1、BRITISH STANDARD BS EN 14569:2004 Foodstuffs Microbiological screening for irradiated food using LAL/GNB procedures The European Standard EN 14569:2004 has the status of a British Standard ICS 07.100.30 BS EN 14569:2004 This British Standard was published under the authority of the Standards Policy
2、and Strategy Committee on 15 October 2004 BSI 15 October 2004 ISBN 0 580 44604 2 National foreword This British Standard is the official English language version of EN 14569:2004. The UK participation in its preparation was entrusted to Technical Committee AW/-/3, Horizontal analysis, which has the
3、responsibility to: A list of organizations represented on this committee can 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 Catalogue under the section entitle
4、d “International Standards Correspondence Index”, or by using the “Search” facility 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 w
5、ith a British Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related intern
6、ational and European developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the EN title page, pages 2 to 18, an inside back cover and a back cover. The BSI copyright notice displayed in this document indicates when the document was
7、 last issued. Amendments issued since publication Amd. No. Date CommentsEUROPEANSTANDARD NORMEEUROPENNE EUROPISCHENORM EN14569 October2004 ICS07.100.30 Englishversion FoodstuffsMicrobiologicalscreeningforirradiatedfoodusing LAL/GNBprocedures ProduitsalimentairesDpistagesmicrobiologiquedes alimentsio
8、nissenutilisantlatechniqueLAL/GNB LebensmittelMikrobiologischesLAL/GNB ScreeningverfahrenzumNachweisvonbestrahlten Lebensmitteln ThisEuropeanStandardwasapprovedbyCENon29July2004. CENmembersareboundtocomplywiththeCEN/CENELECInternalRegulationswhichstipulatetheconditionsforgivingthisEurope an Standard
9、thestatusofanationalstandardwithoutanyalteration.Uptodatelistsandbibliographicalreferencesconcernings uchnational standardsmaybeobtainedonapplicationtotheCentralSecretariatortoanyCENmember. ThisEuropeanStandardexistsinthreeofficialversions(English,French,German).Aversioninanyotherlanguagemadebytra n
10、slation undertheresponsibilityofaCENmemberintoitsownlanguageandnotifiedtotheCentralSecretariathasthesamestatusast heofficial versions. CENmembersarethenationalstandardsbodiesofAustria,Belgium,Cyprus,CzechRepublic,Denmark,Estonia,Finland,France, Germany,Greece,Hungary,Iceland,Ireland,Italy,Latvia,Lit
11、huania,Luxembourg,Malta,Netherlands,Norway,Poland,Portugal, Slovakia, Slovenia,Spain,Sweden,SwitzerlandandUnitedKingdom. EUROPEANCOMMITTEEFORSTANDARDIZATION COMITEUROPENDENORMALISATION EUROPISCHESKOMITEEFRNORMUNG ManagementCentre:ruedeStassart,36B1050Brussels 2004CEN Allrightsofexploitationinanyform
12、andbyanymeansreserved worldwideforCENnationalMembers. Ref.No.EN14569:2004:EEN 14569:2004 (E) 2 Contents page Foreword3 1 Scope 4 2 Normative references 4 3 Principle4 4 Procedures .4 5 Evaluation.4 6 Limitations5 7 Validation5 8 Test report 6 Annex A (normative) Enumeration of total viable Gram nega
13、tive bacteria (GNB).7 Annex B (normative) Determination of endotoxin concentration using the Limulus amoebocyte lysate (LAL) test.11 Annex C (informative) Figures 15 Bibliography 18 EN 14569:2004 (E) 3 Foreword This document (EN 14569:2004) has been prepared by Technical Committee CEN/TC 275 “Food a
14、nalysis - 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 of an identical text or by endorsement, at the latest by April 2005, and conflicting national standards shall be withdrawn at the lat
15、est by April 2005. 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, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland
16、, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EN 14569:2004 (E) 4 1 Scope This document specifies a microbiological screening method comprising two procedures, which are carried out in parallel
17、. It permits the identification of an unusual microbiological profile in poultry meat. The presence of a large excess population of dead micro-organisms can under certain circumstances be presumptive of irradiation treatment, which means, that the results of the procedure of the determination of end
18、otoxin concentration in the test sample using the Limulus amoebocyte lysate (LAL) test and of the procedure of the enumeration of total Gram negative bacteria (GNB) in the test sample are not radiation specific. Therefore, it is recommended that a positive result be confirmed using a standardized re
19、ference method for the detection of irradiated food, e.g. EN 1784, EN 1785 or EN 1786 1 to 3. This screening method has been successfully tested by inter-laboratory trials 4, 5, 6 and the procedure is generally applicable to whole or parts of poultry, e.g. breast, legs, wings of fresh, chilled or fr
20、ozen carcasses with or without skin. The method can also provide information about the microbiological quality of a product prior to irradiation. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition c
21、ited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ENV ISO 11133-1, Microbiology of food and animal feeding stuffs Guidelines on preparation and production of culture media Part 1: General guidelines on quality assurance for the pr
22、eparation of culture media in the laboratory (ISO/TS 11133-1:2000). ISO 7218, Microbiology of food and animal feeding stuffs General rules for microbiological examinations. 3 Principle The method determines the number of viable Gram negative bacteria present in the test sample and the concentration
23、of bacterial endotoxin present on the surfaces of Gram negative bacteria as lipopolysaccharides (LPS) serving as a measure for the estimation of the amount of total Gram negative bacteria, both viable and dead (Figure C.1). If the difference is high, it is assumed that the sample has been treated by
24、 a method of preservation, possibly by treatment with ionising radiation. 4 Procedures The two procedures that are required to be carried out are: Procedure 1: Enumeration of total Gram negative bacteria (GNB) in the test sample (according to Annex A) Procedure 2: Determination of endotoxin concentr
25、ation in the test sample using the Limulus amoebocyte lysate (LAL) test (according to Annex B). Samples should be analysed immediately upon receipt (by both GNB and LAL) to reduce any further microbial proliferation. If this is not possible, samples should be stored at 20 C until examination, which
26、should be carried out as soon as possible. 5 Evaluation In accordance with the methods specified in Annexes A and B the number of GNB forming units shall be expressed as log 10colony forming unit (cfu) GNB per gram and the concentration of endotoxin expressed as log 10Endotoxin Units (EU) per gram.
27、The difference between the GNB and the endotoxin results shall be calculated as (log 10EU/g - log 10cfu GNB/g). EN 14569:2004 (E) 5 Samples that are identified as having high endotoxin levels but low levels or no detectable GNB show an unusual microbiological profile. Thus, when log 10EU/g - log 10c
28、fu GNB/g is greater than 0 the sample is suspected of having been irradiated and should be subjected to further radiation specific tests. Samples that are identified as having high endotoxin levels and similarly high Gram negative bacteria counts show a normal microbiological profile. Thus, when log
29、 10EU/g - log 10cfu GNB/g is lower or equal 0, the sample is not suspected of having been irradiated. Samples that are identified as having endotoxin titres log 10EU/g of lower than 2,0 and low levels of GNB, i.e. log 10cfu GNB/g of lower than 2,0 should be considered inconclusive and should be subj
30、ected to further radiation specific tests. 6 Limitations This method can give only an indication of a possible treatment by ionising radiation. A high amount of dead microorganisms in comparison to the viable fraction can be due to several other reasons. It is therefore necessary to confirm a possib
31、le treatment by ionising radiation by a standardized reference method for the detection of irradiated foods. The method is of particular use to routine microbiological laboratories, which may be involved in the examination of foods. It should be noted that freezing after irradiation can influence th
32、e ratio of GNB to EU due to the loss of viability of microorganisms. Conversely, re-growth of bacterial flora can occur in irradiated samples which are stored unfrozen. 7 Validation The method was developed by the UK Ministry of Agriculture, Fisheries and Food, Food Science Laboratory, Norwich (FSL)
33、 and validated by collaborative trial 4, 5, 6. Twenty UK laboratories participated in the trial (16 Public Analyst Laboratories, 3 Public Health Laboratories and FSL). A commercial poultry processor supplied a single batch of boneless chicken breasts with skin and a single batch of boneless chicken
34、breast fillets. Within 24 h of slaughter, both batches of chicken were transported from the processing plant, under refrigeration conditions (5 C), to a commercial irradiation facility. The batch of chicken breasts were subdivided; one third of the batch were retained as control (unirradiated) sampl
35、es; one third were irradiated at an overall average dose of 2,5 kGy and the remainder irradiated at 5 kGy. The skinless chicken breast fillets were sub-divided into two. Half were retained as controls and the remaining half were irradiated at 2,5 kGy. Samples were irradiated using a Cobalt 60 source
36、. Amber perspex dosimeters 1)were used to estimate the dose received by measuring spectrometrically a change in absorbance at 530 nm. All chicken pieces were randomly coded and packaged in insulated containers under chill conditions. Samples were then delivered by overnight carrier to arrive at part
37、icipating laboratories the following morning. Participants were instructed to commence the analysis immediately upon arrival of the samples. Each participant received 10 samples for analysis comprising control samples of chicken breasts with and without skin; chicken breasts with skin irradiated at
38、2,5 kGy and 5 kGy and skinless breasts irradiated at 2,5 kGy. All samples were dispatched randomly as blind pairs. Participants were asked to examine each sample only once and report the results obtained from the LAL test and enumeration of GNB. Using the evaluation criteria detailed in Clause 5, 10
39、0 % of all unirradiated samples, 97 % of chicken with skin irradiated at 2,5 kGy, 82 % of those irradiated at 5 kGy and 88 % of the skinless fillets irradiated at 2,5 kGy were correctly identified. All other samples were reported as inconclusive due to the very low EU titres obtained. 1) Amber persp
40、ex dosimeter is an example of a suitable product commercially available from Harwell, UK which was used for the interlaboratory test. This information is given for the convenience of users and does not constitute an endorsement by CEN of this product. Equivalent products may be used if they can be s
41、hown to lead to the same results. EN 14569:2004 (E) 6 8 Test report The test report shall contain at least the following: a) information necessary for identification of the sample; b) a reference to this document; c) date of sampling and sampling procedure (if known); d) date of receipt and thermal
42、status of the sample when received; e) date of test; f) storage conditions of the sample in the laboratory after receipt; g) the result; h) any particular points observed in the course of the test; i) any operations not specified in the method or regarded as optional which might have affected the re
43、sults. EN 14569:2004 (E) 7 Annex A (normative) Enumeration of total viable Gram negative bacteria (GNB) A.1 Principle The enumeration of GNB which form colonies aerobically at 21 C 1 C for 24 h 1 h on a selective medium containing nisin, penicillin-G and crystal violet is carried out in four success
44、ive stages. Preparation of initial test suspension in endotoxin-free water; Surface inoculation of non-selective nutrient solid agar medium to allow resuscitation of stressed cells; pre- incubation at ambient temperature for 90 min; Overlaying of the non-selective medium following the resuscitation
45、period with selective agar medium containing nisin, penicillin-G and crystal violet and incubation at 21 C 1 C for 24 h 1 h; Enumeration. A.2 Diluents, culture media, and reagents A.2.1 General For uniformity of results, it is recommended that media be prepared from dehydrated commercial formulation
46、s where possible. All chemicals used in the preparation of culture media shall be of analytical quality. Water shall be distilled or de-ionised and free from inhibitory substances. Applicable provisions of ISO 7218 shall be respected. A.2.2 Endotoxin-free water 2)A.2.3 Peptone salt solution Sodium c
47、hloride 8,5 g Enzymatic digest of casein 1,0 g Water 1 000 ml Dissolve the constituents in the water. Mix well and if necessary adjust pH so that after sterilisation it is 7,0 0,2 at 25 C. Sterilise by autoclaving at 121 C 1 C for 15 min. The diluent may be stored for maximum two weeks at 4 C 1 C. A
48、.2.4 Nutrient agar (NA) medium (non-selective) Enzymatic digest of casein 5,0 g Sodium chloride 5,0 g Yeast extract 2,0 g Meat extract 1,0 g Agar 9 g to 18 g aWater 1 000 ml aDepending on the gel strength of the agar. 2) Endotoxin (pyrogen) free water can generally be obtained from manufacturers supplying the pharmaceutical industry. EN 14569:2004 (E) 8 Dissolve the constituents in the water by boiling. Adjust the pH so that after sterilization it is 7,4 0,2 at 20 C to 25 C. Sterilise by autoclaving at 121 C 1 C for 1
