Microbiology of the food chain - Requirements and guidelines for conducting challenge tests of food and feed products - Part 1: Challenge tests to study growth potential, lag time and maximum growth rate (ISO 20976-1:2019)

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 Challengetests bei Lebensmitteln und Futtermitteln - Teil 1: Challengetests zur Untersuchung von Wachstumspotential, Zeit der lag-Phase und maximaler Wachstumsrate (ISO 20976-1:2019)

Dieses Dokument gibt Verfahrensanweisungen für die Durchführung von mikrobiologischen Challenge-Tests für Wachstumsstudien bei vegetativen und sporenbildenden Bakterien in Ausgangsstoffen sowie in Zwischen  oder Endprodukten an.
Die Verwendung dieser Internationalen Norm 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 1: Tests de croissance pour étudier le potentiel de croissance, le temps de latence et le taux de croissance maximal (ISO 20976-1:2019)

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 izzivnega preskusa pri kmetijskih pridelkih in živilskih proizvodih - 1. del: Izzivni preskus za potencial rasti, čas prilagajanja in najvišjo stopnjo rasti (ISO 20976-1:2019)

Ta dokument določa protokole za izvajanje mikrobioloških izzivnih preskusov za potencial rasti
vegetativnih in sporastih bakterij v surovinah in vmesnih ali končnih proizvodih.
Uporabo tega dokumenta je mogoče razširiti na kvasovke, ki ne tvorijo micelija.

General Information

Status
Published
Publication Date
16-Apr-2019
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
17-Apr-2019
Completion Date
17-Apr-2019

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SLOVENSKI STANDARD
SIST EN ISO 20976-1:2019
01-julij-2019
Mikrobiologija v prehranski verigi - Zahteve in smernice za vodenje izzivnega

preskusa pri kmetijskih pridelkih in živilskih proizvodih - 1. del: Izzivni preskus za

potencial rasti, čas prilagajanja in najvišjo stopnjo rasti (ISO 20976-1:2019)

Microbiology of the food chain - Requirements and guidelines for conducting challenge

tests of food and feed products - Part 1: Challenge tests to study growth potential, lag

time and maximum growth rate (ISO 20976-1:2019)

Mikrobiologie der Lebensmittelkette - Leitfaden zur Durchführung von Challengetests bei

Lebensmitteln und Futtermitteln - Teil 1: Challengetests zur Untersuchung von
Wachstumspotential, der Verzögerungszeit und maximaler Wachstumsrate (ISO 20976-
1:2019)

Microbiologie de la chaîne alimentaire - Exigences et lignes directrices pour la réalisation

des tests d'épreuve microbiologique - Partie 1: Tests de croissance pour étudier le

potentiel de croissance, le temps de latence et le taux de croissance maximal (ISO

20976-1:2019)
Ta slovenski standard je istoveten z: EN ISO 20976-1:2019
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 20976-1:2019 en

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

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SIST EN ISO 20976-1:2019
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SIST EN ISO 20976-1:2019
EN ISO 20976-1
EUROPEAN STANDARD
NORME EUROPÉENNE
April 2019
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 1: Challenge tests to study growth
potential, lag time and maximum growth rate (ISO 20976-
1:2019)

Microbiologie de la chaîne alimentaire - Exigences et Mikrobiologie der Lebensmittelkette - Leitfaden zur

lignes directrices pour la réalisation des tests Durchführung von Challengetests bei Lebensmitteln

d'épreuve microbiologique - Partie 1: Tests de und Futtermitteln - Teil 1: Challengetests zur

croissance pour étudier le potentiel de croissance, le Untersuchung von Wachstumspotential, der

temps de latence et le taux de croissance maximal (ISO Verzögerungszeit und maximaler Wachstumsrate (ISO

20976-1:2019) 20976-1:2019)
This European Standard was approved by CEN on 21 January 2019.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,

Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,

Turkey 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

© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20976-1:2019 E

worldwide for CEN national Members.
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SIST EN ISO 20976-1:2019
EN ISO 20976-1:2019 (E)
Contents Page

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

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SIST EN ISO 20976-1:2019
EN ISO 20976-1:2019 (E)
European foreword

This document (EN ISO 20976-1:2019) has been prepared by Technical Committee ISO/TC 34 "Food

products" in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”

the secretariat of which is held by DIN.

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 October 2019, and conflicting national standards shall

be withdrawn at the latest by October 2019.

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.

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, Former Yugoslav Republic of Macedonia,

France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,

Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,

Turkey and the United Kingdom.
Endorsement notice

The text of ISO 20976-1:2019 has been approved by CEN as EN ISO 20976-1:2019 without any

modification.
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SIST EN ISO 20976-1:2019
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SIST EN ISO 20976-1:2019
INTERNATIONAL ISO
STANDARD 20976-1
First edition
2019-03
Microbiology of the food chain —
Requirements and guidelines for
conducting challenge tests of food and
feed products —
Part 1:
Challenge tests to study growth
potential, lag time and maximum
growth rate
Microbiologie de la chaîne alimentaire — Exigences et lignes
directrices pour la réalisation des tests d'épreuve microbiologique —
Partie 1: Tests de croissance pour étudier le potentiel de croissance, le
temps de latence et le taux de croissance maximal
Reference number
ISO 20976-1:2019(E)
ISO 2019
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SIST EN ISO 20976-1:2019
ISO 20976-1:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019

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
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
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SIST EN ISO 20976-1:2019
ISO 20976-1:2019(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction ................................................................................................................................................................................................................................vi

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

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

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

4 Principle ........................................................................................................................................................................................................................ 4

4.1 General ........................................................................................................................................................................................................... 4

4.2 Estimation of the growth potential ....................................................................................................................................... 6

4.3 Estimation of the growth kinetics parameters (lag time and maximum growth rate) ............. 7

5 Apparatus ..................................................................................................................................................................................................................... 7

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

7 Study design and sampling ........................................................................................................................................................................ 8

7.1 General .......................................................................................................................................................................................................... 8

7.2 Setting decision criteria for growth potential .............................................................................................................. 8

7.3 Number of batches and selection criteria ........................................................................................................................ 8

7.4 Preparation of the test units ........................................................................................................................................................ 8

7.5 Number of test units to be inoculated................................................................................................................................. 9

8 Selection of strains ............................................................................................................................................................................................. 9

9 Preparation of the inoculum .................................................................................................................................................................10

9.1 General .......................................................................................................................................................................................................10

9.2 Preparation of the vegetative cell suspensions ........................................................................................................10

9.3 Preparation of the spore suspensions .............................................................................................................................10

10 Inoculation of the tests units ................................................................................................................................................................10

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

11.1 Food controls .........................................................................................................................................................................................11

11.2 Control units...........................................................................................................................................................................................11

12 Storage of the test units ..............................................................................................................................................................................12

12.1 General ........................................................................................................................................................................................................12

12.2 Estimation of growth potential ..............................................................................................................................................12

12.3 Estimation of growth kinetics parameters (lag time and growth rate) ..............................................12

13 Analysis .......................................................................................................................................................................................................................12

14 Expression of the results ...........................................................................................................................................................................13

14.1 General ........................................................................................................................................................................................................13

14.2 Growth potential (Δ) .......................................................................................................................................................................13

14.3 Growth kinetics parameters (lag time and growth rate) .................................................................................14

15 Test report ................................................................................................................................................................................................................14

15.1 General ........................................................................................................................................................................................................14

15.2 Aim of the study and type of challenge test ................................................................................................................14

15.3 Experimental protocol ...................................................................................................................................................................15

15.4 Sample analysis ...................................................................................................................................................................................15

15.5 Results .........................................................................................................................................................................................................15

15.6 Conclusions .............................................................................................................................................................................................16

15.7 Reference documents .....................................................................................................................................................................16

Annex A (informative) Inter-batch variability assessment based on pH and a .................................................17

Annex B (normative) Minimum number of units to prepare for the challenge test study ........................18

Annex C (informative) Examples of protocols to prepare inocula ......................................................................................19

© ISO 2019 – All rights reserved iii
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SIST EN ISO 20976-1:2019
ISO 20976-1:2019(E)
Annex D (informative) Examples of how to estimate growth potential, lag time and

maximum growth rate from results of challenge tests ..............................................................................................22

Annex E (informative) Use of simulation to assess a microbial population under different

temperature conditions .............................................................................................................................................................................26

Bibliography .............................................................................................................................................................................................................................27

iv © ISO 2019 – All rights reserved
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SIST EN ISO 20976-1:2019
ISO 20976-1:2019(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.
A list of all the 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 2019 – All rights reserved v
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SIST EN ISO 20976-1:2019
ISO 20976-1:2019(E)
Introduction

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

business operators (FBOs) to control microbiological hazards in foods and to manage microbial risks.

[11]

Therefore, FBOs 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 for consumers.

This document provides technical rules, calculations and approaches to investigate the ability of

inoculated microorganism(s) of concern to grow, survive or be inactivated in raw materials and

intermediate or end products under reasonably foreseeable food processes, storage and use conditions.

The objective and the scope of the document are to determine the experimental design and the selection

of the study conditions. Regulatory authorities can have different recommendations, and these

differences have been included as much as possible. It is, however, possible that specific requirements

should be incorporated to get regulatory approval of the challenge test.

As growth and inactivation kinetics 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 growth potential, lag time and maximum growth rate

— Part 2: Challenge tests to study inactivation potential and kinetics parameters (to be developed)

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. Even

though many mathematical models are available to describe and predict bacterial growth, the gamma-

[22]

concept (γ-concept) is particularly useful for further simulations using the data generated from

the challenge test, e.g. to assess the growth at storage temperatures other than the one(s) tested, or in

helping to design better food formulations and storage conditions, and thus improving the microbial

quality and/or safety of the food under consideration.
For practical reasons, the term “food” includes feed.
vi © ISO 2019 – All rights reserved
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SIST EN ISO 20976-1:2019
INTERNATIONAL STANDARD ISO 20976-1:2019(E)
Microbiology of the food chain — Requirements and
guidelines for conducting challenge tests of food and feed
products —
Part 1:
Challenge tests to study growth potential, lag time and
maximum growth rate
1 Scope

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.
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 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
ISO 18787:2017, Foodstuffs — Determination of water activity
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:

— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1
bacterial spore
resistant form of bacteria that is dormant until the germination (3.9) step
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: Commission Regulation (EC) No 2073/2005]
© ISO 2019 – All rights reserved 1
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SIST EN ISO 20976-1:2019
ISO 20976-1:2019(E)
3.3
cardinal value

estimated minimal, optimal and maximal values of physico-chemical factors (e.g. temperature, pH, a )

that characterize the growth of a given microbial strain
3.4
control unit

unit of food identical to the test unit (3.24) but not artificially contaminated (used as a blank)

3.5
challenge test

study of the growth or inactivation of microorganism(s) artificially inoculated in food

3.6
experimental datapoint

result of analysis of a test unit (3.24) per unit weight (log cfu/g), per unit volume (log cfu/ml), or per

10 10
unit area (log cfu/cm )

Note 1 to entry: For specific cases, the enumeration results may be expressed in log MPN.

3.7
exponential growth phase

phase in which the microbial population is exponentially multiplying as rapidly as possible; growth is

dependent on the growth medium and environment (temperature, humidity, etc.)

Note 1 to entry: Figure 1 describes the three phases of microbial growth kinetics.

3.8
generation time

time it takes for the microorganisms to increase by a factor 2, also known as doubling time

3.9
germination

mechanism in which a bacterial spore (3.1) starts becoming a vegetative cell (3.25)

3.10
growth potential

difference between the decimal logarithm of the highest concentration of the target microbial population

(log ) and the decimal logarithm of the initial concentration of this microbial population (log )

max i

Note 1 to entry: The log and log refer to concentrations and are expressed in log cfu/g or log cfu/ml or

max i 10 10
log cfu/cm
3.11
maximum growth rate

kinetics parameter to characterize the exponential growth phase (3.7), represented by the slope of

the curve showing the evolution of the natural logarithm (μ ) or decimal logarithm (V ) of the

max max
population as a function of time, under constant growth conditions
3.12
inoculum

microbial suspension at the desired concentration used to contaminate test units (3.24)

3.13
lag phase

phase, directly after inoculation, during which the microbial population is adapting to the environment,

before it enters the exponential growth phase (3.7)

Note 1 to entry: Figure 1 describes the three phases of microbial growth kinetics.

2 © ISO 2019 – All rights reserved
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SIST EN ISO 20976-1:2019
ISO 20976-1:2019(E)
3.14
lag time
kinetics parameter in time unit to characterize the lag phase (3.13)
3.15
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 — Notes 1 and 2 to entry have been removed.]

3.16
primary model

mathematical model describing the changes of microbial counts as a function of time

3.17
organizing laboratory
laboratory with responsibility for managing the challenge tests (3.5)
3.18
sampling

selection of one or more units or portions of food such that the units or portions selected are

representative of that food
3.19
sampling point

time at which the test units (3.24) are analysed and which are represented as experimental datapoints

(3.6) on the kinetics graph
3.20
secondary model

mathematical model describing the effects of the environmental factors (e.g. temperature, pH, a ) on

the parameters of the primary model (3.16) (e.g. growth rate)
3.21
sporulation
mechanism by which vegetative cell (3.25) forms spore
3.22
stationary phase
phase in which the microbial population is at its maximum level

Note 1 to entry: Figure 1 describes the three phases of microbial growth kinetics.

3.23
test portion

measured (volume or mass) representative sample taken from the test unit (3.24) for use in the analysis

[SOURCE: ISO 6887-1:2017, 3.5, modified — The end of the definition has been changed from “taken

from the laboratory sample for use in the preparation of the initial suspension” and the Note 1 to entry

has been removed.]
3.24
test unit
measured (volume or mass) amount of the food used for inoculation
3.25
vegetative cell

state of microbial form that is capable of growing under favourable environmental conditions

© ISO 2019 – All rights reserved 3
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SIST EN ISO 20976-1:2019
ISO 20976-1:2019(E)
3.26
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, 3.1, modified — The definition has been condensed and the formula and

Notes 1 and 2 to entry have been removed.]
4 Principle
4.1 General

The aim of the study shall be clearly defined (e.g. assessment/validation of the food shelf-life as a control

measure, assessment of microbial stability). The experimental design shall be in accordance with that

purpose and shall take into account the steps of the food chain for which microbial growth is assessed.

The decision criteria shall be clearly defined (see 7.2).

Knowledge from the FBO on its products (e.g. characteristics or production proces

...

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