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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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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
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.
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.
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%
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.
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.
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
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
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
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
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
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
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. ●
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.
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
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.
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.
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.
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
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
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.
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
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
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.
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.
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 =
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
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
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.
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.
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
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?