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1、BRITISH STANDARD BS EN 13783:2002 The European Standard EN 13783:2001 has the status of a British Standard ICS 67.050 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW Licensed Copy: sheffieldun sheffieldun, na, Mon Oct 30 01:53:14 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS E
2、N 13783:2002 This British Standard, having been prepared under the direction of the Consumer Products and Services Sector Policy and Strategy Committee, was published under the authority of the Standards Policy and Strategy Committee on 24 January 2002 BSI 24 January 2002 ISBN 0 580 38936 7 National
3、 foreword This British Standard is the official English language version of EN 13783:2001. The UK participation in its preparation was entrusted to Technical Committee AW/-/3, Food analysis Horizontal methods, which has the responsibility to: A list of organizations represented on this committee can
4、 be obtained on request to its secretary. Cross-references The British Standards which implement international or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by using the
5、“Find” facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from l
6、egal obligations. aid enquirers to understand the text; present to the responsible European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. Summary of pa
7、ges This document comprises a front cover, an inside front cover, the EN title page, pages 2 to 15 and a back cover. The BSI copyright date displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. DateComments Licensed Copy: sheffieldun she
8、ffieldun, na, Mon Oct 30 01:53:14 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 13783 November 2001 ICS 07.100.30; 67.050 English version Foodstuffs - Detection of irradiated food using Direct Epifluorescent Filter Technique/Aerobic Plate Count (DEFT
9、/APC) - Screening method Produits alimentaires - Dtection daliments ioniss en utilisant la technique dpifluorescence aprs filtration et dnombrement de la flore arobie sur milieu glos (DEFT/APC) - Mthode par criblage Lebensmittel - Nachweis der Bestrahlung von Lebensmitteln mit Epifluoreszenz-Filtert
10、echnik/aerober mesophiler Keimzahl (DEFT/APC) - Screeningverfahren This European Standard was approved by CEN on 29 September 2001. 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 stan
11、dard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the Management Centre or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other lan
12、guage made by translation under the responsibility of a CEN member into its own language and notified to the Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, I
13、celand, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: rue de Stassart, 36 B-1050 Brussels 2001 CENAll rights of exploita
14、tion in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 13783:2001 E Licensed Copy: sheffieldun sheffieldun, na, Mon Oct 30 01:53:14 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EN 13783:2001 (E) 2 Contents page Foreword2 1Scope 3 2Normative references 3 3Principle3
15、4Reagents.3 5Apparatus .6 6Sampling technique.7 7Procedure .8 8Evaluation.9 9Limitations10 10Validation10 11Test report 11 Annex A (informative) Further information on the applicability.12 Annex B (informative) Practical example for calculation of the microscope factor.13 Annex C (informative) Flow
16、diagram of the procedure14 Bibliography15 Foreword This European Standard has been prepared by Technical Committee CEN /TC 275 “Food Analysis - Horizontal Methods“, the secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication
17、 of an identical text or by endorsement, at the latest by May 2002, and conflicting national standards shall be withdrawn at the latest by May 2002. This European Standard was elaborated on the basis of a protocol developed following a concerted action supported by the Commission of European Union (
18、XII C.) (BCR), and a screening investigation carried out by the Danish National Food Agency and the local Environment and food Agency, MLK FYN, DK Odense. Experts and laboratories from E.U. and EFTA countries, contributed jointly to the development of the concerted action protocol. WARNING: The use
19、of this standard may involve hazardous materials, operations and equipment. This standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicab
20、ility of regulatory limitations prior to use. The annexes A, B and C are informative. This standard includes a Bibliography. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belg
21、ium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Licensed Copy: sheffieldun sheffieldun, na, Mon Oct 30 01:53:14 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EN 13783:2
22、001 (E) 3 1 Scope This European Standard specifies a microbiological screening method for the detection of irradiation treatment of herbs and spices, using the combined direct epifluorescent filter technique (DEFT) and aerobic plate count (APC). The DEFT/APC technique is not radiation specific, ther
23、efore, it is recommended to confirm positive results using a standardised method (e.g. EN 1788, prEN 13751) to specifically prove an irradiation treatment of the suspected food. The method has been successfully tested in interlaboratory tests with herbs and spices 1 to 5. 2 Normative references This
24、 European Standard incorporates by dated or undated reference, provisions from other publications. These normative references are cited at the appropriate places in the text, and the publications are listed hereafter. For dated references, subsequent amendments to or revisions of any of these public
25、ations apply to this European Standard only when incorporated in it by amendment or revision. For undated references the latest edition of the publication referred to applies (including amendments). ISO 4833, Microbiology General guidance for the enumeration of microorganisms - Colony count techniqu
26、e at 30 C. ISO 7218, Microbiology of food and animal feeding stuffs General rules for microbiological examinations. 3 Principle The method is based on the comparison of the APC with the count obtained using DEFT. The APC gives the number of viable microorganisms in the sample after a possible irradi
27、ation and the DEFT count indicates the total number of microorganisms, including non-viable cells, present in the sample. The difference between the DEFT count and the APC count in spices treated with doses of 5 kGy to 10 kGy is generally about or above 3 to 4 log units. Similar differences between
28、DEFT and APC counts can be induced by other treatments of the foods leading to death of microorganisms, e. g. heat, thus positive results shall be confirmed. A known volume of sample is filtered through a membrane filter at reduced pressure in order to concentrate the microorganisms on the filter. T
29、he microorganisms are stained with a fluorochrome, acridine orange (AO), resulting in an orange and orange-yellow fluorescence under illumination with blue light at 450 nm to 490 nm. These microorganisms are counted using an epifluorescence microscope to give the DEFT count. However, microorganisms
30、which were non-viable before irradiation show green fluorescence and are not counted. In parallel, the APC is determined from a second portion of the same test sample 6 to 10. 4 Reagents 4.1 General During the analysis use only reagents of recognized analytical grade. All the reagents used in the DE
31、FT and APC determinations should be sterilized by membrane filtration through 0,2 m pore size membrane filters or by autoclaving. 4.2 Peptone saline diluent 4.2.1 Composition Sodium chloride8,5 g Peptone1,0 g Distilled or demineralized water1000 ml Licensed Copy: sheffieldun sheffieldun, na, Mon Oct
32、 30 01:53:14 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EN 13783:2001 (E) 4 4.2.2 Preparation Dissolve the components in the water. Adjust the pH, if necessary, so that after sterilization the final pH is 7,2 0,2 at 20 C to 25 C. Sterilize in the autoclave (5.13) at 121 C 1C for 15 min. The diluent
33、may be stored in a glass bottle at 4 C to 6C for not more than two weeks. 4.3 Buffer, pH 3,0 4.3.1 Citric acid solution, substance concentration c(C6H8O7 H2O) = 0,1 mol/l 4.3.1.1 Composition Citric acid monohydrate21 g Distilled or demineralized water1000 ml 4.3.1.2 Preparation Dissolve the citric a
34、cid monohydrate in the water. The solution may be stored in a sterilized glass bottle at 4 C to 6 C for not more than three months. 4.3.2 Sodium hydroxide solution, c(NaOH) = 0,1 mol/l 4.3.2.1 Composition Sodium hydroxide 4,0 g or Sodium hydroxide solution, 1 mol/l100 ml Distilled or demineralized w
35、ater900 ml 4.3.2.2 Preparation Dissolve the sodium hydroxide or dilute the sodium hydroxide solution in the water. The solution may be stored in a glass bottle at 4 C to 6 C for not more than three months. 4.3.3 Complete buffer, pH 3,0 4.3.3.1 Composition Citric acid solution (4.3.1)100 ml Sodium hy
36、droxide solution (4.3.2)54 ml 4.3.3.2 Preparation Mix the citric acid solution and the sodium hydroxide solution. Adjust pH to 3,0 0,2 with citric acid solution or sodium hydroxide solution. Sterilize the buffer through a membrane filter of pore size 0,2 m (5.3) before use. The solution may be store
37、d in a glass bottle at 4 C to 6 C for not more than three weeks. Licensed Copy: sheffieldun sheffieldun, na, Mon Oct 30 01:53:14 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EN 13783:2001 (E) 5 4.4 Acridine orange solution 4.4.1 Buffer solution, pH 6,6 4.4.1.1 Composition Citric acid solution (4.3.1)3
38、5,5 ml Sodium hydroxide solution (4.3.2)100 ml 4.4.1.2 Preparation Mix the citric acid solution and the sodium hydroxide solution. Adjust pH to 6,6 0,2 with citric acid solution or sodium hydroxide solution. Sterilize the buffer through a membrane filter of pore size 0,2 m (5.3) before use. The buff
39、er solution may be stored in a glass bottle at 4 C to 6 C for not more than three weeks. 4.4.2 Complete acridine orange solution 4.4.2.1 Composition Acridine orange0,025 g Buffer, pH 6,6 (4.4.1)100 ml 4.4.2.2 Preparation Dissolve the acridine orange in the buffer solution (4.4.1). Sterilize the acri
40、dine orange solution through a 0,2 m pore size membrane filter (5.3) before use. The solution may be stored in a brown glass bottle at 4 C to 6 C for not more than one week. NOTE 1 Concentrated acridine orange solution is commercially available and is recommended for safety reasons. NOTE 2 As acridi
41、ne orange is regarded as a mutagenic substance, disposable gloves and face masks should be used when weighing the stain. 4.5 2-Propanol 4.6 Triton X-1001), 1 % cleaning solution 4.6.1 Composition Triton X 10010 ml Distilled or demineralized water1000 ml 4.6.2 Preparation Mix Triton X-100 with warm (
42、80C) water. Sterilize the solution through a 0,2 m pore size membrane filter (5.3). The solution may be stored in a glass bottle at 4 C to 6 C for not more than three weeks. 1) Triton X-100 is an example of a suitable product available commercially. This information is only given for the convenience
43、 of users of this International Standard and does not constitute an endorsement by CEN of this product. Licensed Copy: sheffieldun sheffieldun, na, Mon Oct 30 01:53:14 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EN 13783:2001 (E) 6 4.7 Tryptone-Yeast Extract-Glucose-Agar (Plate count agar) 4.7.1 Comp
44、osition Tryptone5,0 g Yeast Extract2,5 g Dextrose (Glucose)1,0 g Agar (according to the gel strength of the agar used) 12 g to 18 g Distilled or demineralized water1000 ml 4.7.2 Preparation Dissolve the components or dehydrated complete medium in the water while heating until boiling. Adjust the pH,
45、 if necessary, so that after sterilization the final pH is 7,2 0,2 at 20 C to 25 C. Sterilize in the autoclave (5.13) at 121 C 1 C for 15 min. 5 Apparatus Usual laboratory apparatus in accordance with ISO 7218 and, in particular, the following: 5.1 Apparatus for membrane filtration of sample suspens
46、ions Filtration equipment made of stainless steel or glass should be used. The bottom filter should be of sintered glass or stainless steel intended for filters with a diameter of 25 mm (5.5). The filter tower volume should be at least 10 ml. The filtration equipment is placed vertically on a suctio
47、n flask or a manifold connected to a water pump or vacuum pump with a pressure regulator. The vacuum during filtration should usually be approximately 70 kPa. NOTEThe use of a water pump is not very suitable if the water pressure cannot be regulated. 5.2 Filter funnel and suitable suction flasks mad
48、e of glass for sterile filtration of reagents and diluent. 5.3 Membrane filters, cellulose ester or similar, pore size 0,2 m, e.g. diameter 30 mm and/or 47 mm, for sterile filtration of reagents. 5.4 Membrane filters, polypropylene, diameter 25 mm, pore size 10 m, for prefiltration of samples. 5.5 M
49、embrane filters, white polycarbonate, diameter 25 mm, pore size 0,6 m for membrane filtration of sample test solution. 5.6 Sterile fast filter paper, for filtration of spice samples. 5.7 Epifluorescence microscope, with suitable light and filter combination (450 nm to 490 nm ) 5.8 Optics, 100 x immersion objective, ocular with magnification of 10 x. (Using a tube magnification of 1,25 a total magnification of 1250 x is achieved.) 5.9 Microscope slide, e. g. 76 mm x 26 mm. Licensed Copy: sheffieldun sheff
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