SIST EN ISO 20976-2:2023
(Main)Microbiology of the food chain - Requirements and guidelines for conducting challenge tests of food and feed products - Part 2: Challenge tests to study inactivation potential and kinetic parameters (ISO 20976-2:2022)
Microbiology of the food chain - Requirements and guidelines for conducting challenge tests of food and feed products - Part 2: Challenge tests to study inactivation potential and kinetic parameters (ISO 20976-2:2022)
This document specifies protocols for conducting microbiological challenge tests for growth studies on vegetative and spore-forming bacteria in raw materials and intermediate or end products.
The use of this document can be extended to yeasts that do not form mycelium.
Mikrobiologie der Lebensmittelkette - Anforderungen und Leitfaden zur Durchführung von Challenge-Tests bei Lebensmitteln und Futtermitteln - Teil 2: Challenge-Tests zur Untersuchung von Inaktivierungspotenzial und kinetischer Parameter (ISO 20976-2:2022)
Dieses Dokument legt Verfahrensanweisungen für die Durchführung von mikrobiologischen Challenge-Tests für Inaktivierungsuntersuchungen an vegetativen Bakterien und Bakteriensporen in den Ausgangsstoffen und Inhaltsstoffen sowie in Zwischen- oder Endprodukten fest.
Die Verwendung dieses Dokuments kann auf Hefen erweitert werden, die kein Myzel bilden.
Microbiologie de la chaîne alimentaire - Exigences et lignes directrices pour la réalisation des tests d'épreuve microbiologique - Partie 2: Tests d’inactivation pour étudier le potentiel d’inactivation et les paramètres de kinétique (ISO 20976-2:2022)
Le présent document spécifie les protocoles de mise en œuvre de tests de croissance sur les bactéries végétatives et sporulées dans les matières premières, les produits intermédiaires ou produits finis.
L'utilisation du présent document peut être étendue aux levures qui ne forment pas de mycélium.
Mikrobiologija v prehranski verigi - Zahteve in smernice za vodenje preskusa ustreznosti kmetijskih pridelkov in živilskih proizvodov - 2. del: Preskus ustreznosti za študijo inaktivacijskega potenciala in kinetičnih parametrov (ISO 20976-2:2022)
Ta dokument določa protokole za vodenje preskusov ustreznosti za študije rasti vegetativnih in sporastih bakterij v surovih živilih in vmesnih ali končnih proizvodih.
Uporabo tega dokumenta je mogoče razširiti na kvasovke, ki ne tvorijo micelija.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-januar-2023
Mikrobiologija v prehranski verigi - Zahteve in smernice za vodenje izzivnega
preskusa pri kmetijskih pridelkih in živilskih proizvodih - 2. del: Izzivni preskus za
raziskavo inaktivacijskega potenciala in kinetičnih parametrov (ISO 20976-2:2022)
Microbiology of the food chain - Requirements and guidelines for conducting challenge
tests of food and feed products - Part 2: Challenge tests to study inactivation potential
and kinetic parameters (ISO 20976-2:2022)
Mikrobiologie der Lebensmittelkette - Anforderungen und Leitfaden zur Durchführung
von Challenge-Tests bei Lebensmitteln und Futtermitteln - Teil 2: Challenge-Tests zur
Untersuchung von Inaktivierungspotenzial und kinetischer Parameter (ISO 20976-
2:2022)
Microbiologie de la chaîne alimentaire - Exigences et lignes directrices pour la réalisation
des tests d'épreuve microbiologique - Partie 2: Tests d’inactivation pour étudier le
potentiel d’inactivation et les paramètres de kinétique (ISO 20976-2:2022)
Ta slovenski standard je istoveten z: EN ISO 20976-2:2022
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN ISO 20976-2
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2022
EUROPÄISCHE NORM
ICS 07.100.30
English Version
Microbiology of the food chain - Requirements and
guidelines for conducting challenge tests of food and feed
products - Part 2: Challenge tests to study inactivation
potential and kinetic parameters (ISO 20976-2:2022)
Microbiologie de la chaîne alimentaire - Exigences et Mikrobiologie der Lebensmittelkette - Anforderungen
lignes directrices pour la réalisation des tests und Leitfaden zur Durchführung von Challenge-Tests
d'épreuve microbiologique - Partie 2: Tests bei Lebensmitteln und Futtermitteln - Teil 2:
d'inactivation pour étudier le potentiel d'inactivation Challenge-Tests zur Untersuchung von
et les paramètres de kinétique (ISO 20976-2:2022) Inaktivierungspotenzial und kinetischer Parameter
(ISO 20976-2:2022)
This European Standard was approved by CEN on 19 September 2022.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20976-2:2022 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
European foreword
This document (EN ISO 20976-2:2022) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with Technical Committee CEN/TC 463 “Microbiology of the food chain” the
secretariat of which is held by AFNOR.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by May 2023, and conflicting national standards shall be
withdrawn at the latest by May 2023.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 20976-2:2022 has been approved by CEN as EN ISO 20976-2:2022 without any
modification.
INTERNATIONAL ISO
STANDARD 20976-2
First edition
2022-10
Microbiology of the food chain —
Requirements and guidelines for
conducting challenge tests of food and
feed products —
Part 2:
Challenge tests to study inactivation
potential and kinetic parameters
Microbiologie de la chaîne alimentaire — Exigences et lignes
directrices pour la réalisation des tests d'épreuve microbiologiques —
Partie 2: Tests d’inactivation pour étudier le potentiel d’inactivation
et les paramètres de la cinétique d’inactivation
Reference number
ISO 20976-2:2022(E)
ISO 20976-2:2022(E)
© ISO 2022
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
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CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
ISO 20976-2:2022(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 5
5 Apparatus . 7
6 Culture media and reagents .7
7 Study design and sampling . 7
7.1 General . 7
7.2 Setting target reduction level for the inactivation study . 8
7.3 Number of batches. 8
7.4 Preparation of the test units . 8
7.5 Number of control units and test units . 9
8 Selection of strains . 9
9 Preparation of the inoculum . 9
9.1 General . 9
9.2 Preparation of the vegetative cells . 10
9.3 Preparation of the spores . 10
10 Inoculation of the test units .10
11 Controls . .12
11.1 Uninoculated controls .12
11.2 Inoculated controls . 12
12 Treatment of the test units .12
13 Analysis . .12
14 Expression of the results .13
14.1 General .13
14.2 Inactivation potential .13
14.3 Inactivation kinetics parameters. 14
14.3.1 General . 14
14.3.2 Primary inactivation kinetics parameters . 14
14.3.3 Secondary inactivation kinetics parameters . 15
14.4 Simulation of inactivation .15
15 Test report .16
15.1 General . 16
15.2 Aim of the study, type of challenge test and target reduction level . 16
15.3 Experimental protocol . . 17
15.4 Sample analysis . . 17
15.5 Results . . 17
15.6 Conclusions . 18
15.7 Reference documents . 18
Annex A (informative) Selection of the type and the location of inactivation study .19
Annex B (normative) Minimum number of units to prepare for the inactivation studies .20
Annex C (informative) Examples of inoculation techniques .21
Bibliography .23
iii
ISO 20976-2:2022(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology, in collaboration with the European Committee for Standardization (CEN) Technical
Committee CEN/TC 463, Microbiology of the food chain, in accordance with the Agreement on technical
cooperation between ISO and CEN (Vienna Agreement).
A list of all parts in the ISO 20976 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
ISO 20976-2:2022(E)
Introduction
Under the general principles of the Codex Alimentarius on food hygiene, it is the responsibility of the
food business operators (FBOs) to control microbiological hazards in foods and to manage microbial
risks. Therefore, FBOs implement validated control measures, within the hazard analysis and critical
control point (HACCP) system, and conducts studies in order to investigate compliance with the food
safety criteria throughout the food chain.
In the framework of microbial risk assessment (MRA), several complementary approaches are developed
to estimate risks posed by pathogens or spoilage microorganisms in the food chain. MRA is adopted by
regulators under the auspices of the international agency for setting food standards. Challenge testing
is one of the recognized approaches used to validate control measures within the HACCP system, as
well as to assess microbiological safety and quality of food, food production processes, food storage
conditions, and food preparation recommendations dedicated to consumers.
Therefore, this document provides technical rules, calculations and approaches to investigate the ability
of an inoculated microorganism of concern to grow, survive or be inactivated in the raw materials,
intermediate or end products under reasonably foreseeable food processes, storage and use conditions.
The objective and the scope of the study are to determine the experimental design and the selection
of the study conditions, and to assess the extent of microbial inactivation. Regulatory authorities can
have different recommendations, and these differences have been included as much as possible. It is,
however, possible that specific requirements need to be incorporated to get a regulatory approval of
the challenge test.
As the growth and inactivation studies are clearly different, the ISO 20976 series consists of two parts,
under the general title Microbiology of the food chain — Requirements and guidelines for conducting
challenge tests of food and feed products:
— Part 1: Challenge tests to study the growth potential, lag time and the maximum growth rate;
— Part 2: Challenge tests to study inactivation potential and kinetic parameters.
The use of the ISO 20976 series involves expertise in relevant areas such as food microbiology, food
science, food processing and statistics. The statistical expertise encompasses an understanding of
sampling theory and design of experiments, statistical analysis of microbiological data, and overview of
scientifically recognized and available mathematical concepts used in predictive modelling.
For practical reasons, the term “food” includes feed.
v
INTERNATIONAL STANDARD ISO 20976-2:2022(E)
Microbiology of the food chain — Requirements and
guidelines for conducting challenge tests of food and feed
products —
Part 2:
Challenge tests to study inactivation potential and kinetic
parameters
1 Scope
This document specifies the protocols for conducting microbiological challenge tests for inactivation
studies on vegetative bacteria and bacterial spores in the raw materials and ingredients, intermediate
or end products.
The use of this document can be extended to yeasts which do not form mycelium.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 6887 (all parts), Microbiology of the food chain — Preparation of test samples, initial suspension and
decimal dilutions for microbiological examination
ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations
ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture media
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
bacterial spore
resistant form of bacteria which is dormant until the germination (3.9) step
[SOURCE: ISO 20976-1:2019, 3.1]
ISO 20976-2:2022(E)
3.2
batch
group or set of identifiable food obtained through a given process under practically identical
circumstances and produced in a given place within one defined production period
Note 1 to entry: The batch is determined by parameters established beforehand by the organization and may be
described by other terms, e.g. lot.
[10]
[SOURCE: Commission Regulation (EC) No 2073/2005, Article 2 (e) , modified — “food obtained
through” has replaced “products obtained from” and Note 1 to entry has been added.]
3.3
bulk products
products that are not separated into individual items or units
[SOURCE: ISO/TS 17728:2015, 3.3.1]
3.4
challenge test
study of the growth or inactivation of microorganism(s) artificially inoculated in a food
[SOURCE: ISO 20976-1:2019, 3.5]
3.5
control unit
unit of food identical to the test unit (3.34) but not artificially inoculated (used as a blank)
[SOURCE: ISO 20976-1:2019, 3.4, modified — “inoculated” has replaced “contaminated”.]
3.6
D value
decimal reduction
time or dose required to achieve reduction of 90 % of the tested microorganism under stated conditions
(e.g. temperature, pH or chemical composition) in case of log linear inactivation kinetics (3.10)
3.7
δ value
first decimal reduction
time or dose required to achieve the first reduction of 90 % of the tested microorganism under stated
conditions (e.g.: temperature, pH or chemical composition) in case of non-log linear inactivation kinetics
(3.10)
3.8
experimental datapoint
result of analysis of a test unit (3.34) per unit mass, per unit volume, or per unit area
Note 1 to entry: The enumeration results may be expressed in log or most probable number (MPN).
[SOURCE: ISO 20976-1:2019, 3.6, modified — In the definition, “mass” has replaced “weight” and the
units have been deleted. In Note 1 to entry, “for specific cases” has been deleted and “or most probable
number (MPN)” has replaced “MPN”.]
3.9
germination
mechanism in which a bacterial spore (3.1) initiates its transformation into a vegetative cell (3.36)
[SOURCE: ISO 20976-1:2019, 3.9, modified — “initiates its transformation into” has replaced “starts
becoming”.]
3.10
inactivation kinetics
change over time in the concentration of the target microorganism subjected to an inactivation process
ISO 20976-2:2022(E)
3.11
inactivation parameter
mathematical estimate that describes the resistance/sensitivity of the target organism to the treatment
(3.35), obtained by fitting primary models (3.18) and secondary models (3.24)
Note 1 to entry: Examples of these parameters are D, δ and p for the primary models and z for the secondary
models.
3.12
inactivation potential
Δ value
log kill
log reduction
difference in the log concentration (log cfu/g or ml or cm ) of the target microorganism between an
earlier and a later time point expressed as log
Note 1 to entry: In this document, the term “inactivation potential” refers to the type of inactivation study, and
the terms “log kill” and “log reduction” refer to the result obtained.
3.13
inactivation treatment
process used to kill or inactivate the target microorganism
3.14
inoculum
microbial suspension at the desired concentration used to contaminate test units (3.34)
[SOURCE: ISO 20976-1:2019, 3.12]
3.15
k value
slope of the inactivation curve
3.16
p value
parameter describing the shape of the inactivation curve
3.17
pH value
measure of the concentration of acidity or alkalinity of a material in an aqueous solution
[SOURCE: ISO 5127:2017, 3.12.2.29, modified — The notes to entry have been deleted.]
3.18
primary model
mathematical model describing the changes of microbial counts as a function of time
[SOURCE: ISO 20976-1:2019, 3.16]
3.19
organizing laboratory
laboratory with responsibility for managing the challenge tests (3.4)
[SOURCE: ISO 20976-1:2019, 3.17]
3.20
pilot facility
manufacturing location used to run an experiment or test before introducing more widely
3.21
processing facility
location where products are made on a larger scale
ISO 20976-2:2022(E)
3.22
sampling
selection of one or more units or portions of food such that the units or portions selected are
representative of that food
[SOURCE: ISO 20976-1:2019, 3.18]
3.23
sampling point
time at which the test units (3.34) are taken for analyses
Note 1 to entry: When assessing inactivation kinetics (3.10), these are represented as experimental datapoints
(3.8) on the inactivation graph.
[SOURCE: ISO 20976-1:2019, 3.19, modified — “taken for analyses” has replaced “analysed and which
are represented as experimental datapoints on the kinetics graph” and Note 1 to entry has been added.]
3.24
secondary model
mathematical model describing the effects of the inactivation process factors (e.g. temperature, pH, a )
w
on the parameters of the primary model (3.18) (e.g. D, δ)
[SOURCE: ISO 20976-1:2019, 3.20, modified — “inactivation process” has replaced “environmental” and
“(e.g. D, δ)” has replaced “(e.g. growth rate)”.]
3.25
sporulation
mechanism by which vegetative cell (3.36) forms spore
[SOURCE: ISO 20976-1:2019, 3.21]
3.26
surrogate
non-pathogenic microorganism that has similar or more robust survival (3.27) capability compared to
the pathogen of concern both in the matrix and under the processing conditions being studied
3.27
survival
state of continuing to live or exist without significant increase or decrease in viability
3.28
target reduction level
target inactivation level expressed in log
3.29
t
time at which the treatment starts
3.30
t
end
time at which the treatment is finished
3.31
t
inoc
time at which the microorganism is inoculated in the food
3.32
t
xD
time of treatment needed for x log reduction of the target microorganism
ISO 20976-2:2022(E)
3.33
test portion
measured (volume or mass) representative sample taken from the test unit (3.34) for use in the analysis
[SOURCE: ISO 6887-1:2017, 3.5, modified — “test unit for use in the analysis” has replaced “laboratory
sample for use in the preparation of the initial suspension” and Note 1 to entry has been deleted.]
3.34
test unit
measured (volume or mass) amount of the food used for inoculation, subsequent treatment (3.35) and
analysis
[SOURCE: ISO 20976-1:2019, 3.24, modified — “subsequent treatment and analysis” has been added.]
3.35
treatment
any process, formulation or product characteristics, or a combination thereof, intended to inactivate
the target microorganism
3.36
vegetative cell
state of microbial form that is capable of growing under favourable environmental conditions
[SOURCE: ISO 20976-1:2019, 3.25]
3.37
water activity
a
w
ratio of the water-vapour pressure in the foodstuff to the vapour pressure of pure water at the same
temperature
[SOURCE: ISO 18787:2017, 3.1 modified — “water-vapour pressure in the foodstuff to the vapour
pressure of pure water at” has replaced “partial water-vapour pressure in equilibrium with the product
analysed to the water-vapour saturation pressure in equilibrium with”, and the formula and the notes
to entry has been deleted]
3.38
z value
change in treatment (3.35) (e.g. temperature, pH, a ,) that induces a 10-fold change in the D value (3.6)
w
Note 1 to entry: Temperature, pH and a can be indexed to the z value to denote the treatment being assessed.
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4 Principle
Inactivation studies are designed to determine the changes in the concentration of the target
microorganism during the challenge test. These studies can be used to assess whether there is significant
microbial inactivation in a foodstuff and to quantify the decrease in the target microorganism under a
given set of processing conditions and/or formulation of the food product. The scope and the aim of
the study shall be clearly specified (e.g. assessment/validation of the food process efficacy as a control
measure, assessment of microbial stability and survival) including the target reduction level and the
decision criteria. The experimental design shall be in accordance with that purpose and shall take
into account the steps of the process and the food chain for which microbial inactivation is assessed.
Knowledge from the FBO (e.g. on their products characteristics and production process) shall be
combined with the expertise in food microbiology and analytical sciences to ensure the robustness of
the study (see 14.3.2). Ideally, inactivation studies employ the target microorganism. In the challenge
tests studies conducted within food production facilities, a validated surrogate shall be used in place of
[26]
the target pathogen .
ISO 20976-2:2022(E)
The organizing laboratory shall have knowledge and skills in food microbiology, food science and
technology, and statistics to design and conduct the studies, interpret the results and draw the
conclusions. The analyses shall be conducted under a quality assurance system (e.g. ISO/IEC 17025).
To conduct an inactivation challenge study, the inoculum should be prepared such that the microbial
cells or spores have been adapted to the environmental conditions that mimic the food processing
environment, thereby encouraging natural microbial response once inoculated into the food.
[14]
The same microbial strain could exhibit various shapes as a function of the treatment (see Figure 1) .
The heterogeneous shapes of microbial inactivation curves are the results of the microbial resistance
[16]
or adaptive response or cell heterogeneity .
Key
X inactivation treatment (time or dose)
Y log (N)
a
Log-linear.
b
Concave.
c
Convex or with a shoulder.
d, e
With a tail or biphasic.
f
Sigmoïdal curve.
Figure 1 — Examples of microbial inactivation curve types
There are two types of inactivation study: the inactivation potential and the inactivation kinetics of a
target microorganism.
The inactivation potential studies are most appropriate for process validation and/or product
formulation. Inactivation potential results are specific to the conditions and matrix under study. To
extrapolate to other conditions, inactivation kinetic parameters shall be used, or a new inactivation
potential study conducted.
The inactivation kinetics studies are used to characterize the inactivation of the microorganism
through the determination of inactivation parameters such as D, δ and z values. Those studies are more
complex in terms of study design, execution, results interpretation and exploitation, particularly in the
ISO 20976-2:2022(E)
case of nonlinearity (see Figure 1) but allow to extrapolate the results to non-tested conditions within
the range of the study.
5 Apparatus
Routine microbiology labware specified in ISO 7218 shall be used. Specific labware, including the
following, can also be needed to prepare the test portions, to store them under suitable conditions, or
monitor how their characteristics change during the challenge test study.
5.1 Apparatus for packaging the samples under air, under vacuum or under a protective modified
atmosphere.
5.2 Climate-control chamber, able to reach and hold setpoint temperature to ± 1 °C and to adjust
relative humidity to ± 10 %.
5.3 pH meter, able to perform measurements to a tolerance of ± 0,1 pH units. pH meters shall give
readings to a resolution of 0,01 pH units.
5.4 a meter, meeting the requirements of ISO 18787.
w
5.5 Headspace gas analyser, to measure gas composition (e.g. O , CO ).
2 2
5.6 Logger for measuring temperature conditions of the test unit.
5.7 Logger for measuring relative humidity conditions of the test unit.
5.8 Device for measuring the inactivation treatment (e.g. dosimeter for irradiation treatment,
probe for heat treatment) of the test unit.
5.9 Apparatus (pilot facility or equipment in a laboratory) to run the inactivation treatment, which
mimic foreseeable conditions of production leading to microbial inactivation.
5.10 Industrial piece of equipment to conduct the inactivation study at the factory level.
6 Culture media and reagents
Follow current laboratory practices as specified in ISO 7218.
For the preparation and performance testing of culture media and reagents, follow the procedures
as specified in ISO 11133 and in the International Standard specific to the target microorganism. Use
relevant internationally recognized and widely accepted methods for the detection or enumeration
of injured target microorganisms (e.g. using a selective agar and non-selective agar with a dual layer
method).
7 Study design and sampling
7.1 General
The study design and sampling will depend on the target level of reduction, the process, the formulation
and the product characteristics. The factors impacting the inactivation shall be clearly identified and
their range shall be specified.
ISO 20976-2:2022(E)
There are two types of challenge tests to study inactivation: inactivation potential and inactivation
kinetics. The aim of the study and the technologies used will determine the type (see Annex A).
For inactivation potential, test units shall be taken at a minimum at the beginning and the end of
treatment.
For inactivation kinetics, test units shall be taken at each sampling point throughout the treatment
duration. When sampling access during processing is not possible due to the equipment and/or
technology assessed, independent treatments of varying lengths can be required.
The associated expected variability will determine the number of batches and test units to be
considered (see Annex B).
The study design shall consider various sources of variability linked to:
— process characteristics (e.g. flow rate, residence time, temperature, moisture, equipment design);
— formulation and product characteristics (e.g. fat, sugar, salt, a , protein, acid, pH, particle size,
w
chemical composition, background microorganisms, preservatives concentrations, gas atmosphere);
— where relevant (e.g. studies on preservatives), transportation and storage conditions (e.g. humidity,
packaging) during the shelf-life.
The study design shall also consider the target reduction level.
7.2 Setting target reduction level for the inactivation study
Depending on the aim of the challenge test, the target reduction level shall be specified at the start
of the study. It may be related to regulatory requirements or to internal requirements. Laboratories
conducting inactivation studies in products that are subject to regulations should be aware of the most
current requirements. The target reduction level influences the inoculum level to be used. It can vary
depending on the food type (e.g. for Salmonella inactivation, a 4 log reduction has been required for
[12] [13]
the treatment of almonds and a 7 log reduction for poultry products ). To be able to analyse the
target reduction level, the limit of quantification of the analytical method used and the inoculum level
shall be appropriate.
7.3 Number of batches
The number of batches to be included in the study depends on the variability of the food production
process and food characteristics (see Annex B). The characteristics of the studied batches shall be
representative of the variability of the production process based on historical data.
A minimum number of three batches should be used for both inactivation potential and inactivation
kinetics studies. Generally, the number of batches should be increased in situations of higher variability
or uncertainty.
The use of a single batch shall be clearly justified, for example:
a) evaluating the impact of a new formulation of the food;
b) using a batch representing the most unfavourable inactivation conditions (worst case);
c) when multiple levels of the treatment will be assessed in inactivation kinetics studies.
7.4 Preparation of the test units
Test units representative of the matrix (raw materials and ingredients, intermediate or end products)
before the inactivation treatment can be either:
— aseptically-sampled portions of the bulk products from the production line;
ISO 20976-2:2022(E)
— aseptically-sampled portions or complete content of the packaging unit(s).
The test units shall be maintained at appropriate conditions before inoculation, preferably inoculated
as close as possible to the time of production unless otherwise specified by the aim of the study. Once
inoculated, all test units, including t (see Figure 2), shall be maintained under the same appropriate
conditions, including transport and storage where necessary.
7.5 Number of control units and test units
The minimum number of units to be analysed will depend on the type of the inactivation study, the
nature of the food and the processing conditions (see Annex B).
For inactivation potential studies, the number of sample points depends on the accessibility of the
samples. In some cases, it is only possible to get samples at the start and the end of the process. In other
cases, it is possible to get intermediate samples.
For inactivation kinetics studies, it is important to have enough experimental datapoints available to
get an accurate estimation of the curve type (see Figure 1). For linear curve types, a minimum of six
sampling points is required. For nonlinear curve types, usually more sampling points are needed.
The number of units should be increased in situations of high variability or uncertainty related to
food and process characteristics. It is recommended to prepare additional units to cover unforeseen
incidents.
8 Selection of strains
Each strain used shall be characterized biochemically and/or serologically and/or genetically in
sufficient detail for its identity to be known. Strains previously isolated from the food matrix (raw
materials, ingredients, end products) or from the production environment or from clinical/food/
environmental samples in outbreaks involving the food are preferred.
The original source of all isolates should be known and they should be held in a local (e.g. laboratory
which runs the challenge test), national or international culture collection to enable them to be used in
future testing, if required. Use strains for which the inactivation parameter(s) (e.g. D, k or δ values) have
been previously determined in a similar matrix (e.g. heat, pressure, acid resistance) so as to ensure that
the selected strain(s) is/are fit for purpose. If the matrix is not similar, a preliminary study needs to be
[15]
performed to measure the resistance .
Cocktails of three to five fit for purpose strains are appropriate for inactivation potential studies. For
the inactivation kinetics, strains shall only be tested individually.
Ideally, inactivation studies would employ the target microorganism. In case of pathogens, this is
possible when the study is performed in a laboratory or in a pilot plant. However, this is not possible
in factories or food processing facilities (see Annex A). In such cases, a surrogate shall be used. The
suitability of the selected surrogate shall be justified and documented in relation to the pathogen,
food matrix and treatment under assessment. Otherwise, a specific surrogate validation study shall be
[26]
conducted and documented .
9 Preparation of the inoculum
9.1 General
In order to take into account the dilution effect when inoculating the test units, the inoculum
concentration shall be greater than the targeted log reduction. In some cases, preliminary studies need
to be taken to achieve the target inoculum level such as concentrating by centrifugation or by growing
on solid medium to minimize volume.
ISO 20976-2:2022(E)
9.2 Preparation of the vegetative cells
Prepare the strains to obtain a standardised inoculum with physiological conditions and concentration
which are fit for purpose.
The culture should be initially grown in non-selective media and under conditions suitable for optimal
growth. This culture can be used under optimum conditions or be adapted in order to mimic conditions
of potential contamination. Examples of adaptation are pH, acid type, temperature, a , salt content,
w
sugar content, liquid or solid media and atmosphere.
The microbial concentration of the inoculum shall be estimated using internationally recognized and
widely accepted methods or alternative methods validated according to internationally accepted
protocols. This inoculum can be concentrated by centrifugation to meet
...
SLOVENSKI STANDARD
01-januar-2023
Mikrobiologija v prehranski verigi - Zahteve in smernice za vodenje preskusa
ustreznosti kmetijskih pridelkov in živilskih proizvodov - 2. del: Preskus
ustreznosti za študijo inaktivacijskega potenciala in kinetičnih parametrov (ISO
20976-2:2022)
Microbiology of the food chain - Requirements and guidelines for conducting challenge
tests of food and feed products - Part 2: Challenge tests to study inactivation potential
and kinetic parameters (ISO 20976-2:2022)
Mikrobiologie der Lebensmittelkette - Anforderungen und Leitfaden zur Durchführung
von Challenge-Tests bei Lebensmitteln und Futtermitteln - Teil 2: Challenge-Tests zur
Untersuchung von Inaktivierungspotenzial und kinetischer Parameter (ISO 20976-
2:2022)
Microbiologie de la chaîne alimentaire - Exigences et lignes directrices pour la réalisation
des tests d'épreuve microbiologique - Partie 2: Tests d’inactivation pour étudier le
potentiel d’inactivation et les paramètres de kinétique (ISO 20976-2:2022)
Ta slovenski standard je istoveten z: EN ISO 20976-2:2022
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN ISO 20976-2
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2022
EUROPÄISCHE NORM
ICS 07.100.30
English Version
Microbiology of the food chain - Requirements and
guidelines for conducting challenge tests of food and feed
products - Part 2: Challenge tests to study inactivation
potential and kinetic parameters (ISO 20976-2:2022)
Microbiologie de la chaîne alimentaire - Exigences et Mikrobiologie der Lebensmittelkette - Anforderungen
lignes directrices pour la réalisation des tests und Leitfaden zur Durchführung von Challenge-Tests
d'épreuve microbiologique - Partie 2: Tests bei Lebensmitteln und Futtermitteln - Teil 2:
d'inactivation pour étudier le potentiel d'inactivation Challenge-Tests zur Untersuchung von
et les paramètres de kinétique (ISO 20976-2:2022) Inaktivierungspotenzial und kinetischer Parameter
(ISO 20976-2:2022)
This European Standard was approved by CEN on 19 September 2022.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20976-2:2022 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
European foreword
This document (EN ISO 20976-2:2022) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with Technical Committee CEN/TC 463 “Microbiology of the food chain” the
secretariat of which is held by AFNOR.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by May 2023, and conflicting national standards shall be
withdrawn at the latest by May 2023.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 20976-2:2022 has been approved by CEN as EN ISO 20976-2:2022 without any
modification.
INTERNATIONAL ISO
STANDARD 20976-2
First edition
2022-10
Microbiology of the food chain —
Requirements and guidelines for
conducting challenge tests of food and
feed products —
Part 2:
Challenge tests to study inactivation
potential and kinetic parameters
Microbiologie de la chaîne alimentaire — Exigences et lignes
directrices pour la réalisation des tests d'épreuve microbiologiques —
Partie 2: Tests d’inactivation pour étudier le potentiel d’inactivation
et les paramètres de la cinétique d’inactivation
Reference number
ISO 20976-2:2022(E)
ISO 20976-2:2022(E)
© ISO 2022
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
ISO 20976-2:2022(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 5
5 Apparatus . 7
6 Culture media and reagents .7
7 Study design and sampling . 7
7.1 General . 7
7.2 Setting target reduction level for the inactivation study . 8
7.3 Number of batches. 8
7.4 Preparation of the test units . 8
7.5 Number of control units and test units . 9
8 Selection of strains . 9
9 Preparation of the inoculum . 9
9.1 General . 9
9.2 Preparation of the vegetative cells . 10
9.3 Preparation of the spores . 10
10 Inoculation of the test units .10
11 Controls . .12
11.1 Uninoculated controls .12
11.2 Inoculated controls . 12
12 Treatment of the test units .12
13 Analysis . .12
14 Expression of the results .13
14.1 General .13
14.2 Inactivation potential .13
14.3 Inactivation kinetics parameters. 14
14.3.1 General . 14
14.3.2 Primary inactivation kinetics parameters . 14
14.3.3 Secondary inactivation kinetics parameters . 15
14.4 Simulation of inactivation .15
15 Test report .16
15.1 General . 16
15.2 Aim of the study, type of challenge test and target reduction level . 16
15.3 Experimental protocol . . 17
15.4 Sample analysis . . 17
15.5 Results . . 17
15.6 Conclusions . 18
15.7 Reference documents . 18
Annex A (informative) Selection of the type and the location of inactivation study .19
Annex B (normative) Minimum number of units to prepare for the inactivation studies .20
Annex C (informative) Examples of inoculation techniques .21
Bibliography .23
iii
ISO 20976-2:2022(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology, in collaboration with the European Committee for Standardization (CEN) Technical
Committee CEN/TC 463, Microbiology of the food chain, in accordance with the Agreement on technical
cooperation between ISO and CEN (Vienna Agreement).
A list of all parts in the ISO 20976 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
ISO 20976-2:2022(E)
Introduction
Under the general principles of the Codex Alimentarius on food hygiene, it is the responsibility of the
food business operators (FBOs) to control microbiological hazards in foods and to manage microbial
risks. Therefore, FBOs implement validated control measures, within the hazard analysis and critical
control point (HACCP) system, and conducts studies in order to investigate compliance with the food
safety criteria throughout the food chain.
In the framework of microbial risk assessment (MRA), several complementary approaches are developed
to estimate risks posed by pathogens or spoilage microorganisms in the food chain. MRA is adopted by
regulators under the auspices of the international agency for setting food standards. Challenge testing
is one of the recognized approaches used to validate control measures within the HACCP system, as
well as to assess microbiological safety and quality of food, food production processes, food storage
conditions, and food preparation recommendations dedicated to consumers.
Therefore, this document provides technical rules, calculations and approaches to investigate the ability
of an inoculated microorganism of concern to grow, survive or be inactivated in the raw materials,
intermediate or end products under reasonably foreseeable food processes, storage and use conditions.
The objective and the scope of the study are to determine the experimental design and the selection
of the study conditions, and to assess the extent of microbial inactivation. Regulatory authorities can
have different recommendations, and these differences have been included as much as possible. It is,
however, possible that specific requirements need to be incorporated to get a regulatory approval of
the challenge test.
As the growth and inactivation studies are clearly different, the ISO 20976 series consists of two parts,
under the general title Microbiology of the food chain — Requirements and guidelines for conducting
challenge tests of food and feed products:
— Part 1: Challenge tests to study the growth potential, lag time and the maximum growth rate;
— Part 2: Challenge tests to study inactivation potential and kinetic parameters.
The use of the ISO 20976 series involves expertise in relevant areas such as food microbiology, food
science, food processing and statistics. The statistical expertise encompasses an understanding of
sampling theory and design of experiments, statistical analysis of microbiological data, and overview of
scientifically recognized and available mathematical concepts used in predictive modelling.
For practical reasons, the term “food” includes feed.
v
INTERNATIONAL STANDARD ISO 20976-2:2022(E)
Microbiology of the food chain — Requirements and
guidelines for conducting challenge tests of food and feed
products —
Part 2:
Challenge tests to study inactivation potential and kinetic
parameters
1 Scope
This document specifies the protocols for conducting microbiological challenge tests for inactivation
studies on vegetative bacteria and bacterial spores in the raw materials and ingredients, intermediate
or end products.
The use of this document can be extended to yeasts which do not form mycelium.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 6887 (all parts), Microbiology of the food chain — Preparation of test samples, initial suspension and
decimal dilutions for microbiological examination
ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations
ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture media
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
bacterial spore
resistant form of bacteria which is dormant until the germination (3.9) step
[SOURCE: ISO 20976-1:2019, 3.1]
ISO 20976-2:2022(E)
3.2
batch
group or set of identifiable food obtained through a given process under practically identical
circumstances and produced in a given place within one defined production period
Note 1 to entry: The batch is determined by parameters established beforehand by the organization and may be
described by other terms, e.g. lot.
[10]
[SOURCE: Commission Regulation (EC) No 2073/2005, Article 2 (e) , modified — “food obtained
through” has replaced “products obtained from” and Note 1 to entry has been added.]
3.3
bulk products
products that are not separated into individual items or units
[SOURCE: ISO/TS 17728:2015, 3.3.1]
3.4
challenge test
study of the growth or inactivation of microorganism(s) artificially inoculated in a food
[SOURCE: ISO 20976-1:2019, 3.5]
3.5
control unit
unit of food identical to the test unit (3.34) but not artificially inoculated (used as a blank)
[SOURCE: ISO 20976-1:2019, 3.4, modified — “inoculated” has replaced “contaminated”.]
3.6
D value
decimal reduction
time or dose required to achieve reduction of 90 % of the tested microorganism under stated conditions
(e.g. temperature, pH or chemical composition) in case of log linear inactivation kinetics (3.10)
3.7
δ value
first decimal reduction
time or dose required to achieve the first reduction of 90 % of the tested microorganism under stated
conditions (e.g.: temperature, pH or chemical composition) in case of non-log linear inactivation kinetics
(3.10)
3.8
experimental datapoint
result of analysis of a test unit (3.34) per unit mass, per unit volume, or per unit area
Note 1 to entry: The enumeration results may be expressed in log or most probable number (MPN).
[SOURCE: ISO 20976-1:2019, 3.6, modified — In the definition, “mass” has replaced “weight” and the
units have been deleted. In Note 1 to entry, “for specific cases” has been deleted and “or most probable
number (MPN)” has replaced “MPN”.]
3.9
germination
mechanism in which a bacterial spore (3.1) initiates its transformation into a vegetative cell (3.36)
[SOURCE: ISO 20976-1:2019, 3.9, modified — “initiates its transformation into” has replaced “starts
becoming”.]
3.10
inactivation kinetics
change over time in the concentration of the target microorganism subjected to an inactivation process
ISO 20976-2:2022(E)
3.11
inactivation parameter
mathematical estimate that describes the resistance/sensitivity of the target organism to the treatment
(3.35), obtained by fitting primary models (3.18) and secondary models (3.24)
Note 1 to entry: Examples of these parameters are D, δ and p for the primary models and z for the secondary
models.
3.12
inactivation potential
Δ value
log kill
log reduction
difference in the log concentration (log cfu/g or ml or cm ) of the target microorganism between an
earlier and a later time point expressed as log
Note 1 to entry: In this document, the term “inactivation potential” refers to the type of inactivation study, and
the terms “log kill” and “log reduction” refer to the result obtained.
3.13
inactivation treatment
process used to kill or inactivate the target microorganism
3.14
inoculum
microbial suspension at the desired concentration used to contaminate test units (3.34)
[SOURCE: ISO 20976-1:2019, 3.12]
3.15
k value
slope of the inactivation curve
3.16
p value
parameter describing the shape of the inactivation curve
3.17
pH value
measure of the concentration of acidity or alkalinity of a material in an aqueous solution
[SOURCE: ISO 5127:2017, 3.12.2.29, modified — The notes to entry have been deleted.]
3.18
primary model
mathematical model describing the changes of microbial counts as a function of time
[SOURCE: ISO 20976-1:2019, 3.16]
3.19
organizing laboratory
laboratory with responsibility for managing the challenge tests (3.4)
[SOURCE: ISO 20976-1:2019, 3.17]
3.20
pilot facility
manufacturing location used to run an experiment or test before introducing more widely
3.21
processing facility
location where products are made on a larger scale
ISO 20976-2:2022(E)
3.22
sampling
selection of one or more units or portions of food such that the units or portions selected are
representative of that food
[SOURCE: ISO 20976-1:2019, 3.18]
3.23
sampling point
time at which the test units (3.34) are taken for analyses
Note 1 to entry: When assessing inactivation kinetics (3.10), these are represented as experimental datapoints
(3.8) on the inactivation graph.
[SOURCE: ISO 20976-1:2019, 3.19, modified — “taken for analyses” has replaced “analysed and which
are represented as experimental datapoints on the kinetics graph” and Note 1 to entry has been added.]
3.24
secondary model
mathematical model describing the effects of the inactivation process factors (e.g. temperature, pH, a )
w
on the parameters of the primary model (3.18) (e.g. D, δ)
[SOURCE: ISO 20976-1:2019, 3.20, modified — “inactivation process” has replaced “environmental” and
“(e.g. D, δ)” has replaced “(e.g. growth rate)”.]
3.25
sporulation
mechanism by which vegetative cell (3.36) forms spore
[SOURCE: ISO 20976-1:2019, 3.21]
3.26
surrogate
non-pathogenic microorganism that has similar or more robust survival (3.27) capability compared to
the pathogen of concern both in the matrix and under the processing conditions being studied
3.27
survival
state of continuing to live or exist without significant increase or decrease in viability
3.28
target reduction level
target inactivation level expressed in log
3.29
t
time at which the treatment starts
3.30
t
end
time at which the treatment is finished
3.31
t
inoc
time at which the microorganism is inoculated in the food
3.32
t
xD
time of treatment needed for x log reduction of the target microorganism
ISO 20976-2:2022(E)
3.33
test portion
measured (volume or mass) representative sample taken from the test unit (3.34) for use in the analysis
[SOURCE: ISO 6887-1:2017, 3.5, modified — “test unit for use in the analysis” has replaced “laboratory
sample for use in the preparation of the initial suspension” and Note 1 to entry has been deleted.]
3.34
test unit
measured (volume or mass) amount of the food used for inoculation, subsequent treatment (3.35) and
analysis
[SOURCE: ISO 20976-1:2019, 3.24, modified — “subsequent treatment and analysis” has been added.]
3.35
treatment
any process, formulation or product characteristics, or a combination thereof, intended to inactivate
the target microorganism
3.36
vegetative cell
state of microbial form that is capable of growing under favourable environmental conditions
[SOURCE: ISO 20976-1:2019, 3.25]
3.37
water activity
a
w
ratio of the water-vapour pressure in the foodstuff to the vapour pressure of pure water at the same
temperature
[SOURCE: ISO 18787:2017, 3.1 modified — “water-vapour pressure in the foodstuff to the vapour
pressure of pure water at” has replaced “partial water-vapour pressure in equilibrium with the product
analysed to the water-vapour saturation pressure in equilibrium with”, and the formula and the notes
to entry has been deleted]
3.38
z value
change in treatment (3.35) (e.g. temperature, pH, a ,) that induces a 10-fold change in the D value (3.6)
w
Note 1 to entry: Temperature, pH and a can be indexed to the z value to denote the treatment being assessed.
w
4 Principle
Inactivation studies are designed to determine the changes in the concentration of the target
microorganism during the challenge test. These studies can be used to assess whether there is significant
microbial inactivation in a foodstuff and to quantify the decrease in the target microorganism under a
given set of processing conditions and/or formulation of the food product. The scope and the aim of
the study shall be clearly specified (e.g. assessment/validation of the food process efficacy as a control
measure, assessment of microbial stability and survival) including the target reduction level and the
decision criteria. The experimental design shall be in accordance with that purpose and shall take
into account the steps of the process and the food chain for which microbial inactivation is assessed.
Knowledge from the FBO (e.g. on their products characteristics and production process) shall be
combined with the expertise in food microbiology and analytical sciences to ensure the robustness of
the study (see 14.3.2). Ideally, inactivation studies employ the target microorganism. In the challenge
tests studies conducted within food production facilities, a validated surrogate shall be used in place of
[26]
the target pathogen .
ISO 20976-2:2022(E)
The organizing laboratory shall have knowledge and skills in food microbiology, food science and
technology, and statistics to design and conduct the studies, interpret the results and draw the
conclusions. The analyses shall be conducted under a quality assurance system (e.g. ISO/IEC 17025).
To conduct an inactivation challenge study, the inoculum should be prepared such that the microbial
cells or spores have been adapted to the environmental conditions that mimic the food processing
environment, thereby encouraging natural microbial response once inoculated into the food.
[14]
The same microbial strain could exhibit various shapes as a function of the treatment (see Figure 1) .
The heterogeneous shapes of microbial inactivation curves are the results of the microbial resistance
[16]
or adaptive response or cell heterogeneity .
Key
X inactivation treatment (time or dose)
Y log (N)
a
Log-linear.
b
Concave.
c
Convex or with a shoulder.
d, e
With a tail or biphasic.
f
Sigmoïdal curve.
Figure 1 — Examples of microbial inactivation curve types
There are two types of inactivation study: the inactivation potential and the inactivation kinetics of a
target microorganism.
The inactivation potential studies are most appropriate for process validation and/or product
formulation. Inactivation potential results are specific to the conditions and matrix under study. To
extrapolate to other conditions, inactivation kinetic parameters shall be used, or a new inactivation
potential study conducted.
The inactivation kinetics studies are used to characterize the inactivation of the microorganism
through the determination of inactivation parameters such as D, δ and z values. Those studies are more
complex in terms of study design, execution, results interpretation and exploitation, particularly in the
ISO 20976-2:2022(E)
case of nonlinearity (see Figure 1) but allow to extrapolate the results to non-tested conditions within
the range of the study.
5 Apparatus
Routine microbiology labware specified in ISO 7218 shall be used. Specific labware, including the
following, can also be needed to prepare the test portions, to store them under suitable conditions, or
monitor how their characteristics change during the challenge test study.
5.1 Apparatus for packaging the samples under air, under vacuum or under a protective modified
atmosphere.
5.2 Climate-control chamber, able to reach and hold setpoint temperature to ± 1 °C and to adjust
relative humidity to ± 10 %.
5.3 pH meter, able to perform measurements to a tolerance of ± 0,1 pH units. pH meters shall give
readings to a resolution of 0,01 pH units.
5.4 a meter, meeting the requirements of ISO 18787.
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5.5 Headspace gas analyser, to measure gas composition (e.g. O , CO ).
2 2
5.6 Logger for measuring temperature conditions of the test unit.
5.7 Logger for measuring relative humidity conditions of the test unit.
5.8 Device for measuring the inactivation treatment (e.g. dosimeter for irradiation treatment,
probe for heat treatment) of the test unit.
5.9 Apparatus (pilot facility or equipment in a laboratory) to run the inactivation treatment, which
mimic foreseeable conditions of production leading to microbial inactivation.
5.10 Industrial piece of equipment to conduct the inactivation study at the factory level.
6 Culture media and reagents
Follow current laboratory practices as specified in ISO 7218.
For the preparation and performance testing of culture media and reagents, follow the procedures
as specified in ISO 11133 and in the International Standard specific to the target microorganism. Use
relevant internationally recognized and widely accepted methods for the detection or enumeration
of injured target microorganisms (e.g. using a selective agar and non-selective agar with a dual layer
method).
7 Study design and sampling
7.1 General
The study design and sampling will depend on the target level of reduction, the process, the formulation
and the product characteristics. The factors impacting the inactivation shall be clearly identified and
their range shall be specified.
ISO 20976-2:2022(E)
There are two types of challenge tests to study inactivation: inactivation potential and inactivation
kinetics. The aim of the study and the technologies used will determine the type (see Annex A).
For inactivation potential, test units shall be taken at a minimum at the beginning and the end of
treatment.
For inactivation kinetics, test units shall be taken at each sampling point throughout the treatment
duration. When sampling access during processing is not possible due to the equipment and/or
technology assessed, independent treatments of varying lengths can be required.
The associated expected variability will determine the number of batches and test units to be
considered (see Annex B).
The study design shall consider various sources of variability linked to:
— process characteristics (e.g. flow rate, residence time, temperature, moisture, equipment design);
— formulation and product characteristics (e.g. fat, sugar, salt, a , protein, acid, pH, particle size,
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chemical composition, background microorganisms, preservatives concentrations, gas atmosphere);
— where relevant (e.g. studies on preservatives), transportation and storage conditions (e.g. humidity,
packaging) during the shelf-life.
The study design shall also consider the target reduction level.
7.2 Setting target reduction level for the inactivation study
Depending on the aim of the challenge test, the target reduction level shall be specified at the start
of the study. It may be related to regulatory requirements or to internal requirements. Laboratories
conducting inactivation studies in products that are subject to regulations should be aware of the most
current requirements. The target reduction level influences the inoculum level to be used. It can vary
depending on the food type (e.g. for Salmonella inactivation, a 4 log reduction has been required for
[12] [13]
the treatment of almonds and a 7 log reduction for poultry products ). To be able to analyse the
target reduction level, the limit of quantification of the analytical method used and the inoculum level
shall be appropriate.
7.3 Number of batches
The number of batches to be included in the study depends on the variability of the food production
process and food characteristics (see Annex B). The characteristics of the studied batches shall be
representative of the variability of the production process based on historical data.
A minimum number of three batches should be used for both inactivation potential and inactivation
kinetics studies. Generally, the number of batches should be increased in situations of higher variability
or uncertainty.
The use of a single batch shall be clearly justified, for example:
a) evaluating the impact of a new formulation of the food;
b) using a batch representing the most unfavourable inactivation conditions (worst case);
c) when multiple levels of the treatment will be assessed in inactivation kinetics studies.
7.4 Preparation of the test units
Test units representative of the matrix (raw materials and ingredients, intermediate or end products)
before the inactivation treatment can be either:
— aseptically-sampled portions of the bulk products from the production line;
ISO 20976-2:2022(E)
— aseptically-sampled portions or complete content of the packaging unit(s).
The test units shall be maintained at appropriate conditions before inoculation, preferably inoculated
as close as possible to the time of production unless otherwise specified by the aim of the study. Once
inoculated, all test units, including t (see Figure 2), shall be maintained under the same appropriate
conditions, including transport and storage where necessary.
7.5 Number of control units and test units
The minimum number of units to be analysed will depend on the type of the inactivation study, the
nature of the food and the processing conditions (see Annex B).
For inactivation potential studies, the number of sample points depends on the accessibility of the
samples. In some cases, it is only possible to get samples at the start and the end of the process. In other
cases, it is possible to get intermediate samples.
For inactivation kinetics studies, it is important to have enough experimental datapoints available to
get an accurate estimation of the curve type (see Figure 1). For linear curve types, a minimum of six
sampling points is required. For nonlinear curve types, usually more sampling points are needed.
The number of units should be increased in situations of high variability or uncertainty related to
food and process characteristics. It is recommended to prepare additional units to cover unforeseen
incidents.
8 Selection of strains
Each strain used shall be characterized biochemically and/or serologically and/or genetically in
sufficient detail for its identity to be known. Strains previously isolated from the food matrix (raw
materials, ingredients, end products) or from the production environment or from clinical/food/
environmental samples in outbreaks involving the food are preferred.
The original source of all isolates should be known and they should be held in a local (e.g. laboratory
which runs the challenge test), national or international culture collection to enable them to be used in
future testing, if required. Use strains for which the inactivation parameter(s) (e.g. D, k or δ values) have
been previously determined in a similar matrix (e.g. heat, pressure, acid resistance) so as to ensure that
the selected strain(s) is/are fit for purpose. If the matrix is not similar, a preliminary study needs to be
[15]
performed to measure the resistance .
Cocktails of three to five fit for purpose strains are appropriate for inactivation potential studies. For
the inactivation kinetics, strains shall only be tested individually.
Ideally, inactivation studies would employ the target microorganism. In case of pathogens, this is
possible when the study is performed in a laboratory or in a pilot plant. However, this is not possible
in factories or food processing facilities (see Annex A). In such cases, a surrogate shall be used. The
suitability of the selected surrogate shall be justified and documented in relation to the pathogen,
food matrix and treatment under assessment. Otherwise, a specific surrogate validation study shall be
[26]
conducted and documented .
9 Preparation of the inoculum
9.1 General
In order to take into account the dilution effect when inoculating the test units, the inoculum
concentration shall be greater than the targeted log reduction. In some cases, preliminary studies need
to be taken to achieve the target inoculum level such as concentrating by centrifugation or by growing
on solid medium to minimize volume.
ISO 20976-2:2022(E)
9.2 Preparation of the vegetative cells
Prepare the strains to obtain a standardised inoculum with physiological conditions and concentration
which are fit for purpose.
The culture should be initially grown in non-selective media and under conditions suitable for optimal
growth. This culture can be used under optimum conditions or be adapted in order to mimic conditions
of potential contamination. Examples of adaptation are pH, acid type, temperature, a , salt content,
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sugar content, liquid or solid media and atmosphere.
The microbial concentration of the inoculum shall be estimated using internationally recognized and
widely accepted methods or alternative methods validated according to internationally accepted
protocols. This inoculum can be concentrated by centrifugation to meet
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