1、 g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58foodstuffs Determination of the transfer of antimicrobial constituentsThe European Standard EN 1104
2、:2005 has the status of a British StandardICS 67.250; 85.060Paper and board intended to come into contact with BRITISH STANDARDBS EN 1104:2005BS EN 1104:2005This British Standard was published under the authority of the Standards Policy and Strategy Committee on 25 November 2005 BSI 25 November 2005
3、ISBN 0 580 46690 6request to its secretary.Cross-referencesThe British Standards which implement international or European publications referred to in this document may be found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Search” f
4、acility of the BSI Electronic Catalogue or of British Standards Online.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application.Compliance with a British Standard does not of itself confer immunity from legal obligatio
5、ns.Summary of pagesThis document comprises a front cover, an inside front cover, the EN title page, pages 2 to 10, an inside back cover and a back cover.The BSI copyright date displayed in this document indicates when the document was last issued.Amendments issued since publicationAmd. No. Date Comm
6、entsA list of organizations represented on this subcommittee can be obtained on enquiries on the interpretation, or proposals for change, and keep UK interests informed; monitor related international and European developments and promulgate them in the UK.National forewordThis British Standard is th
7、e official English language version of EN 1104:2005. It supersedes BS EN 1104:1996 which is withdrawn.The UK participation in its preparation was entrusted by Technical Committee CW/47, Materials in contact with food, to Subcommittee CW/47/3, Paper and board in contact with food, which has the respo
8、nsibility to: aid enquirers to understand the text; present to the responsible international/European committee any EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 1104August 2005ICS 67.250; 85.060 Supersedes EN 1104:1995 English VersionPaper and board intended to come into contact with foodstuffs
9、 -Determination of the transfer of antimicrobial constituentsPapier et carton destins entrer en contact avec desdenres alimentaires - Dtermination du transfer desconstituants antimicrobiensPapier und Pappe vorgesehen fr den Kontakt mitLebensmitteln - Bestimmung des bergangsantimikrobieller Bestandte
10、ileThis European Standard was approved by CEN on 27 June 2005.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical refer
11、ences concerning such nationalstandards may be obtained on application to the Central Secretariat or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into
12、its own language and notified to the Central Secretariat has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxe
13、mbourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 2005 CEN All rights of explo
14、itation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 1104:2005: EEN 1104:2005 (E) 2 Contents page Foreword 3 1 Scope .4 2 Normative references .4 3 Terms and definitions.4 4 Principle.4 5 Apparatus 4 5.1 Punch iron, 4 5.2 Pressing device,4 5.3 Zone reading devi
15、ce, 4 5.4 Ordinary microbiological laboratory apparatus .5 6 Reagents5 6.1 Water,.5 6.2 Non ionic wetting agent,.5 6.3 Nutrient medium for Bacillus subtilis5 6.4 Sabouraud modified mould nutrient medium for Aspergillus niger .6 6.5 Nutrient medium for inhibition test with Bacillus subtilis .6 6.6 Sa
16、lt peptone solution6 6.7 Test micro-organisms.7 6.7.1 General 7 6.7.2 Preparation of inoculating spore suspension of Bacillus subtilis 7 6.7.3 Preparation of inoculating spore suspension of Aspergillus niger 8 6.7.4 Concentrations of spores for inhibition test.8 6.8 Positive controls .8 6.8.1 Penici
17、llin, G 0,03 units 8 6.8.2 Isothiazolinon-solution.8 7 Sampling and preparation of test pieces 8 8 Procedure 9 8.1 Sterilisation .9 8.2 Preparation of plates 9 8.2.1 General 9 8.2.2 Bacillus subtilis.9 8.2.3 Aspergillus niger.9 8.3 Incubation9 9 Evaluation10 10 Test report .10 EN 1104:2005 (E) 3 For
18、eword This European Standard (EN 1104:2005) has been prepared by Technical Committee CEN/TC 172 “Pulp, paper and board”, the secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at
19、the latest by February 2006, and conflicting national standards shall be withdrawn at the latest by February 2006. This European Standard supersedes EN 1104:1995. With regard to EN 1104:1995 the following changes have been made: a) description of the preparation of some reagents were partly modified
20、; b) more detailed description of the procedure was formulated; c) editorial updating. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic, Denmark,
21、Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EN 1104:2005 (E) 4 1 Scope This European Standard specifies a method for the de
22、termination of transfer of antimicrobial constituents from paper and board materials and articles intended for food contact. 2 Normative references The following referenced documents are indispensable for the application of this European Standard. For dated references, only the edition cited applies
23、. 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 186:2002) 3 Terms and definitions For the purposes of this European Standard, the following term and definition applies.
24、3.1 inhibition zone zone formed around the test pieces, placed on nutrient medium, which has been seeded with a pre-selected test organism that releases water-soluble antimicrobial constituents. 4 Principle A prepared nutrient medium is mixed with an appropriate inoculum and poured into Petri dishes
25、. The test pieces are placed on the semi solid nutrient medium 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, Aspergillu
26、s niger. NOTE The result is based on a visual inspection. 5 Apparatus 5.1 Punch iron, d = 10 mm to 15 mm, sterilizable. 5.2 Pressing device, suitable for pressing the test pieces on the agar plate (e.g. Drygalski spatula). 5.3 Zone reading device, to measure the diameter of inhibition. NOTE Measurin
27、g the diameter of the inhibition zone is not compulsory. EN 1104:2005 (E) 5 5.4 Ordinary microbiological laboratory apparatus 6 Reagents 6.1 Water, freshly distilled or water purified by ion exchange and freshly boiled (deionised water). 6.2 Non ionic wetting agent, for example polyoxyethylenesorbit
28、ane monooleate. 6.3 Nutrient medium for Bacillus subtilis A typical formulation of the nutrient medium is: beef extract 3,0 g; tryptone (peptone of casein) 5,0 g; sodium chloride, pure 5,0 g; agar-agar 12,0 g water 1000,0 ml. NOTE The addition of 10 mg/l MnSO4to the nutrient medium for Bacillus subt
29、ilis (6.7.1) will support the formation of spores. Prepare the nutrient medium as follows: Dissolve the components, or a ready-made medium of a comparable composition, in water by boiling. The pH of the ready prepared nutrient medium shall be (7,2 0,2) referred at a temperature of 45 C. Adjust the p
30、H to (7,2 0,2) as required either with NaOH approx. 0,01 M or HCl approx. 0,01 M. Separate the nutrient medium into two parts. Dispense one part in 300,0 ml portions into nutrient medium flasks or 600,0 ml flasks e.g. Roux flasks and stopper them with caps, e.g. Kapsenberg caps. Use the other part f
31、or 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, e.g. cellulose stoppers. Sterilise flasks and test tubes for 15 min at (121 1) C. After sterilisation position the test tubes immediately in such a way tha
32、t the nutrient medium solidifies with a sloping surface. Store them at 4 C to 8 C not longer than 14 d. Cool the nutrient medium flasks to approx. 45 C for the preparation of the inoculating suspension of Bacillus subtilis (6.7) or allow to solidify. Cool the flasks to solid. EN 1104:2005 (E) 6 6.4
33、Sabouraud modified mould nutrient medium for Aspergillus niger A typical formulation of the Sabouraud modified mould nutrient medium is: tryptone (peptone of casein) 5,0 g peptone (peptone of meat) 5,0 g; D (+) glucose C6H12O6 H2O 10,0 g; maltose C12H22O11 H2O 10,0 g; agar-agar 10,0 g to 15,0 g; wat
34、er 1000,0 ml Prepare the Sabouraud modified mould nutrient medium as follows: Dissolve the components, or a ready-made medium of a comparable composition, by boiling. The pH of the ready prepared nutrient medium shall be (5,4 0,1) referred at a temperature of 45 C. Adjust the pH to (5,4 0,1) as requ
35、ired either with NaOH approx. 0,01 M or HCl approx. 0,01 M. Proceed as described in 6.3 dispensing the medium into nutrient medium flasks or flasks e.g. Roux flasks and test tubes and the sterilising and cooling procedures. 6.5 Nutrient medium for inhibition test with Bacillus subtilis The compositi
36、on of the nutrient medium for inhibition test with Bacillus subtilis shall be as follows: tryptone (peptone of casein) 3,45 g; peptone (peptone of meat) 3,45 g; sodium chloride, pure 5,1 g; agar-agar 13,0 g; water 1000,0 ml. Prepare the nutrient medium as follows: Dissolve the components, or a ready
37、-made medium of a comparable composition, in water by boiling. The pH of the ready made nutrient medium shall be (6,0 0,1) referred at a temperature of 45 C. Adjust the pH to (6,0 0,1) as required either with NaOH approx. 0,01 M or HCl approx. 0,01 M. Dispense portions of nutrient medium flasks or t
38、est tubes and stopper them with caps. e.g. Kapsenberg caps and sterilise for 15 min at (121 1) C. Cool the flasks to below 60 C for the preparation of the inoculation medium (8.2.2) or allow to solidify. 6.6 Salt peptone solution The composition of the salt peptone solution shall be as follows: EN 1
39、104:2005 (E) 7 peptone (peptone of meat) 1,0 g; sodium chloride, pure 8,5 g; water 1000,0 ml. Prepare the salt peptone solution as follows: Dissolve the components in water of pH between 6 and 7. Dispense equal volumes into three flasks, stopper with caps, e.g. Kaspensberg caps and sterilise for 15
40、min at (121 1) C. The solution shall be used within 8 d if stored at room temperature and within 14 d when stored at 4 C to 8 C. 6.7 Test micro-organisms 6.7.1 General The following are used: Bacillus subtilis DSM 347 (ATCC 6633) and Aspergillus niger DSM 1957 (ATCC 6275) or other corresponding stra
41、ins. As different strains may also have different sensitivity, a comparative examination would have to be performed when using other strains than ATTC 6633 and ATTC 6275. Working cultures of Bacillus subtilis are obtained by inoculating onto the test tubes (6.3) and incubating for 7 d at 30 C. After
42、 incubation the test tubes are stored at 4 C to 8 C. Working cultures of Aspergillus niger are obtained by inoculating onto the test tubes (6.4) and incubating for 5 d at 25 C. After incubating the test tubes are stored at 4 C to 8 C. 6.7.2 Preparation of inoculating spore suspension of Bacillus sub
43、tilis Transfer aliquots of about 15,0 ml of the liquefied nutrient medium (6.3) cooled to approximately 45 C to 10 sterile Petri dishes (d = 90 mm) and allow to solidify. The nutrient medium in flasks (6.3) is ready for inoculation. Wash off the colonies of ten test tubes with the working culture of
44、 Bacillus subtilis (6.7.1) with 2,0 ml to 3,0 ml sterile salt, peptone solution (6.6). Spread the washings over the surface of the ten Petri dishes (each dish is incubated from a separate tube) or all the washings over the surface of the Roux flask. Incubate for 7 d at 30 C. Wash off the colonies fr
45、om the Petri dishes with 3,0 ml salt peptone solution (6.6) and the flask with 30,0 ml salt peptone solution (6.6). Bring the suspension over into a sterile flask by using a sterile funnel and close the flask with a sterile stopper. Heat the solution with occasional shaking, for 30 min in a water ba
46、th at approx. 85 C in order to kill the vegetative forms. After heating transfer the spore suspension to a sterile centrifuging flask of 40,0 ml and centrifuge for 10 min at 10000 g. Eliminate the liquid. Wash the residue with 30,0 ml salt peptone solution (6.6) and centrifuge again. Repeat the wash
47、ing 3 times. Suspend the spores in 20,0 ml of the salt peptone solution (6.6). The spore suspension may be stored at 4 C to 8 C not longer than 4 weeks. NOTE The spore suspension is also commercially available. EN 1104:2005 (E) 8 6.7.3 Preparation of inoculating spore suspension of Aspergillus niger
48、 Transfer aliquots of about 15,0 ml of the liquefied modified Sabouraud medium (6.4), cooled to approx. 45 C to at least 5 sterile Petri dishes (d = 90 mm) and allow to solidify. The nutrient medium in flask (6.4) is ready for inoculation. Inoculate the Aspergillus niger strain from the working cult
49、ures (6.7.1) with an inoculation loop onto the Petri dish. Each Petri dish is inoculated from a separate tube. The flask is incubated from at least five test tubes. Incubate for 8 d to 10 d at 25 C. Transfer the conidia with an inoculating ring moistened with salt peptone solution (6.6) to a sterile test tube containing 10,0 ml of salt peptone solution (6.6) mixed with 0,01 ml of a non ionic wetting agent (6.2) and seal with a sterile stopper. Shake
