EURL TECHNICAL GUIDANCE DOCUMENT For Conducting Shelf-life .
EURL LmEuropean Union Reference Laboratory forListeria monocytogenesEURL Lm TECHNICAL GUIDANCE DOCUMENTfor conducting shelf-life studies on Listeria monocytogenes in ready-to-eatfoodsVersion 3 of 6 June 2014 – Amendment 1 of 21 February 2019Annie Beaufort, Hélène Bergis, Anne-Laure Lardeux, Unit Modelling of Bacterial Behaviour, BertrandLombard, Manager EU Reference Laboratory for Listeria monocytogenes Anses-Food SafetyLaboratory, Maisons-Alfort, FranceIn collaboration with representatives of 10 National Reference Laboratories (NRLs) for Listeriamonocytogenes and 1 associated National Reference Laboratory for Listeria monocytogenes:-Marie Polet and Nadine Botteldoorn, Scientific Institute of Public Health, Belgium;George Papageorgiou, State General Laboratory, Cyprus;Jens Kirk Andersen and Jeppe Boel, National Food Institute, Danish Technical University,Denmark;Bernadette Hickey, Dairy Science Laboratory, Republic of Ireland;Vincenza Prencipe, Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G.Caporale”, Italy;Wilma Jacobs-Reitsma, National Institute for Public Health and the Environment (RIVM),The Netherlands (NL-NRL);Ife Fitz-James, Netherlands Food and Consumer Product Safety Authority (NVWA), TheNetherlands (associated NL-NRL);Celcidina Maria Pires Gomes, Instituto Nacional De Investigação Agrária e Veterinária(INIAV), Portugal;Lenka Cabanova, State Veterinary and Food Institute, Slovakia;Cristina Acebal Sarabia, Institute for Hygiene and Veterinary Public Health, Spain;Taran Skjerdal, Norwegian Veterinary Institute, Norway.And in collaboration with a representative of another laboratory: Gail Betts, Campden & ChorleywoodFood Research Association, Gloucestershire, United Kingdom (for the UK-NRL).1/47TGD Lm shelf-life studiesEURL Listeria monocytogenes21/02/2019
CONTENTSForeword . 41Introduction . 41.1Listeria monocytogenes . 41.2Ready-to-eat foods . 61.3Legislative background . 61.4EU guidance document dedicated to food business operators . 62Scope . 73Challenge test . 93.1Review of data . 93.2Protocols for challenge test. 103.2.13.2.1.1General aspects . 103.2.1.2Protocol of a challenge test to assess growth potential . 113.2.24Challenge test assessing growth potential . 10Challenge test assessing maximum growth rate . 25203.2.2.1General aspects . 25203.2.2.2Protocol of a challenge test to assess maximum growth rate . 2520Durability study . 32204.1Food sampling procedure. 32204.1.1Introduction . 32204.1.2Simple random sampling . 32204.1.2.1Description . 32204.1.2.2Example of a method used to select randomly 10 test units from a batch . 32204.2Storage conditions . 33204.3Microbiological analyses . 33204.4Calculation . 34205References . 36206Glossary . 38207Annexes . 40207.1Flow diagram describing schematically the steps from review of data to test in thelaboratory . 40207.2EURL Lm set of L. monocytogenes strains with their growth characteristics. 41207.3Example of preparation of the inoculum for the challenge test . 42207.3.1Preparation of subcultures for strain 1 . 42202/47TGD Lm shelf-life studiesEURL Listeria monocytogenes21/02/2019
7.3.2Preparation of the inoculum for challenge test assessing growth potential . 42207.3.3Preparation of the inoculum for challenge test assessing maximum growth rate . 42207.3.4Method to obtain the targeted concentration of the inoculum with a numericalexample: 43207.4Examples of total number of test units required per batch in the frame of a challenge testto assess a growth potential . 44207.5Some examples of contamination techniques . 44207.6Example of the impact of storage temperature on the shelf-life . 45207.7Example of preparation of the initial suspension . 46207.8Examples of the total number of test units to be prepared in the frame of a challenge testto assess a maximum growth rate, per strain, per batch. 47203/47TGD Lm shelf-life studiesEURL Listeria monocytogenes21/02/2019
ForewordThis document is the third version of the Technical Guidance Document for conducting shelf-life studieson Listeria monocytogenes in ready-to-eat foods of the European Union Reference Laboratory forListeria monocytogenes (EURL Lm). It replaces the 2nd version of November 2008.This document is complementary to the EC/DG SANCO document, entitled “Guidance document onListeria monocytogenes shelf-life studies for ready-to-eat foods, under Regulation (EC) No. 2073/2005of 15 November 2005 on microbiological criteria for foodstuffs”.This document has been approved by the Standing Committee of the Food Chain & AnimalHealth/Section “Biological safety of the food chain”, at its meeting of 16 June 2014.An amendment to this third version has been prepared by EURL Lm and a working group of 8 NRLsLm. It has been approved by the Standing Committee of the Food Chain & Animal Health/Section“Biological safety of the food chain”, at its meeting of 12 February 2019. The changes concern thestorage temperature of the test units at retail level to conduct a challenge test assessing the growthpotential of Listeria monocytogenes, see clause 3.2.1.2.e, p 18-19.References are given in clause 5 and annexes in clause 7.1 Introduction1.1 Listeria monocytogenesThe genus Listeria contains ten species including monocytogenes, which may be pathogenic for humansand animals. Listeria monocytogenes may cause a disease called listeriosis that may affect humans andanimals.Listeria is a small (0.5-2 μm x 0.5 μm), Gram-positive bacillus, isolated or arranged in small chains,motile at 20-25 C and non-spore-forming. It is aerobic and facultatively anaerobic, catalase-positiveexcept for a few rare strains, oxidase negative and hydrolyses esculin. Listeria ferments manycarbohydrates without producing gas. Strains of L. monocytogenes are always D-xylose negative andproduce lecithinase. They are generally β-haemolytic and L-rhamnose positive. The speciesmonocytogenes is divided into 13 serovars based on somatic and flagellar antigens. Since 2005, theseserovars have been replaced by 5 genoserogroups determined by PCR: IIa (serovars 1/2a and 3a), IIb(serovars 1/2b and 3b), IIc (serovars 1/2c and 3c), IVb (serovars 4b, 4d and 4e) and L (other serovars).Of these, IVb followed by IIa and IIb are the genoserogroups most frequently implicated in human cases.Table 1 describes some growth and inactivation characteristics of L. monocytogenes.4/47TGD Lm shelf-life studiesEURL Listeria monocytogenes21/02/2019
Table 1: Some growth and inactivation characteristics of L. monocytogenes (variable depending onthe strains and the food matrix)1GrowthMin.OptimumMax.(lower growth limit)(fastest growth)(upper growth limit)Temperature ( C)-1.530.0-37.045.0pH4.2-4.37.09.4-9.50.99 0.990.712-160.93 (0.90 withawglycerol)Salt concentration (%)2Atmosphere 0.5Facultative anaerobe (it can grow in the presence or absence of oxygen,e.g. in a vacuum or modified atmosphere package)Thermal inactivationD65 C0.2 to 2 minz7.5 C (4 to 11 C)High pressure inactivation400 MPa for 10 min at 20 C 2 log10 reductions in phosphate buffer (pH 7)400 MPa for 10 min at 20 C 8 log10 reductions in citrate buffer (pH 5.6)400 to 500 MPa for 5 to 10 min at 20 C 3 to 5 log10 reductions in meat products.350 MPa for 5 to 10 min at 20 C 3 to 5 log10 reductions in acidic products (e.g. fruit juices, jams).Sources: Anses datasheet on biological hazards “Listeria monocytogenes”, 2011 and Guidance document on Listeriamonocytogenes shelf-life studies for ready to eat foods, under Regulation (EC) No 2073/2005 of 15 November 2005 onmicrobiological criteria for foodstuffs1:The growth and inactivation data from L. monocytogenes presented in this table are based on research carried out primarilyin lab media under optimum conditions and may vary depending on the strain and food matrix2:Based on percent sodium chloride, water phaseL. monocytogenes is a psychrotrophic bacterion which is able to grow at -1.5 C, and thus may grow wellat refrigeration temperatures. The microorganism has the ability to persist in food-processing areas andequipment.Listeriosis occurs in two forms: invasive or non-invasive. For the entire population, listeriosis can causebacteremia, septicemia, meningitis, meningoencephalitis, rhombencephalitis, brain abscess, localinfections. In addition, for pregnant women, listeriosis can provoke flu-like symptoms (fever, chills,back pain), spontaneous abortion, death in utero, prematurity and neonatal infection. Susceptiblepopulation groups are people most likely to develop a severe form of listeriosis. These groups are5/47TGD Lm shelf-life studiesEURL Listeria monocytogenes21/02/2019
composed of pregnant women, people aged over 65 years or those with cancer or blood disorders,dialysis patients, other immunocompromised diseases (e.g. HIV, ). Non-invasive forms are rare: theyare essentially febrile gastroenteritis, for which some outbreaks have been recorded. Although rare,listeriosis is a foodborne infection with extremely high lethality (25 to 30%) and hospitalisation rates( 92%).1.2 Ready-to-eat foodsThe definition of ready-to-eat food is given in the glossary (clause 6) and is taken from Regulation (EC)2073/2005 of 15 November 2005 (modified) on microbiological criteria for foodstuffs and DG SANCO“Guidance document on Listeria monocytogenes shelf-life studies for ready-to-eat foods, underRegulation (EC) No. 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs”.1.3 Legislative backgroundThe first version of this technical guidance document (2008) was prepared at the request ofDirectorate General Health & Consumers (DG SANCO) of European Commission (EC) in response tothe needs expressed by EU Member States. EC/DG SANCO acknowledged that a document wasrequired, providing both detailed and practical information on how to conduct shelf-life studies onListeria monocytogenes (L. monocytogenes) in ready-to-eat foods to ensure conformance to themicrobiological criteria set out in Article 3.2 of Regulation (EC) No. 2073/2005.Annex I of Regulation (EC) No. 2073/2005 sets out the microbiological criteria for foodstuffs, includingthe criteria for L. monocytogenes in RTE foods (criteria 1.1 to 1.3). Annex II of this regulation specifiesthat food business operators (FBOs) shall conduct, as necessary, studies to evaluate the growth ofL. monocytogenes that may be present in the product during the shelf-life under reasonably foreseeablestorage conditions. Annex II does not describe the technical procedure to conduct such studies.1.4 EU guidance document dedicated to food business operatorsThe EC/DG SANCO document, entitled “Guidance document on Listeria monocytogenes shelf-lifestudies for ready-to-eat foods, under Regulation (EC) No. 2073/2005 of 15 November 2005 onmicrobiological criteria for foodstuffs”, is directed at FBOs who produce ready-to-eat foods. In thisdocument, a decision tree shows a schematic approach for the steps of shelf-life studies, giving the FBOan indication of when additional specific studies are needed in order to investigate the growth of L.monocytogenes in the product.6/47TGD Lm shelf-life studiesEURL Listeria monocytogenes21/02/2019
2 ScopeThis EURL Lm technical guidance document is basically intended for laboratories conducting challengetests and durability studies on L. monocytogenes in RTE foods, on behalf of the FBOs. Theselaboratories should have the required expertise for such studies and demonstrate good laboratorypractices.This document describes laboratory studies, challenge tests and durability studies related to the growthof L. monocytogenes in RTE foods.It is mainly dedicated to packaged products1. For unpackaged products, other additional factors, such ashygrometry, have to be considered for the storage of the product under reasonably foreseeable storageconditions; it is thus necessary to adapt the review of data and the experimental protocol to this type ofproducts.The shelf-life is determined for the product as marketed by the producer. Once the product is openedand stored by a retailer, restaurant owner, , a new shelf-life has to be assessed.This document details the relevant information required before implementing a challenge test andrecommendations on how to implement and perform the challenge tests required: challenge testassessing growth potential or challenge test assessing maximum growth rate.This document also provides recommendations on how to implement and perform durability studies.The choice of the test to be implemented should be done by the FBO, if necessary with the collaborationof the laboratory which will conduct it. The choice should be based on the information to be obtained,as illustrated in Figure 1.1Challenge tests for packaged products should be conducted using the product in its final packaged formatincluding gas atmosphere if present. For products which are intended to be displayed in bulk (i.e. largeblocks of cheese, pieces of ham or tubs of deli-salads), the tests should be conducted using the typicalpackaging which is expected to be supplied to consumers (e.g ham may be overwrapped with packagingfilm, salads may be filled into plastic pots). The aim of a challenge test is to simulate as closely as possiblethe likely storage conditions of the product. The challenge test report should record what packaging andstorage conditions were used as the results are not applicable to different storage conditions.7/47TGD Lm shelf-life studiesEURL Listeria monocytogenes21/02/2019
Figure 1 describes microbiological procedures for determining the growth of L. monocytogenes usingchallenge tests and durability studies.Challenge-tests« »(artificial contamination)Durability studies(natural contamination)Challenge-tests« max » or « ratemax »(artificial contamination)« max » or « ratemax »(maximum growth rate)RTE not able to supportthe growth of L. mRTE able to support thegrowth of L. mGrowth of L. m duringthe shelf-lifeFinal concentrationof L. m« »(growth potential)Initial concentrationof L. mFinal concentrationof L. mProportion of unitsabove 100 cfu/g at theend of the shelf-lifeInitialconcentrationof L. mConcentration ofL. m at a givendayFigure 1. Data obtained from shelf-life studiesChallenge tests aim to provide information on the behaviour of L. monocytogenes which have beenartificially inoculated into a food, under given storage conditions. They may take into account thevariability of the batches, of the food samples and of strains. The level of contamination, theheterogeneity of the contamination and the physiological state of the bacteria are difficult to mimic in achallenge test study; the contamination method cannot always enable to fully imitate the naturalcontamination.Durability studies allow an assessment of the shelf-life of the food regarding L. monocytogenes in anaturally contaminated food during its storage according to reasonably foreseeable conditions.Durability studies may be considered more realistic than a challenge test, as the contamination isnaturally occurring. But the implementation of durability studies is limited in case of low prevalenceand low level of contamination.8/47TGD Lm shelf-life studiesEURL Listeria monocytogenes21/02/2019
3 Challenge testIf the challenge test is related to a range of products, only the product which is expected to give theworse-case scenario for L. monocytogenes growth is tested. This product is selected by the FBO,possibly with the help of the laboratory.A flow diagram describing schematically the different steps, from review of data to laboratoryprocedure, is given in clause 7.1.3.1 Review of dataPrimarily, the FBO is responsible for setting the shelf-life under defined conditions, which should takeinto account reasonably foreseeable conditions during transportation2, storage at retail and at consumerlevels.The FBO should be able to provide the laboratory with the following relevant product information inorder to define the experimental challenge testing procedure when a challenge test is needed:-Description of the product.-Composition of the RTE food.-Product characteristics; it is important to note if the values of certain characteristics changeduring the shelf-life.-Packaging condition of the end-product.-The main steps of the production process.-Characterisation of the cold chain.2From manufacturer to retail, including storage in the warehouses.9/47TGD Lm shelf-life studiesEURL Listeria monocytogenes21/02/2019
3.2 Protocols for challenge testA challenge test should be performed using the conditions which would be the most favourable forgrowth of L. monocytogenes within the possible range of each parame
1/47 TGD Lm shelf-life studies 21/02/2019 EURL Listeria monocytogenes EURL Lm TECHNICAL GUIDANCE DOCUMENT for conducting shelf-life studies on Listeria monocytogenes in ready-to-eat foods Version 3 of 6 June 2014 – Amendment 1 of 21 February 2019 Annie Beaufort, Hélène Bergis, Anne-Laure Lardeux, Unit Modelling of Bacterial Behaviour, Bertrand
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