strength of the agar.

NOTE When using commercially available, ready-to-use media, please take note that variations in composition

and pH can occur between products from different manufacturers and could therefore give results different from

the ones obtain with the medium as specified in this document.
5.2.2.2 MRS agar supplemented with TTC (0,01 %)

Sterilize MRS agar (5.2.2.1) by autoclaving at 121 °C ± 3 °C for 15 min. Supplement with 1 ml of a filter

sterilized 1 g/100 ml water solution of TTC per 100 ml MRS agar.
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 with

10 µg/ml vancomycin and 0,1 µg/ml novobiocin.
5.2.3 Preparation
5.2.3.1 MRS agar

Dispense the medium (Table 3) into suitable containers, e.g. bottles or flasks with non-toxic metal screw-

caps. Dissolve all components specified in Table 3 in water by boiling. If necessary, adjust to a final pH of

6,2 ± 0,2 after sterilization. Sterilize at 121 °C ± 3 °C for 15 min. Excessive heating during sterilization

shall be avoided.
5.2.3.2 MRS agar supplemented with TTC

Prepare 1 g TTC in 100 ml water and filter sterilize. Add 1 ml per 100 ml MRS agar medium (5.2.2.1)

which is tempered at 44 °C to 47 °C after autoclaving.

TTC is destroyed by autoclaving. Protect the solution from light, and discard it if a pink tinge develops.

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EN 15786:2021 (E)
5.2.3.3 AMRSA

Prepare the medium as specified in 5.2.3.1. Adjust the pH of MRS agar with HCl to 5,4 ± 0,1 prior to

autoclaving. Sterilize at 121 °C ± 3 °C for 15 min.
5.2.3.4 Selective medium

Prepare MRS medium with 500 mg/l cysteine hydrochloride as specified in 5.2.3.1. Autoclave the medium

for 15 min at 121 °C ± 3 °C and temper it at 44 °C to 47 °C in a water bath or incubator.

Prepare 100 mg vancomycin in 100 ml water and sterilize by sterile filtration. Add 1 ml per 100 ml MRS

medium after cooling. This procedure results in a final vancomycin concentration of 10 mg/l.

Prepare a solution of 10 µg/ml of novobiocin through a series of ten-fold dilutions from an initial 1 mg/ml

stock and sterilize by sterile filtration. Add 1 ml novobiocin per 100 ml MRS medium after cooling. This

procedure results in a final novobiocin concentration of 100 µg/l.

Care is needed in preparing the novobiocin stock as levels above 100 µg/l can be inhibitory to pediococci.

Add the selective agents from sterile-filtered stocks to the medium immediately before pouring.

6 Apparatus
Usual microbiological laboratory equipment and, in particular, the following:

6.1 Equipment for dry sterilization (oven) and wet sterilization (autoclave), for example

according to EN ISO 7218 [5].

6.2 Incubator, capable of maintaining a temperature of 37 °C ± 1 °C. Optionally also capable of

maintaining a temperature of 40 °C ± 1 °C and/or between 44 °C and 47 °C.

6.3 Water bath, capable of maintaining a temperature of 40 °C ± 1 °C and between 44 °C and 47 °C.

6.4 Blending equipment.
The following apparatus may be used according to EN ISO 7218 [5]:
-1 -1

— a rotary homogenizer (blender) with a notional variable speed of 3 000 min to 10 000 min , as

well as aseptic glass or metals bowls equipped with covers; or

— a peristaltic homogenizer with sterile bags (paddle homogenizer), possibly with the option to adjust

blending speed and time; or
— a vibrational mixer with sterile bags; or

— any other homogenizing system with equivalent efficiency (e.g. a hand blender with aseptic beaker).

6.5 Mechanical stirrer.

A mechanical stirrer (e.g. Vortex Mixer) facilitates the homogenous mixing of decimal dilutions, as

described in e.g. EN ISO 7218 [5].

6.6 Balances, of the required range and accuracy, for example according to EN ISO 7218 [5], for the

different products to be weighed.
6.7 Flasks or screw-cap bottles, of appropriate capacities.
6.8 Test tubes, of appropriate capacities.
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EN 15786:2021 (E)
6.9 Pipettes or pipettor and sterile tips, to dispense 0,1 ml to 1 ml.

6.10 Sterile pipettes, to dispense 5 ml, for full outlet with wide (approx. 3 mm) tips (e.g. serological

pipette.
NOTE As alternative, 5 ml graduated pipettes without tips can be used.

6.11 Spreading spatula, sterile L- or triangular-shaped spreaders from glass or metal or sterile

disposable plastic spreaders.

NOTE As alternatives, a spiral plater with a sanitized dispensing system or disposable one-way micro syringes

can be used.
6.12 Sterile Petri dishes, with triple vents (plates), 90 mm in diameter.
6.13 Laminar flow cabinet.

6.14 Microscope, capable of phase-contrast microscopy at a magnification of 600× to 1 000×.

6.15 pH meter, having an accuracy of calibration of ± 0,1 pH unit at 20 °C to 25 °C.

6.16 Equipment for anaerobic incubation.

Appropriate anaerobic jars or chambers with a system for the generation of anaerobic conditions.

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 [1] for official control sampling of

animal feeds.

NOTE Sampling can be done according to EN ISO 6497 [6]. Although EN ISO 6497 is not specifically applicable

for microorganisms, due to the lack of other references it seems the most suitable protocol to be taken into account

for microorganisms as feed additives.

Take precautions to avoid potential cross-contamination of samples with microorganisms, particularly

after sampling additives and premixtures supplemented with microorganisms. Where required, clean

and disinfect the sampling equipment between each sample, particularly after sampling additives and

premixtures.
Put the sample in a sterile container.
8 Preparation of test sample

The test sample preparation shall be done in accordance with EN ISO 6498 and the congruent product

standard.
NOTE EN ISO 6498 gives general guidelines on test sample preparation.
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EN 15786:2021 (E)
9 Procedure
9.1 Samples with a single or multi-component added microflora

If pediococci are the only bacterial component in the sample, use MRS agar in a spread plate or pour plate

method. If they are dominant with additional microflora, enumeration can proceed on AMRSA or

MRS+TTC in the spread plate method. If pediococci are not dominant, use the selective antibiotic medium

in the spread plate method.
9.2 Preparation of poured agar plates for spread plate method

The culture media are prepared as specified in 5.2.3 or according to the manufacturer’s directions. Cool

after autoclaving in a water bath to a temperature between 44 °C and 47 °C and add TTC solution (5.2.3.2)

or the vancomycin and novobiocin solutions (5.2.3.4) if necessary. Pour portions of approximately 15 ml

into each plate (6.12) under sterile conditions and spread to give a homogeneous layer.

Vancomycin, novobiocin and TTC are heat sensitive ingredients, therefore do not re-melt solidified agar.

When the medium has solidified, pile quantities of four inverted plates on each other and dry at room

temperature or in an incubator at 37 °C ± 1 °C for approximately 12 h or overnight. Alternatively, spread

the plates out in a laminar flow cabinet and dry the agar surface with the lids partially removed for about

30 min.

Check the dried plates for sterility. If correctly protected against dehydration, dried plates may be stored

in a refrigerator at 5 °C ± 3 °C for up to two weeks. Bring the plates to room temperature before use.

9.3 Preparation of MRS agar for pour plate method

After sterilization, hold the medium at a temperature between 44 °C and 47 °C in a water bath or

incubator until the preparation of the pour plates. The homogeneity of the ingredients shall be ensured.

9.4 Preparation of the initial suspension and decimal dilutions

Table 4 gives recommended sample quantities and corresponding volumes of tempered diluent for the

preparation of initial suspensions for various feeds and treatment of the initial suspension.

Table 4 — Recommended sample quantities and corresponding volumes of tempered diluent for

the preparation of initial suspensions for various feeds and treatment of initial suspension

Critical Diluent for
Dilution
Sample
Treatment of
copper suspension
Nature of factor of
quantity
initial
content (5.1.1)
sample initial
suspension
g or ml
suspension
mg/kg
Additives n.a.
5,0 ± 0,25 (495 ± 9,9) ml 1 : 100
Premixtures > 2 000
Mixer or paddle
blender 5 min
Compound feeds > 200
50,0 ± 2,5 (450 ± 9) ml 1 : 10
Milk replacers n.a.
(90 ± 1,8) ml +
Paste-like and Paddle blender
> 400 5,0 ± 0,25 5 g of 1 : 20
oleaginous feed 5 min
Tween 80 [7]

Weigh the recommended quantity of the sample according to Table 4 into an aseptic blender bowl, beaker

or plastic bag. Add the corresponding amount of tempered tPBS (5.1.1) according to Table 4. If

...

SLOVENSKI STANDARD
oSIST prEN 15786:2020
01-marec-2020

Krma: metode vzorčenja in analize - Izolacija in štetje prisotnih Pediococcus spp

Animal feeding stuffs: Methods of sampling and analysis - Isolation and enumeration of

Pediococcus spp.
Futtermittel: Probenahme‑ und Untersuchungsverfahren - Trennung und Zählung von
Pediococcus spp.
Aliments des animaux - Méthodes d’échantillonnage et d’analyse - Isolement et
dénombrement des souches de Pediococcus spp.
Ta slovenski standard je istoveten z: prEN 15786
ICS:
65.120 Krmila Animal feeding stuffs
oSIST prEN 15786:2020 en,fr,de

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

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oSIST prEN 15786:2020
DRAFT
EUROPEAN STANDARD
prEN 15786
NORME EUROPÉENNE
EUROPÄISCHE NORM
January 2020
ICS 65.120 Will supersede EN 15786:2009
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Isolation and enumeration of Pediococcus spp.

Aliments des animaux - Méthodes d'échantillonnage et Futtermittel: Probenahme- und

d'analyse - Isolement et dénombrement des souches de Untersuchungsverfahren - Trennung und Zählung von

Pediococcus spp. Pediococcus spp.

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

© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 15786:2020 E

worldwide for CEN national Members.
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Contents Page

European foreword ....................................................................................................................................................... 3

Introduction .................................................................................................................................................................... 4

1 Scope .................................................................................................................................................................... 5

2 Normative references .................................................................................................................................... 5

3 Terms and definitions ................................................................................................................................... 5

4 Principle ............................................................................................................................................................. 6

5 Diluents and selective media ...................................................................................................................... 6

6 Apparatus and glassware ............................................................................................................................. 9

7 Sampling .......................................................................................................................................................... 10

8 Preparation of test sample ....................................................................................................................... 11

9 Procedure........................................................................................................................................................ 11

9.1 Samples with a single or multi-component added microflora .................................................... 11

9.2 Preparation of poured agar plates ......................................................................................................... 11

9.3 Preparation of MRS agar for pour plate method .............................................................................. 11

9.4 Preparation of the initial suspension and decimal dilutions ....................................................... 12

9.5 Inoculation and incubation of plates .................................................................................................... 13

9.6 Counting of colonies .................................................................................................................................... 14

9.7 Confirmation .................................................................................................................................................. 14

10 Expression of results ................................................................................................................................... 15

11 Precision .......................................................................................................................................................... 15

11.1 General ............................................................................................................................................................. 15

11.2 Interlaboratory study ................................................................................................................................. 15

11.3 Repeatability .................................................................................................................................................. 15

11.4 Reproducibility ............................................................................................................................................. 16

12 Test report ...................................................................................................................................................... 16

Annex A (informative) Notes on procedure ..................................................................................................... 17

Annex B (informative) Results of the interlaboratory study ..................................................................... 18

Bibliography ................................................................................................................................................................. 20

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European foreword

This document (prEN 15786:2019) 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.

This document is currently submitted to the CEN Enquiry.
This document will supersede EN 15786:2009.
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Introduction

This methodology has been developed to enumerate pediococci used as feed additives to enable the

European Commission to control proper labelling of animal feeding products (EU project SMT4-CT98-

2235 – “Methods for the official control of probiotics (microorganisms) used as feed additives”) [1]. The

described methodology was validated in an interlaboratory study [2]. The method is validated in this

project for one strain of Pediococcus acidilactici. It may be assumed that the method is suitable also for

other Pediococcus strains used as feed additives.
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1 Scope

This document defines general rules for the enumeration of pediococci in feeding stuffs (additives,

premixtures and compound feeds excluding mineral feeds) that contain pediococci as a single

microorganism component or in a mixture with other microorganisms. Applying the method to

compound feeds with critical amounts of copper demands a special procedure (see Annex A). The

document is not applicable to mineral feeds which are defined as complementary feeding stuffs

composed mainly of minerals and containing at least 40 % crude ash (Regulation R767/2009) [3].

There are different categories of feed samples:
a) Additives containing about 10 (colony forming units) CFU/g;
b) Premixtures containing about 10 CFU/kg;
c) Compound feeds, meal or pellets, which contain about 10 CFU/kg.
The detection limit is as defined in EN ISO 7218.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements 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 ISO 6498, Animal feeding stuffs - Guidelines for sample preparation (ISO 6498)

EN ISO 7218, Microbiology of food and animal feeding stuffs - General requirements and guidance for

microbiological examinations (ISO 7218)

EN ISO 6887-1, Microbiology of the food chain - 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)
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:

• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at http://www.iso.org/obp
3.1
pediococci

gram-positive catalase negative immobile cocci that grow under aerobic as well as under anaerobic

conditions

Note1 to entry This description is based on their characteristics as used for this standard.

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 [6] (see 9.7).

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4 Principle

a) Preparation of sterile and dry poured agar plates or preparation of sterile liquid selective medium

tempered at 44 °C to 47 °C;
b) A representative test sample is taken under aseptic conditions;

c) An initial suspension is prepared with a tempered diluent to obtain a homogeneous distribution of

Pediococcus cells from the test portion;

d) The number of microorganisms per unit volume is reduced by the preparation of further decimal

dilutions from the initial suspension to obtain a countable number of colonies on the selective

enumeration media;

e) Inoculation of prepared poured plates with an aliquot of the optimum dilutions and dispersion of the

inoculum using a sterile spreader or inoculation of blank petri dishes with an aliquot of the optimum

dilutions and pouring of the molten agar medium into each Petri dish, mixing and solidification;

f) The inoculated plates are incubated for 48 h to 72 h at 37 °C ± 1 °C under aerobic or anaerobic

conditions;

g) Counting of typical colonies, considering the specific properties of Pediococcus spp. as listed in 3.1;

h) Morphological verification of isolates by use of microscopy;

i) Calculation of the colony forming units of Pediococcus spp. per g or kg of feed sample.

5 Diluents and selective media
5.1 Diluents
5.1.1 Diluent for initial suspension

This diluent is used for the preparation of the initial suspension and may also be used for the preparation

of further decimal dilutions.
Table 1 — Phosphate buffered saline supplemented with Tween® 80 (tPBS)
Sodium chloride NaCl 8,00 g
Potassium chloride KCl 0,20 g
Disodium hydrogen phosphate anhydrous Na HPO 1,15 g
2 4
Potassium dihydrogen phosphate anhydrous KH PO 0,20 g
2 4
Polyoxyethylensorbitanmonooleate (Tween 80) 1 ml
Water, distilled or deionized 1 000 ml

Dissolve the components in water. If necessary, adjust to a final pH of 7,3 ± 0,2 at 25 °C after sterilization.

The solution is filled into appropriate containers (e.g. bottles or flasks, test tubes) and sterilized at

121 °C ± 1 °C for 15 min. To avoid loss during autoclaving, screw cap bottles are recommended.

Tween® is an example of a suitable product available commercially. This information is given for the convenience

of users of this document and does not constitute an endorsement by CEN of this product.

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Temper to 40 °C ± 1°C in a water bath or incubator immediately before usage.

NOTE The use of commercially available PBS buffer tablets is acceptable. However, please take note that

variations in composition and pH can occur between products from different manufacturers and could therefore

give results different from the ones obtained with the medium as specified in this International Standard.

5.1.2 Diluents for serial dilutions

For serial dilutions, the diluent for initial suspension (5.1.1) or alternatively peptone salt solution (PSS)

according to EN ISO 6887-1 can be used.
Table 2 — Peptone salt solution PSS according to EN ISO 6887-1
Enzymatic digest casein 1,0 g
Sodium chloride (NaCl) 8,5 g
Water, distilled or deionized 1 000 ml

Dissolve the components in the water in flasks, bottles or test tubes. Adjust the pH if necessary so that,

after sterilization, it is 7,0 ± 0,2 at 25 °C. For decimal dilutions, prepare test tubes containing

9,0 ml ± 0,1 ml after sterilization or use screw cap bottles to avoid weight loss during autoclaving.

Sterilize at 121 °C ± 1 °C for 15 min. Bring the diluent to room temperature before use.

NOTE Commercially available, ready-to-use PSS tubes of 9 ml are suitable.
5.2 Enumeration Media
5.2.1 General
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 and

novobiocin.

For routine enumeration of pediococci 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 designed to

encourage the growth of 'lactic acid bacteria' such as pediococci, enterococci and lactobacilli. Selection

can be made by pH adjustment, as pediococci 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 TTC allows differentiation of colonies by different colouration

after anaerobic incubationbut can exert a negative influence on the colony amount. The MRS medium

supplemented with two 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.

NOTE An antifungal agent like nystatin (50 U/ml) can be added to MRS agar, AMRSA or MRS+TTC to inhibit

moulds and yeasts.
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5.2.2 Composition
5.2.2.1 MRS agar
Table 3 — Composition of the MRS agar
Glucose 20 g
Peptone 10 g
Meat extract 8 g
Yeast extract 4 g
Sodium acetate 3 H O 5 g
Dipotassium hydrogen phosphate 2 g
Triammonium citrate 2 g
Sorbitan mono-oleate (Tween® 80) 1 ml
Magnesium sulphate 7 H O 0,2 g
Manganese sulphate 4 H O 0,05 g
Agar agar 10–15 g
Water, distilled or deionized 1 000 ml
pH 6,2 ± 0,2 at 25 °C
a Depending on the gel strength of the agar.

NOTE The use of commercially available, ready-to-use media is acceptable. However, please take note that

variations in composition and pH can occur between products from different manufacturers and could therefore

given results different from the ones obtain with the medium as specified in this International Standard.

5.2.2.2 MRS agar supplemented with TTC (0,01 %)

Sterilize MRS agar (5.2.2.1) by autoclaving at 121 °C ± 1 °C for 15 min. Supplement with 1 ml of a filter

sterilized 1 g/100 ml water solution of Triphenyl Tetrazolium Chloride (TTC) per 100 ml MRS agar.

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 with

10 µg/ml vancomycin and 0,1 µg/ml novobiocin.
5.2.3 Preparation
5.2.3.1 MRS agar

Dispense the agar medium into suitable containers (bottles or flasks with non-toxic metal screw-caps

may be used). Dissolve all components described in 5.2.2.2 in water by boiling. If necessary adjust the pH

so that after sterilization it is pH 6,2 ± 0,2 or 5,4 ± 0,1. Sterilize at 121 °C ± 1 °C for 15 min. Excessive

heating shall be avoided.
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5.2.3.2 MRS agar supplemented with TTC

Prepare 1 g Triphenyl Tetrazolium Chloride (TTC) in 100 ml water and filter sterilize. Add 1 ml per

100 ml MRS agar medium which is tempered at 44 °C to 47 °C after autoclaving.

NOTE TTC is destroyed by autoclaving. Protect the solution from light, and discard it if a pink tinge develops.

5.2.3.3 AMRSA

Prepare the medium as described in 5.2.3.1. Adjust the pH of MRS agar with HCl to 5,4 ± 0,1 prior to

autoclaving. Sterilize at 121 °C ± 1 °C for 15 min.
5.2.3.4 Selective medium

Prepare MRS medium with 500 mg/l cysteine hydrochloride as described in 5.2.3.1. Autoclave the

medium for 15 min at 121 °C ± 1 °C and temper it at 44 °C - 47 °C in a water bath or incubator.

Prepare 100 mg Vancomycin in 100 ml water and sterilize by sterile filtration. Add 1 ml per 100 ml MRS

medium after cooling. This procedure results in a final vancomycin concentration of 10 mg/l.

Prepare a solution of 10 µg/ml of Novobiocin through a series of ten-fold dilutions from an initial 1 mg/ml

stock and sterilize by sterile filtration. Add 1 ml Novobiocin per 100 ml MRS medium after cooling. This

procedure results in a final Novobiocin concentration of 100 µg/l.

WARNING — Care is needed in preparing the novobiocin stock as levels above 100 µg/l may be inhibitory

to pediococci.

Add the selective agents from sterile-filtered stocks to the medium immediately before pouring.

6 Apparatus and glassware
Usual microbiological laboratory equipment and, in particular, the following:
6.1 Equipment for dry sterilization (oven) and wet sterilization (autoclave)
According to EN ISO 7218.
6.2 Incubator

Capable of maintaining a temperature of 30 °C ± 1 °C. Optionally also capable of maintaining a

temperature of 40 ± 1°C and/or 44 °C to 47 °C.
6.3 Water bath
Capable of maintaining a temperature of 44 °C to 47 °C and 40 °C ± 1 °C.
6.4 Blending equipment
The following apparatus may be used (EN ISO 7218):

— a rotary homogenizer (blender) with a notional variable speed of 3 000 rpm to 10 000 rpm, as well

as aseptic glass or metals bowls equipped with covers; or

— a peristaltic blender with sterile bags (paddle homogenizer), possibly with the option to adjust

blending speed and time; or
— a vibrational mixer with sterile bags; or

— any other homogenizing system with equivalent efficiency (e.g. a hand blender with aseptic beaker).

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6.5 Mechanical stirrer
A mechanical stirrer e.g. Vortex Mixer (see EN ISO 7218), or equivalent.
6.6 Balance

Balances of the required range and accuracy according to EN ISO 7218 for the different products to be

weighed.
6.7 Microscope
Capable of phase-contrast microscopy at a magnification of 600x to 1 000x.
6.8 Flasks or screw-cap bottles of appropriate capacities
6.9 Test tubes of appropriate capacities
6.10 Sterile 5 ml graduated pipettes

For full outlet with wide (approx. 3 mm) tips (e.g. serological pipette; alternatively: 5 ml-graduated

pipettes without tips).
6.11 Pipettes or Pipettor and sterile tips to dispense 0,1 ml to 1 ml
6.12 pH meter
Having an accuracy of calibration of ± 0,1 pH unit at 20 °C to 25 °C.
6.13 Sterile petri dishes, 90 mm in diameter
6.14 Bacterial Cell spreaders

Sterile L- or triangular-shaped spreaders from glass or metal or sterile disposable plastic spreaders.

Alternatively a spiral plater with a sanitized dispensing system or disposable one–way microsyringes can

be used.
6.15 Laminar flow cabin
...

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