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CRL L. monocytogenes Annie Beaufort, Hélène Bergis, Anne-Laure Lardeux

Shelf-life laboratory durability and challenge studies for Listeria monocytogenes in ready-to-eat foods: a presentation of the European technical guidance intended for laboratories. CRL L. monocytogenes Annie Beaufort, Hélène Bergis, Anne-Laure Lardeux.

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CRL L. monocytogenes Annie Beaufort, Hélène Bergis, Anne-Laure Lardeux

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  1. Shelf-life laboratory durability and challenge studies for Listeria monocytogenesin ready-to-eat foods: a presentation of the European technical guidance intended for laboratories CRL L. monocytogenes Annie Beaufort, Hélène Bergis, Anne-Laure Lardeux

  2. EC provided 2 guidance documents related to shelf-life for RTE foods 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?" Technical guidance document on shelf-life studies for L. monocytogenes in RTE foods, provided by CRL for Listeria monocytogenes • It helps the laboratories to implement: • - challenge tests • - durability studies.

  3. Listeria monocytogenes • L. monocytogenes is able to grow in hard conditions: •  temperature: -2°C •  pH: 4.2 •  aw: 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 • 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. EC Regulation on microbiological criteria for foodstuffs (No 2073/2005) • EU Regulation specifies that: “…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 Regulation has fixed the limit of L.m for RTE foods at 100 cfu/g at the market.

  6. First step of a shelf-life study regarding L. m: to collect information related to the characteristics of the food and research data ANNEX II of thisregulationspecificies studiesrelated to the evolution of L. m • Characteristics of the product: physical-chemical characteristics, preservatives content, type of packaging, process, foreseen shelf-life. • Availablescientific literature and research dataregarding the growth and survival characteristics of the micro-organisms of concern. And, when necessary, • Predictivemicrobiology, • Challenge tests: - challenge test assessing growth potential () • - challenge test assessing the maximum growth rate (µmax), • Durability studies.

  7. The characteristics of the food include: • the ingredients • the process • the shelf-life Each of these factors has an impact on the growth of L. m • pH • aw or salt content • 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 = 5.5 pH = 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 undervacuum 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 without associated flora with associated flora

  12. Studiesspecified in ANNEX II • Characteristics of the product: physical-chemical characteristics, preservatives content, type of packaging, process, foreseen shelf-life. • Availablescientific literature and research dataregarding the growth and survival characteristics of the micro-organisms of concern. Second step of a shelf-life study regarding L. m: to collect data from predictive microbiology software And, when necessary, • Predictivemicrobiology. • Challenge tests: - challenge test assessing growth potential () • - challenge test assessing the maximum growth rate (µmax). • Durability studies

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

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

  15. Studiesspecified in ANNEX II • Characteristics of the product: physical-chemical characteristics, preservatives content, type of packaging, process, foreseen shelf-life. • Availablescientific literature and research dataregarding the growth and survival characteristics of the micro-organisms of concern. And, when necessary, Third step of a shelf-life study regarding L. m: to implement laboratory tests. This is the scope of the technical guidance document. • Predictivemicrobiology. • Challenge tests: - challenge test assessing growth potential () • - challenge test assessing the maximum growth rate (µmax). • Durability studies

  16. 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. • Growth potentialiscalculated 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 concentrationat the production according to the limit of 100 cfu/g at the end of the shelf life.

  17. The challenge test assessing  needs the preparation of at least 14 test units for analyses at "Day 0" and "Day end". • At least 3 different batches are tested to take into account the variability of the production.

  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: • One of them is a reference strain • The others are isolated from the same food matrix or a similar food matrix The inoculation is made with or without depackaging.

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

  20. Or, if no information is available, the test units are stored according to conditions fixed by the EC. • Shelf-life ≤ 21 days 1/3 of the total shelf-life 8°C 7days 1/3 of the total shelf-life ½ (shelf-life – 7 days) 12°C ½ (shelf-life – 7 days) 1/3 of the total shelf-life 12°C

  21. Results 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.

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

  23. Studiesspecified in ANNEX II • Characteristics of the product: physical-chemical characteristics, preservatives content, type of packaging, process, foreseen shelf-life. • Availablescientific literature and research dataregarding the growth and survival characteristics of the micro-organisms of concern. And, when necessary, • Predictivemicrobiology. • Challenge tests: - challenge test assessing growth potential () • - challenge test assessing the maximum growth rate (µmax). Third step of a shelf-life study: to implement laboratory tests • Durability studies

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

  25. The challenge test to assess µmax needs the preparation of at least 20 tests units/batch. Most of the test units are used to draw the growth curve of L. m with a fast strain The experiment is repeated using another fast strain * 1 unit is enough is the product is homogeneous • At least 3 batches are tested to take into account the variability of the production. • The storage of the test units is made at a fixed temperature.

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

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

  28. Studiesspecified in ANNEX II • Characteristics of the product: physical-chemical characteristics, preservatives content, type of packaging, process, foreseen shelf-life. • Availablescientific literature and research dataregarding the growth and survival characteristics of the micro-organisms of concern. And, when necessary, • Predictivemicrobiology. Exploitation of existing results using durability studies • Challenge tests: - challenge test assessing growth potential () • - challenge test assessing the maximum growth rate (µmax). • Durability studies

  29. A durability study • 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.

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

  31. Results • All the results may be pooled. • The result is the proportion of units: • Exceeding 100 cfu/g • At the end of the shelf-life. The more units that are analysed, the narrower is the confidence interval

  32. Practically, according to the regulation, for a new product • If pH ≤ 4.4 or aw≤ 0.92 • If pH ≤ 5 and aw≤ 0.94 L. monocytogenes can’t grow For other conditions related to pH and aw • The ability of L. mto 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 (µmax) • or by predictive microbiology. For a product already commercialised • Durability studies give some information about the bacteria growth. How to combine the different tools?

  33. Thank you for your attention !

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