1. ISOPOL XVII, Porto 2010 CRL L. monocytogenes Annie Beaufort, Hélène Bergis, Anne-Laure Lardeux

2. Guidance document on L. monocytogenes shelf-life studies for RTE foods, provided by DG SANCO It helps the FBOs to answer to the question: "When and which shelf-life studies are needed?" It helps the laboratories to implement: - challenge tests - durability studies. Technical guidance document on shelf-life studies for L. monocytogenes in RTE foods, provided by CRL for Listeria monocytogenes

3. L. monocytogenes is able to grow in hard conditions: temperature: -2°C pH: 4.2 a w : 0.90 (in laboratory media, under optimum conditions) And, L. monocytogenes is a concern for RTE foods because RTE foods: may be contaminated by this bacteria may support the growth of L. m will be eaten without cooking.

4. Listeriosis is a severe disease that may cause septicemia or meningetis and mainly affects: unborn chidren the elderly persons with compromised immune system. For pregnant women, infection can lead to: miscarriage stillbirth premature delivery infection of the newborn. Listeriosis is associated to a high rate of morbidity: ~25%

5. …the FBOs….shall conduct studies … to investigate compliance with the criteria …. In particular, …for RTE foods able to support the growth of Listeria monocytogenes…. EU R egulation has fixed the limit of L.m for RTE foods at 100 cfu/g at the market. EU R egulation specifies that:

6. Characteristics of the product: physical-chemical characteristics, preservatives content, type of packaging, process, foreseen shelf-life. A vailable scientific literature and research data regarding the growth and survival characteristics of the micro-organisms of concern. And, when necessary, Challenge tests: - challenge test assessing growth potential ( ) - challenge test assessing the maximum growth rate (µ max ), Durability studies. Predictive microbiology, First step of a shelf-life study regarding L. m: to collect information related to the characteristics of the food and research data

7. The characteristics of the food include: the ingredients pH a w or salt content Each of these factors has an impact on the growth of L. m the process the shelf-life packaging atmosphere

8. The growth of L. m is influenced by the initial pH of the food. Evolution of L. m in jelly according to pH pH = 5 pH = 5.5

9. The growth of L. m is influenced by preservatives. Evolution of L. m in jelly according to potassium lactate [lactate] = 0 g/l [lactate] = 7 g/l

10. The growth of L. m is influenced by packaging atmosphere. Evolution of L. m in poultry ham dices according to gas atmosphere under vacuum gas atmosphere

11. The growth of L. m is influenced by the associated microflora. Evolution of L. m in raw diced bacon according to associated microflora with associated flora without associated flora

12. Characteristics of the product: physical-chemical characteristics, preservatives content, type of packaging, process, foreseen shelf-life. A vailable scientific literature and research data regarding the growth and survival characteristics of the micro-organisms of concern. And, when necessary, Challenge tests: - challenge test assessing growth potential ( ) - challenge test assessing the maximum growth rate (µ max ). Durability studies Predictive microbiology. Second step of a shelf-life study regarding L. m: to collect data from predictive microbiology software

13. To collect data related to growth probability For ex: the growth probability of L. m in a product at pH = 5.6 and a w = 0.958 is high.

14. Ex: growth simulation of L. m in a food (pH = 6; a w = 0,98) stored first at 4°C then at 8°C. To collect data related to growth simulations

15. Characteristics of the product: physical-chemical characteristics, preservatives content, type of packaging, process, foreseen shelf-life. A vailable scientific literature and research data regarding the growth and survival characteristics of the micro-organisms of concern. And, when necessary, Challenge tests: - challenge test assessing growth potential ( ) - challenge test assessing the maximum growth rate (µ max ). Durability studies Predictive microbiology. Third step of a shelf-life study regarding L. m: to implement laboratory tests. This is the scope of the technical guidance document. Third step of a shelf-life study regarding L. m: to implement laboratory tests. This is the scope of the technical guidance document.

16. Growth potential is calculated according to the formula: = ( L. m] at the end of the test) – ( L. m] at the beginning of the test) The growth potential can be used: To determine if a food permits the growth of L. m To set up the concentration of L. m at the end of the shelf life according to the concentration at the plant To set up the concentration at the production according to the limit of 100 cfu/g at the end of the shelf life. The challenge test assessing growth potential ( ) Is a laboratory test based on the growth of a bacteria in a food: Artificially contaminated Stored under foreseeable conditions from production to consumption.

17. Day 0Day end Determination of the concentration of L. m33 Detection/enumeration of L. m in blank samples (optional)33 Determination of physical-chemical characteristics1 to 3 Determination of the concentration of the associated flora33 At least 3 different batches are tested to take into account the variability of the production. The challenge test assessing needs the preparation of at least 14 test units for analyses at "Day 0" and "Day end".

18. The Inoculation of the test units used (to follow the evolution of L. m) is made with a mixture of at least 3 strains: The inoculation is made with or without depackaging. One of them is a reference strain The others are isolated from the same food matrix or a similar food matrix

19. The test units are stored according to collected information: 4 °C/12 d 8°C/26 d 8°C/ 26 days to mimic the storage at retail and atv the consumer Percentages Temperature 4°C/12 days to mimic transportation from plant to the display cabinet Percentages Temperature For example:.

20. Part of cold chain Storage temperature Storage duration Shelf-life > 21 days From plant to the display cabinet At retail At consumer 8°C 1/3 of the total shelf-life 12°C 1/3 of the total shelf-life 7 days ½ (shelf-life – 7 days) Shelf-life 21 days Or, if no information is available, the test units are stored according to conditions fixed by the EC.

21. For each batch, the growth potential is the difference between the median of the 3 results at "Day end"and the median of the 3 results at "Day 0". For further calculations, the highest growth potential (among 3) is considered. Results

22. How the growth potential is used? < 2 log cfu/g Initial concentration of L. m = 1 log cfu/g Final concentration of L. m = 1.88 log cfu/g Growth potential (δ) = 0.88 log cfu/g

23. Characteristics of the product: physical-chemical characteristics, preservatives content, type of packaging, process, foreseen shelf-life. A vailable scientific literature and research data regarding the growth and survival characteristics of the micro-organisms of concern. And, when necessary, Challenge tests: - challenge test assessing growth potential ( ) - challenge test assessing the maximum growth rate (µ max ). Predictive microbiology. Third step of a shelf-life study: to implement laboratory tests Durability studies

24. The challenge test assessing the maximum growth rate (µ max ) It may be considered as the daily growth rate of the bacteria. Lag phase Exponential phase Is a laboratory test based on the growth of a bacteria in a food: Artificially contaminated Stored at a fixed temperature.

25. Test units Growth curve of L. m10 to 15 Detection at day 0 and enumeration at day end of L.m in blank samples 3 + 3 Determination of physical-chemical characteristics3* + 3* Determination of the concentration of the associated flora2 Or 10 to 15 * 1 unit is enough is the product is homogeneous Most of the test units are used to draw the growth curve of L. m with a fast strain Test units Growth curve of L. m10 to 15 Detection at day 0 and enumeration at day end of L.m in blank samples 3 + 3 Determination of physical-chemical characteristics3* + 3* Determination of the concentration of the associated flora2 Or 10 to 15 The experiment is repeated using another fast strain The storage of the test units is made at a fixed temperature. The challenge test to assess µ max needs the preparation of at least 20 tests units/batch. At least 3 batches are tested to take into account the variability of the production.

26. MicroFit shows the experimental points, the fitted curve and assesses the µ max with its confidence interval. The calculation of µ max may be made with a sotfware (ex: MicroFit) Then, it is possible to deduce µ max at any other temperature T. µ maxT =

27. 0.20 log cfu/g For a RTE with a shelf-life of 10 days D0 D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 [ L. m] = 1.8 log cfu/g [L. m] = 2.4 log cfu/g [ L. m] = 2.0 log cfu/g [L. m] = 2.2 log cfu/g 0.20 log cfu/g µ max = 0.20 log cfu/g > 2 log cfu/g How the µ max is used?

28. Characteristics of the product: physical-chemical characteristics, preservatives content, type of packaging, process, foreseen shelf-life. A vailable scientific literature and research data regarding the growth and survival characteristics of the micro-organisms of concern. And, when necessary, Challenge tests: - challenge test assessing growth potential ( ) - challenge test assessing the maximum growth rate (µ max ). Durability studies Predictive microbiology. Exploitation of existing results using durability studies

29. Is a laboratory test based on the growth of a L. m in a food: Naturally contaminated Stored at foreseeable conditions. The different stages of a durability study are: Food sampling Storage conditions Microbiological analyses Calculation. A durability study

30. Is to select randomly n samples out of all the samples of a batch. Either on the "numbered units" Simple random sampling may be implemented with a software (e.g. Excel): Or on the "numbered production times". The aim of food sampling The storage of test units Is made at foreseeable conditions of temperature and duration.

31. The result is the proportion of units: Exceeding 100 cfu/g At the end of the shelf-life. Results All the results may be pooled. n Number of analysed units r Number of units >100 cfu/g p Estimated proportion CI Confidence Interval at 95 % 20 0 0 %[0 % - 16 %] 1000 %[0 % - 4 %] 20 1 5 %[1 % - 24 %] 1001 %[0.2 % - 5 %] 20 2 10 %[3 % - 30 %] 1002 %[0.6 % - 7 %] The more units that are analysed, the narrower is the confidence interval

32. If pH 4.4 or a w 0.92 If pH 5 and a w 0.94 L. monocytogenes cant grow Practically, according to the regulation, for a new product For a product already commercialised Durability studies give some information about the bacteria growth. For other conditions related to pH and a w The ability of L. m to grow in a food And the range of growth of L. m during the shelf-life may be assessed: by challenge test assessing growth potential ( ) or by predictive microbiology. The L. m concentration day by day may be assessed: by challenge test assessing the maximum growth rate (µ max ) or by predictive microbiology. How to combine the different tools?

33. Thank you for your attention !