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

Status
Published
Public Enquiry End Date
29-Nov-2021
Publication Date
14-Dec-2022
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
05-Dec-2022
Due Date
09-Feb-2023
Completion Date
15-Dec-2022

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Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 20976-2:2023
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
SIST EN ISO 20976-2:2023 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
SIST EN ISO 20976-2:2023
---------------------- Page: 2 ----------------------
SIST EN ISO 20976-2:2023
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.
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SIST EN ISO 20976-2:2023
EN ISO 20976-2:2022 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST EN ISO 20976-2:2023
EN ISO 20976-2:2022 (E)
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.
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SIST EN ISO 20976-2:2023
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SIST EN ISO 20976-2:2023
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 2022
---------------------- Page: 7 ----------------------
SIST EN ISO 20976-2:2023
ISO 20976-2:2022(E)
COPYRIGHT PROTECTED DOCUMENT
© 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
© ISO 2022 – All rights reserved
---------------------- Page: 8 ----------------------
SIST EN ISO 20976-2:2023
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 2022 – All rights reserved
---------------------- Page: 9 ----------------------
SIST EN ISO 20976-2:2023
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.
© ISO 2022 – All rights reserved
---------------------- Page: 10 ----------------------
SIST EN ISO 20976-2:2023
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.
© ISO 2022 – All rights reserved
---------------------- Page: 11 ----------------------
SIST EN ISO 20976-2:2023
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SIST EN ISO 20976-2:2023
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 2022 – All rights reserved
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SIST EN ISO 20976-2:2023
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 2022 – All rights reserved
---------------------- Page: 14 ----------------------
SIST EN ISO 20976-2:2023
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 2022 – All rights reserved
---------------------- Page: 15 ----------------------
SIST EN ISO 20976-2:2023
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 )

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
time at which the treatment starts
3.30
end
time at which the treatment is finished
3.31
inoc
time at which the microorganism is inoculated in the food
3.32
time of treatment needed for x log reduction of the target microorganism
© ISO 2022 – All rights reserved
---------------------- Page: 16 ----------------------
SIST EN ISO 20976-2:2023
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 68
...

SLOVENSKI STANDARD
SIST EN ISO 20976-2:2023
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
SIST EN ISO 20976-2:2023 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
SIST EN ISO 20976-2:2023
---------------------- Page: 2 ----------------------
SIST EN ISO 20976-2:2023
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.
---------------------- Page: 3 ----------------------
SIST EN ISO 20976-2:2023
EN ISO 20976-2:2022 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

---------------------- Page: 4 ----------------------
SIST EN ISO 20976-2:2023
EN ISO 20976-2:2022 (E)
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.
---------------------- Page: 5 ----------------------
SIST EN ISO 20976-2:2023
---------------------- Page: 6 ----------------------
SIST EN ISO 20976-2:2023
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 2022
---------------------- Page: 7 ----------------------
SIST EN ISO 20976-2:2023
ISO 20976-2:2022(E)
COPYRIGHT PROTECTED DOCUMENT
© 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
© ISO 2022 – All rights reserved
---------------------- Page: 8 ----------------------
SIST EN ISO 20976-2:2023
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 2022 – All rights reserved
---------------------- Page: 9 ----------------------
SIST EN ISO 20976-2:2023
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.
© ISO 2022 – All rights reserved
---------------------- Page: 10 ----------------------
SIST EN ISO 20976-2:2023
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.
© ISO 2022 – All rights reserved
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SIST EN ISO 20976-2:2023
---------------------- Page: 12 ----------------------
SIST EN ISO 20976-2:2023
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 2022 – All rights reserved
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SIST EN ISO 20976-2:2023
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 2022 – All rights reserved
---------------------- Page: 14 ----------------------
SIST EN ISO 20976-2:2023
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 2022 – All rights reserved
---------------------- Page: 15 ----------------------
SIST EN ISO 20976-2:2023
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 )

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
time at which the treatment starts
3.30
end
time at which the treatment is finished
3.31
inoc
time at which the microorganism is inoculated in the food
3.32
time of treatment needed for x log reduction of the target microorganism
© ISO 2022 – All rights reserved
---------------------- Page: 16 ----------------------
SIST EN ISO 20976-2:2023
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 688
...

SLOVENSKI STANDARD
oSIST prEN ISO 20976-2:2021
01-november-2021
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/DIS 20976-
2:2021)

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/DIS 20976-2:2021)

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/DIS 20976-

2:2021)

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/DIS 20976-2:2021)
Ta slovenski standard je istoveten z: prEN ISO 20976-2
ICS:
07.100.30 Mikrobiologija živil Food microbiology
oSIST prEN ISO 20976-2:2021 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
oSIST prEN ISO 20976-2:2021
---------------------- Page: 2 ----------------------
oSIST prEN ISO 20976-2:2021
DRAFT INTERNATIONAL STANDARD
ISO/DIS 20976-2
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2021-09-13 2021-12-06
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 microbiologique —

Partie 2: Tests d’inactivation pour étudier le potentiel d’inactivation et les paramètres de kinétique

ICS: 07.100.30
THIS DOCUMENT IS A DRAFT CIRCULATED
This document is circulated as received from the committee secretariat.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
ISO/CEN PARALLEL PROCESSING
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 20976-2:2021(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION. ISO 2021
---------------------- Page: 3 ----------------------
oSIST prEN ISO 20976-2:2021
ISO/DIS 20976-2:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

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 2021 – All rights reserved
---------------------- Page: 4 ----------------------
oSIST prEN ISO 20976-2:2021
ISO/DIS 20976-2:2021(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 5

5 Apparatus ..................................................................................................................................................................................................................... 6

6 Culture media and reagents ...................................................................................................................................................................... 7

7 Study design and sampling ........................................................................................................................................................................ 7

7.1 Setting target reduction level for the inactivation study .................................................................................... 7

7.2 Number of batches ............................................................................................................................................................................... 8

7.3 Preparation of the test units ........................................................................................................................................................ 8

7.4 Number of control units and test units .............................................................................................................................. 8

8 Selection of strains ............................................................................................................................................................................................. 8

9 Preparation of the inoculum .................................................................................................................................................................... 9

9.1 Preparation of the vegetative cells ......................................................................................................................................... 9

9.2 Preparation of the spores .............................................................................................................................................................. 9

10 Inoculation of the test units ...................................................................................................................................................................10

11 Controls .......................................................................................................................................................................................................................11

11.1 Uninoculated controls ....................................................................................................................................................................11

11.2 Inoculated controls ..........................................................................................................................................................................11

12 Treatment of the test units .....................................................................................................................................................................11

13 Analysis .......................................................................................................................................................................................................................11

14 Expression of the results ...........................................................................................................................................................................12

14.1 Inactivation potential .....................................................................................................................................................................13

14.2 Inactivation kinetics parameters ..........................................................................................................................................13

14.2.1 Primary inactivation kinetics parameters ..............................................................................................13

14.2.2 Secondary inactivation kinetics parameters ........................................................................................14

14.3 Simulation of inactivation...........................................................................................................................................................14

15 Test report ................................................................................................................................................................................................................15

15.1 Aim of the study, type of challenge test and target reduction level ........................................................15

15.2 Experimental protocol ...................................................................................................................................................................15

15.3 Sample analysis ...................................................................................................................................................................................16

15.4 Results .........................................................................................................................................................................................................16

15.5 Conclusions .............................................................................................................................................................................................17

15.6 Reference documents .....................................................................................................................................................................17

Annex A (informative) Selection of the type and the location of inactivation study ........................................18

Annex B (normative) Minimum number ofunits to prepare for the inactivation studies ..........................19

Annex C (informative) Examples of inoculation techniques .....................................................................................................21

Bibliography .............................................................................................................................................................................................................................23

© ISO 2021 – All rights reserved iii
---------------------- Page: 5 ----------------------
oSIST prEN ISO 20976-2:2021
ISO/DIS 20976-2:2021(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 meaning of ISO specific terms and expressions related to conformity

assessment, as well as information about ISO's adherence to the WTO principles in the Technical

Barriers to Trade (TBT) see www .iso .org/ iso/ foreword .html.

The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 9,

Microbiology.
A list of all the parts in the ISO 20976 series can be found on the ISO website.
iv © ISO 2021 – All rights reserved
---------------------- Page: 6 ----------------------
oSIST prEN ISO 20976-2:2021
ISO/DIS 20976-2:2021(E)
Introduction

Under the general principles of the Codex Alimentarius on food hygiene, it is the responsibility of the

Food Business Operators (FBO) to control microbiological hazards in foods and to manage microbial

risks. Therefore, the FBO shall implement validated control measures, within the hazard analysis and

critical control point (HACCP) system, and conduct 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 may

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, this International Standard consists of

two parts, under the general title Microbiology of the food chain — Requirements and guidelines for

conducting challenge-test:

— 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.
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DRAFT INTERNATIONAL STANDARD ISO/DIS 20976-2:2021(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 sets out 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 terminological databases for use in standardization at the following addresses:

— IEC Electropedia: available at https:// www .electropedia .org/
— ISO Online browsing platform: available at https:// www .iso .org/ obp
3.1
bacterial spore
resistant form of bacteria which is dormant until the germination step
[SOURCE: ISO 20976-1: 2019, 3.1]
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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.
[SOURCE: Regulation (CE) n°2073/2005 [7]]
3.3
bulk product
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- “contaminated” has been replaced by “inoculated”]

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

3.7
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

3.8
experimental datapoint

result of analysis of a test unit per unit weight, per unit volume, or per unit area

Note 1 to entry: Note to entry: the enumeration results may be expressed in log or Most Probable Number

(MPN)
[SOURCE: ISO 20976-1:2019, 3.6]
3.9
germination

mechanism in which a bacterial spore (3.1) initiates its transformation to a vegetative cell (3.31)

[SOURCE: ISO 20976-1:2019, 3.9 modified “starts becoming a vegetative cell” replaced by “initiates its

transformation”]
3.10
inactivation kinetics

change over time in the concentration of the target microorganism subjected to an inactivation process

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3.11
inactivation parameters

mathematical estimates that describe the resistance/sensitivity of the target organism to the treatment,

obtained by fitting primary and secondary models.

Note 1 to entry: Note to entry examples of these parameters are D, δ, p for the primary models and z for the

secondary models.
3.12
inactivation potential
△ 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
3.13
inactivation process
treatment used to kill or inactivate the target microorganism
3.14
inoculum
microbial suspension at the desired concentration used to contaminate test units
[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 5127:2001:2019, 6.2.29]
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
[SOURCE: ISO 20976-1:2019, 3.17]
3.20
pilot facility

a 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
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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(s)
time(s) at which the test units are taken for analyses

Note 1 to entry: Note to entry: when assessing inactivation kinetics these are represented as experimental

datapoints on the inactivation graph
[SOURCE: ISO 20976-1:2019, 3.19]
3.24
secondary model

mathematical model describing the effects of the inactivation process factors (such as temperature, pH,

a ) on the parameters of the primary model (3.18) (e.g.: D, δ)

[SOURCE: ISO 20976-1:2019, 3.20 modified – environmental factors have been changed by inactivation

process factors]
3.25
sporulation
mechanism by which vegetative cell forms spore
[SOURCE: ISO 20976-1:2019, 3.21]
3.26
surrogate

non pathogenic microorganism that has similar or more robust survival 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
time at which the treatment starts
3.30
end
time at which the treatment is finished
3.31
inoc
time at which the microorganism is inoculated in the food
3.32
txD
time of treatment needed for x log reduction of the target microorganism
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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]
3.34
test unit

measured (volume or mass) amount of the food used for inoculation, subsequent treatment and analysis

[SOURCE: ISO 20976-1:2019, 3.24]
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 which is capable of growing under favourable environmental conditions

[SOURCE: ISO 20976-1:2019, 3.25]
3.37
water activity

ratio of the water-vapour pressure in the foodstuff to the vapour pressure of pure water at the same

temperature
[SOURCE: ISO 18787:2017]
3.38
z value

change in treatment (e.g.: temperature, pH, a ,) that induces a 10-fold change in the D value

Note 1 to entry: Note to entry: T, pH, a , can be indexed to the z value to denote the treatment being assessed

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 defined (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 subclause 14.2.1). Ideally, inactivation studies would employ the target microorganism.

In the challenge tests studies conducted within food production facilities, a validated surrogate shall be

used in place of the target pathogen [32].

The organizing laboratory (see 3.19) 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 [2]).

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.

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The same microbial strain could exhibit various shapes as a function of the treatment (see Figure 1) [14].

The heterogeneous shapes of microbial inactivation curves are the results of the microbial resistance

or adaptive response or cell heterogeneity [13].
Inactivation treatment (time or dose)

Figure 1 — Examples of microbial inactivation curve types: log-linear (a), concave (b), convex or

with a shoulder (c), with a tail or biphasic (d,e) and sigmoïdal curve(f)

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

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 is required. Specific labware may 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. These include:

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 the ISO 18787.
5.5 Headspace gas analyser, to measure gas composition (e.g. O , CO ).
2 2
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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 c
...

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