SIST EN 1104:2019

SLOVENSKI STANDARD
oSIST prEN 1104:2017
01-september-2017
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DQWLPLNUREQLKVQRYL
Paper and board intended to come into contact with foodstuffs - Determination of the
transfer of antimicrobial constituents
Papier und Pappe vorgesehen für den Kontakt mit Lebensmitteln - Bestimmung des
Übergangs antimikrobieller Bestandteile
Papier et carton destinés à entrer en contact avec les denrées alimentaires -
Détermination du transfert des constituants antimicrobiens
Ta slovenski standard je istoveten z: prEN 1104
ICS:
67.250 Materiali in predmeti v stiku z Materials and articles in
živili contact with foodstuffs
85.060 Papir, karton in lepenka Paper and board
oSIST prEN 1104:2017 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN 1104:2017

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oSIST prEN 1104:2017


DRAFT
EUROPEAN STANDARD
prEN 1104
NORME EUROPÉENNE

EUROPÄISCHE NORM

May 2017
ICS 67.250; 85.060 Will supersede EN 1104:2005
English Version

Paper and board intended to come into contact with
foodstuffs - Determination of the transfer of antimicrobial
constituents
Papier et carton destinés à entrer en contact avec les Papier und Pappe vorgesehen für den Kontakt mit
denrées alimentaires - Détermination du transfert des Lebensmitteln - Bestimmung des Übergangs
constituants antimicrobiens antimikrobieller Bestandteile
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 172.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, 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: Avenue Marnix 17, B-1000 Brussels
© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 1104:2017 E
worldwide for CEN national Members.

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Contents Page
European foreword . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 5
5 Apparatus . 5
6 Reagents and media . 6
6.1 General . 6
6.2 Non ionic wetting agent . 6
6.3 Nutrient medium for the preparation of Bacillus subtilis spores . 6
6.3.1 Composition . 6
6.3.2 Preparation . 6
6.4 Nutrient medium of Sabouraud for the preparation of Aspergillus niger spores . 7
6.4.1 Composition . 7
6.4.2 Preparation . 7
6.5 Nutrient medium for the inhibition test with Bacillus subtilis . 7
6.5.1 Composition . 7
6.5.2 Preparation . 7
6.6 Nutrient medium of modified Sabouraud for the inhibition test with Aspergillus
niger . 7
6.6.1 Composition . 7
6.6.2 Preparation . 8
6.7 Tryptone salt solution . 8
6.7.1 Composition . 8
6.7.2 Preparation . 8
6.8 Test microorganisms . 8
6.8.1 General . 8
6.8.2 Preparation of working cultures . 8
6.8.3 Preparation of inoculating spore suspension of Bacillus subtilis . 8
6.8.4 Preparation of inoculating spore suspension of Aspergillus niger . 9
6.8.5 Concentrations of spores for inhibition test . 9
6.9 Positive controls . 9
6.9.1 Antibiotic . 9
6.9.2 Antifungal agent . 9
7 Sampling and preparation of test pieces . 10
8 Procedure. 10
8.1 General . 10
8.2 Preparation of plates . 10
8.2.1 General . 10
8.2.2 Test with Bacillus subtilis . 10
8.2.3 Test with Aspergillus niger . 10
8.3 Incubation . 11
9 Evaluation . 11
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10 Test report . 11
Annex A (informative) Guidelines for interpreting results . 13
A.1 Negative and positive controls . 13
A.2 Paper and board samples . 15
3

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European foreword
This document (prEN 1104:2017) has been prepared by Technical Committee CEN/TC 172 “Pulp, paper
and board”, the secretariat of which is held by DIN.
This document is currently submitted to the CEN Enquiry.
This document will supersede EN 1104:2005.
With regards to EN 1104:2005 the following changes have been made:
a) the definition of the inhibition zone has been clarified;
b) modified Sabouraud nutrient medium for the preparation of Aspergillus niger spores has been
replaced by 4 % Sabouraud;
c) guidelines for interpreting the results in Annex A have been added, including figures: these
guidelines are intended to aid the interpretation of results obtained in the framework of application
of EN 1104 standard for the various controls performed and for the samples tested;
d) editorial updating.
4

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1 Scope
This European Standard specifies a method for the determination of transfer of antimicrobial
constituents from paper and board materials and articles intended for food contact.
NOTE The need of using this Standard can be specified by the legislation regarding paper and board intended
to come into contact with foodstuffs.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
EN ISO 186, Paper and board - Sampling to determine average quality (ISO 186)
EN ISO 7218, Microbiology of food and animal feeding stuffs - General requirements and guidance for
microbiological examinations (ISO 7218)
EN ISO 11133, Microbiology of food, animal feed and water - Preparation, production, storage and
performance testing of culture media (ISO 11133)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
inhibition zone
obvious area in which growth is absent and which forms around test pieces placed on a nutrient
medium inoculated with a preselected test microorganism, due to the release of water-soluble
antimicrobial constituents
Note 1 to entry: Proof of the presence of an inhibition zone is provided by the absence of test micro-organism
growth (translucent zone) in a minimum of 2 mm width zone at the edges of the test pieces.
4 Principle
A prepared nutrient medium is mixed with an appropriate inoculum and poured into Petri dishes. The
test pieces are placed on the nutrient medium before its complete solidification and then incubated.
When incubation is terminated, the existence of an inhibition zone is an indicator of the release of
antimicrobial constituents.
The test is performed with a bacterium, Bacillus subtilis, and with a fungus, Aspergillus niger.
NOTE The result is based on a visual decision.
5 Apparatus
All laboratory equipment and parts of the equipment shall be as described in EN ISO 7218.
5.1 Punch iron:
Diameter = 10 mm to 15 mm, sterilizable.
5.2 Pressing device suitable for pressing the test pieces on the agar plates (e.g. Drigalski spatula).
5

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5.3 Zone reading device (facultative) allowing the measurement of the diameter of the inhibition
zone.
NOTE Measuring the diameter of the inhibition zone is not compulsory.
6 Reagents and media
6.1 General
When water is mentioned in a formula, use distilled water or purified water, as mentioned in
EN ISO 11133.
For preparing culture media, general requirements are described in EN ISO 11133. Culture media shall
be prepared as follows, or from commercially available dehydrated culture media according to the
manufacturer's instructions. Ready-to-use medium may be used when its composition and/or growth
performance is comparable to that of formula given hereafter. (according to EN ISO 11133).
6.2 Non ionic wetting agent
For example polyoxyethylenesorbitane monooleate.
6.3 Nutrient medium for the preparation of Bacillus subtilis spores
6.3.1 Composition
— Beef extract 3,0 g
— Peptone from casein, tryptic digest 5,0 g
— Sodium chloride, pure 5,0 g
— Agar-agar 12,0 g
— Water 1 000,0 ml
NOTE The addition of 10 mg/l MnSO to the nutrient medium for Bacillus subtilis will support the formation
4
of spores.
6.3.2 Preparation
Dissolve the components, or a dehydrated ready-to-use medium, in the water by mixing while heating.
Separate the nutrient medium into two parts:
— Dispense one part in 300,0 ml portions into 600,0 ml sterile flasks, for example Roux flasks, closed
with filter screw caps.
— Use the other part for the preparation of the working culture media into test tubes: dispense
10,0 ml portions into 15 to 20 test tubes and seal them with stoppers, for example cellulose
stoppers.
Sterilize flasks and test tubes in an autoclave for 15 min at 121 °C. After sterilization, the pH shall be
equivalent to 7,2 ± 0,2 when measured at 45 °C. Before solidification, position the test tubes in such a
way that the nutrient medium solidifies with a sloping surface.
Medium may be stored according to EN ISO 11133 prescriptions.
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6.4 Nutrient medium of Sabouraud for the preparation of Aspergillus niger spores
6.4.1 Composition
— Peptone from casein, tryptic digest 5,0 g
— Meat peptone 5,0 g
— D (+) glucose C H O H O (Dextrose) 40,0 g
6 12 6 2
— Agar-agar 10,0 g to 15,0 g
— Water 1 000,0 ml
6.4.2 Preparation
Dissolve the components, or a dehydrated ready-to-use medium, in the water by mixing while heating.
Proceed as described in 6.3 by dispensing the medium into sterile flasks or Roux flasks and sterile test
tubes.
Sterilize in an autoclave for 15 min at 121 °C. After sterilization, the pH shall be equivalent to 5,6 ± 0,2,
when measured at 25 °C.
6.5 Nutrient medium for the inhibition test with Bacillus subtilis
6.5.1 Composition
— Peptone from casein, tryptic digest 3,45 g
— Meat peptone 3,45 g
— Sodium chloride, pure 5,1 g
— Agar-agar 13,0 g
— Water 1 000,0 ml
6.5.2 Preparation
Dissolve the components, or a dehydrated ready-to-use medium, in the water by mixing while heating.
Dispense the medium into sterile flasks and close them with filter screw caps.
Sterilize in an autoclave for 15 min at 121 °C. After sterilization, the pH shall be equivalent to 6 ± 0,2,
when measured when measured at 45 °C.
Cool the flasks to below 60 °C for the preparation of the inoculation medium (8.2.2) or allow to solidify.
Medium may be stored according to EN ISO 11133 prescriptions.
6.6 Nutrient medium of modified Sabouraud for the inhibition test with Aspergillus niger
6.6.1 Composition
— Peptone from casein, tryptic digest 5,0 g
— Meat peptone 5,0 g
— D (+) glucose C H O H O (Dextrose) 10,0 g
6 12 6 2
— Maltose C H O H O 10,0 g
12 22 11 2
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— Agar-agar 10,0 g to 15,0 g
— Water 1 000,0 ml
6.6.2 Preparation
Dissolve the components, or a dehydrated ready-to-use medium, in the water by mixing while heating.
Dispense the medium into sterile flasks and close them with filter screw caps.
Sterilize in an autoclave for 15 min at 121 °C. After sterilization, the pH shall be equivalent to 5,4 ± 0,2,
when measured at 45 °C.
6.7 Tryptone salt solution
6.7.1 Composition
— Peptone from casein, tryptic digest 1,0 g
— Sodium chloride, pure 8,5 g
— Water 1 000,0 ml
6.7.2 Preparation
Dissolve the components in the water by mixing while heating.
Dispense into flasks or test tubes. Sterilize in an autoclave for 15 min at 121 °C. After sterilization, the
pH shall be equivalent to 7,0 ± 0,2, when measured at room temperature.
6.8 Test microorganisms
6.8.1 General
The following strains are used:
Bacillus subtilis DSM 347 (ATCC 6633) and Aspergillus niger DSM 1957 (ATCC 6275).
6.8.2 Preparation of working cultures
Working cultures of Bacillu
...