EURL Lm European Union Reference Laboratory for Listeria monocytogenes. Version 0 03/02/2012

Transcription

1 EURL Lm European Union Reference Laboratory for Listeria monocytogenes Guidance document to evaluate the competence of laboratories implementing challenge tests on the growth potential of Listeria monocytogenes in ready-to-eat foods Version 0 03/02/2012 Annie BEAUFORT, Hélène BERGIS, Laurent GUILLIER, Anne-Laure LARDEUX (Unit Modelling of bacterial behaviours), Bertrand LOMBARD, EURL Manager In collaboration with the representatives of 12 National Reference Laboratories for Listeria monocytogenes: Marie POLET Institute of Public Health, Belgium; Nadine BOTTELDOORN Institute of Public Health, Belgium; George PAPAGEORGIOU Food Microbiology Laboratory State General Laboratory, Cyprus; Hana BULAWOVA Department of Food State Veterinary Institute, Czech Republic; Jens KIRK ANDERSEN National Food Institute Technical University of Denmark; Bernadette HICKEY Dairy Science Laboratory, Ireland; Gita TUPE Institute of Food Safety Animal Health and Environment, Latvia; Taran SKJERDAL Norwegian Veterinary Institute Section Feed and Food Microbiology, Norway; Celcidina PIRES GOMES Laboratório Nacional de Investigação Veterinária, Portugal; Majda BIASIZZO University of Ljubljana Veterinary faculty National Veterinary Institute, Slovenia; Cristina ACEBAL SARABIA Agencia Española de Seguridad Alimentar y Nutrición Centro Nacional de Alimentación, Spain; Susanne THISTED-LAMBERTZ National Food Administration, Sweden; Ife FITZ-JAMES Laboratory of Food and Consumer Product Safety Authority, the Netherlands. French Agency for Food, Environmental and Occupational Health Safety Maisons-Alfort Laboratory for Food Safety 23, avenue du Général de Gaulle - F94706 Maisons-Alfort Cedex - Telephone: Fax:

2 CONTENT 1 INTRODUCTION Scope References Challenge tests assessing growth potential of Listeria monocytogenes GENERAL INFORMATION RELATED TO THE LABORATORY AND THE FBO Information and requirements related to the laboratory Information related to the FBO REVIEW OF DATA PROVIDED BY THE FBO ASSESSMENT ON THE TECHNICAL SKILLS OF THE LABORATORY CONDUCTING CHALLENGE TESTS Experimental challenge testing procedure Use of the results of challenge tests assessing the growth potential (optional) Test report ASSESSMENT Annex 1: Definitions Annex 2: FBO information EURL for Listeria monocytogenes 2/24 03/02/2012

3 1 INTRODUCTION 1.1 Scope Regulation (EC) No 2073/ of 15 November 2005 on microbiological criteria for foodstuffs sets out specific food safety criteria for L. monocytogenes in ready-to-eat (RTE) foods (Annex I of the Regulation). Particularly for RTE foods, other than those intended for infants and for special medical purposes, placed on the market during their shelf-life, a quantitative limit of 100 cfu/g for L. monocytogenes has been laid down. According to article 3 point 2 of the Regulation, Food Business Operators (FBO) producing RTE foods, shall conduct studies in accordance with Annex II in order to investigate compliance with the criteria throughout the shelf-life. In order to guide RTE producers in identifying the L. monocytogenes risk in their RTE foods and laboratories in implementing the shelf-life studies, two guidance documents have been produced: - Guidance document on Listeria monocytogenes shelf-life studies for ready to eat foods, under Regulation (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs, DG-Sanco 1628/2008 ver. 9.3, (DG-SANCO guidance document), intended for FBO, - Technical guidance document on shelf-life studies for Listeria monocytogenes in RTE foods CRL for Listeria monocytogenes version.2 - November 2008, (TGD Nov. 2008), intended for laboratories. The laboratory shelf-life studies, especially durability studies and challenge tests, should be implemented properly and carried out in laboratories having the required expertise for such studies and demonstrating good laboratory practices. The purpose of this document is to provide guidance for evaluating the competence of laboratories implementing shelf-life studies according to Annex II of the Regulation, especially challenge tests aimed at assessing the growth potential of Listeria monocytogenes in RTE foods: - to evaluate the required expertise for shelf-life studies, - to ensure that challenge tests are implemented according to the Technical Guidance Document (TGD, Nov. 2008). After an introduction and general information related to the laboratory and the FBO (parts 1 and 2), the part 3 of this document covers the justification of challenge tests, based on the DG-SANCO Guidance document (SANCO/1628/2008 ver.9.3). The FBO is guided on the necessity or not to implement shelflife studies (e.g. durability and challenge tests). The purpose of this part is also to ensure that the laboratory has collected the relevant information to be used to implement the challenge test. The part 4 of this document aims at ensuring that challenge tests are conducted in compliance with the EURL Lm Technical guidance document on shelf-life studies for Listeria monocytogenes in RTE foods. The present document, based on the aforementioned guidance documents (SANCO/1628/2008 ver.9.3 and TGD, Nov. 2008) may be used as a reference document, in order to conduct uniform assessment of laboratories performing challenge tests. Tables in the document provide check-lists as support for the assessment. 1 OJ L 338, , p.1. EURL for Listeria monocytogenes 3/24 03/02/2012

4 1.2 References - EN ISO 7218 (2007) Microbiology of food and animal feeding stuffs General requirements and guidance for microbiological examinations - EN ISO (1996), amended (2005) Microbiology of food and animal feeding stuffs Horizontal method for the detection and enumeration of Listeria monocytogenes Part 1: Detection method - EN ISO (1998), amended (2005) Microbiology of food and animal feeding stuffs Horizontal method for the detection and enumeration of Listeria monocytogenes Part 2: Enumeration method - EN ISO/CEN (2005) General requirements for the competence of testing and calibration of laboratories - Guidance document on Listeria monocytogenes shelf-life studies for ready to eat foods, under Regulation (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs, Commission of the European Communities, SANCO/1628/2008 ver. 9.3 ( ) - Regulation (EC) No. 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs amended (2007) - Technical guidance document on shelf-life studies for Listeria monocytogenes in ready-to-eatfoods, CRL for Listeria monocytogenes, version 2 November X (1982) Environment and basic resources. Vocabulary for the channel of raw materials. 1 - Main terms and vulnerability analysis. 1.3 Challenge tests assessing growth potential of Listeria monocytogenes A challenge test assessing the growth potential is a microbiological laboratory-based study that measures the growth of L. monocytogenes in artificially contaminated food stored under foreseeable storage conditions from the end of production until consumption. The growth potential ( ) is defined as the difference between log 10 cfu of L. monocytogenes per gram of food at the end of the test and at the beginning of the test. The growth potential ( ) depends on many factors, the most important being: - intrinsic properties of the food (e.g. ph, NaCl content, a w, associated microflora, preservatives), - extrinsic properties (e.g. time-temperature profile, packaging conditions), - the physiological state of the inoculated strain(s), - the inoculated strain(s). EURL for Listeria monocytogenes 4/24 03/02/2012

5 To comply with the microbiological criteria for Listeria monocytogenes set out in Annex I (food categories 1.2 and 1.3) of Regulation (EC) No. 2073/2005, the growth potential ( ) at a given timetemperature scenario can be used: - To classify a food: when 0.5 log 10 cfu/g, the RTE food is classified as Ready-to-eat foods unable to support the growth of L. monocytogenes (category 1.3), when > 0.5 log 10 cfu/g, the RTE food is classified as Ready-to-eat foods able to support the growth of L. monocytogenes (category 1.2). - To assess the growth of L. monocytogenes in a RTE food of category 1.2 according to defined reasonably foreseeable conditions of storage between production and consumption. - To permit the calculation of the concentration of L. monocytogenes at production, taking into account the limit of 100 cfu/g at the end of shelf-life. EURL for Listeria monocytogenes 5/24 03/02/2012

6 2 GENERAL INFORMATION RELATED TO THE LABORATORY AND THE FBO 2.1 Information and requirements related to the laboratory The laboratory conducting challenge tests must be EN ISO accredited for the detection and enumeration of L. monocytogenes in food. For other bacterial parameters, such as other bacteria useful to better interpret the results of the challenge test, validity of analyses through regular participation to proficiency testing (PT) trials for other bacteria than L. monocytogenes is sufficient. For physico-chemical parameters, such as ph, a w, preservatives, validity of analyses through regular participation to PT trials or regular metrological checking is acceptable. - Identification of the laboratory Laboratory name: Contact person and his/her position: Address: Phone: - Technical skills of the laboratory Accreditation according to EN ISO 17025, for: the detection of L. monocytogenes the enumeration of L. monocytogenes Validity of analyses through regular participation to PT trials for other bacteria (useful for the test interpretation) Validity of analyses through regular participation to ph PT trials or regular metrological ph checking Validity of analyses through regular participation to a w PT trials or regular metrological a w checking Validity of analyses through regular participation to PT trials or regular metrological ph checking for other parameter (only the ones being useful for the test) Bacteria: Parameter: - Experience in conducting challenge tests assessing the growth potential of L. monocytogenes Challenge tests conducted since: (year) Frequency of challenge tests achieved per year to determine growth potential: 2.2 Information related to the FBO - Identification of the FBO Company name: Business activity: Contact person: Address: Phone: EURL for Listeria monocytogenes 6/24 03/02/2012

7 3 REVIEW OF DATA PROVIDED BY THE FBO The necessary product information for conducting shelf-life studies is stated in the DG-SANCO Guidance document (SANCO/1628/2008 ver. 9.3). The FBO should provide relevant product information (product characteristics e.g. ph, a w, storage temperature, shelf-life, ) and all necessary data (e.g. appropriate historical data for the growth of L. monocytogenes in the product and/or from previous shelf-life studies). These data must provide the laboratory sufficient information to define the experimental challenge testing procedure. Annex 2 lists the most relevant information of the RTE food to be provided by the FBO in order to set up the appropriate experimental challenge testing procedure in the laboratory. DESCRIPTION OF THE PRODUCT Name of the product Comments Shelf-life Labeled storage temperature (range) Weight of the sale unit Annual or seasonal production COMPOSITION OF THE RTE FOOD Expected information Information provided Comments List of ingredients on the label. EURL for Listeria monocytogenes 7/24 03/02/2012

8 PRODUCT CHARACTERISTICS Expected information Information provided Comments ph in the end-product 2 (or ph dynamic profile) a w in the end-product 2 (or a w dynamic profile) Salt content (%) Humidity (%) Sugar content (%) Preservative concentration in the end-product Technological microflora (e.g. starter cultures, probiotics) added Total microflora, naturally present in the end-product Lactic acid bacteria in the endproduct Other specific spoilage microflora, e.g. Pseudomonas, in the endproduct PACKAGING CONDITION OF THE END-PRODUCT Expected information Information provided Comments Packaging conditions: gas atmosphere (air, vacuum packaging, modified atmosphere packaging) Control of the gas atmosphere: is the composition of the gas atmosphere checked? PROCESS Expected information Information provided Comments Different stages of the process 2 If the product is heterogeneous, ph and/or aw must be measured in the part(s) that is (are) most likely to be contaminated by Listeria monocytogenes EURL for Listeria monocytogenes 8/24 03/02/2012

9 CHARACTERISATION OF THE COLD CHAIN Expected information Information provided Comments Temperature Time Storage temperature and time: At manufacturing level During distribution At retail level At household level SCIENTIFIC LITERATURE OR RESEARCH DATA (OPTIONAL) Expected information Information provided Comments Data from scientific literature or research data such as: - Values related to factors (or combination of factors) affecting the survival and growth of L. m. - Evolution of L. m. in products identical or similar to the studied product - Other data HISTORICAL DATA Expected information Information provided Comments Prevalence of L. m in the specific RTE food Data exceeding the limit of 100 cfu/g for L. monocytogenes at the end of shelf-life, if initially present in the food PREDICTIVE MICROBIOLOGY (MODELLING) (OPTIONAL) Expected information Information provided Comments Software used or Model used Module/Modelling toolbox used Factor values introduced in the model Growth probability and/or growth simulation EURL for Listeria monocytogenes 9/24 03/02/2012

10 OUTCOME OF THE FIRST STEP (see the flow chart below): Outcome based on the review of data (collected in Annex 2) Are the collected data relevant to perform a challenge test? No Has the laboratory performed the challenge test? No Yes Has the laboratory performed the challenge test? 1 No Yes Are the given reasons satisfactory? 3 No 2 Yes 4 Yes Comments of the assessor related to answers 1 to 4, from the flow chart. Answers 1 Comments EURL for Listeria monocytogenes 10/24 03/02/2012

11 4 ASSESSMENT ON THE TECHNICAL SKILLS OF THE LABORATORY CONDUCTING CHALLENGE TESTS 4.1 Experimental challenge testing procedure The following items, identified in the EURL Lm Technical guidance document on shelf-life studies for Listeria monocytogenes in ready-to-eat-foods, (TGD Nov. 2008), should be specified in the challenge testing experimental design implemented by the laboratory. Particular points to be checked 1. Number of batches tested 2. Batches representative of the production period Specifications of the TGD Nov At least 3 batches, with the same ingredients, physicochemical characteristics, packaging conditions, process, etc. Repartition representative of the batches tested all over the production period BATCHES Information provided Deviations Comments Yes Planned Unplanned No EURL for Listeria monocytogenes 11/24 03/02/2012

12 Particular points to be checked 1. Number of strains used 2. Source of strains (food origin or reference) 3. Number of subcultures performed 4. T of the 1 st subculture Specifications of the TGD Nov PREPARATION OF THE INOCULUM Information provided Deviations Comments Yes Planned Unplanned At least 3 different strains Source known (2 strains from the same food or a food similar to the one being tested and one reference strain) 2 subcultures At optimal T (between C) No 5. T of the 2 nd subculture T near the storage T of the product 6. Phase of the 2 nd subculture 7. Concentration of the 2 nd subculture 8. Concentration of the mixture of the strains 9. Dilution of the inoculum 10. Enumeration of the inoculum Early stationary phase Determined Determined Diluted in physiological water Determined EURL for Listeria monocytogenes 12/24 03/02/2012

13 Particular points to be checked 1. Choice of the part of the RTE food to be contaminated (for an heterogeneous product) 2. Number of contaminated test units per batch INOCULATION OF SAMPLES Specifications of the TGD Information provided Deviations Comments Nov Yes Planned Unplanned No Identification of the ingredient potentially contaminated by L. m At least 6 samples 3. Method of inoculation Reflecting the likely type of contamination during the process 4. With or without depackaging 5. Volume of inoculation 6. Level of targeted inoculation Vol inoc. < 1% Vol sample Between cfu/g Particular points to be checked 1. Choice of the storage temperature STORAGE CONDITIONS Specifications of the TGD Information provided Deviations Comments Nov Yes No Planned Unplanned Justified by detailed information Based on the T set in the table 2 of TGD Nov Choice of the storage duration Justified by detailed information Based on the duration set in the table 2 of TGD Nov EURL for Listeria monocytogenes 13/24 03/02/2012

14 Particular points to be checked 1. Measurement of physicochemical parameters 2. Measurement of gas atmosphere Specifications of the TGD Nov ph a w Preservatives Methods used (reference methods) for determination of: - ph - a w - - CHARACTERISATION OF THE RTE FOOD Information provided Deviations Comments Yes Planned Unplanned No 3. Enumeration of associated microflora Total microflora Lactic acid bacteria Other bacteria specified 4. Detection and / or enumeration of L. m in blank test units Reference method for detection Alternative method (ISO validated) for detection Reference method for enumeration Alternative method (ISO 16140validated) for enumeration Particular points to be checked 1. Enumeration method ENUMERATION METHODS Specifications of the TGD Information provided Deviations Comments Nov Yes No Planned Unplanned EN ISO method amended Alternative method validated according to Article 5 of Regulation No 2073/ Enumeration threshold Limit of enumeration EURL for Listeria monocytogenes 14/24 03/02/2012

15 Particular points to be checked 1. Calculation of the concentration values at D 0 2. Calculation of the standard deviation Specifications of the TGD Nov Values expressed in log 10 cfu/g Standard deviation between the 3 log 10 -results of the 3 different samples at D 0 is < 0.3 log 10 cfu/g CALCULATIONS Information provided Deviations Comments Yes Planned Unplanned No 3. Calculation of the concentration values at D end Values expressed in log 10 cfu/g 4. Calculation of the growth potential per batch Difference between the median values at D 0 and at D end OUTCOME OF THE SECOND STEP: Assessment of the technical skills of the laboratory conducting challenge test on the growth potential of L. monocytogenes, based on the EURL Lm Technical guidance document on shelf-life studies for Listeria monocytogenes in RTE foods. Assessor s comments related to: Items Batches Comments Preparation of the inoculum Inoculation of samples Storage conditions Characterisation of the RTE food Enumeration methods Calculation EURL for Listeria monocytogenes 15/24 03/02/2012

16 4.2 Use of the results of challenge tests assessing the growth potential (optional) This part may be fulfilled by the laboratory according to previous agreement with the FBO. Particular points to be checked 1. The maximal value is derived from at least the 3 tested batches Expected information Information provided Comments 2. Classification of RTE food Based on δ obtained from the challenge-tests The RTE food is able to support growth of L. m 3. Calculation of the initial concentration to respect the limit of 100 cfu/g Limit of 100 cfu/g exceeded at the end of the shelf-life 4. Other purposes for the analyses EURL for Listeria monocytogenes 16/24 03/02/2012

17 4.3 Test report Points to be checked Expected information Information provided Comments 1. Report reference Reference number 2. Accreditation number 3. Purpose of study 4. Identification of the RTE food Characteristics of the RTE food The stated shelf-life of the product, including a safety margin Identification of the batches 5. Data relating to the challenge test Number of batches tested Number of test units used per batch Mass or volume of the test unit inoculated Strains used Preparation of the inoculum Inoculum concentration Volume of inoculum introduced per test unit Contamination method Contamination level targeted Day of inoculation Justification of the storage temperature Justification of the storage duration Enumeration and detection methods used Limit of the enumeration Physico-chemical values at D 0 and at D end Concentration of associated microflora at D 0 and at D end EURL for Listeria monocytogenes 17/24 03/02/2012

18 Concentration of specific microflora at D 0 and at D end Concentration of L. m at D 0 and at D end Growth potential per batch Conclusion EURL for Listeria monocytogenes 18/24 03/02/2012

19 5 ASSESSMENT The laboratory is able to perform challenge tests assessing the growth potential based on relevant information provided by the FBO and according to TGD Nov YES NO Comments The laboratory should be able to perform challenge tests provided that improvements are made on certain items YES NO Comments Description of the items which need to be improved EURL for Listeria monocytogenes 19/24 03/02/2012

20 ANNEX 1: Definitions (Sources: DG-SANCO Guidance document (SANCO/1628/2008 ver. 9.3) and X30-010) Batch A group or set of identifiable products obtained from a given process under practically identical circumstances in a given place within one defined production period. Day 0 (D 0 ) Date of production or packaging. Day End (D end ) Last day of the shelf-life as defined by FBO and expressed at a product by "use by" date. End-product Product collected at the end of the process, ready-to-be sold Food additive Substance added to food to preserve flavour or enhance its taste and appearance. ph A measure of the acidity or alkalinity of a food. The ph 7 is defined as neutral. Values of a ph less than seven are considered acidic and those with greater than seven are considered basic (alkaline). Preservative A naturally occurring or synthetic substance added to products such as food, pharmaceuticals, paints, biological samples, wood, to prevent decomposition by microbial growth or by undesirable chemical changes. Raw material Mineral or organic material of natural origin, not used as such, at the moment of consumption and generally requiring treatment to extract the component materials. Ready to eat (RTE) food Food intended by the producer or the manufacturer for direct human consumption without the need for cooking or other processing effective to eliminate or reduce to an acceptable level microorganisms of concern. Shelf life Either period corresponding to the period preceding the 'use by' or the minimum durability date, as defined respectively in Articles 9 and 10 of Directive 2000/13/EC concerning, among others, the labelling of foodstuffs. Water activity (a w ) The term refers to the unbound and available water in a food and is not the same as the water content of the food. Water in food which is not bound to other molecules can support the growth of microbes. The water activity scale extends from 0 to 1.0 (pure water) but most foods have a water activity level in the range of 0.2 for very dry foods to 0.99 for moist fresh foods. EURL for Listeria monocytogenes 20/24 03/02/2012

21 ANNEX 2: FBO information Identification of the FBO Company name: Business activity: Contact person and his/her position: Address: Phone: Expected data on the product The laboratory needs to review data on the concerned RTE food provided by the FBO: - to propose the FBO to implement or not the challenge test and - to define the challenge testing experimental procedure. Note: All tables below must apply to end-products with identical formulation and process. Name of the product Shelf-life DESCRIPTION OF THE PRODUCT Labeled storage temperature (range) Weight of the sale unit Annual or seasonal production - Raw materials (e.g: pork, salmon) - Processed products (e.g: cooked ham, cheese) - Preservatives (name and E-number) COMPOSITION OF THE RTE FOOD List of ingredients EURL for Listeria monocytogenes 21/24 03/02/2012

22 VARIABILITY OF THE PRODUCT CHARACTERISTICS ph in the end-product 3 (or ph dynamic profile) Minimal value Maximal value Mean and standard deviation Number of results Period (per year) a w in the end-product 3 (or a w dynamic profile) Minimal value Maximal value Mean and standard deviation Number of results Period (per year) Salt content in the end-product (%) Minimal value Maximal value Mean and standard deviation Number of results Period (per year) Humidity in the end-product (%) Minimal value Maximal value Mean and standard deviation Number of results Period (per year) Sugar content in the end-product (%) Minimal value Maximal value Mean and standard deviation Number of results Period (per year) Name Preservatives concentration in the end-product Mean and Number of results E-number standard (approximately) deviation Period (per year) Equally distributed in the heterogeneous food or not (if known) Yes No 3 If the product is heterogeneous, ph and/or aw must be measured in the part(s) that is (are) most likely to be contaminated by Listeria monocytogenes EURL for Listeria monocytogenes 22/24 03/02/2012

23 Addition of technological microflora (e.g: starter cultures, probiotics) If Yes, please specify Total microflora, naturally present in the product, (cfu/g) in the end-product Minimal value Maximal value Mean and standard deviation Number of results Period (per year) Lactic acid bacteria (cfu/g) in the end-product, if relevant Minimal value Maximal value Mean and standard deviation Number of results Period (per year) Other specific spoilage microflora, e.g. Pseudomonas, (cfu/g), in the end-product Minimal value Maximal value Mean and standard deviation Number of results Period (per year) Packaging condition of the end-product Gas atmosphere (air, vacuum, modified atmosphere packaging) Type of packaging Is the composition of the gas atmosphere checked? Please specify PROCESS Present the different stages of the process. CHARACTERISATION OF THE COLD CHAIN Please specify information on storage temperature and time during different stages of the cold chain: At manufacturing level During the distribution At retail level At household level EURL for Listeria monocytogenes 23/24 03/02/2012

24 SCIENTIFIC LITERATURE OR RESEARCH DATA (OPTIONAL) Give the relevant information related to: factors (or combination of factors) affecting the growth of L. m, evolution of L. m in products identical or similar to the studied product, RTE food involved in outbreaks, others. Expected information Prevalence of L. m in the specific RTE food HISTORICAL DATA Period (per year) Number of data Number of L. m positive results Expected information Data exceeding the limit of 100 cfu/g for L. monocytogenes at the end of the shelflife, if initially present in the food Period (per year) Number of data Number of results > 100 cfu/g Consequences? Corrective actions? PREDICTIVE MICROBIOLOGY (OPTIONAL) Expected information Information provided Software used (e.g.: Pathogen Modeling Program, Growth Predictor, Seafood Safety Spoilage Predictor, Sym Previus, ) or model used Module/Modelling toolbox used Factor values introduced in the model Result for growth probability and/or for growth simulation EURL for Listeria monocytogenes 24/24 03/02/2012