ISOPOL XVII, Porto 2010

CRL L. monocytogenesAnnie Beaufort, Hélène Bergis, Anne-Laure Lardeux

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

• 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 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%

“…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.

• EU Regulation specifies that:

Characteristics of the product: physical-chemical characteristics, preservatives content, type of packaging, process, foreseen shelf-life.

Available 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 (µmax),

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

The characteristics of the food include:

the ingredients

pH

aw or salt content

Each of these factors has an impact on the growth of L. m

the process

the shelf-life

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

pH = 5.5

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

under vacuum

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

with associated flora

without associated flora

Characteristics of the product: physical-chemical characteristics, preservatives content, type of packaging, process, foreseen shelf-life.

Available 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 (µmax). Durability studies

Predictive microbiology.

Second step of a shelf-life study regarding L. m: to collect data from predictive microbiology software

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.

●

Ex: growth simulation of L. m in a food (pH = 6; aw = 0,98) stored first at 4°C then at 8°C.

To collect data related to growth simulations

Characteristics of the product: physical-chemical characteristics, preservatives content, type of packaging, process, foreseen shelf-life.

Available 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 (µmax). 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.

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.

Day 0 Day end

Determination of the concentration of L. m 3 3

Detection/enumeration of L. m in blank samples (optional) 3 3

Determination of physical-chemical characteristics 1 to 3 1 to 3

Determination of the concentration of the associated flora 3 3

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".

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

The test units are stored according to collected information:

0

2

4

6

8

10

0 4 8 12 16 20 24 28 32 36 40

Days

4°

C/1

2 d

8°C

/26

d

• 8°C/ 26 days to mimic the storage at retail and atv the consumer

Histogramme

Moyenne de la température

Pou

rcen

tage

s

0 3 6 9 12 15 180

4

8

12

16

20

Per

cent

ages

Temperature

• 4°C/12 days to mimic transportation

from plant to the display cabinet

Histogramme

Moyenne de la température

Pourc

enta

ges

0 2 4 6 8 100

4

8

12

16

20

24

Per

cent

ages

Temperature

For example:.

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

12°C

1/3 of the total shelf-life

1/3 of the total shelf-life

7 days

½ (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.

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

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

Characteristics of the product: physical-chemical characteristics, preservatives content, type of packaging, process, foreseen shelf-life.

Available 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 (µmax).

Predictive microbiology.

Third step of a shelf-life study: to implement laboratory tests

Durability studies

The challenge test assessing the maximum growth rate (µmax)

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.

Test units

Growth curve of L. m 10 to 15

Detection at “day 0” and enumeration at “day end” of L.m in blank samples

3 + 3

Determination of physical-chemical characteristics 3* + 3*

Determination of the concentration of the associated flora 2

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. m 10 to 15

Detection at “day 0” and enumeration at “day end” of L.m in blank samples

3 + 3

Determination of physical-chemical characteristics 3* + 3*

Determination of the concentration of the associated flora 2

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 µmax 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.

MicroFit shows the experimental points, the fitted curve and assesses the µmax with its confidence interval .

The calculation of µmax may be made with a sotfware (ex: MicroFit)

Then, it is possible to deduce µmax at any other temperature T. 

µmaxT =

2minref

2min

maxTT

TTμ

ref

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

µmax = 0.20 log cfu/g

> 2 log cfu/g

How the µmax is used?

Characteristics of the product: physical-chemical characteristics, preservatives content, type of packaging, process, foreseen shelf-life.

Available 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 (µmax).

Durability studies

Predictive microbiology.

Exploitation of existing results using durability studies

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

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.

The result is the proportion of units:

• Exceeding 100 cfu/g

• At the end of the shelf-life.

ResultsAll 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 %

200

0 % [0 % - 16 %]

100 0 % [0 % - 4 %]

201

5 % [1 % - 24 %]

100 1 % [0.2 % - 5 %]

202

10 % [3 % - 30 %]

100 2 % [0.6 % - 7 %]

The more units that are analysed,

the narrower is the confidence interval

If pH ≤ 4.4 or aw≤ 0.92 If pH ≤ 5 and aw≤ 0.94 L. monocytogenes can’t 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 aw

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 daymay be assessed:

by challenge test assessing the maximum growth rate (µmax)

or by predictive microbiology.

How to combine the different tools?

Thank you for your attention !Thank you for your attention !