SIST EN 15787:2022

SLOVENSKI STANDARD
01-februar-2022
Nadomešča:
SIST EN 15787:2009
Krma: metode vzorčenja in analize - Določanje in štetje prisotnih Lactobacillus
spp., uporabljenih kot krmni dodatek
Animal feeding stuffs: Methods of sampling and analysis - Detection and enumeration of
Lactobacillus spp. used as feed additive
Futtermittel: Probenahme- und Untersuchungs-verfahren - Nachweis und Zählung von
Lactobacillus spp. als Futtermittelzusatzstoff
Aliments des animaux: Méthodes d’échantillonnage et d’analyse - Détection et
dénombrement des souches de Lactobacillus spp. utilisées comme additifs pour
l’alimentation animale
Ta slovenski standard je istoveten z: EN 15787:2021
ICS:
65.120 Krmila Animal feeding stuffs
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 15787
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2021
EUROPÄISCHE NORM
ICS 65.120 Supersedes EN 15787:2009
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Detection and enumeration of Lactobacillus 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 Lactobacillus
Lactobacillus 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 15787:2021 E
worldwide for CEN national Members.

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. 11
9.1 Samples with a single or multi-component added microflora . 11
9.2 Preparation of poured agar plates for spread plate method . 11
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 . 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 the procedure . 17
A.1 General . 17
A.2 Critical copper concentration . 17
Annex B (informative) Results of the interlaboratory study . 19
B.1 General . 19
B.2 Results of the interlaboratory study . 19
Bibliography . 20

European foreword
This document (EN 15787:2021) 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 15787:2009.
The main changes compared to the previous edition are as follows:
— Amendment of the title;
— Extension of the scope of application to all Lactobacilli 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;
— Relocation of the use of LAMVAB media from the normative part of the document to the informative
Annex A;
-1
— Replacement of the required laboratory mixer with a rotation speed of 18 000 min to
-1
22 000 min by homogenization devices, for example according to EN ISO 7218, with a maximal
-1
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;
— 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, 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.
Introduction
This methodology has been developed to enumerate lactobacilli 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 Lactobacillus acidophilus and one strain of Lactobacillus
rhamnosus. It can be assumed that the method is suitable also for other Lactobacillus strains used as
feed additives.
This method is not selective for lactobacilli used as feed additives, but can be applied to enumerate
Lactobacilli spp. in additives, premixtures and compound feeds assuming that the added lactobacilli are
present in far higher numbers than any other lactobacilli.
This method is not applicable for the detection of ubiquitous contaminants of Lactobacillus spp. and any
other lactic acid bacteria in food and animal feeding stuffs.
1 Scope
This document specifies general rules for the enumeration of lactobacilli in feeding stuffs (additives,
premixtures and compound feeds excluding mineral feeds) that contain lactobacilli 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) No
767/2009) [3].
There are different categories of feed samples:
colony forming units (CFU)/g;
a) Additives containing about 10
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
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).
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 bacterial cells from the test portion;
d) Preparation of further decimal dilutions of the initial suspension in order to reduce the number of
microorganisms per unit volume to allow, after incubation, the counting of colonies;
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 inverted plates for 48 h to 72 h at 37 °C ± 1 °C under anaerobic conditions;
g) Counting of typical colonies, considering the specific properties of lactobacilli;
h) Morphological verification of isolates within the Lactobacillus genus through the use of microscope
analysis if necessary;
i) Calculation of the colony forming units of lactobacilli 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
Disodium hydrogen phosphate anhydrous Na HPO 1,15 g
2 4
Potassium dihydrogen phosphate anhydrous KH PO 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.

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.
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.
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
Three 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).
For routine enumeration of lactobacilli 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 the 'lactic acid bacteria' such as pediococci, enterococci and lactobacilli.
Selection can be made by pH adjustment, as lactobacilli will tolerate a lower pH than enterococci
(pH 5,0 to pH 6,5), with pediococci growing best in this range. When enterococci are expected to be
present in similar concentrations as lactobacilli, acidified MRS agar (AMRSA) should be used. When
lactobacilli in combination with pediococci 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.
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.
Table 3 — Composition of the MRS agar
Glucose 20,0 g
Peptone 10,0 g
Meat extract 8,0 g
Yeast extract 4,0 g
Sodium acetate trihydrate 5,0 g
Dipotassium hydrogen phosphate 2,0 g
Triammonium citrate 2,0 g ®
1,0 ml
Polyoxyethylen (20) sorbitan monooleate (Tween 80)
Magnesium sulphate heptahydrate 0,2 g
Manganese sulphate tetrahydrate 0,05 g
a
Agar
10 g to 15 g
Water, distilled or deionized 1 000 ml
a
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.
5.2.3 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.
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.
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.
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 an 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.
...