Como Puedo Aumentar La Seguridad Alimentaria de Mis Productos

Cmo puedo aumentar la seguridad alimentaria de mis productos?
How to improve the food safety of my products?
Cmo puedo aumentar la seguridad

alimentaria de mis productos?
How to improve the food
safety of my products?
INSTITUTO TOMS PASCUAL SANZ

para la nutricin y la salud
P de la Castellana 178 - 3 Dcha. 28046 Madrid
Tel.: 91 703 04 97. Fax: 91 350 92 18
webmasterinstituto@institutotomaspascual.es www.institutotomaspascual.es
Universidad de Burgos
Hospital del Rey s/n. 09001 Burgos
Coordinacin editorial:
Alberto Alcocer, 13, 1 D. 28036 Madrid
Tel.: 91 353 33 70. Fax: 91 353 33 73. imc@imc-sa.es
Reservados todos los derechos. Ninguna parte de esta publicacin puede ser reproducida, transmitida en ninguna forma o medio alguno, electrnico o mecnico, incluyendo
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ISBN: 978-84-7867-054-3
ISBN: 978-84-92681-13-6
Depsito Legal: M-20018-2010

Autores/Authors
B. Biavati
University of Bologna. Italy.
S. Braun
Department of Economic Policy. University of Stuttgart. Germany.
N. Carlini
Probiotical S.p.A., Novara, Italy.
B. Carpentier
AFSSA, Research Laboratory on Food Quality and Processes. Maisons-Alfort Cedex, France.
R. Cocker
Cocker Consulting Ltd. Ireland.
F. Devlieghere
Department of Food Safety and Food Quality, Laboratory of Food Preservation
and Food Microbiology, Faculty of Bioscience Engineering, Ghent University. Belgium.
A. Friis
National Food Institute, Technical University of Denmark. Lyngby, Denmark.
F. Gagga
University of Bologna. Italy.
L. Gram
DTU Aqua. Lyngby, Denmark.
K. Hadwiger
Department for Economic Policy. University of Stuttgart. Germany.
K. Hruska
Veterinary Research Institute, Brno, Czech Republic.
L. Jacxsens
V. Jasson
RA. Juste
NEIKER-Tecnalia, Bizkaia. Spain.
M. Kousta
Agricultural University of Athens, Department of Food Science and Technology,
Laboratory of Food Quality Control and Hygiene. Athens, Greece.
J. Kussaga
Product Design and Quality Management Group, Department of Agrotechnology and Food Sciences,
Wageningen University. The Netherlands.

H. Lje
National Food Institute, Technical University of Denmark. Lyngby, Denmark.
PA. Luning
M. I. Llorca
AINIA Centro Tecnolgico. Spain.
WJ. Marcelis
Management Studies Group, Department of Social Sciences, Wageningen University. The Netherlands.
V. Martnez
AINIA Centro Tecnolgico. Spain.
A. Martnez-Lpez
Instituto de Agroqumica y Tecnologa de Alimentos, CSIC. Burjassot, Valencia. Spain.
L. Mogna
S. Oss
Department of Biotechnology and Food Science. University of Burgos. Spain.
I. Pavlik
Veterinary Research Institute, Brno, Czech Republic.
MC. Pina-Prez
A. Rajkovic
Laboratory of Food Microbiology and Food Preservation, Department of Food Safety and Food Quality, Faculty of
Bioscience Engineering, Ghent University, Ghent, Belgium.
D. Rodrigo
J. Rovira
Department of Biotechnology and Food Science. University of Burgos. Spain.
AB. Silva-Angulo
N. Smigic
GP. Strozzi
M. Uyttendaele
M. Van der Spiegel
J. Verran
Manchester Metropolitan University, School of Biology, Chemistry and Health Science. United Kingdom.
ndice/Index
7
Prlogo
D. Ricardo Mart Flux
Prologue
Prof. Mogens Jakobsen
15
Obstacles and solutions for the knowledge transfer between

science and industry
S. Braun, K. Hadwiger
23
La seguridad alimentaria en el siglo XXI

MC. Pina-Prez, AB. Silva-Angulo, D. Rodrigo, A. Martnez-Lpez
31
Tools to support the self assessment of the performance of Food

Safety Management Systems
PA. Luning, L. Jacxsens, V. Jasson, WJ. Marcelis, J. Kussaga,
M. Van der Spiegel, M. Kousta, S. Oss, J. Rovira,
F. Devlieghere, M. Uyttendaele
45
Prevention of biofilm formation and foodborne infections by control of moisture management

R. Cocker
55
How can the food industry contribute to decrease the risk of

contamination by mycobacteria: a hypothetical case discussion
K. Hruska, I. Pavlik, RA. Juste
63
New processing technologies that can reduce the presence of

pathogens in foods
A. Rajkovic, M. Uyttendaele, N. Smigic, F. Devlieghere
73
Reducing foodborne pathogens in the food chain by the use of

protective and probiotic cultures
N. Carlini, L. Mogna, GP. Strozzi
89
The application of PathogenCombat research results in practice

F. Gagga, B. Biavati
93
EHEDG: mtodo para la comprobacin de la evaluacin de la

limpieza in situ de los equipos para el procesado de los alimentos
M. I. Llorca
103
New cleaning and disinfection methods and summary of methods

applied for verification of their efficiency
H. Lje, A. Friis, L. Gram, B. Carpentier, J. Verran
117
Aplicacin de nuevas tcnicas eco eficientes de limpieza y

desinfeccin en sistemas CIP, basadas en el uso del ozono.
Proyecto OZONECIP
V. Martnez
Prlogo
PathogenCombat es la abreviatura del proyecto Control y prevencin a niveles

celulares y moleculares de patgenos emergentes y futuros dentro de la cadena
alimentaria. El ttulo es bien expresivo y demuestra la importancia que la Unin
Europea concede a la seguridad alimentaria.
Las nuevas tcnicas de procesado y los envases con atmsfera modificada procuran conservar al mximo las propiedades sensoriales y nutritivas de los alimentos pero, al mismo tiempo, posibilitan la generacin de nuevos nichos ecolgicos donde especies que antes no eran deterioradoras o patgenas van a
serlo en esas circunstancias. La ciencia debe abrir caminos a la industria alimentaria, describiendo estos fenmenos y enseando como detectarlos y controlarlos.
Esta publicacin recoge las actas del Seminario Cmo puedo aumentar la seguridad alimentaria de mis productos?, una de las actividades acadmicas programadas en la Ctedra Toms Pascual Sanz, Universidad de Burgos, aprovechando la reunin en Burgos de cientficos participantes en el proyecto que
expusieron sus investigaciones y resultados. Organizado por la Universidad de
Burgos y dirigido por su Vicerrector de Investigacin, el Dr. Jordi Rovira, en este
seminario se trataron temas relacionados con la desinfeccin y limpieza de
plantas, mejora del nivel de calidad de los alimentos y equipos agroalimentarios y sistemas de gestin de la seguridad alimentaria, todo ello desde un enfoque multidisciplinar y colaborativo entre los centros europeos que forman
parte del Proyecto PathogenCombat.
Quizs sorprenda al lector que publiquemos este libro en el idioma ingls. Pero
la naturaleza cientfica del proyecto y lo plural de sus participantes nos aconsejan esta decisin. Este es un libro para todos los participantes del proyecto
y su trascendencia va ms all del mbito nacional.
Para nuestro Instituto es un orgullo contribuir a la difusin de los resultados
de este proyecto, interesante para la ciencia y para la empresa, abriendo caminos a la ciencia espaola y en particular a los equipos de la Universidad de
Burgos, cuyo trabajo merece todo nuestro apoyo.
D. Ricardo Mart Flux
Presidente Instituto Toms Pascual Sanz
para la nutricin y la salud
Prologue
Overview
The PathogenCombat-project is an integrated project under the 6th Framework
Program of the European Union. It started in 2005 and will run till April 2010. The overall objectives are summarised in table 1.
Table 1. Overall, the objetives of PathogenCombat can be describeb as follows:

1. Production of safe food with none or acceptably low levels of pathogens.
2. Determination of factors in the food chain, which enable the viability, persistence and
virulence of pathogens.
3. Detection and prediction of the occurrence and virulence of pathogens in the food chain
with molecular biology based culture independent techniques and microarrays.
4. Determination of host-pathogen interaction with functional cell model replacing the use of
experimental animals.
5. Prevention of pathogen transmission along the food chain by new processing technologies
and systems, protective cultures and new information on host-pathogen interaction.
6. Application in the food chain/SMEs of PathogenCombat deliverables.
7. Pathogen control throughout the food chain by new mathematical models.
8. Food Safety Management System, which incorporates the deliverables of PathogenCombat.
9. SME Network including dissemination of knowledge and dissemination of results.
10. Training of SMEs.
11. Promoting consumer awareness of food safety.
The Pathogen Combatproject deal with the issue of food safety, which becomes
more and more important for consumers and industry. With the media coverage of food
born diseases and consistently occurring food scandals, the public becomes more aware
and interested in these problems. So, while the investment in research in this broad spectrum is rising, the problem still doesnt seem to fade. The PathogenCombatproject
including the problem of food pathogens by a multidisciplinary approach, dealing with
known and upcoming food borne pathogens making use of a variety of methods throughout the entire food chain.
The project developed a several of platforms to investigate the occurrence, virulence and
survival of pathogens in the food chain, from primary production to the consumer. The
platforms rest upon fluorescent ratio imaging microscopy (FRIM), laser tweezers, phage
display, functional cell models, functional genomics and microarrays. Emerging food
10
Coordinator
Mogens Jakobsen, LIFE
Deputy Coordinator
Effiue Tskalidou AUA
Project manager
Vicki Lei, LIFE
Consortium meetings
External Advisory Board

Larry Beuchat, USA
Grahame Gould, UK
Karel Hruska, CZ
Wihelm Holzapfel, DE
Niels Skovgaard, DK
Serv Notermans, NL
Project Management Group

Member of External Advisory Board (Larry Beuchat)
Coordinator (Mogens Jakobsen)
Administration
NTC team
RTD team
Francois Lefvre, INRA
Susanne Braun, STUITT
Jrn D. Mikkelsen, DANISCO Clare HALL, SAC
Luca Cocoln, UNIUD
Bruno Biavati, UNIBO
Knowledge
management
& IPR
Reporting
& communication
Leader:Clare Hall, SAC
III
Consumer awareness
Application and management

Leader: Bruno Biavati, BU
WP: 13, 14 and 15
Leader: Susanne Braun, STUTT
Control and prevention

Leader: Luca CXocolin, UNIUD
WP: 8, 9, 10, 11 and 12
II
Training activities
Leader: Susanne Braun, STUTT
SME network
II
Detection and virulence traits
Leader:Jrn D.Mikkelsen, Danisco
WP: 4, 5, 6 and 7
NTC Pillars
I
Discovery and development
Leader: Franois Lefevre, INRA
WP: 1, 2 and 3
RTD Pillars
Finance
Figure 1. Organizational chart of the PathogenCombat-project.
borne bacteria, yeast, fungi and viruses are targeted for milk and dairy products, ruminants, poultry and pigs and their meat products.
The pillars of RTD (Research, Technology and Development) and NTC (Network, Training
and Consumer) are the two supporting legs of the project.
In overal terms the RTD Pillars are dealing with basic research, scientific development
and invention of new techniques. In details they can be described as follows.
RTD I Discovery and development
This pillar addresses pathogen-matrix interaction. Measurements were carried out to investigate the factors that enable a pathogen to establish itself in food and on feed matrices and food, feed and water contact surfaces. Microbial surface molecules responsible for microbial adhesion to matrices and surfaces were determined and subsequently
used as target for prevention of adhesion. PCR techniques and microarrays were developed for determination and prediction of microbial expression of virulence, expression
of stress and for expression of genes responsible for adhesion. Microarrays for gene expressions have been developed to study the behaviour of pathogens in the food chain
at the molecular level i.e. pork, poultry and ruminants and enable host pathogen inte-
Prologue
11
ractions to be studied at the molecular level. Functional cell models were established for
pork, poultry and ruminants to be used in studies of host-pathogen interactions.
RTD II Detection and Virulence traits
PathogenCombat developed reliable and cost-effective sampling schemes as well as rapid
and specific methods methods for detecting new and emerging pathogens. This is important in relation to developments in the food industry in which raw materials and ingredients originate from many different countries and different production systems. Such
external sources of new and emerging pathogens are also a likely source of pathogens
with unknown patterns of resistance towards control methods and with unknown virulence traits. Stress adaptation and new virulence traits may develop within the particular
food chain and in both situations call for appropriate control and detection methods to
be applicable throughout the chain.
Based upon the functional genomics studies, culture independent techniques to detect
new and emerging pathogens were developed. DNA-arrays for detection of pathogens
were developed to predict the occurrence of pathogens as well as their virulence at a
given time in the food chain and at the time of consumption. The methods developed
not only report numbers of pathogens, but most importantly they give information on
their virulence and relevance in the food chain.
RTD III Control and Prevention
Attachment and establishment of viable and virulent pathogens in or on solid matrices
is considered the most important route of transmission along the food chain. Detachment
e.g. by cleaning or exposure to various food processing stresses leading to inactivation
as well as loss of virulence will break this transmission.
Studies to determine and improve the hygienic design and close the present gap between hygiene and technology were conducted. The optimal characteristics of food contact surfaces and efficient cleaning and desinfection procedures have been addressed.
Protective and probiotic bacteria, which can prevent attachment by competitive exclusion or inactive pathogens in the food chain including the intestinal tract of pigs, chicken
and ruminants as estimated by use of the functional cell model, were identified. Finally
inactivation of pathogens by mild processing techniques, e.g. intense UV light pulses
and hydro-static pressure was included.
RTD IV Application and Management
The food processing SME Partners and the food chain they belong to were the targets
and collaborators in application of the results obtained in RTD I, II and III and for the development of the generic and specific Food Safety Management Systems. This is the transition of research and development into practical application in the food industry fully
integrated with networking of SMEs and the training activities described in NTC I and
NTC II respectively. The SMEs involved were dealing with milk and dairy products, poultry,
12
pigs, beef and lamb and their meat products. Modelling of microbial food safety risk estimation played a major role in the development of the Food Safety Management System
by identification of CCPs of the HACCP based system developed.
The NTC Pillars are dealing with the tasks of transferring the new information and technologies to the users. This is done by creating a network of enterprises that would profit from
these findings, providing, processing and distributing knowledge in appropriate form, offering training courses and increasing consumer awareness to food borne pathogens.
The tasks are in detail:
NTC I SME Network
The integration of all SMEs and industrial Partners in PathogenCombat from the very beginning and the continuous exchange of knowledge and experiences gained are essential for the project.
Through contacts to SMEs having experience from participating in EU projects in depth
information are obtained on the optimal method to integrate SMEs in PathogenCombat.
The network is operational through a net-based system of communication. The function of the network is supported by the training activities and the dissemination of Project
results is described in NTC II. The network is permanently expanded with new partners,
government agencies and associations.
NTC II Training
It is the main objective of training to ensure the transition from research to practical application. Transition from research to practical application is carried out to allow a European
wide production of food with no or acceptably low level of pathogens. This training is organized in three levels. Level one addresses the food producing SME Partners of
PathogenCombat. At Level two SMEs outside PathogenCombat are included in several regional workshops to teach the participants how to apply the deliverables of
PathogenCombat including the Food Safety management System developed. Level three
is pan-European and based upon the SME network created in NTC I including national and
European SME organizations as well as regulatory agencies.
NTC III Consumer Awareness
This activity involves the participation of consumers and consumer groups and organizations. It establishes methods for effectively communicating accurate information regarding
food safety issues to European consumers. It seeks to strengthen our understanding of the
role of food safety in consumer behavior and thereby develop best practice in communicating with consumers to enhance their awareness of food safety issues. It is an objective of
this activity to raise awareness of food safety issues throughout Europe and to strengthen
the activities in NTC Pillars I and II.
The unique achievements of PathogenCombat can be summarised as shown in table 2.
Prologue
13
Table 2. Achievements: examples.

I. New biotechnological platforms
Novel approaches to analyse the interactions at cellular and molecular level between pathogens
and food and feed matrices and contact surfaces in the food chain including the intestinal tract
of farm animals. To understand the mechanisms, by which pathogens enter, adapt, persist and
express virulence in the food chain:
Fluorescence Ratio Imaging Microscopy, atomic force microscopy and bio-imaging.
Laser tweezer technology.
Convergent evolution.
Functional genomics.
Functional mammalian cell models.
II. Novel information on emerging pathogens
Listeria monocytogenes, Mycobacterium avium subsp. paratuberculosis, Campylobacter jejuni,
Escherichia coli (STEC), Saccharomyces cerevisiae, Penicillium nordicum, Hepatitis E (HEV) &
tickborne encephalitis (TBEV) virus and Staphylococcus aureus.
III. Rapid and meaningful detection methods
Microarrays and molecular biology culture-independent techniques, have been developed for
pathogens included in PathogenCombat. The methods will not only report numbers, but include
a new approach to estimate viability and virulence of pathogens throughout the food chain.
IV. Virulence expression in matrices
A novel strategy for food formulation, food preservation and quantitative risk assessment.
V. Methods for breaking the transmission of pathogens along the food chain
Prevention of cross contamination by hygienic design and development of cleaning and
disinfection procedures to remove bio-films in the food chain.
Inactivation of pathogens by mild processing techniques (organic acids, chlorine dioxide, intense
UV light pulses, and hydrostatic pressure).
New protective and probiotic cultures for elimination of pathogens in the food chain.
VI. Mammalian functional cell models
Models developed for pigs, chicken and ruminants. Applied in host-pathogen interactions and
selection of protective and probiotic cultures. A new opportunity has been made available for
determinations of dose-response and risk assessment.
VII. Food Safety Management
Diagnostic instruments and tools have been developed for SMEs for identification of technological
and managerial interventions which can improve food safety management systems (FSMS) and
lead to integration of the new knowledge and methods developed in PathogenCombat. Concept
of web-based FSMS support systems for SMEs has been developed.
VIII. Interaction with food producing SMEs
For new knowledge obtained and methods and tools developed, validation and testing of
applicability in SMEs are in progress.
IX. The consumer, the food industry and the regulatory bodies in Europe
The consumers understanding for key food safety issues has been evaluated and the most effective
ways of communicating results is being assessed. A strategy for interaction and exchange of
information with the food industry and regulatory agencies has been established.
Prof. Mogens Jakobsen
Coordinator of PathogenCombat. Faculty of Life Sciences, University of Copenhagen, Denmark
Obstacles and solutions for the knowledge

transfer between science and industry
S. Braun, K. Hadwiger
Overview
Knowledge transfer
Knowledge transfer is defined as the problem of transferring knowledge between
different organizations or from one part
of an organization to another part of the
same organization. In other words, knowledge transfer is the gateway for the
transfer of knowledge between donating
and receiving entities. In most cases, the
donor is a public organization, like a university or research institute, while the receiving entity, which often has interest in
commercializing the knowledge, is a company. Furthermore, knowledge transfer
can be seen as a more general term, describing the distribution of knowledge through human beings. This approach focuses on characteristics of communication
between experts and the person that receive the knowledge.
Despite the different views, knowledge
transfer is often hindered by a variety of
issues. Nowadays, removal of these barriers and the improvement of knowledge
transfer are seen as a main driver of economic growth. The present society has
been defined as a knowledge society. Due
to increasing importance of knowledge,
a close relationship between science and
technology became more important.
Generally, the increased competition on a
global scale led to an increasing rate of
technology changes, shorter product life

cycles and increasing product quality. The
finding and utilization of advanced knowledge and new technologies is more than
ever substantial for the survival and prosperity of any firm.
Many large enterprises have their own
scientific department. Therefore, the importance of distribution of knowledge
created by public bodies has became
more important, especially, for small and
medium enterprises (SMEs) that cannot
afford the cost of research and are dependent on external knowledge to improve
their production processes. Although it is
an important advantage for large enterprises that they are able to conduct research, it has become increasingly difficult
to rely solely on internal knowledge production.
Companies are classified as small and medium-sized enterprises (SMEs) by the
European Union if the number of their
employees does not exceed 250 and if
they are independent from large companies. In addition, their annual turnover
may not be beyond 50 million, or their
annual balance sheet beyond 43 million. SMEs are divided into three subcategories: micro-enterprises with 10 or less
employees, small enterprises with 10 to
49 employees and medium enterprises
with 50 to 249 employees.
16
29,69
32,9
Micro
Small
Medium
Large
16,8
20,6
In this context, the distribution of knowledge transfer has become an important

subject of scientific research, as well as in
economic and public policy. An important
role of the PathogenCombat project is the
support of SMEs of the food sector in this
process and helps with the implementation of new technologies.
Figure 1. Classification of enterprises based on

number of employees, non-financial business sector
(Source: Eurostat).
16
14
12
10
%
For a successful knowledge transfer from

any knowledge donor to a SME, it is not
sufficient to provide knowledge or technology, considering their limited possibilities, also the financial support of the
SME is necessary to make the implementation of new technology possible.
The different industrial branches depend
on this external knowledge on a varying
degree. The food industry in the European
Union, which consists mainly of SMEs
with 10 or less employees, is heavily dependent on this knowledge due to a lack
of resources to carry out any research and
development activity. It is estimated that
70% of all SMEs of the food sector do
not carry out any kind of research activity.
In addition to that, this industry has to
deal with the demand for safer products
and higher quality, as well as a high competition. To cope with these challenges,
new technologies have to be introduced.
6
4
2
Pulp and paper
Wood products
Textiles
Food and drink
All NACE branches
Computer services
ICT
Automobile
Chemicals
Electronical and
optical equipment
The importance of technology transfer is

obvious when one takes into account that
98.8% of all European enterprises are in
fact SMEs and they provide 67% of all
jobs in the private sector. Moreover,
91.5% of SMEs are in fact micro-enterprises with less than 10 employees.
Figure 2. Research activity as percentage of added

value by industrial sector.
The food sector is the largest manufacturing sector in the European Union, with
an annual turnover of 910 Billions in
2008. Its importance is also shown in its
high level of legal regulation. Many national and international regulations determine the form of production and food
handling as well as food quality itself.
New regulations are added at a regular
basis and the demand is still increasing.
SMEs have to survive in an overregulated
marked, with high requirement and
strong competition. The implementation
of new knowledge in the food industry is
not only optional, but necessary for all
companies. Its assistance should be considered as highest priority.
The task of supporting innovation in food
producing SMEs becomes even more important when taking into consideration
Obstacles and solutions for the knowledge transfer between science and industry
17
that exports of the food industry have shrunken in the last years. The market share
of world food and drink exports of the
European Union diminished from 24.8%
in 1998 to 19.2% in 2008. New innovations in the food sector could help averting a further decline and improve competitiveness on the world market.
1,4
1,2
1
0,8
USA
EU15
Japan
%
0,6
0,4
prises regarding their research potential.

Contrary to the knowledge transfer between two enterprises, knowledge created
by universities is widely available.
Moreover, there are many advantages for
enterprises and universities to collaborate.
The enterprise can benefit from the highly
trained staff of a public institution, and
can also improve their corporate image
due to this joint venture. The university
can benefit from additional fund for research, the opportunity to research close to
the market and an increased opportunity
of employment for students and graduates.
0,2
0
1996
1997
1998
1999
2000
2001
2002
2003
2004
Figure 3. Business expenditure on research by food

industries in different countries (percentage of industry output).
The focus of knowledge transfer to SMEs

is on the distribution of knowledge created at universities, which is mostly described as being largely underexploited.
However, recent studies indicate a positive trend in university-industry interaction. The reasons for this improvement
are changes in public policies and institutional environment and a strong encouragement of commercialization of scientific discoveries. Prior to this improvement,
scientific discoveries arose directly from
the ivory tower of science, and there
was no specific aim to utilize and commercialize them. At present, academic research is more focused on future industrialization and exploitation of their
discoveries.
Universities and other public research institutions are not comparable with enter-
The cooperation of public research bodies

like universities and private enterprises
can work in a variety of ways. The most
common way is the support of research,
mostly by financial contributions. This funding was often unspecific and unrestricted in the past, but nowadays it is
bound to specific research areas and activities that presumably will benefit the financer by bringing in new knowledge
and technology. Another possibility of cooperation is cooperative research. This
postulates a close teamwork in the field
of research. The research teams, constituted by scientists of both partners, have
to work together on a topic to maximize
efficiency and prevent interference of research fields.
There is also a possibility of direct knowledge or technology transfer in both directions. With this method both teams
need also to have a broad understanding
of the particular research field and full insight in each others work. However, the
implementation of new technology has to
be conducted with full support of the donating partner.
18
The formerly low levels of knowledge distribution of universities were attributed to

a lack of knowledge structures at the enterprise side and a lack of stimulant gratification for the distribution of scientific
knowledge by the academic side.
Different approaches have been tried to
eliminate this problems. Universities have
accumulated a lot of experience in the last
years on transfer of technologies, and public organizations which focus on the distribution and transfer of knowledge between science and industry have been
founded.
PathogenCombat tries to contribute to
the knowledge transfer in the area of
food safety. Later in this article, the problems concerning knowledge transfer
between science and industry will be described, and the role of PathogenCombat
will be presented in the interaction between science and industry and its contribution to technology and knowledge
transfer.
Obstacles in knowledge transfer
Some problems concerning knowledge
transfer have already been mentioned in
this article (see overview). The most
common problems are:
The lack of willingness to share knowledge by the donor, due to:
Mistrust.
Assumed benefits of possessing
knowledge exclusively.
Absence of ability to transfer knowledge to a non-specialist.
Language and culture barriers.
Lacking of rewards for knowledge
transfer.
The lack of willingness to accept knowledge by the receiving entity, due to:
Lack of structures for knowledge processing.
Missing contacts to knowledge producers.
Lack of knowledge concerning the
know-how transfer process.
Lack of ability to understand and implement knowledge.
Language and culture barriers.
Knowledge transfer is hampered by a variety of ways. Both donors and receiving
entities of knowledge can be the reason
for stopping the transfer and implementation of knowledge and technology.
Firstly, the knowledge donor should have
the willingness to share his knowledge to
a third party. Knowledge, as a property of
some worth, is nothing to give away easily because it is often perceived as earned
by hard work and years of research. A
scientist who publishes his findings in
scientific papers will clearly receive a reward in form of reputation. In the process
of technology transfer, the benefits can
be less evident. Whichever form of cooperation is chosen, important research results have to be given away, often, before
publishing. As a consequence, it is very
important to provide an appropriate reward for sharing knowledge. It has to be
very clear that the receiving entity and the
knowledge donor will equally benefit
from this process. It is important that both
partners build up their relationship based
on trust. Knowledge has a considerable
value and, unlike other goods, it can be
given away quite easily, even unknowingly. Consequently, the partners in a
19
While many large enterprises have dedicated structures that deal with knowledge
transfer, like scientific trained employees
or research departments, most SMEs lack
employees which have an understanding
Employees with higher education
Manufacture of pulp paper

and paper products;
publishing and printing
Manufactur of textiles and

textile products
Products of wood and cork;

manufacture of articles of
straw and plaiting materials
Motor vehicles, trailers and

semi-trailers
Manufacture of chemicals and

chemical products
Computer services and

related activities
450,00
400,00
350,00
300,00
250,00
200,00
150,00
100,00
50,00
0,00
ICT
Scientific information is easily available,

often for free. While some scientists pay
to the scientific journals for publishing
their scientific articles, the author himself provides these papers willingly for
free. Despite this, research results will
not find its way to the enterprises that
may need them; especially small and
medium enterprises (SMEs) that cannot
afford to conduct research on their own.
The reason for this is the barrier between entrepreneur and specialists. Lets
take a look at the food sector, for
example, which consists mainly of SMEs
of five to ten employees. Most of entrepreneurs and managers of these SMEs
do not have an academic degree and
most of them would not have the
knowledge on how to obtain scientific
information. Even if they know how to
obtain new scientific findings, either by
scientific articles or personal communication, it is unlikely that a non-specialist
can understand and digest this information - partly due to the fact that scientific language is very hard to comprehend without appropriate training. In
addition, to comprehend a scientific article, good background knowledge is required that only a specialist in this field
of activity can have.
Electrical and optical

equipment
Another important problem arises when

both partners are on a different level of
insight to the matter in hand.
of the process of knowledge transfer, as

well as knowledge processing. In fact, enterprises relying mostly on external knowledge have many problems receiving and
processing it. This disadvantage can be
counteracted by choosing the right presentation form.
Food products
knowledge transfer process should have

a high degree of reliance.
Figure 4. Number of employees with higher education per sector (Source: Hollanders & Arundel 2005).
For a successful knowledge transfer, information has to be processed into a form

that is understandable for anyone. It has
to be easily readable and understandable
and, at best, easy to implement into the
existing production process. Nevertheless,
it still has to contain all the critical information needed for the full and successful
know-how transfer. While this is easily
manageable with small efforts in some
areas, this can be almost impossible in
other fields of science with state of the
art developments or very specialized research. Here, the only possibility is the
knowledge transfer through extensive
workshops and intensive assistance to the
receiving partner.
An additional problem is the language barrier, which often represents the main barrier of knowledge transfer. Currently,
English is the language for science and
business, and most of scientific articles,
20
workshops or meetings are either written

or hold in English. The English language
is widespread across the world; however,
only people with a relatively high level of
education are able to read and fluently
communicate in English. This ability is almost absent in less well educated people,
except for those with English is their
mother tongue. The first step to ease and
stimulate the knowledge transfer process
would be the translation of the English
works into the local language. This would
give the possibility for many SMEs with
employees of lower educational background to obtain critical knowledge.
Both partners share the responsibility for
a successful knowledge transfer. On one
hand, the donor entity has to process the
knowledge to make it understandable for
non-specialists. On the other hand, the receiving entity has to be willing to accept
to work with a new process, to be open
to new circumstances, and to have a
commitment to the project.
The contribution of
PathogenCombat to the
knowledge transfer
PathogenCombat has made a variety of
contributions to the transfer of knowledge
and technology to SMEs throughout the
European Union and beyond its borderline.
The implementation and transfer of new
scientific findings during the project has
been a priority task from the beginning.
The distribution of knowledge was accomplished through different approaches.
A widespread network of contacts throughout the European Union was established, consisting of SMEs, industry, research institutions, government agencies
and associations related to the food

sector. A database with several thousands
of addresses is created, maintained, and
constantly extended during the course of
the project.
The PathogenCombat-Newsflash was created and published throughout the
Network as a source of up-to-date information for the food industry. This includes
results of state of the art research, current
national and Pan-European developments
and upcoming events like workshops and
meetings.
In addition, a web portal was established
as contact point for SMEs. The webpage
offers a wide range of useful information.
Amongst others, visitors can find the latest
research results and publications in the
field of food quality and safety from the
PathogenCombat consortium members,
information about upcoming workshops
as well as outcomes of the previous ones,
and information about PathogenCombat
project and its partners.
Workshops organized by PathogenCombat
are greatly contributing to the process of
knowledge transfer. These are open to
SMEs, scientists and organizations interested in the topics of food safety, food
quality and food processing. They allow for
personal contact and communication between knowledge donors and receiving entities. Special attention was paid to the problem of the language barrier and the
specific requirements of SMEs in the food
sector. The collaboration with local partners and associations provided valuable insight in the hot topics for the local SMEs
and their expectations. Workshops held
entirely in the local language added to a
successful transfer of research.
21
With more than 40%, the Pathogen

Combat project consortium has a very high
proportion of members that are SMEs.
Though not all results are immediately applicable for the industry, especially at the
beginning of the project where fundamental research has been conducted, the
project SMEs were able to transform many
new findings into countable results.
A good example of a successful knowledge
transfer to a PathogenCombat SME is Pittas
Dairy Industries, based in Cyprus. The company was founded in 1939 and produces
over 100 different dairy products, from
cheese to yogurt. Pittas is a small but international operating company, and they
know that innovation is an important factor
if one wants to stay ahead of competitors.
Therefore, when the Agricultural University
of Athens contacted Pittas and asked for
their participation in the project, they did
not hesitate to join it. The role of Pittas,
along with other SMEs, was to apply new
research results in their production process.
This included developing a predictive model
for the shelf life of yoghurt, using probiotic
strains in animal feed, developing new products with protective cultures and comparing their own food safety systems against
national and international standards.
For Andreas Hadjipetrou of Pittas, the cooperation was a full success. In the five years
of participation in the project, the company
was able to gain new insight in the process
of food production. Additionally, they were
able to meet experts from different fields of
expertise, like microbiology, food safety and
nutritional science. In cooperation with
PathogenCombat, Pittas was able to increase food quality and safety, while launching new products and gaining new customers European-wide.
Further information about knowledge

transfer and PathogenCombat can be obtained by visiting www.pathogencombat.com
Bibliography
Baardseth P, Dalen GA, Tandberg A.
Innovation/technology transfer to food SMEs;
Trends in food science & technology, 1999;
10:234-8.
Debackere K, Veugelers R. The role of academic technology transfer organizations in improving industry science links; Research Policy,
Number of Employees with higher education
per sector (Source: Hollanders & Arundel
2005; 34 (3):321-42.
European Commission, SME Success Stories
in the area of Food Agriculture and Fisheries
and Biotechnology (EUR 23612EN), 2008;
14-5.
Feller I. Universities as engines of R&D based
economic growth: they think they can,
Research Policy, 1990; 19:349-55.
Kaufmann A, Tdling F. Science-industry interaction in the process of innovation: the importance of boundary-crossing between systems. Research Policy; 30:791-04.
Kingsley G, Bozeman B, Coker K. Technology
transfer and absorption: an R&D value mapping approach. Research Policy, 1996;
25:967-95.
Mansfield E, Lee JY. The modern university:
contributor to industrial innovation and recipient of industrial R&D support. Research
Policy, 1996; 25:1047-58.
Mansfield E. Academic research and industrial
innovations, Research Policy, 1991; 26:773-6.
Meyer-Krahmer F, Schmoch U. Sciencebased technologies: universityindustry interactions in four fields. Research Policy,
1998; 27:835-51.
Mowery DC, Sampat BN. Universities in national innovation systems, The Oxford
Handbook of Innovation, Oxford University
Press, Oxford, 2005; 209-39.
22
Paula E. Stephan. Educational Implications of

University-Industry Technology Transfer. The
Journal of Technology Transfer, 2001; 26
(3):199-205.
Rothwell R. Successful industrial innovation Critical factors for the 1990s, R&D Management,
1992; 22:221-39.
Santoro and Chakrabarti. Firm size and technology centrality in industry-university interactions,
Research Policy, 2002; 31:1163-80.
Traill W, Meulenberg M. Innovation in the food
industry. Agribusiness, 2002; 18 (1):1-21.
Von Hippel E. The Sources of Innovation; Oxford
University Press, Oxford. 1988.

MC. Pina-Prez, AB. Silva-Angulo, D. Rodrigo, A. Martnez-Lpez
Introduccin
Dada la magnitud y el alcance de la cadena de suministro de alimentos en las
sociedades modernas, es muy complejo
garantizar que todos los alimentos distribuidos y consumidos estarn alejados
de fuentes de contaminacin potenciales. En su lugar, la seguridad alimentaria se debe incrementar concentrando
los esfuerzos de forma sistemtica de
manera que se minimicen las oportunidades de contaminacin en cada punto
de la cadena de produccin, procesado
y distribucin hasta su preparacin y
consumo. A pesar de que ha habido importantes avances en la conservacin y
control de alimentos, las enfermedades
transmitidas por los mismos permanecen como la principal causa de enfermedad y mortalidad en el mundo. En
consecuencia, las polticas de la Unin
Europea y de los Estados miembro,
debe enfocarse para alcanzar el nivel
ms alto de seguridad alimentaria con
objeto de reducir la incidencia de las
enfermedades producidas por el consumo de alimentos. Para alcanzar este
objetivo, el anlisis de riesgos debe ser
el pilar en el cual se base la poltica de
seguridad alimentaria, complementando esta herramienta con la creacin
de la Autoridad Europea para la
Seguridad Alimentaria y las distintas
agencias nacionales de los Estados
miembro (AESAN, por ejemplo). Todas
estas estructuras deben trabajar de

forma cercana de manera que el esfuerzo vaya en la misma direccin y sea
eficaz en el control de los alimentos y
en reducir la incidencia de enfermedades transmitidas por los mismos.
Contaminacin microbiana
Los alimentos, dada su naturaleza biolgica, proporcionan a los consumidores los
nutrientes necesarios para cubrir sus necesidades; a la vez, son sustratos en los
que el crecimiento de microorganismos es
posible en un amplio rango de condiciones. Desde que un alimento se produce (agrcolas, ganaderos, pesqueros,
etc.) o fabrica (cualquier alimento manufacturado: pan, queso, entre otros), tiene
riesgos de contaminarse.
La presencia de microorganismos patgenos potenciales en nuestro ambiente,
la habilidad de algunos de ellos para sobrevivir, crear resistencias o proliferar en
condiciones de refrigeracin y a bajas
concentraciones de oxgeno, y en ciertos
casos, las bajas concentraciones necesarias para producir enfermedad, agravan el
problema al que nos enfrentamos en
cuanto a seguridad alimentaria (de Swarte
and Donker, 2005).
El proceso de infeccin por patgenos alimentarios se produce de acuerdo a la relacin alimento-patgeno-consumo:
24
Alimento
Microorganismo
Consumo
Las enfermedades de origen alimentario

provocadas por los microorganismos son
de tres tipos:
Intoxicaciones.
Infecciones.
tualmente se considera la principal causa

de ETA en Estados Unidos. Hay dos principales especies de Campylobacter que
son responsables de la mayora de las enfermedades en seres humanos. C. jejuni
representa a la mayora (90%) y C. coli representa slo el 10%, estos pertenecen a
la flora natural de los intestinos de muchos animales, incluyendo aves y animales
domsticos. La mayora de estas infecciones estn asociadas con el consumo de
aves de corral mal preparadas o en mal
estado, el consumo de leche cruda y agua
no clorada.
Toxicoinfecciones.
Entre los principales microorganismos patgenos presentes en alimentos, destacan
por la gravedad de los sntomas y altas
tasas de incidencia, los siguientes:
Salmonella spp
Salmonella spp es una de las causas ms
comunes de enfermedades transmitidas
por los alimentos en los seres humanos, la
duplicacin de la incidencia de los casos de
salmonelosis en las dos ltimas dcadas ha
acompaado la modernizacin de las industrias alimentarias (Altekruse et al,
1997).
Este grupo se puede dividir en dos grandes
categoras: los que causan la fiebre tifoidea y los que no: S. typhy, S. paratyphi
y S. enteritidis, sta ltima como causante
de gastroenteritis por consumo de huevos,
leche, alimentos que contienen huevos
crudos, carne, aves de corral y productos
frescos (Acheson, 2003).
Campylobacter jejuni
Campylobacter jejuni es un patgeno
nuevo transmitido por los alimentos, ac-
Escherichia coli O157:H7

E. coli O157:H7 es una de las principales
causas de diarrea sanguinolenta en todo
el mundo, adems es la causa del sndrome urmico hemoltico, la principal
causa de insuficiencia renal aguda en
nios de Estados Unidos con una tasa de
muerte del 5%. Este microorganismo se
ha asociado con el consumo de carne de
res molida, lechuga, leche cruda y agua
no tratada, y tambin se han documentado casos de transmisin persona a persona (McCabe and Beattie, 2004).
Listeria monocytogenes
L. monocytogenes es un microorganismo
comn. Se sabe que entre 1 a 10% de
la poblacin mundial son portadores de
L. monocytogenes. Los alimentos donde
frecuentemente se encuentra incluyen
leches contaminadas despus de su pasteurizacin, pat, quesos, vegetales
crudos y carne mal cocida. Aunque es un
microorganismo que fcilmente muere
con el calor, la recontaminacin de alimentos es muy frecuente, adems de soportar y ser capaz de crecer a tempera-
25
turas de refrigeracin. Las mujeres embarazadas son un grupo altamente susceptible a este microorganismo, debido
a que tiene la capacidad de llegar al feto,
provocando aborto espontneo, nacimiento prematuro, sepsis neonatal y meningitis. Adems, la listeriosis puede
causar muertes, meningitis o sepsis en
personas inmunocomprometidas; teniendo una tasa de letalidad del 40%.
Clostridium perfringens
y Bacillus cereus
Estos dos microorganismos son las principales causas de brotes alimentarios en
banquetes, debido a su fcil proliferacin
cuando los alimentos se mantienen a una
temperatura ambiente por un largo periodo de tiempo, causando gastroenteritis
muy leve. Adems, comparten caractersticas comunes: formacin de esporas,
produccin de enterotoxinas que causan
gastroenteritis, estn presentes en alimentos previamente sometidos a tratamientos trmicos como salsas y arroz.
Los alimentos ms susceptibles de estar
implicados en los brotes con B. cereus incluyen carnes y verduras cocidas, productos lcteos pasteurizados, cereales y
arroz cocido.
Yersinia spp
De los tres miembros de este genero, Y.
enterocolitica y Y. pseudotuberculosis son
considerados como patgenos transmitidos por alimentos. No es un microorganismo comnmente causante de enfermedades transmitidas por los alimentos
comparado con Salmonella spp o
Campylobacter spp; sin embargo, se sabe
que es transmitido por los alimentos a los
seres humanos en los que causa una

grave enfermedad gastrointestinal; el alimento comnmente implicado en estos
brotes es el cerdo. La leche es otra fuente
frecuentemente asociada con este microorganismo.
Control y reduccin del riesgo

El control y reduccin del riesgo causado
por el consumo de alimentos contaminados puede llevarse a cabo actuando
sobre tres factores: el patgeno, el consumidor, o el vehculo de transmisin, en
este caso, los alimentos en los cuales vive
e interacta (Havelaar et al, 2002).
Estos tres factores son necesarios para
que ocurra la enfermedad transmitida por
los alimentos y existen interacciones complejas entre ellos. Cambios producidos en
estos tres factores pueden reducir la probabilidad de adquirir una enfermedad por
consumo de alimentos, por ejemplo la esterilizacin comercial de un alimento destruye a los microorganismos patgenos
eliminando el factor exposicin, lo que
hace al alimento ms seguro. Por el contrario, los cambios en estos tres factores
pueden favorecer que emerjan nuevos
patgenos, este es el caso de individuos
inmuno-comprometidos por haber sufrido
un transplante. Microorganismos que habitualmente no producen enfermedades
pueden colonizar el tracto intestinal de los
bebes prematuros, personas mayores, etc.
Se pueden producir cambios de virulencia
como consecuencia de los tratamientos
de conservacin, en este caso el patgeno
adquiere caractersticas que le ayudan a
invadir el cuerpo humano. Estos tres factores son una pieza clave en la reduccin
de la enfermedad de origen alimentario.
26
Como se ver ms adelante, la industria

y agricultura han introducido una serie
de medidas de control efectivas enfocadas a limitar alguno de los factores
claves de la enfermedad por consumo de
alimentos.
En 1896, el U.S. Public Health Service
(USDA/FSIS, 1996) comenz a investigar
la calidad sanitaria de la leche, y mediante
estudios epidemiolgicos sobre enfermedades humanas relacionadas con la poblacin consumidora de leche, estableci
el primer criterio especfico para pasteurizacin de leche a 161 F y 15 s, con el
objetivo de eliminar los microorganismos
ms resistentes al calor, no formadores de
esporas.
Tambin en la dcada de los 90 se establece una regulacin en lo referente a conservas de productos de baja acidez, exigiendo en las conserveras el alcance de la
esterilidad comercial con el objetivo de destruir Clostridium botulinum. La esterilidad
comercial puede ser definida como la
aplicacin de un tratamiento trmico suficientemente intenso como para que el
producto esterilizado est libre de microorganismos capaces de reproducirse en las
condiciones normales de almacenamiento
sin refrigeracin y libre de microorganismos
incluyendo esporas, que puedan comprometer la salud del consumidor.
Actualmente existe una amplia gama de
opciones para prevenir y controlar las
enfermedades transmitidas por alimentos; a nivel de produccin, las
Buenas Prcticas Agroalimentarias (BPA)
pueden aplicarse y reforzarse, mejorando la explotacin y el saneamiento,
la adopcin del sistema Hazard
Analysis and Critical Control Points
(HACCP) durante toda la cadena de produccin y el control de la contaminacin

y temperatura durante el transporte y almacenamiento deben ser capaces de
garantizar la inocuidad de los alimentos;
a nivel de los manipuladores de alimentos se deben mantener las condiciones de higiene adecuadas, Buenas
Prcticas Higinicas (BPH). Por otra
parte, las tecnologas de control microbiolgico como la pasteurizacin y tecnologas emergentes se ha confirmado
que pueden evitar muchas enfermedades. Su objetivo final es claro: ayudar
a producir alimentos ms seguros y en
consecuencia, reducir el nmero de
brotes de las enfermedades transmitidas
por ellos (Tauxe, 2002; Panisello, 2000;
Pina, 2006).
El sistema y concepto del HACCP se desarroll hace 30 aos pero slo hace una
dcada que se ha incluido en el programa
de control de los alimentos de las autoridades de Salud Pblica, en la Unin
Europea el trmino HACCP se present
por primera vez en la Directiva 93/43/CE
(1993); reconocindose como un mtodo
de referencia para el aseguramiento de la
inocuidad de los alimentos y un sistema
de regulacin en los sistemas de control
de los alimentos. El sistema HACCP se
basa en siete principios: 1) anlisis de
riesgos, identificar los peligros y especificar las medidas de control; 2) identificar
los puntos crticos de control (PCC); 3) establecer lmites crticos; 4) establecer procedimientos de vigilancia; 5) establecer
medidas correctivas; 6) establecer los procedimientos de verificacin y; 7) documentar los procedimientos (Motarjemi,
1996; Motarjemi, 1999).
27
Control dentro de la Unin

Europea
De acuerdo con el Libro Blanco para la seguridad alimentaria (Comisin of the
European Communities 2000), los consumidores deben disponer de una oferta amplia de alimentos seguros y de alta calidad
procedentes de los Estados miembro. Esto
es esencial en el mercado interno de la
Unin Europea y significa que cada Estado
miembro tiene una responsabilidad no slo
hacia sus propios ciudadanos sino tambin
hacia todos los ciudadanos de la Unin y
terceros pases por los alimentos producidos
en su territorio. Las acciones estratgicas a
tomar para reducir los peligros deben por
lo tanto comenzar a nivel nacional en cada
Estado miembro incluyendo una implicacin a nivel regional, local e internacional.
Para garantizar la seguridad de los alimentos procesados se debe aplicar el anlisis de riesgos, cuya misin es mejorar los
actuales sistemas de control de alimentos
(HACCP).
El anlisis de riesgos es un proceso complejo, exige un enfoque multidisciplinar e
incluye tres componentes bsicos: la evaluacin, la gestin y la comunicacin del
riesgo (Hugas et al, 2007). La evaluacin
de riesgos es una evaluacin cientfica de
los efectos adversos, potenciales o efectivos, resultantes de la exposicin a un
consumo de alimentos en los que se encuentran presentes agentes patgenos; la
gestin es el proceso que comprende la
valoracin de las posibles alternativas para
minimizar o reducir el riesgo o mejorar las
alternativas en aplicacin; y la comunicacin es el proceso interactivo de intercambio de informacin y opinin entre
asesores y evaluadores del riesgo.
Durante la pasada dcada, el anlisis de

riesgos en seguridad alimentaria fue mejorado, llevado a cabo en gran cantidad de estudios (EFSA, 2009) e integrado dentro del
proceso de vigilancia de la seguridad a nivel
nacional, internacional y comunitario, como
una herramienta basada en el conocimiento
cientfico, herramienta en la que se apoya
el proceso de toma de decisin, para mejorar los sistemas de control de alimentos, y
para fijar prioridades sobre los diferentes
problemas en seguridad alimentaria.
Para asegurar la salud del consumidor, en
el proceso de anlisis se proponen una
serie de objetivos o criterios de seguridad,
as aparece el concepto de nivel de proteccin apropiado (ALOP), el concepto
de objetivo de seguridad o Food Safety
Objective (FSO). Adicionalmente, el
Comit de Higiene Alimentaria (Codex
Alimentarius Commission, 2003b) define
criterios microbiolgicos especficos,
desde el procesado de alimentos hasta el
momento del consumo, como sigue:
El nivel apropiado de proteccin (ALOP)
puede ser definido como la expresin
cuantitativa de que se produzca un efecto
adverso sobre la salud de los consumidores o la probabilidad de incidencia de
enfermedad.
Objetivos de seguridad (FSO): la frecuencia o concentracin mxima de la
presencia de un riesgo en un producto en
el momento de consumo que contribuye
a establecer un adecuado nivel de proteccin (Appropiate Level of Protection,
ALOP).
Objetivo de consecucin (Perfor-mance
Objective, PO): la frecuencia mxima y/o
concentracin de un riesgo en un producto en un momento especfico de la
28
cadena de produccin antes del momento de consumo y que contribuye al

establecimiento de un FSO, ALOP como
aplicable.
Criterio de realizacin (Perfor-mance
Criteria, PC): el efecto en frecuencia y/o
concentracin de un riesgo en un alimento que debe ser conseguido por la
aplicacin de una o ms medidas de
control para asegurar o contribuir a un
PO o a un FSO.
Criterio microbiolgico (Microbiological Criteria, MC): define la aceptabilidad
de un producto o un lote de productos,
basada en la ausencia o presencia, o nmero de microorganismos, incluidos por
unidad de masa, volumen o lote.
En ciertos casos, y para microorganismos
de gran virulencia, como el caso de E. coli
0157:H7 se establecen criterios de tolerancia zero. En 1996, el FISIS (USDA/FISIS,
1996) desarroll un criterio estndar para
la carne de vaca cruda, a consecuencia de
un brote por E. coli 0157:H7 en el que se
dieron 700 casos de enfermedad y 4
muertes, por consumo de comida rpida
en un restaurante (Bell et al, 1994). Este
criterio es nico y reconoce la prctica frecuente de consumidores de este tipo de
producto de cocinar insuficientemente el
producto crudo no destruyendo as los patgenos presentes. Esta prctica aplica
temperaturas que son inadecuadas para
destruir los microorganismos patgenos
del interior del producto, siendo el calentamiento superficial, y por ello puede
causar enfermedad, colitis hemorrgica y
sndrome urmico hemoltico, por lo que
FISIS estableci el nivel zero de tolerancia
en 1994.
Conclusiones
Se aplicarn criterios de control de microorganismos en la fuente y en la seleccin
de la materia prima, a continuacin en el
diseo y ejecucin de proceso, mediante la
aplicacin de buenas prcticas higinicas, y
la mejora de los sistemas de anlisis y control de puntos crticos a lo largo de la cadena de produccin/consumo (FAO/WHO,
2001; ICMSF, 2002).
Las autoridades de Salud Pblica deben
promover la seguridad alimentaria en la
sociedad y, en particular, entre los consumidores, para que stos no slo
adopten prcticas seguras de manipulacin de los alimentos en sus hogares,
sino que tambin sean capaces de demandar Buenas Prcticas Higinicas
(BPH) y aprecien los esfuerzos de las empresas alimentarias por ofrecer alimentos inocuos, siendo estos esfuerzos
innovadores y continuos por parte de
las industrias alimentarias de todo el
mundo (Motarjemi, 1999). Al mismo
tiempo deben impulsar el desarrollo de
las agencias o autoridades de seguridad
alimentaria con la misin de llevar a
cavo una evaluacin de riesgos que permita a las autoridades sanitarias llevar a
cabo polticas de seguridad alimentaria
eficaces en Espaa y en los distintos
Estados miembro de la Unin Europea.
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Altekruse S, Cohen M, Swerdlow D. Perspectives.
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Bell BP, Goldoft M, Griffin PM, Davis MA, Gordon
DC, Tarr PI, Bartleson CA, Lewis JH, Barrett TJ,
Wells JG, et al. A multistate outbreak of
Escherichia coli O157:H7 associated with bloody
diarrhea and hemolytic uremic syndrome. 1994.
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Tools to support the self assessment of the

performance of Food Safety Management
Systems
PA. Luning, L. Jacxsens, V. Jasson, WJ. Marcelis, J. Kussaga, M. Van der Spiegel,
M. Kousta, S. Oss, J. Rovira, F. Devlieghere, M. Uyttendaele
Introduction
Changes in food supply chains, health and
demographic situations, lifestyle and social
situations, environmental conditions, and increased legislative requirements have led to
significant efforts in the development of
quality and safety management systems in
agribusiness and food industry worldwide
(Ropkins and Beck, 2000; Efstratiadis, Karirti,
and Arvanitoyannis, 2000; Jacxsens, et al,
2009a, Luning and Marcelis, 2009a).
Nowadays, companies have implemented
various quality assurance (QA) guidelines
and standards, such as GMP and HACCP
guidelines (like General Principles of food
hygiene (Codex Alimentarius 2003), GFSI
guidance document (GFSI (2007), and quality assurance standards (like ISO 9001:2008
(2008), ISO22000:2005 (2005), BRC (2008),
and IFS (2007) into their company own food
safety management system. The performance of such systems in practice is, however, still variable. Moreover, the continuous pressure on food safety management
system (FSMS) performance and the dynamic environment wherein the systems
operate (such as emerging pathogens,
changing consumer demands, developments in preservation techniques) require
that they can be systematically analysed to
determine opportunities for improvement

(Wallace, et al, 2005; Manning et al, 2006;
Van der Spiegel et al, 2006; Cornier et al,
2007; Luning et al, 2009a). Within the
European project entitled PathogenCombatEU FOOD-CT-2005-007081 various tools
have been developed to support food companies and establishments in systematically
analysing and judging their food safety management system and its microbiological
performance as basis for strategic choices
on interventions to improve the FSMS performance. This chapter describes briefly
principles of the major tools that have been
developed and some others, which are still
under still under construction.
Quality assurance evaluation

grids
The wide range of quality assurance standards and guidelines commonly leads to difficulties for small and medium enterprises
(SME) to select and implement them into
their company specific Quality Management
System (QMS) and or Food Safety
Management System (FSMS). It is often hard
for SMEs to understand the detailed differences between various QA standards and
guidelines and to judge the possible consequences of implementation, because they
32
not always have the necessary expertise, experience, and resources (e.g. financial, staffing capabilities) (Yapp and Fairman, 2006;
Aggelogiannopoulos et al, 2007; Karipidis,
et al, 2009). Therefore, quality assurance
evaluation grids have been developed,
which show the major differences between
acknowledged QA standards and guidelines
on distinct features. The QA evaluation grids
may support companies in the agri-food
chain to balance the benefits of implementing certain QA standards (and guidelines)
against the efforts that are required.
Moreover, it might serve as a compact overview of possibilities and consequences of
implementing QA standards and guidelines
when supporting companies to improve
their own FSMS. The features that have
been included in the grids are in table 1
summarised. For details the reader is referred to Kussaga and co-authors (2009).
Food Safety Management

System Diagnostic Instrument
(FSMS-DI)
Stakeholders (like government, branch organisations, customers, retail, etc) put demands on the design of a companys FSMS
by requiring the implementation of certain
(sets) of quality assurance (QA) standards
and or guidelines. However, each company
or establishment has a unique companyspecific FSMS depending on how standards
and guidelines have been translated into
the own situation (Jacxsens, et al, 2009a;
Luning and Marcelis, 2009a). Recently, a
Table 1. Features on which acknowledged QA standards and guidelines have been

evaluated (modified from Kussaga et al, 2009b).
Features related to position of QA Standards and Guidelines
Focus
QA standards and guidelines have been (are being) developed for
different purposes, they may have a different focus (like, safety, quality,
organisation).
Scope
Scope refers to the range and applicability of the standard or guideline,
which can be restricted or broad.
Legislative Status
Legislative status refers to being compulsory or voluntary.
Combined
The feature combined implies if a standard or guideline is a primary one
or is typically a combination of more standards/guidelines.
GFSI status
GFSI status refers to the benchmarking position of the Global Food
Safety Initiative of the QA standard/guideline.
Acknowledgement
Acknowledgement of QA standards and guidelines indicates whether
they are nationally (e.g. one country), regionally (e.g., the whole
region/continent like Europe, Middle East), or worldwide recognized.
Features related to type of requirements of QA Standards and Guidelines
Comprehensiveness
Extent validity
requirements
Degree of
organisational
demands
Comprehensiveness refers to the extent of detail of the requirements,

requirements and to how they are in the document formatted.
Extent of validity requirements refers to what degree demands are put
on assuring that the system is really effective in practice.
Degree of organisational demands refers to what extent QA standard
and guideline set requirements on typical organisational issues
(like training of personnel, setting procedures).
Tools to support the self assessment of the performance of Food Safety Management Systems
33
Table 1. Features on which acknowledged QA standards and guidelines have been

evaluated (modified from Kussaga et al, 2009b) (continuation).
Features related to Certification of QA standards
Scope of certification Certification scope refers to what the certification process covers.
Gradation in
Differences in gradation refers to fact that QA standards vary in the way
certification
certification requirements can be fulfilled.
Frequency
Frequency of certification refers to how often certification audits must
be by third parties carried out.
diagnostic tool has been developed using

a techno-managerial research approach to
consider both technological factors and
people behaviour in the performance of
food safety management systems (Luning
and Marcelis, 2006, 2007, 2009b). The tool
is called food safety management system
diagnostic instrument (FSMS-DI). The
FSMS-DI is a tool that enables a systematic
analysis and assessment of a companys
unique food safety management system independent of the QA standards and or guidelines that have been implemented
(Luning et al, 2008, 2009a, b, c; Jacxsens
et al, 2009c). The instrument consists of
comprehensive lists with sets of indicators
to analyse respectively which core control
and core assurance activities are addressed
in the company specific FSMS, which major
contextual factors could affect FSMS performance, and to analyse the microbiological safety performance of the system.
Moreover, the FSMS-DI encompasses grids
to assess respectively levels of control and
assurance activities (i.e. more or less advanced), contextual situations (i.e. more or
less risky) wherein the FSMS has to operate, and the microbiological safety level.
For each indicator, to assess core control or
assurance activities or food safety performance, four different levels have been described (i.e. 0, 1, 2, and 3 representing a low,
basic, average, and advanced level respectively). Similarly, for each indicator, to assess
contextual factors, three different risk levels
have been described (i.e. 1, 2, and 3 representing low, moderate and high-risk context respectively).
The elements of the FSMS-DI are summarised in figure 1, it starts with introductory
questions followed by defining a representative production unit for which a QA manager can do the self assessment (part I).
Part II includes the indicators and grids to
assess the major contextual factors product
characteristics, process characteristics, organisational characteristics, and chain environment characteristics. Part III is for assessment of the core control activities
design of preventive measures, design of
intervention measures, design of monitoring systems, and actual operation of control measures, whereas the core assurance
activities setting system requirements, validation, verification, and documentation
and record-keeping are covered in part IV.
Part V includes the indicators (called the
Food Safety Performance Indicators FSPI)
and grids to assess internal and external
food safety performance (Jacxsens et al,
2009c). The assumption behind the FSMSDI is that companies operating in a highrisk context (due to highly risky product and
34
PART I: introductory section for Food Safety Management System (FSMS)

A. Introduction questions
B. Selection of Representative Production Unit (RPU) for self-assessment
PART II: assessment of contextual factors
A. Assessment of product characteristics
B. Assessment of process characteristics
C. Assessment of organisation characteristics
D. Assessment of chain environment characteristics
(1 -11)
(12-20)
(A1-3)
(B4-6)
(C7-13)
(D14-17)
PART III: assessment of core safety control activities

E. Assessment of preventive measures design
F. Assessment of intervention processes design
G. Assessment monitoring system design
H. Assessment of operation of preventive measures, intervention process and
monitoring systems
(E18-23)
(F24-27)
(G28-34)
(H35-41)
PART IV: assessment of core assurance activities

I. Assessment of setting system requirements activities
J. Assessment validation activities
K Assessment of verification activities
L Assessment of documentation and record-keeping to support food
assurance
(I42-43)
(J44-46)
(K47-48)
(L49-50)
PART V: assessment of food safety performance

M. EXTERNAL Food Safety Performance
N. INTERNAL Food Safety Performance
(M51-54)
(N55-57)
Figure 1. Overview of elements of food safety management system diagnostic instrument.
processes, less supporting organisational

conditions, highly vulnerable and depend
chain position) need to have an advanced
FSMS (i.e. based on precise information,
scientifically underpinned, critically
analysed, procedure-based, systematic, and
independent) to realise a predictable and
controllable food safety performance. In a
moderate-risk context an average FSMS is
expected to be sufficient to realise a good
FS performance, while in a low-risk context
even a basic FSMS would be adequate to
realise a good FS performance (Luning et

al, 2009c). At the other hand, a good FS
performance is an indication for a well
functioning FSMS (Jacxsens, et al, 2009c).
Figure 2 illustrates this assumption. The FS
performance can be analysed by using the
food safety performance indicators
(Jacxsens et al, 2009c) and can be measured by experiments using the microbial
Assessment Scheme (MAS) of Jacxsens and
co-authors (2009b) (Section 4).
35
Context
FSMS
FS
3=most
dangerous
3=highest
level
3=best
performance
Figure 2. Principle assumption behind the research

work of tools to measure the performance of Food
Safety Management Systems.
The FSMS-DI has been tested and validated in pre-tests and a pilot study with
15 food producing companies in the area
of dairy, pork, beef & lamb, and poultry
products. Moreover, the instrument has
been slightly adapted and applied in the
catering sector (50 food service establishments) in Spain (Chinchilla, 2009).
Recently, the paper based instrument
has been transformed to a web based
application, i.e. the FSMS self assessment tool. This self assessment tool is
now used for a quantitative study to assess food safety management systems in
dairy, pork, beef & lamb, and poultry
companies in Europe. For details about
the diagnostic tool, the reader is referred
to Luning and co-authors (2008, 2009 a,
b, c), Jacxsens and co-authors (2009c),
and the Pathogen Combat website
(www.pathogencombat.com).
Microbiological Assessment
Scheme (MAS)
As previously stated, the actual microbiological performance of FSMS in practice is still
variable (e.g. Cormier et al, 2007; Manning
et al, 2006; Tsalo et al, 2007). In fact, attention has been shifted from implementing
QA standards to better understanding the
performance of an FSMS (Domnech et al,
2008; Luning et al, 2008; Stringer and Hall,
2007) and various audit tools have been
developed to determine performance towards certain QA standards (e.g. Wallace
et al, 2005; CIES, 2007; Cormier et al,
2007). However, these audit tools basically
check on compliance to the set requirements, for instance, during internal or external auditing (Van der Spiegel et al, 2005),
whereas the FSMS-DI focuses on crucial
control and assurance activities (not linked
to specific QA standards). Although, the
FSMS-DI can give an indication about the
microbiological safety performance, it gives
restricted insight in the actual microbiological performance.
In practice, food processing companies
commonly use microbial testing of final
products to assess if their products meet
food safety criteria (e.g. ICMSF, 2002;
Legan, 2001). These criteria are set by different stakeholders or regulatory bodies
(like EU and/or country regulations and/or
customers requirements), but can also be
used to guide the evaluation of a manufacturing process to define preventive actions (Kvenberg and Schwalm, 2000;
Martins and Germano, 2008). However,
no procedure to systematically evaluate
the microbiological performance of a
FSMS was yet available. Therefore, the
Microbial Assessment Scheme (MAS)
36
tool has been developed to support a systematic analysis of microbial counts to assess the current microbial performance of
an implemented FSMS.
The MAS tool is a procedure that defines
the identification of critical sampling locations (CSL), the selection of microbiological
parameters, the assessment of sampling frequency, the selection of sampling method
and method of analysis, and finally data
processing and interpretation (figure 3).
Based on the MAS assessment, microbial
safety level profiles can be derived, indicating which microorganisms and to what
extent they contribute to microbiological
safety for a specific food processing company. A microbial safety level can be classified from 1 to 3, where level 3 reflects a
Identification Critical
Sampling locations CSL)
Selection microbiological
parameters
Assessment sampling
frequency
Selection sampling method

& method of analysis
Data processing
& interpretation of data
good performance (legal criteria or guidelines are respected, no improvements are

needed current level of FSMS is high
enough to cover this hazard), level 2 corresponds with a moderate performance
(legal criteria or guidelines are exceeded,
improvements need to be made on a
single control activity of the FSMS) and
level 1 represents a poor performance
(legal criteria or guidelines are exceeded,
improvements need to be made on multiple control activities of the FSMS). The
sum of the levels is resulting in the microbial food safety level profile. The principle
behind the MAS tool is that low numbers
of microorganisms and small variations in
microbiological counts imply a well functioning FSMS (Jacxsens et al, 2009b).
Locations provide info about microbial performance of product flow

(e.g. raw materials, intermediated food products and final food
products) and core control strategies (e.g. contact surfaces, hands of
personnel, after pasteurisation as intervention method)
Relevant pathogens (e.g. Listeria, Salmonella)

Hygiene indicators (e.g. E. coli, S. aureus)
Utility parameter (total mesophilic count)
Obtain picture of actual microbial performance e.g. Three months, 3

times (days), 3 times a day
Product sampling & surface sampling per CSL

Use of ISO methods for sampling & analysis (reliable, accurate,
robust) or via acknowledged and validated alternative methods
Show variability in raw data

Use legal (available) criteria to judge outcome
Development food safety level profiles
Figure 3. Steps of the MAS scheme (modified from Jacxsens et al, 2009b).
37
A MAS scheme will differ depending on the

production processes and food type that
are addressed in the specific company.
MAS-schemes have been specified for microbiological analyses in respectively poultry,
dairy, beef & lamb, and pork companies.
Depending on the product and production
processes, specific microorganisms have
been selected to indicate respectively safety
(e.g. Listeria monocytogenes, Salmonella
spp, Campylobacter), hygiene indicators
(e.g. E. coli and Enterobacteriaceae, Staphyloccocus aureus), and overall performance
(total aerobic count). Data provided indepth insight in microbiological counts in
product flows (both raw materials, intermediate, and final products), contact surface
areas (like at critical cutting areas, knives,
conveyor belts, etc), and people (hands and
gloves).
The detailed MAS data provide insight in
which indicator microorganisms exceed limits and at which critical locations, but also
reveals the extent of variation in microbiological counts. The microbial safety level
profiles give an immediate insight in the
room o improvement and or which microbiological parameters. These profiles can
also be used to compare the microbiological performance of different companies
with the same type of production processes
and food products as benchmarking tool.
As such, microbiological problems in a
sector can be identified, independent of the
type of company. The food safety performance indicators (FSPI) have been analysed
on their indicative value by comparing data
with the extensive MAS data for nine
European companies. (Jacxsens et al,
2009c). The food safety performance diagnosis can be a useful tool to have a first indication about the microbiological perfor-
mance of an operational food safety management system.

For more details the reader is referred to
Jacxsens and co-authors (2009a, 2009c),
and Sampers and co-authors (2009), and
Kussaga and co-authors (2009b).
MAS analysis method

selection tool
Different authors recommended the use of
microbial testing to evaluate critical control
points (e.g.), to evaluate procedures for
Good Hygienic practices (GHPs) and
Standard Operating Procedures (SOPs) (e.g.
Brown et al, 2000; Swanson and
Anderson, 2000; Kvenberg and Schwalm,
2000; Gonzalez-Miret et al, 2001; Cormier
et al, 2007; Martins and Germano, 2008).
The MAS-scheme can support food safety
experts in systematically designing a tailored scheme to asses the microbiological
performance of implement ted food safety
management systems (Jacxsens et al,
2009b). According to the MAS protocol
appropriate methods for sampling and
analyses of pathogens and other micro-organisms (to indicate hygiene or total performance) need to be selected. In the current MAS protocol, the authors refer to
the use of internationally acknowledged
sampling and analysis methods according
to ISO standards. However, nowadays a
wide range of methods to sample and or
analyse micro-organisms (and specifically
pathogens) are existing or have been recently developed. Each method has its own
specific characteristics, which may affect
the choice of a certain method.
Therefore a MAS analysis method selection tool has been developed, which can
aid in the process of decision-making regar-
38
ding selection of microbial analysing methods in specific situations. A comprehensive review of literature regarding different
enumeration and detection method was
performed. Based on this review specific
method characteristics were determined
that have used as a parameter in the selection tool. The major characteristics on
which the methods have been analysed are
shown in table 2. Moreover, a decision tree
was made that allows classifying the MA
Regarding the selection tool it should be

noticed that no method exist that is a
100% sensitive, 100% specific, that can be
performed in real-time and that is completely without costs. All methods have advantages and disadvantages. The challenge is
to select the method that fulfils the most of
the characteristics of the ideal method in a
specific situation. Advantages of a method
should be optimally exploited and the disadvantages should be recognized. The se-
Table 2. Some examples of characteristics on which microbiological methods have been

evaluated as basis of the MAS method analysis selection tool (modified from Jasson et
al, 2009).
Characteristics
Alternative method
Time
Matrix
Validation
certificate
Type of microbial
parameters
Brief description
An alterative method is a method of analysis that demonstrates or
estimates, for a given category of products, the same analyte as is
measured by using the corresponding reference method. This
alternative method can be proprietary or non-commercial and covers
an entire analysis procedure, that is, from the preparation of samples
to the test results either as such or may include references to other
procedures in order to be complete. The alternative method exhibits
attributes appropriate to the user needs, e.g.: speed of analysis and/or
response, ease of execution and/or automation, analytical properties,
miniaturisation or reduction of cost
Total time to result is the time needed from sample until counting
results or presence/absence result (confirmation not included)
Food matrix. A method should be applicable for the food matrix of
interest
Users of commercially available kits (proprietary methods) need
guarantees regarding the performance of these kits. Validation of
alternative methods is a process that determines if an alternative
method can obtain the same analyte as is measured by using the
corresponding reference method
Multi-functionality of the method regarding different microorganisms
that needs to be performed. The decision

tree is based on a techno-managerial point
of view.
lection tool can aid in finding the most appropriate method for a specific situation in
need of microbial analysis.
39
For details about the MAS analysis

method selection tool, the reader is referred to Jasson et al, (2009).
Improvement roadmap for

FSMS
After companies have analysed their
system by using the self assessment tool
FSMS-DI alone or in combination with
MAS, they have detailed insight in the levels at which they execute their core control and assurance activities (ranging from
absent, low, medium to high (= level 3).
They also have an idea about the typical
contextual situation wherein their system
has to operate (ranging from highly, to restricted and not vulnerable, ambiguous and
uncertain (situation 3-1) moreover, they
have an indication about the (actual) microbiological performance. As previously,
stated the principle behind the diagnosis is
that companies that operate in a more
risky context (i.e. more vulnerable, ambiguous and uncertainly) require a more advanced (high level) FSMS to be able to realise and ensure safety requirements
(Luning et al, 2009b). If the assessment
data reveal food safety levels below 3
(Jacxsens et al, 2009b,c) and this is perceived as a problem, then a company
could first consider those core control and
assurance activities that are at level 1 (is associated with aspect, like not scientifically
underpinned, general, not structured, incomplete, not independent) or at level 0
(absent, not used, unknown), to consider
possible interventions in the FSMS to improve the performance. However, one can
also consider those contextual factors that
are allocated in situation 3, to identify possible interventions in the contextual situa-
tion, which are commonly long-term interventions (like changing production process,
increasing competence level of operators,
improving information system, enhancing
supplier relationships, etc) (Luning and
Marcelis, 2009a; Luning et al, 2009c).
To support the improvement process, generic roadmaps have been made showing
how to go through the different steps of
an improvement process The systematic approach is based on the principles of the
food quality relationship model (food quality = f (food behaviour, human behaviour),
the food quality management decisions
grid, and the principles of improvement
processes (Luning and Marcelis, 2006,
2007, 2009a, b). The basic steps of an improvement cycle are: 1) map problem area,
i.e. collecting information and documentation, 2) analyse problem area: i.e. identification of causes and effects, and 3) redesign: i.e. development and implementation
of solutions as depicted in Figure 4.
Improvement processes are characterised
by a gradual nature, it is a step-by-step ongoing process. Depending on the starting
situation, improvements can vary from
simple measures to reduce variation in products and decision-making on the short
term, to changes in the infrastructure on
the long-term. Using the food quality relationship we have defined three levels of increasing improvement efforts, i.e. a)
changes in product and people behaviour,
b) changes in technological and decisionmaking process conditions, and c) changes
in the technological and organisational infrastructure. After each improvement cycle
the new situation should be reassessed in
order to judge the effect of the improvement. Subsequently, the new situation
must be assured (Luning and Marcelis,
40
Level of improvemenet efforts

a. Change product
and people behaviour
b. Change technological
and decision-making
process conditions
c. Change technological
and organisational
infrastructure
1. Map problem
situation
2. Analyse
problem
3. Redesign
Roadmap
Figure 4. Generic approach to develop roadmaps.
2009). Using above approach, an example

of a roadmap has been elaborated indicating typical activities that could be done by
food companies when they want to improve problems with aw materials, table 3
shows typical activities in the three improvement steps for the different levels of improvement. The activities are a selection of
information gathering, analyses methods,
and improvement measures addressing
both technological and managerial issues
to demonstrate how food companies can
systematically improve their FSMS. Companies have to select themselves which tools,
techniques, and methods are most suitable
for their own situation.
still not yet well worked out in practice.

Protocols have been developed to support
companies in improving their validation and
verifications activities, and a protocol to improve design of sampling plans.
Additional supporting tools
To support companies in improving their

FSMS they need to have access to information, knowledge, and experience about
these tools. In this perspective, a food safety management support system has
been developed to provide in a systematic
way information about control and assurance principles, supporting tools (like new
enumeration, detection and monitoring
techniques for pathogens, new intervention techniques and methods, protocols
and procedures on sanitation, validation,
verification, microbial assessment, etc), principles and structure of acknowledged quality assurance standards and guidelines, and
legislative requirements.
Data from the pre-tests and pilot studies indicated that validation and verification activities but also design of sampling plans are
The FSMS support system is available via

the Pathogen Combat website (www.pathogencombat.com).
The principle of generic roadmaps for improvement will be further in the near future (Luning et al, 2009c).
41
Table 3. Typical activities in the improvement steps for problems with Raw materials
a. Change product and

people behaviour
b. Change technological
and decision-making
process conditions
c. Change technological
and organisational
infrastructure
1. Map
problem
situation:
gathering
information
Gather materials
information, like
rejections, incidence
reports, complaints,
microbial load
products, % realised
inspections
Gather process condition

information, like storage
temperatures, complaints,
actual availability of and
compliance to procedures,
actual availability of
materials and supplier
information
Gather information on
storage facilities and
suppliers, like microbial
load walls and floors,
supplier performance,
communication
problems, quality system
performance
2. Analyse
problems:
methods
and tools
Use basic statistic

tools, and
brainstorming for
analysing structural
deviations
Use CCP analysis, risk

analysis, Total Productive
Maintenance, and literature
analysis
Use CCP analysis,

hygienic design
methods/principles, risk
analysis, predictive
modelling, and literature
analysis
Possible measures for

improvement: change
incoming material
inspection, change storage
temperature control,
change corrective measures,
change procedures,
training, intensify support
quality department,
intensify information supply
Possible measures for

improvement: building
conditioned storage
rooms, change suppliers,
intensify supply chain
requirements, change
supplier agreements,
change organisational
responsibilities
3. Redesign: Possible measures for

improvemen improvement: change
t options
corrective actions,
change inspection
frequency, change
frequency of recording
data, change
instructions, intensify
supervision
Final considerations
It is evident that an implemented FSMS in
a company in the agri-food chain must be
seen as a dynamic system, which needs to
be frequently analysed, judged, improved,
and tailored to the actual and changing situation with respect to the control and assurance activities and the contextual factors
affecting the performance of the companys
unique FSMS. The FSMS self assessment
tool in combination with the FSMS support
system (including all relevant tools deve-
loped in PathogenCombat, useful guidelines, legislative requirements, scientific

knowledge) can be used to search for
knowledge, information and tools to
analyse, judge, and improve an implemented FSMS.
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Prevention of biofilm formation

and foodborne infections by control
of moisture management
R. Cocker
Introduction
Conclusions
This article is based on a presentation

given at the PathogenCombat/EHEDG
SME Workshop at the University of
Burgos, Spain, on 18th June 2009. During
the PathogenCombat project, Cocker
Consulting Ltd had the objective of working with SMEs on hygienic engineering
and design, with a secondary responsibility in the objective of minimising the formation of biofilms and assisting in the development of practical advice on how to
remove biofilms once they had formed.
The advice contained in the presentation
at the PathogenCombat workshops in
Ljubljana, Copenhagen, Burgos and in future in Copenhagen and Budapest was
based on my background as first, a graduate microbiologist, followed by a doctorate and extensive practical learning in
the field of aseptic and hygienic design
and engineering. This work included visits
in Spain and Ireland to SMEs in the meat,
poultry and dairy sectors and visits to
these sectors, plus fish processing, on behalf of Tesco and many other clients, both
SMEs and those with world famous
brands.
To a greater or lesser extent, food-processing organisations operate with hazards

and risks to food safety. Risk is the product of the magnitude of a hazard and its
probability. In my extensive visits and audits, many food-processing organisations
did not have the insight in hygienic design and engineering to assign a probability to the various hazards. They unknowingly accepted an excessive number and
magnitude of hygiene risks because the
occurrence had also been zero. This was
despite that fact that a future occurrence
of any one of these hazards could wreck
their business and seriously injure or kill
consumers.
The importance of good moisture management and hygienic design was not
understood. The following examples
were repeated (in large organisations
and SMEs alike:
Regulators and auditors demanding
washing of a dry process area.
An emphasis on performing the ritual
of cleaning, rather than on prevention
by design.
Cleaning that was not working properly and leaving a biofilm. Some of
these biofilms were not visible in
normal light, but they were visible
using U.V. light.
This article concentrates mainly on issues

applying to open equipment, which is
used extensively in meat, poultry, fish and
dairy processing.
46
Equipment left wet overnight or for a

whole weekend after cleaning.
Footwear poorly cleaned on exiting
production areas and on re-entering.
Poor drainage, poor access for inspection and cleaning, wet films, condensation and aerosols.
There was often a mistaken confidence
in the effectiveness of cleaning-most
open equipment had many unreachable crevices, formed by unsealed
joints, tack welds and threaded fittings
yet owners felt that their cleaning was
effective.
Many suppliers, inspectors, veterinarians and auditors lacked hygiene
knowledge.
The users were not very aware either.
There were both product safety and
occupational hazards, stemming from
poor control of moisture.
The poor moisture control and poor hygienic design was associated with excessive environmental and cleaning costs.
Most new, CE-marked food processing
equipment (estimate: over 70%) did not
comply with the hygiene provisions of the
Machinery Directive, 98/37/EC (The
Directive).
The user organisations did not know
this and did not understand their rights
to have equipment and instructions that
could allow them to produce safe food.
Claims related to alleged deficiencies in
hygienic design which Cocker Consulting
Ltd and fellow consultants had been involved with were settled out-of-court,
with confidentiality conditions. This
meant that not only were the hygiene
provisions of 98/37/EC not policed, there

was also a lack of publicity to warn other
equipment suppliers of the size of the risk
to their businesses. Note: The Directive,
98/37/EC will be replaced by Directive
2006/42/EC from December 2009.
Aggressive fluxes of energy, thermal treatments and chemicals were thought
unavoidable. However, hygienic buildings and equipment were needed in
order to realise the benefits of ecological cleaning methods, longer process
times, increased safety and lower costs.
A common target was visually clean.
This could be misleading.
Enforcement and customer demands
often suggested poor knowledge.
Inspectors, regulators and auditors needed better training in hygienic design.
Operators, fitters, quality assurance personnel, engineers and designers also needed better training in hygienic design.
Apart from the knowledge of the design
principle, engineers, designers and others
needed an outline of the functional properties of microbes that were relevant to
the design principles, together with the
knowledge of how these functional properties relate to hygienic design. This applied not just in the specific area of equipment design, but also to training and to
the whole food production systems, organisations and procedures.
The reasons and evidence for the designs
in EN 1672-2 are not explained. More
knowledge was needed in the industry of
the hygiene provisions of The Machinery
Directive and EN 1672-2, the standard
that was written to explain how to meet
these provisions.
47
It was concluded that at best the engineers and designers at equipment manufacturers lack the microbiological
knowledge mentioned above, which
may make the hygienic design principles
in EN 1672-2 credible to them and also
the skills in food technology necessary
to carry out a hygiene risk assessment,
as required by The Directive.
A scientific basis for designs and for the
validation of equipment, such as that of
the European Hygienic Engineering and
Design Group (EHEDG), was needed.
Explanations
The Role of Moisture in the establishment of Biofilms:
Many microbes, including bacteria,
can either swim or grow via films of
liquid. Stagnant liquid provides a
ready breeding-ground for microbes
and cleaning sprays may redistribute
pathogens from such pools.
Unwittingly, manufacturers may assist
this by promoting a wet environment.
Even the distinctions of product and
non productcontact surfaces, on
which the designs may rely for their
hygienic performance, can become
meaningless under such conditions.
In case further motivation is needed, the report of the United Kingdom Health and
Safety Executive established that 30% of all
major injuries were slips and that 90% of
these slips were caused by wet floors. 95%
resulted in broken bones and 1,000,000
days were lost per annum, at an average
compensation cost of STG 4,000 per accident.
The transport of microbes through the
air:
Because of electrostatic attraction, microbes in the air are inevitably attached to other particles. They have no
active means of flying, but are instead
always passengers, either in liquid or
solid aerosols, or in sprays.
Dry conditions and products:
These do not support the growth or
propagation of microbes.
Moisture Management:
Water is a major component of most food
products and processing often involves
transfer of this water from the food.
In cleaning, water is the dominant carrier
and solvent for soil and detergents.
There are two forms of water removal,
which may be viewed as (a) Passive
(better) and (b) Active (poorer).
48
Passive moisture removal is the result of

a preventive strategy, embodied in the
basic principles of hygienic design (See
the free-to-download Document #8
www.ehedg.org). The key point is that
equipment and its surroundings need to
shed liquid quickly and to retain it only
on demand, for example by closure of
a drain valve or by turning a vessel
upright. There was poor recognition of
the ability of horizontal surfaces to retain liquids, especially downwards-facing surfaces. Many of these downwards-facing surfaces were not easily
accessible for inspection or cleaning.
This adaptation of a schematic from the
EHEDG Trainers Toolbox illustrates the
point for framework.
The image shows the effect underneath a ventilator and underneath

adjacent cylindrical and square-section piping:
And below are such areas immediately
above a conveyer belt:
Active moisture removal usually consumes energy, for example ventilation,

extraction, heating and the use of absorbent materials, rubber blades, and,
for closed systems, vacuum. It is often
necessary after wet cleaning.
Protection of microorganisms by soil
Residues of food products and biofilms
on equipment have been shown to
protect microorganisms from respectively thermal and chemical treatments
designed to kill them.
49
It has also been demonstrated that the

deeper the crevice, the greater the protection that is afforded by the soil.
There are plenty of examples of such

crevices in recentlypurchased equipment, such as this at an SME dairy:
The dairy in question was considering
purchase of a further scraped surface
heat exchanger, so advice was provided
on improved designs.
The Phoenix
A principle difference between microbial contaminants and other contaminants such as chemicals and foreign bodies is that microbes are capable of
re-growing after any setback. Dilution
is perfectly effective for controlling chemical contaminants, for example, but

usually offers only temporary relief,
where microbes are concerned.
A typical sequence is that crevices and
hidden surfaces collect proteins, fats and
microbes, then escape effective cleaning
and detection, even though 99.99% of
the rest of the surfaces are very well cleaned and disinfected. The managers involved conclude on the basis of visual inspection and possibly, point-sampling of
accessible surfaces, that they have clean
equipment. Each crevice is then a ready
locus of contamination, which can lead
to dissemination and biofilm formation,
and then in turn to more frequent contamination events and to increased contamination-levels. This is especially so if the
equipment does not have dry surfaces.
It is worth ensuring that all concerned understand the importance and mechanics
of biofilm consolidation, which is measured in hours and days and is characterised by physiological and metabolic
changes that lead to increased resistance
to lethal agents, increased adhesion and
an ability to survive in the presence of low
nutrient concentrations. As we all know
from the biofilms that form on our teeth,
mechanical force is needed to remove
biofilms, once consolidation has taken
place, and because of the kinetics of biofilm consolidation, the frequency of cleaning is crucial to maintaining control.
In the presence of nutrients, the
growth-rate of microbes can vary from
exponential down to apparent stasis,
depending on nutrient sufficiency. The
green curves in the following schematics represent a hygienic design, versus
a non-hygienic design (yellow).
50
This can allow significant advantages to

be gained from increasing levels of hygienic design, until ultimately for aseptic
systems, the absence of contaminating
microbes means that there is no growth
of contaminants, and extremely long
processing times are possible, without
the need for cleaning or sterilization.
Aerosol Formation
An appreciation of necessary hygienic design considerations for open systems requires some understanding of aerobiology, or the mechanisms by which
microorganisms survive and spread whilst
in the air. Even a drop of liquid falling
from a pipette onto a lab bench generates a large quantity of micro-droplets.
(Dimmick & Akers, 1969). The rate and
intensity is proportional to the mechanical
energy-flux (Dimmick factor). In a foodprocessing environment, examples include high-pressure jets from cleaning,
high-speed fans and high-speed slicers.
Direct and reflected spray can carry microorganisms and nutrients a few feet
away, but the associated aerosol can be
copious and can spread much further.
The finest aerosols have neutral buoyancy
in the air and hang in the air for a very
long time. This is why, for example, jet
cleaning should never be performed in
rooms where food or clean equipment is
within reach of the aerosol cloud. It is also
why high-speed, water-lubricated, slicerblades can give problems like those at
Maple Foods in Ontario. The spray and
aerosol from the disc disseminates moisture, microbes and food, and there is
ample time between cleaning shifts for
multiplication. There were 22 deaths
from Listeria monocytogenes in this outbreak.
51
High-pressure sprayers have been shown

to become contaminated and to backcontaminate the water supply in food
plants (Gagniere S, Auvray F, Carpentier,
B. Spread of a green fluorescent proteintagged Pseudomonas putida in a water
pipe following airborne contamination. J
Food Prot 2006; 69:2692-6).
This phenomenon is supported by the
finding that water-cooled high-speed
dentist drills also suffer the same problem
(Appl. Env. Microbiol. 66:6.636).
EHEDG Document #13, Open Equipment,
draws attention to the positioning of
common sources of high energy, such as
motor drives and fans, highvoltage insect
traps and static components such as piping and cable trays (which can carry liquid to a point where it can contact such
a device). The following image is taken
from the EHEDG Trainers Toolbox, which
was developed using EU funding within
the HYFOMA project.
The use of high-pressure sprays, especially those targeted near the floor or
near drains with the intention of transporting food materials to the drain. It is
easy to see how even the coarse, visible
mist can spread contamination, let

alone the invisible fine aerosol.
During PathogenCombat visits to
Spanish food processors, it was found
that for the ripening rooms of jamon
iberico, both veterinarians and customer representatives (auditors) had demanded wet cleaning of the floor.
52
Hoses & mops locked during production.

Rubber blades with scoops and bins
only, for the removal of waste that
falls to the floor.
Good ventilation.
Controlled wet cleaning where necessary, for example, use of impregnated
wet wipes.
To prepare for this, the processor had

to remove all the hams from the (very
large) ripening rooms and then a rideon floor scrubber/dryer was used.
Unfortunately, this type of machine
sucks the remaining fluid from the floor
and exhausts it as an aerosol.
We recommended against using the
floor scrubber or wet cleaning. The ripening-rooms have an ambient relative
humidity of around 40%, so it was
counterproductive to add water.
Instead, we recommended using a
sheet of material under each row of
ham frames, rolling this up when it was
time to clean. It could be recyclable,
reusable or disposable. If reusable, it
could be cleaned elsewhere, without
the need to move all the hams out of
the room and then move them back
again.
For moisture-management, it is recommended to implement a dry floor policy. This means:
Removing waste at source and spiriting liquid waste straight to drain.
NO rubber boots or aprons.
Normal safety shoes.
NO boot-washers at production.
Whilst this discussion is focused on liquid aerosols, it would be misleading to

draw attention away from dust and
solid aerosols, for example from building work or other sources of dust.
In the meat, poultry, fish and dairy industries, poorly-controlled sources of
moisture include:
All chilled equipment in process areas.
Freezers and chill rooms.
Overhead chiller units.
Product itself (especially chilled).
Non-insulated pipes and tanks.
Ice.
Process water.
Washing fluids.
Vapour from internal combustion engines (battery versions OK).
Traffic.
Drafts.
Boot-washers at entrances.
A significant moisture-source was the
movement of lift-trucks between areas.
This should be avoided as far as possible,
by use of roller- and belt-conveyers, passing via windows from one room to
another, and split at the door, so that the
rooms and conveyers can be isolated for
cleaning and when product is not being
transported.
53
process is very contaminated, and the

visitors (yellow group) have just passed
though from the live bird intake, via
slaughter and evisceration.
Note that the conveyer above is also covered, effectively making it a closed
part of the system, unaffected by the
surroundings until opened.
The role of correct integration:
Humidity control and many other factors important for food safety often
broke down once the layout and flow
of products, people, equipment, materials and waste products became disrupted. This could have happened because of poor initial planning and/or
because of badly planned expansions of
production. There should be a systematic, sequential flow of product in one
direction, with air, waste and people
running counter to this. In the
PathogenCombat visits, it was almost
always the case that we were led through the process from beginning to
end, dirty to clean. Many other visits
have been the same.
The slide below is a schematic of the
correct flows. The example is illustrated
with respect to a poultry plant, as an
example where the beginning of the
The last Integration topic in the Burgos

presentation was to re-emphasise that
food safety requires a holistic approach.
A forgotten aspect in many plants was
the Maintenance Department, where
equipment, clothing and people that
find their way into processing areas
(note the hairnets) were not handled in
a hygienic fashion.
How can the food industry contribute to

decrease the risk of contamination by
mycobacteria: a hypothetical case discussion
K. Hruska, I. Pavlik, RA. Juste
Abstract
A case study is presented as a contribution
to the knowledge dissemination from the
PathogenCombat project. A general administrative regulation for Mycobacterium
avium subsp. paratuberculosis in milk and
Mycobacterium spp in drinking water is not
yet available. Dairies and dairy food producers should be interested in the paratuberculosis herd prevalence in the cluster of
their milk suppliers. Their classification as likely MAP free should be based on regular
analysis of bulk tank milk or milk filters by
real time quantitative PCR. Farmer should
be encouraged to control paratuberculosis
in their herds and mycobacteria-free milk
should be used for baby foods production.
The hypothesis
There are some infections that cause in
some individuals an altered immune response that leads to persistent local inflammation at the environment-organism interface. They have protracted incubation
periods since they are caused by low virulence organisms replicating at very low
rates. Also some cell wall components of
bacteria are able to trigger chronic human
illnesses. People in risk are mainly newborn babies and people with a specific
mutation of one gene.
Let researchers assess this hypothesis as

true or false. However, unless the risk
will not be credibly excluded, it should
be taken as a real one and measures for
consumer protection should be applied.
The published data support sufficiently
needs to develop an efficient system
how to eliminate or at least how to decrease this risk. The following case study
is an example, how food industry, from
small to global enterprises, can contribute to the consumer protection. The
basic premise is that a solution does not
consist either in a simple elimination of
a risky foods and tap water from human
consumption, or in pasteurization or boiling. Cattle is the largest producer of
Mycobacterium avium subspecies paratuberculosis (MAP), but milk and beef and
dairy products cannot be generally excluded from human diet.
The implications and the possible economic
impacts on milk demand were analysed
(Groenendaal and Zagmutt, 2008). Three
scenarios were developed based on the effectiveness of possible risk-mitigation strategies. In the first scenario, it was assumed
that an effective strategy exists; therefore,
a negligible demand decrease in the consumption of dairy products was expected.
In the second scenario, it was assumed that
new risk mitigation would need to be im-
56
plemented to minimize the health hazard

for humans. In this case, a small milk demand decrease was expected, but larger
demand decreases were also possible. The
third scenario assumed that no fully effective risk mitigation was available, and this
resulted in a considerable demand decrease
and a potential reduction in milk supply as
a result of regulatory measures. A milk demand reduction of 1 or 5% resulted in a reduction in consumer surplus of $600 million and $2.9 billion, and a reduction in
dairy farm income of $270 million and $1.3
billion, respectively. A decrease in milk
supply would cause a slight increase in total
losses, but would cause the greatest losses
to test-positive dairy farms. Given the current scientific knowledge about MAP and
Crohns disease, the authors conclude that
if a link were established, it is most likely
that the first or second scenario would
occur. Thus, consumer response and economic consequences to the discovery of
such a link are expected to be limited, but
could be large if the consumer's perception
of risk is large or if risk-mitigation strategies
were ineffective (Groenendaal and
Zagmutt, 2008).
The basic terms

The basic terms have to be defined in the
first place before any decisions should be
done. In each item should be known if
knowledge is scientifically confirmed. A
doubtful definition has to be explicitly
marked. In this case study the important
terms are as follows:
Crohns disease
CD is a devastating illness in search of a
cause and a cure. More than 800 000 pe-
ople in North America suffer from CD, a

gastrointestinal disorder characterized by
severe abdominal pain, diarrhoea, bleeding, bowel obstruction, and a variety of
systemic symptoms that can impede the
ability to lead a normal life during chronic
episodes that span months to years.
Researchers and clinicians agree that
onset of CD requires a series of events;
implicated are certain inherited genetic
traits, an environmental stimulus, and an
overzealous immune and inflammatory
response. The combination of these factors contributes to a disease whose
course is variable among patients and
whose symptoms range from mild to devastating on any given day. The economic
and social impact of this disease is substantial for the patient, the family, the
community, and the healthcare system
(Nacy and Buckley, 2008).
Paratuberculosis (Johnes disease)
Paratuberculosis, also known as Johnes disease, is a bacterial infection of the gastrointestinal tract and is characterised by chronic diarrhoea, persistent weight loss,
decreased milk production and eventually
death (Davies et al, 2009). The disease is
not treatable and vaccinations avoids clinical disease but do not completely prevent
infection; therefore, economic loss is substantial in both the dairy and beef industries.
Conflicting opinions have been published
that indicate a potential link between the
causative agent (Mycobacterium avium
subspecies paratuberculosis) and Crohns
disease in humans, via the consumption of
infected dairy products (Chiodini and
Rossiter, 1996; Bakker et al, 2000). In the
United States, economic losses from the disease have been estimated to exceed
How can the food industry contribute to decrease the risk of contamination by mycobacteria
57
$1,500,000,000 per year, mainly from the

effects of reduced milk production (Okafor
et al, 2008).
Mycobacteria (http://en.wikipedia.
org/wiki/mycobacterium)
Mycobacterium is the only genus in the family Mycobacteriaceae belonging to order
Actinomycetales, phyllum Actinobacteria.
The genus includes pathogens known to
cause serious diseases in mammals, including tuberculosis and leprosy. All mycobacterial species share a characteristic cell wall,
thicker than in many other bacteria, which
is hydrophobic, waxy, and rich in mycolic
acids (mycolates). The cell wall consists of
the hydrophobic mycolic layer and a peptidoglycan layer held together by a polysaccharide, arabinogalactan. The cell wall
makes a substantial contribution to the hardiness of this genus. Many mycobacterial
species adapt readily to growth on very
simple substrates, using ammonia or amino
acids as nitrogen sources and glycerol as a
carbon source in the presence of mineral
salts. Optimal growth temperatures vary widely according to the species and range
from 25 C to over 50 C.
With regards to food safety the most important species are Mycobacterium bovis
because of its possible presence in unpasteurized milk and cheese and Mycobacterium avium subspecies paratuberculosis because of the huge amounts shed by cows
irregularly in their faeces (1012 cells per
gram) and that might contaminate foods
and environment. (Anon, 2000). American
Microbiology Academy in its report (Nacy
and Buckley, 2008) quotes a recent study
using culture-dependent methods detected
viable MAP in 2.8% of homogenized milk
cartons sampled from supermarket shelves
in the U.S. The prevalence of 19.7% IS900

PCR-positive bulk-milk samples shows a
wide distribution of subclinical MAP-infections in dairy stock in Switzerland. The prevalence, however, in the different regions
of Switzerland shows significant differences
and ranged from 1.7% to 49.2% (Corti
and Stephan, 2002). Other studies detected
MAP in samples of cheese (Ikonomopoulos
et al, 2005; Stephan et al, 2007). Overall,
different levels of MAP contamination of
milk, meat and food products have been
noted in the U.S. and a variety of countries
around the world (Bosshard et al, 2006;
Clark et al, 2006; Slana et al, 2008). MAP
has also been identified in environmental
sources, including river water and municipal
water (Pickup et al, 2006). MAP has been
detected using IS900 PCR in 49.0 % of
baby foods. These results correspond to the
epidemiological situation in Europe and are
not unexpected (Hruska et al, 2005). A
comprehensive review of mycobacteria in
the environment and their impact on animals and humans health has been published recently (Kazda et al, 2009). Equally,
the demonstration of frequent presence of
MAP in muscle of clinically and subclinically
infected cattle opens a hitherto overlooked
route of exposure of humans through food.
Since consumption of raw cattle meat is
very common in some cultures, this route
could be even more relevant that the milk,
since in the case of raw beef there is not
even a significant thermal treatment.
The beef industry also needs to both require
certification of reduction or eradication of
MAP from its suppliers and to establish a
control of the paratuberculosis status of the
individuals entering its commercial chain. A
general administrative regulation is not expected from several reasons. The herd pre-
58
valence of paratuberculosis is estimated in

some countries as high as 50% to 85%.
The identification of shedders is difficult and
their culling contributes to the disease eradication but does not exclude shedding of
MAP by another animal next week or next
months. A substitution of culled animals
from another herd is not secure because no
herd is absolutely paratuberculosis free. The
eradication certification of herds as MAP
free will be a long and expensive procedure.
Use of vaccines is a common practice in
some countries, it is inexpensive and has
shown a high efficacy in reducing rates of
bacterial excretion for which it could make
a good immediate mitigation strategy.
However, even though it is a practice currently in the increase, especially in small ruminants, still, due to prejudices regarding
to interference with TB control programs,
it remains largely unused for cattle in most
countries. The consequence is that very little
is being done in paratuberculosis control.
Slow infections and microbial
triggers
The term was first used by Bjorn Sigurdson
in 1954 (Sigurdsson, 1954) referring to the
sheep diseases that spread through some
regions of Iceland in the thirties as a consequence of an import of apparently healthy
rams that had even been submitted to long
quarantine periods. This concept has not
been widely used, but fits nicely with a set
of chronic diseases usually not lethal by
themselves if proper care is given. Since the
agents are difficult to grow or detect, are
ubiquitous and do not act directly, but by
way of the own host response, they are very
difficult to causally associate to a series of
diseases for which an infectious cause has
long been suspected but, that do not fully

comply with classical causation postulates.
In the report of American Academy of
Microbiology (Carbone et al, 2005), a microbial trigger is defined broadly to mean
any organism that sets in motion or expedites a disease process. Hence, a microbial trigger can bring on disease in any of
a number of ways, including persistence
as a chronic infection and the induction
of destructive host immune responses.
The priming effect can be mediated also
by the components of bacterial cells, e.g.
peptidoglycan and its minimal motif muramyldipeptide.
In 1974, muramyldipeptide (MDP) was
discovered as the minimal structure responsible for the improved reaction to mycobacteria in Freunds complete adjuvant.
Numerous reports suggest that MDP and
other muropeptides directly induce cytokines, thus activating and modulating immune responses and inflammation (Traub
et al, 2006). Hence not only the life bacteria, containing MDP, but also dead cells
and their components participate in development of some diseases. Timing of
their ingestion or inhalation, the way of
entering, the effective mass and hosts genetic disposition are without a doubt the
important factors in disease pathogenesis.
Food borne diseases
At: http://www.cdc.gov/ncidod/dbmd/diseaseinfo/files/foodborne_illness_FAQ.pdf
a simple but detailed description of food
borne diseases is available. According to
the U.S. Centers for Disease Control and
Prevention a foodborne disease is caused
by consuming contaminated foods or beverages. Many different disease causing
59
microbes, or pathogens, can contaminate

foods, so there are many different food
borne infections. In addition, poisonous
chemicals, or other harmful substances
can cause food borne diseases if they are
present in food. More than 250 different
food borne diseases have been described.
Most of these diseases are infections,
caused by a variety of bacteria, viruses,
and parasites that can be food borne.
Other diseases are poisonings, caused by
harmful toxins or chemicals that have
contaminated the food, for example, poisonous mushrooms or pesticide residues.
Autoimmune and autoinflammatory
diseases (McGonagle and McDermott,
2006)
Triggered by ingested bacterial components can be classified as specific food
borne diseases. The evident difference
from current food borne infections is several or dozens years of latent period and
absence of apparent clusters of diseased
persons usual in food borne infections
outbreaks. Direct proving or disproving
the origin of clinical forms of chronic diseases is impossible and Kochs postulates
cannot be applied. Bacterial cells and their
components should be better describes as
harmful contaminants, closer to toxic xenobiotics or pollutants than to pathogens.
A hypothetical case discussion
It is not possible to take the assignment together as a quite different approach has to
be applied on different mycobacteria considered as risky from the food safety point
of view. Mycobacterium bovis and
Mycobacterium tuberculosis can appear in
milk and meat from animals suffering from

bovine or human tuberculosis if the veterinary inspection fails. The problem can arise
when bioproducts can be sold from the
farm directly to consumers. The food industry enterprises in the European Union
are responsible for using the traceable feedstock from official sources under inspection. Milk and meat primary contamination
thus should be avoided by the veterinary
inspection and food producers have to follow only the possibility of secondary contamination from people suffering from tuberculosis in open form. An apparent
manifestation of the disease, mainly coughing, should not be overlooked.
Mycobacterium avium subspecies paratuberculosis represents another problem, not
yet suitably treated by legislative norms.
Therefore the following recommendations
are not obligatory and should be applied
voluntarily to the consumers benefit. The
recommendations are addressed (1) to farmers (how to decrease economic losses
from paratuberculosis and keep their herds
under control, (2) to water companies (how
to decrease the mycobacterial contamination of water in the pipe systems), (3) to
consumers (how to decrease the risk of mycobacteria ingestion namely is there is
higher risk of their disposition to Crohns disease), (4) to the research and development
sector (how to apply the semiquantitative
analyses of mycobacterial contamination of
water, milk and meat and how to improve
the control of paratuberculosis), and (5) to
administration (how the financial support
to the agriculture, environmental a health
sectors should be directed and the consumer protection legislatively treated). In
agreement with the aim of this workshop
60
the recommendation addressed to the food

industry are described in more detail.
Possible case treatment

All dairies and dairy food producers should
be interested in the paratuberculosis herd
prevalence in the cluster of their milk suppliers. Their classification as likely MAP free
should be based on quarterly analysis of
bulk tank milk or milk filters by real time
quantitative PCR as long as the system of
herd certification would not be used.
Having the results steps should be directed
towards the consumer protection. Entirely
in the enterprise management is securing
the use of tap water free of mycobacteria.
It is necessary to collaborate with the laboratory, specialized in mycobacteria determination both in milk and water analysis. The
appropriate methods are not used in every
routine microbiological or molecular biological laboratory and ready-to-use kits are
not yet available.
The enterprises producing baby food, namely for newborns, pre-term borne and
youngest babies should follow the possibility to introduce the MAP free formulas as
soon as possible. Again, the input check of
milk and selection of suppliers should be
based on interest of food industry itself as
the farmers need not to produce MAP free
milk yet. Similarly, the food industry needs
not to warrant MAP free product but the
comparative advantage can be used in marketing. However, once the product is declared as MAP free, the consumer protection organizations will check the retail
products certainly. A great responsibility is
on the managements of producers of bottled water, designed as suitable for the babies. Beside the currently required quality
the mycobacteria should be tested.

Moreover, the consumers should be well informed about the risk of mycobacteria growing on the inner surface of botles.
Paratuberculosis should be a notifiable disease and the national system for certification of MAP free herds or with infection reduction strategies should be opened. As
long as the certification is not available all
herds have to be assumed as infected. For
commercial purposes the customer (dairy)
could require periodical evaluation of bulk
tank milk or milk filters on its own account
and for its own production management
and HACCP system, which has to consider
mycobacteria as a risk.
The quick, reliable, robust and accreditable semiquantitative or quantitative
tests for MAP DNA in milk or meat and
for mycobacteria DNA in water are available. Nevertheless, a development of
new formats and principles of tests can
be expected.
In summary, consumer protection from the
risk of mycobacteria contamination of food
is at present a voluntary task for the food
industry. The changes of regulations are expected, but both for farmers and state administration they will very difficult, expensive and long time required assignment.
Nevertheless, if only the prevalence of
Crohns disease would decrease by 5%
only, at least 100 000 consumers could be
protected in the USA, Canada and Europe.
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Ayele WY, Roubal P, Lukas J, Cook N, Gazouli
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New processing technologies that can

reduce the presence of pathogens in foods
A. Rajkovic, M. Uyttendaele, N. Smigic, F. Devlieghere
.
Introduction
Number of alternative methods and technologies rose up to replace historically
proven heat treatments in the attempt to
satisfy modern trends in food consumption.
These new trends were induced by the
change in the consumers perception of
food quality and nutrition. The modern consumer seeks fresh looking, convenient and
nutritionally healthy food, which requires
from industry to adopt new strategies in
safe food production. The main change in
terms of microbial food safety is that sterilization and pasteurization as we knew
them are in great extent replaced by very
mild heat treatment, high pressure processing, pulsed electric fields, intense light
pulses, application of organic and natural
preservatives etc. The ability of these techniques, alone and even more in combination, to inactivate and suppress recovery
and growth of surviving microorganisms at
low temperatures is beneficial for applications of heat sensitive foods and ingredients
and to minimize adverse effects on the sensory characteristics of food products. Many
of these novel technologies have been already subject of extensive research, but before actual commercial application number
of technical, economical, and regulatory issues are to be solved. Among the factors
that will determine the success of certain
novel technology is the consumers accep-
tance, which influences their purchase intent. Although many consumers prefer the
non-thermal food processing technologies
to manufacture higher quality, value-added
foods that feature higher vitamin and nutrient retention, and improved sensory attributes the lack of knowledge may pose an
obstacle in their buying behavior (table 1).
Table 1. Percent of (n = 198) respondents
that were very or extremely
concerned with foods processed by
novel food processing techniques
(adopted from Wright et al, 2007).
Food
processing
method
% very or
extremely
concerned % uncertain
Genetically modified
Irradiation
Radio frequency
sterilization
High pressure treatment
Microwave processing
Thermal processing
Heat pasteurization
54
49
40
17
17
21
20
18
18
13
18
12
14
6
The ideal processing technique would be

the one that meets following demands
(Raso et al, 2005):
Improvement of shelf life and safety by
inactivating enzymes and spoilage and
pathogenic microorganisms.
64
No changes in organoleptic and nutritional attributes.

No residues left on food.
Convenient to apply.
Cheap.
No objections from consumers and legislators.
Pulsed electric field (PEF)

Pulsed electric field (PEF) technology is a
non-thermal inactivation technology based
on the use of electric fields instead of heat.
Next to microbial inactivation PEF maintains
quality attributes such as sensory quality
and nutritional value. Successful application
of PEF technology to liquid products such
as fruit juices (Aguilo-Aguayo et al, 2009;
Elez-Martnez et al, 2004; Elez-Martnez et
al, 2005; Marselles-Fontanet et al, 2009;
Mosqueda-Melgar et al, 2008; Riener et al,
2009; Zhang et al, 2007), liquid egg (Dunn,
1996; Jeantet et al, 2004; Jin et al, 2009),
fruit smoothies (Walkling-Ribeiro et al,
2008), and milk (Sampedro et al, 2009;
Walkling-Ribeiro et al, 2009) at laboratory
and pilot plant levels suggests the potential
of this technology as a substitute for traditional thermal pasteurization. While for the
reduction of wine and must spoilage flora
an optimum treatment of 186 kJ/kg at 29
kV/cm has been established (Puertolas et al,
2009), opposite was found for PEF application in beer production (Evrendilek et al,
2004) due to significant degradation in
flavor and mouth feel.
The exact mechanism by which PEF inactivate microorganisms is not yet completely
understood; however, much of the research in the field points toward damage of
the cell membrane as the principal factor
responsible for microbial inactivation.

Other effects resulting from the application
of high-intensity pulsed electric fields, such
as DNA damage and generation of toxic
compounds, have been suggested, although some of the later studies rejected
these hypotheses (Barbosa-Canovas and
Sepulveda, 2005). Although the current
knowledge does not provide ultimate
answer on the antimicrobial mechanism(s)
of PEF it seems that there is reasonable
doubt if the membrane integrity is the only
factor to be considered.
Numbers of factors play a role in the determination of the effectiveness of pulsed
electric field technology as a microbialinactivation process. Among those the
key role can be attributed to the type of
the equipment used, setting of the treatment parameters, the type of media/food
processed, and the target microorganism
(Aronsson et al, 2005; Barbosa-Canovas
and Sepulveda, 2005; de Azeredo et al,
2008; Jin et al, 2009; Wouters et al,
2001). The relationship between these
factors and their overall contribution to
the measured effectives of the PEF still requires further investigation.
High pressure processing

Historically seen food processing with
high pressure goes long back to the end
of 19th century. In 1899 (Heinz and Knorr,
2005 and references therein), Hite subjected milk to high hydrostatic pressure
treatment as an alternative to classic heat
sterilization in an attempt to avoid the
sensory shortcomings of heat sterilized
milk, while maintaining its microbiological
safety and quality. Hite achieved a 4-log
reduction in microbial count in milk with
New processing technologies that can reduce the presence of pathogens in foods
65
a 10-min treatment at approximately 700

MPa at room temperature. Below 200
MPa, the lethal effect of pressure was
found to be significantly reduced. The effect on spores was already than noticed
to be much less effective (Heinz and
Knorr, 2005 and references therein).
Therefore, an increasing interest exists in
producing shelf-stable pressure-treated
foods using HPP in conjunction with heat
(initial temperatures around 80-90 C,
and compression heating temperatures
> 121 C) to kill resistant spores. High
pressure technology (100-1000 MPa, i.e.
1000-10000 bar) is in general of increasing interest to biological and food systems primarily because it permits microbial inactivation at low or moderate
temperature. The commercial production
of pressurized foods has become a reality
in Japan, France, Spain, the USA and
many other countries. This is in a great
deal results of extensive scientific research, technological and technical advances in equipment production and decrease in the processing costs (Cheftel
and Culioli, 1997). Nowadays, commercial application of high hydrostatic pressure has been found its place in the production of juices, smoothies, ready-to-eat
meat products, guacamole, oysters etc
(Hjelmqwist, 2005; Patterson et al, 2007).
A typical high pressure system for food processing consists of a pressure vessel in
which food packages are loaded and into
which the pressure medium, usually water,
is pumped, and a pressure-generating device. In the case of liquids, such as fruit
juices, the vessel is filled with the juice,
which acts as the pressure transmission
fluid. Once the desired pressure is reached,
the pressure can be maintained without
further need for energy input. A fundamental principle underlying HPP is the isostatic process allowing that all regions of the
food are rapidly exposed to a uniform pressure. The work of compression during HPP
treatment also increases the temperature
of foods through a process known as adiabatic heating, and the extent of the temperature increase varies with the composition
of the food (normally 3-9 C/100 MPa). HPP
is traditionally a batch process. A series of
these vessels can work in a staggered sequence for an overall system that is semicontinuous.
HPP treatment is generally considered to act
on bacterial cell membranes and impair
their permeability and ion exchange
(Cheftel, 1995; Hoover et al, 1989;
Yaldagard et al, 2008). Microorganisms vary
widely in their resistance to HPP treatment
(Chung and Yousef, 2008; Scurrah et al,
2006; Whitney et al, 2008). Most often,
bacterial vegetative cells are inactivated at
pressures around 300-400 MPa at ambient
temperature or higher temperatures. In recent study of Alpas et al. (2003) different
juices were inoculated with Alicyclobacillus
acidoterrestris cells to 6 log CFU/ml and
were pressurized at 350 MPa at 50 C for
20 min. More than 4 log cycle reduction
was achieved in all juices studied immediately after pressurization. The inactivation of
spores by HPP is less efficient and requires
higher pressures and higher temperatures.
Bacterial spores were found to survive up
to 1200 MPa at room temperature (San
Martin et al, 2002; Zhang and Mittal, 2008
and references therein) Furthermore, a detailed review of Zhang et al. (2008) compiled much of the published data showing
that there can be significant variations in
the requirements of high pressure and tem-
66
perature among different bacterial spores

species and also among strains of the same
species. In general spores are unlikely to be
(sufficiently) inactivated by HPP at room
temperature. Therefore, optimization of the
HPP conditions or combination with other
treatments and agents may be needed for
a successful inactivation of spores. Most of
the time the inactivation of spores is a twostep process: first, germination of the
spores, and second, subsequent inactivation of the germinated spores. Germination
is the process by which a stimulus is applied
to induce the dormant spores to convert to
a metabolically active vegetative state. Heat
shock is probably the most common stimulus. In principle, if all of the spores present in a food material could be induced to
germinate, the food material could then be
sterilized by a subsequent preservation treatment that would be milder than the treatment needed to inactivate ungerminated
spores (Murad et al, 2007).
Not only bacterial inherent characteristics
and treatment parameters play an important role in effectiveness of HPP, but also
the environment in which bacteria are
found. Patterson et al. (1995) reported that
treating E. coli O157:H7 under the same
conditions of 700 MPa for 30 min at 20 C
resulted in a 6 log reduction in phosphatebuffered saline, a 4 log reduction in poultry
meat, and a < 2 log reduction in UHT milk.
This reason is probably in the protective role
of certain food constituents. However, the
pH and water activity (aw) of foods can also
significantly affect the inactivation of microorganisms by HPP. Most microorganisms
tend to be more susceptible to pressure in
lower pH environments, and pressure-damaged cells are less likely to survive in acidic
environments (Patterson, Linton, and

Doona, 2007).
Regarding inactivation of foodborne viruses
differences in HPP effect were noticed between different viruses, different treatment
parameters and different foods/media were
reported (Baert et al, 2009; Kingsley et al,
2007). In review of Baert et al. (2009) it was
reported that exposure of hepatitis A virus
to pressures of 375 MPa at 21 C for 5 min
induced reduction of respectively 4.3 and
4.7 log in strawberry puree and on sliced
green onions. HPP treatment of oysters with
a pressure of 400 MPa for 1 min (9.0 C) induced 3 log reduction of HAV whereas
MNV-1 was reduced by 4 log (5 C). On the
other hand, Aichivirus and coxsackievirus
B5 remained fully infectious if 600 MPa was
applied for 5 min at ambient temperature
whereas coxsackievirus A9 was reduced by
7.6 log under the same conditions. Similarly
poliovirus was found to be resistant to 600
MPa for 1 h. It can be concluded that the
sensitivity towards HPP does not agree between genetically related taxonomic groups
or even strains. A possible explanation could
be the difference in protein sequence and
structure (Baert et al, 2009).
Intense light pulses

Intense light pulse (ILP) is one of the emerging non-thermal techniques investigated
as an alternative to traditional thermal treatment. ILP is a technique to decontaminate surfaces by killing microorganisms
using short time pulses of an intense broad
spectrum, rich in UV-C light (the portion of
the electromagnetic spectrum corresponding to the band between 200 and 280
nm), which has been proven to be effective
for microbial inactivation on food surfaces
67
and food packages. The mode of action of

the pulsed light process is attributed to the
effect of the high peak power and the UV
component of the broad spectrum of the
flash. Inactivation occurs by several mechanisms, including chemical modification and
cleavage of DNA, protein denaturation and
other cellular materials alteration (Turtoi and
Nicolau, 2007 and Barbosa-Canovas et al,
2004 therein).
tups and technology (equipment) used.

Namely, contradictory findings were noted
between Gmez-Lpez et al. (2005) who
did not observe any sensitivity pattern
among different groups of microorganisms,
after studying 27 bacterial, yeast and mould
species and decreasing order of sensitivity
observed by Anderson et al. (2000): Gramnegative bacteria, Gram-positive bacteria
and fungal spores.
The following units are commonly used

to characterize the ILP treatment (GmezLpez et al, 2007):
Gmez-Lpez et al. (2005) described that

for an industrial implementation: the position and orientation of strobes in an unit
will determine the lethality, that products
to be treated should be flashed as soon as
possible after contamination occurs, that a
cooling system should be used for heatsensitive products and that flashed products
should be light protected. However, it is important to note that latest results have
shown that repetitive cycles of inactivation
with ILP can result in increased resistance,
but will not affect growth characteristics of
resistance cells (Rajkovic et al, Resistance of
Listeria monocytogenes, Escherichia coli
O157:H7 and Campylobacter jejuni after
exposure to repetitive cycles of mild bactericidal treatments Food Microbiology accepted for publication).
Fluence rate: is measured in Watt/ meter2

(W/m2) and is the energy received from
the lamp by the sample per unit area per
second.
Fluence: is measured in Joule/meter2
(J/m2) and is the energy received from
the lamp by the sample per unit area
during the treatment.
Dose: used sometimes as a synonym of
fluence.
Exposure time: length in time (seconds)
of the treatment.
Pulse width: time interval (fractions of seconds) during which energy is delivered.
Pulse-repetition-rate (prr): number of
pulses per second (Hertz [Hz]) or commonly expressed as pps (pulses per second).
Peak power: is measured in Watt (W)
and is pulse energy divided by the pulse
duration.
The trend of susceptibility of different microorganisms towards ILP has been reviewed by Gmez-Lpez (Gmez-Lpez et
al, 2007). The contradiction in reported
data can be based on the experimental se-
Food preservation by
combined processes (hurdle
technology)
For almost all treatments that do not
cause complete inactivation of microorganisms it is characteristic to induce sublethal injury to the present bacterial cells.
Depending on the type of the injury, type
of the organism and surrounding environment these injured microbial cells have
the potential to resuscitate and resume
68
growth under favorable conditions. These

conditions are commonly found in foods
and the safety of foods treated with alternative, non-thermal and less-than sterilizing technologies requires to be well
thought-out. In addition to the inactivation technologies applied to foods, both
microbial growth and survival can be influenced by different intrinsic factors of
the food. This means that intrinsic factors,
alone or combined with the extrinsic factors, can enhance or inhibit recovery and
growth of microbial cells. Therefore, the
safety and stability of food can be improved by a combination of several factors that will prevent surviving and injured
cells to proliferate. These multiple intrinsic
factors are part of a dynamic system that
changes from the moment of application
to the moment of consumption. During
this process, each factor plays a role of
different magnitude and such magnitude
changes over time (Raso, Pagan, and
Condon, 2005 and references therein).
Food preservation by combined processes
(aka hurdle technology) supports the combination of existing and novel preservation
techniques to establish a series of preservation factors (hurdles) that no microorganism
(of concern) present should be able to overcome (Leistner, 1992; Leistner, 1994;
Leistner and Gorris, 1995; Raso and
Barbosa-Canovas, 2003; Raso, Pagan, and
Condon, 2005; Ritz et al, 2002; Shin et al,
2006). This is especially true for the hardy
microorganisms and bacterial spores that
are very resistant to processing treatments.
To apply principles of food preservation by
combined processes correctly, an appropriate understanding of the mechanisms of
action of the individual factors alone and in
combination is needed. This understanding
allows justified and well balanced combination of hurdles to achieve desired level of
safety and quality, avoiding the need to
apply only one factor at such high intensity
that it causes severe changes in the foods
quality (Raso, Pagan, and Condon, 2005).
Instead, using combined hurdles one can
interfere with the microbial homeostasis
and extend the effect of sublethal injury by
breaking the homeostatic mechanisms and
rendering cells incapable of responding to
the stresses produced by the preservation
factors. This can not only results in growth
inhibition, but can also impair survival possibilities leading the death of injured microbial cells. As said before several studies have
indicated that HPP and PEF inflict sublethal
injury. Also for other technologies similar
findings were reported. Van Houteghem et
al. (2008) studied the effects of carbon dioxide in modified atmospheres on the resuscitation of Listeria monocytogenes cells injured by intense light pulses (ILP), chlorine
dioxide (ClO2), lactic acid (LA) and mild heat
mild bactericidal treatments during storage
at 7 C were examined. The results indicated additional bactericidal effects of CO2
on cultures treated with LA, ClO2 and ILP,
with additional reductions in viable L. monocytogenes of 0.5-1.0 log cfu/ml. Lag
phase duration was significantly different
between the different treatments, with
non-treated cells having the shortest lag
phase, followed by that of heat, intense
light pulses, lactic acid and finally ClO2 treated cells. The authors have found relationship between the amount of sub-lethally damaged cells after a mild inactivation
treatment and the lag phase duration in the
CO2 environment. Similarly, Rajkovic et al.
(2009) reported on the effect of partial
inactivation of Listeria monocytogenes with
69
LA, liquid ClO2 and ILP on injury and posttreatment growth under increased NaCl
concentration and reduced pH values. The
results showed that the inactivation levels
and the percentage of sub-lethal were dependent upon strain and type of inactivation technique used. The biggest effect on
the growth retardation was at every pH observed for the cultures treated with ClO2,
followed by LA and ILP. Under increased
NaCl concentration LA treated cells suffered
hardest growth retardation, followed by
ClO2 and ILP, respectively. Recovery of ILP
treated cultures was not always different
from untreated cultures. In general, damaged microorganisms become more
exacting in growth requirements and are
more sensitive to other preservation factors
like low pH, antimicrobial components, etc.
(Raso, Pagan, and Condon, 2005 and
Mackey, 2000 therein).
In foods preserved by combined methods
the microbial homeostasis is threatened on
different multiple sites asking for a complex
and energy demanding microbial response
(Raso, Pagan, and Condon, 2005 and
Montville and Matthews, 2001 therein).
This fact enables the obtaining of safe and
stable foods by balancing different factors
and strategies. Particularly under mildly
lethal stress, the ultimate cause of inactivation is subject of cellular response to additional regulation that integrates information about the global state of the cell and
its environment (Aertsen and Michiels,
2004). It is therefore an art of combining
different suboptimal factors that will push
microbial cell over the thin line between
bacterial growth and inactivation. The extended post-treatment effect based on the
growth retardation or inhibition of injured
cells under sub-optimal conditions can be
utilized as an important tool in conditioning

of microbial food safety.
The risks to be considered

The European and World food industry
aims increasingly at applying novel and
mild preservation techniques for the production of food products that will meet
demands of a modern consumer. Hence,
their product range is shifting more and
more towards refrigerated, microbiologically unstable food products, which results in an increased complexity to control
the microbial safety of their products. As
seen in principles of food preservation by
combined processes for the production of
mildly preserved food products, often an
inactivation step is applied as a first step.
Most often, a mild heat treatment is applied for this purpose. However, the industry resorts more and more to nonthermal alternatives such as high
hydrostatic pressure, decontamination
with organic acids or other decontamination agents (e.g. chlorine dioxide in the
gas phase), electrical inactivation and intense light pulses. When this type of novel
and mild inactivation techniques are applied, incomplete inactivation and sublethal damage of the target organisms
is often obtained, but by the application
of suboptimal inhibiting factors, growth
during preservation is prevented or
further inactivation is promoted.
In the scientific community, but also among
food processors and legislators there is a
concern about the fact that the application
of sublethal stress factors could induce
(cross) resistance mechanisms in the surviving population and change their virulence
characteristics (Rajkovic et al, Resistance of
70
Listeria monocytogenes, Escherichia coli

O157:H7 and Campylobacter jejuni after
exposure to repetitive cycles of mild bactericidal treatments Food Microbiology accepted for publication; Abee and Wouters,
1999; Hill et al, 2002; Lou and Yousef,
1997; Rowan, 1999). This increased resistance stems from the fact that bacteria, as
living organisms, can respond to and harness themselves against stresses to which
they are exposed. A quantification of these
responses as well as understanding the molecular and cellular mechanisms of this
adaptive response is of great interest, because it may lead to improved strategies for
combating microbes, not only in foods, but
also in disease.
Saccharomyces cerevisiae in relation to membrane permeabilization and subsequent leakage

of intracellular compounds due to pulsed electric field processing. International Journal of
Food Microbiology, 2005; 99:19-32.
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Reducing foodborne pathogens in the food

chain by the use of protective and
probiotic cultures
N. Carlini, L. Mogna, GP. Strozzi
Abstract
With a significant impact on trade and
competitiveness, food safety is of fundamental importance to the European consumer, food industry and economy. But
despite significant investment in the area
of food safety, the incidence of foodborne disease is still on the rise in the
European Union.
The use of protective and probiotic cultures
may be a useful and effective strategy to
prevent or reduce the incidence of pathogens in the food chain, improve food safety and enhance consumer health.
The benefits of these protective and probiotic cultures can be effected at any level
of the food process chain, from the farm
animals to the final food product (Farm
to Fork approach). More specifically,
these beneficial bacteria can be used
either as protective cultures (which reduce
or control the growth of pathogens in the
farm environment or in the final food product) or as probiotic cultures (which confer
a beneficial effect upon the host, either a
farm animal through probiotic animal feed
or the final consumer through a probiotic
food product).
Specific research objectives were identified within the PathogenCombat project
to identify and characterise a collection of

protective and probiotic strains which demonstrated the following multifunctional
properties:
Inhibit a variety of pathogenic organisms common in the food industry.
Capable of survival in food processing
conditions or the gastric system of animals and humans.
Safe for consumption by farm animals
and humans.
Of the 1087 strains screened to date, 70 demonstrated inhibitory activity against specific pathogens. Selected strains were then
tested for survival in simulated food processing conditions (high temperatures, presence of salt, lack of nutrients) and animal
and human gastrointestinal tract conditions
(low pH and presence of bile salts).
As a result of this research, a total of 23
promising protective and probiotic strains
have been identified which inhibit in-vitro
pathogenic organisms found in the food
industry and are capable of surviving food
processing conditions and the gastric
system of animals and humans.
Some of these protective and probiotic
cultures were selected for further testing
in specific food chain applications.
74
Preliminary results are encouraging and positive benefits have been demonstrated to
date in specific poultry and dairy applications.
trast, Escherichia coli is readily transmitted

via water, food, and blood, but is not readily transmitted via air or insect bite.
Additional testing with PathogenCombat

protective and probiotic strains in other
applications is recommended to further
understand the impact of these findings
and the potential of these strains to improve food safety.
Zoonoses are infections or diseases which

are transmitted from animals to humans.
The infection can be acquired directly
from animals or by ingestion of contaminated foodstuffs. The severity of these diseases can vary from mild symptoms to
life-threatening conditions.
Introduction to pathogens
and disease
A disease is any condition caused by the
presence of an invading organism or a toxic
component (i.e. pathogen) which causes
damage to the host. In humans, diseases
can be caused by the growth of micro-organisms such as bacteria, viruses, protozoa,
and fungi. However, bacterial growth is not
mandatory to cause disease.
Not all pathogens cause diseases which
have the same severity of symptoms. For
example, an infection with the influenza
virus can cause the short term aches and
fever that are hallmarks of the flu, or it can
cause more serious symptoms, depending
on the type of virus which causes the infection. Bacteria also vary in the damage
they cause. For example, ingestion of food
contaminated with Salmonella enteritica
causes intestinal upset. But consumption
of Escherichia coli O157:H7 can cause a serious disease which can permanently damage the kidneys and even be fatal.
Pathogens can be spread from person to
person in a number of ways and not all
pathogens use all available routes. For
example, the influenza virus is transmitted
from person to person through the air, typically via sneezing or coughing. But the
virus is not transmitted via water. In con-
Pathogens and zoonotic

disease in the European
Community
In order to prevent the occurrence of zoonoses and protect human health, it is important to identify which animals and foodstuffs are the main sources of infection
or disease. For this reason, information is
collected every year from all European
Union Member States and analysed by the
European Food Safety Authority (EFSA) in
collaboration with the European Centre for
Disease Prevention and Control (ECDC).
An annual Community Zoonoses Summary
Report is published, which reports the occurrence of infectious diseases transmitted
from animals to humans. The recently published report for 2007 highlights that
many bacteria are still being transmitted
from animals to our food.
Campylobacter
Campylobacteriosis is an infectious disease
caused by bacteria of the genus
Campylobacter found in animals such as
poultry, cattle, pigs, wild birds and wild
mammals. Most human illness is caused by
one species, Campylobacter jejuni. This species is well adapted to birds, whose body
Reducing foodborne pathogens in the food chain by the use of protective and probiotic cultures
75
temperature (approximately 42 C) allows

for optimal growth of the bacteria. Birds can
carry these bacteria without becoming ill.
Most people who become ill with campylobacteriosis experience diarrhoea, cramping, abdominal pain, and fever within
two to five days after exposure to the organism. The diarrhoea may be bloody and
can be accompanied by nausea and vomiting. The illness typically lasts one
week. In individuals with compromised
immune systems, Campylobacter occasionally spreads to the bloodstream and
causes a serious life-threatening infection.
Campylobacter infection has also been associated with complications such as later
joint inflammation and, on rare occasions,
Guillain-Barr syndrome, a temporary but
severe paralysis which may be total.
In 2007, infections from Campylobacter
were again the most frequently reported
zoonotic disease in humans across the
European Union with a total of 200,507
cases reported. This represents an increase
of 14.2% from 175,561 in the previous
year.
Table 1. Campylobacter in fresh broiler meat.

Samples tested
in EU
7598
% Positive
in EU
26.0%
Sampling point
Samples tested
in Spain
% Positive
in Spain
At slaughter
At processing plant
At retail
147
168
208
55.8%
29.0%
30.8%
Total samples
tested in Spain
36
0
0
96
% Positive
in Spain
0%
Not applicable
Not applicable
0%
Samples
tested in Spain
89
230
163
% Positive
in Spain
46.1%
71.3%
46.0%
Table 2. Campylobacter in other foods.

Food
Pig meat
Bovine meat
Cows milk
Dairy products
Samples
tested in EU
537
695
4,158
520
% Positive
in EU
0.9%
1.2%
0.5%
1.1%
Table 3. Campylobacter in animals.

Animal
Broiler flocks
Pigs
Cattle
Total
tested in EU
10,260
1,102
12,539
% Positive
in EU
25.2%
56.1%
5.9%
76
In foodstuffs, Campylobacter was mostly

found in raw poultry meat with an average
of 26% of samples showing contamination. In live animals, Campylobacter was
found in poultry, pigs and cattle.
Salmonella
Salmonellosis is caused by bacteria belonging to the genus Salmonella. This genus
consists of more than 2,500 serotypes but
the two serotypes Salmonella typhimurium
and Salmonella enteritidis account for the
majority (70-80%) of Salmonellosis cases
reported in Europe.
The infections may occur in small, contained outbreaks in the general popula-
tion or in large outbreaks, particularly in

hospitals, restaurants, or institutions housing children or the elderly.
Salmonellosis is generally contracted by
the consumption of contaminated food
of animal origin that has not been properly heat-treated or is eaten raw (mainly
meat, poultry, eggs and milk). Many other
foods, including green vegetables contaminated from manure, have also been implicated in its transmission.
Food prepared on surfaces previously in
contact with raw meat or meat products
can, in turn, become contaminated with
the bacteria. This is known as cross-contamination.
Table 4. Salmonella in fresh broiler meat.

Samples
tested in EU
28,012
% Positive
in EU
5.5%
Sampling point
Slaughter
Processing
/cutting plant
Retail
Samples
tested in Spain
184
144
% Positive
in Spain
22.3%
2.8%
206
10.2%
Samples
tested in Spain
1,653
98
41
% Positive
in Spain
2.8%
1.0%
2.8%
Samples
tested in Spain
315
63
% Positive
in Spain
4.8%
7.9%
66
6.1%
Table 5. Salmonella in table eggs.

Samples
tested in EU
16,626
% Positive
in EU
0.8%
Sampling point
Packing centre
Retail
Not specified
Table 6. Salmonella in fresh pig meat.

Samples
tested in EU
81,131
% Positive
in EU
1.1%
Sampling point
Slaughter
Processing
/cutting plant
Retail
77
Most people infected with Salmonella

develop diarrhoea, fever, and abdominal
cramps 12 to 72 hours after infection.
The illness usually lasts 4 to 7 days, and
most recover without treatment.
However, in some cases, the diarrhoea
may be so severe that the patient needs
to be hospitalized. In these patients, the
Salmonella infection may spread from
the intestines to the blood stream, and
then to other body sites and can cause
death unless the person is treated
promptly with antibiotics. The elderly, infants, and those with impaired immune
systems are more likely to have a severe
illness.
Although the number of cases showed a
decrease for the fourth successive year, a
total of 151,995 people in the European
Community were still affected by the bacterium Salmonella in 2007 (compared to
164,011 cases reported in 2006).
Poultry and pig meat were reported as
the foods most frequently associated
with Salmonella, and an average of
5.5% of all fresh poultry meat samples
within the European Union was found
to be contaminated. Eggs and egg products were also found to be contaminated, while the bacterium was only rarely detected in raw dairy products,
vegetables and fruits.
In animal populations, Salmonella was
most frequently detected in poultry flocks.
In 2007, the European Commission launched a new control programme against
Salmonella in breeding poultry flocks. At
the end of 2007, 15 Member States had
already met the target of 1% which must
be achieved by the end of 2009.
Listeria
The bacterium Listeria monocytogenes,
commonly referred to as listeria, is found
in soil, vegetation, sewage, water and the
feces of animals and humans. Listeria can
also be found in unpasteurised dairy products, raw vegetables and meats and processed foods including deli meats and hot
dogs.
Listeriosis is a rare but potentially lethal
food-borne infection caused by Listeria monocytogenes. Infected pregnant women
may experience only a mild, flu-like illness.
However, infections during pregnancy can
lead to miscarriage or stillbirth, premature
delivery, or infection of the newborn.
In addition, the elderly and individuals suffering from immuno-compromising diseases such as cancer or HIV are particularly
vulnerable to listeriosis. In these cases, listeriosis may lead to meningitis, brain infection, and severe blood infection.
In 2007, the number of Listeria infections
in humans in the European Union remained
at the same level as 2006 with 1,554 confirmed cases. Although less frequent than
Campylobacter and Salmonella, infections
from Listeria are quite dangerous due to
their high mortality rate (20%).
Cases of Listeria above the legal safety
limit were found in ready-to-eat foods,
most often in smoked fish and other fishery products, followed by meat products
and cheese.
Escherichia coli
Escherichia coli or E. coli is a bacterium
from the family Enterobacteriaceae
which is usually found in the digestive
system of healthy humans and animals.
78
Table 7. Listeria in food.

Food
Samples
tested in EU
932
21,245
2,644
Bovine meat
Pig meat
Red, mixed or
unspecified meat
Poultry meat
2,581
Cheeses from
4,879
cow milk
Cheeses from sheep
1,064
and goat milk
Fish
2,629
Crustaceans, shellfish or
2,328
unspecified fishery products
% Positive
in EU
1.8%
2.2%
2.5%
Samples
tested in Spain
No data
418
No data
% Positive
in Spain
No data
4.1%
No data
2.6%
0.1%
31
No data
6.5%
No data
1.0%
No data
No data
18.3%
2.5%
No data
653
No data
5.2%
Table 8. Listeria in animals.

Animal
Total
tested in EU
% Positive
in EU
Total tested
in Spain
% Positive
in Spain
Gallus gallus (fowl)

Turkeys and ducks
Pigs
Cattle (bovine animals)
Goats
Sheep
4,860
140
5,834
76,376
689
2,973
0.1%
0%
0.1%
0.2%
3.8%
2.4%
No data
No data
No data
68,311
No data
No data
No data
No data
No data
0%
No data
No data
There are hundreds of known E. coli

strains. Many are harmless but some
strains, such as E. coli O157:H7 and E.
coli O121:H19, belong to a group of
Escherichia coli known as verotoxigenic
E. coli (VTEC) or shiga-like toxin producing E. coli (STEC).
VTEC organisms have several characteristics which make them so dangerous. They
produce one or more verocytotoxins (VT)
which cause severe damage to the intestinal tract lining of those infected. VTEC organisms also have a very low infectious
dose, which means that only a relatively

small number of bacteria are needed to
set-up housekeeping in the intestinal tract
and cause infection. Finally, they are quite
hardy and can survive for quite some time,
depending on the environmental conditions. Some organisms (e.g. E. coli
O157:H7) can survive at low temperatures
and in acidic conditions which makes it difficult to eradicate them in nature.
The acute disease associated with E. coli
O157:H7 is named hemorrhagic colitis.
Symptoms characteristic of this disease in-
79
Table 9. VTEC in fresh bovine meat.

Total samples
tested in EU
14,115
% Positive
in EU
0.3%
Sampling point
Samples
tested in Spain
% Positive
in Spain
At slaughter, cutting
/processing plant
At retail
Not specified
201
1.8%
69
No data
1.4%
No data
% Positive
in EU
3.6%
8.1%
Total tested
in Spain
312
No data
% Positive
in Spain
17.0%
No data
Table 10. VTEC in cattle.

Animal
Total animal
Total herd/holding
Total tested
in EU
5,154
559
clude watery and/or bloody diarrhea,

fever, nausea, severe abdominal cramping, and vomiting; these symptoms may
appear within hours or up to several days
after ingestion of the bacteria. Treatment
is usually not necessary as most people recover from the illness on their own after
a duration of 5-10 days.
However, particularly virulent strains of E.
coli can cause serious illness or death in the
elderly or those with weakened immune
systems. Some individuals may develop hemolytic uremic syndrome (HUS). In the very
young, this disorder may cause renal failure,
hemolytic anemia (destruction of red blood
cells), or even permanent loss of kidney
function. In the elderly, these symptoms as
well as thrombotic thrombocytopenic purpura (TTP) can occur and the mortality rate
due to TTP can be as high as 50% in this
population.
Transmission of E. coli usually occurs by
consumption of contaminated food or
water or by contact with an infected animal
or person.
Categories of foodstuffs where VTEC represents a hazard to public health have been
identified and include: raw or undercooked
beef and possibly meat from other ruminants; minced and/or fermented beef and
products thereof; raw milk and raw milk
products; fresh produce, in particular
sprouted seeds, and unpasteurised fruit and
vegetable juices; and water.
In the European Union, VTEC accounted for
a total of 2,905 human infections in 2007.
Among animals and foodstuffs, VTEC was
most often reported in cattle and bovine
meat and very rarely in vegetables.
The utilisation of protective

and probiotic cultures as a
strategy to prevent or reduce
pathogen transmission along
the food chain
The utilisation of protective and probiotic cultures may be a useful and effective strategy to prevent or reduce the
incidence of food-borne pathogens in
80
the food chain, improve food product

safety and enhance consumer health.
The benefits of these protective and probiotic cultures can be effected at any
level of the food process chain, from
farm animals to the final food product
(Farm to Fork approach). More specifically, these beneficial bacteria can be
used in the food process chain as protective cultures which reduce or control the
growth of pathogens in the farm environment and in the final food product or
as probiotic cultures which confer a beneficial effect upon the host, either a
farm animal through probiotic animal
feed or the final consumer through a
probiotic food product.
Specific objectives of the scientific research

within WP 10 of the PathogenCombat project were to identify and characterise a collection of bacterial strains which demonstrated the following multifunctional
properties:
Inhibit a variety of pathogenic organisms
common in the food industry (pathogens
targeted for investigation within the project are identified in table 11).
Capable of survival in food processing conditions or the gastric system of animals or
humans (results are reported in table 12).
Safe for consumption by farm animals
or humans.
Table 11. Pathogens selected for investigation within the scope of the
PathogenCombat project.
Pathogen Category
Gram-positive bacteria
Gram-negative bacteria
Yeast
Ochratoxin A producing filamentous fungus
Viruses
Pathogen
Listeria monocytogenes
Mycobacterium avium subsp.
paratuberculosis (MAP)
Campylobacter jejuni, Escherichia coli
Saccharomyces cerevisiae
Penicillium nordicum
Hepatatis E virus (HEV)
Tickborne encephalitis virus (TBEV)
Table 12. Resistance of PathogenCombat strains to stress conditions (low pH and high
temperature).
Strain
Origin
Survival at low pH
(2.5 for 3 hours)
Lactobacillus
pentosus PCA 227
Enterococcus faecium
PCD 71
Unknown
Medium survival
Survival at high
temperature
(55 C for 15 minutes)
Inconclusive results
Sausage
Low survival
High survival
Lactobacillus pentosus
PCD 101
Sausage
(Austria)
Low survival
High survival
81
Table 12. Resistance of PathogenCombat strains to stress conditions (low pH and high
temperature) (continuation).
Lactobacillus plantarum
PCA 236
PCA 263
PCA 275
PCS 20
PCS 22
Lactobacillus gasseri
PCA 185
Lactobacillus fermentum
PCA 144
Lactobacillus fermentum
PCK 129
Lactobacillus delbrueckii
PCK 103
Leuconostoc
pseudomesenteroides
PCK 18
Leuconostoc mesenteroides
PCD 119
Leuconostoc PCK 73
Pediococcus pentosaceus
PCD 215
Pediococcus pentosaceus
PCD 237
PCK 38
PCK 45
PCK 49
Bifidobacterium longum
PCB 133
Bifidobacterium
longum biovar suis
PCD 733B
Enterococcus durans
PCD 103
Kasseri cheese
Xynotyri cheese Medium survival
Feta cheese
Lowmedium survival
Medium-high survival
Cheese
Cheese
Low survival
Feta cheese
High survival
High survival
Kasseri cheese
High survival
Kunun-zaki
(Nigeria)
Salgam
(Turkey)
Maasai milk
(Kenya)
High survival
Lowmedium survival
High survival
Low survival
Pasta filled
with mince
meat
Coffee
fermentation
(Ethiopia)
Ham
Low survival
High survival
Low survival
Medium survival
High survival
Sausage
High survival
Maasai milk
(Kenya)
Maasai milk
(Kenya)
Maasai milk
(Kenya)
New-born
infant
Pig
(International
Culture
Collection)
Sausage
(Austria)
Low survival
Low survival
High survival
Low survival
High survival
Low survival
High survival
Low survival
High survival
82
PathogenCombat protective
and probiotic strains with invitro activity against foodborne pathogens
As a result of the scientific research conducted in WP 10 of the PathogenCombat
project, twenty-three protective and probiotic strains have been identified which
demonstrate in-vitro antimicrobial activity
against the food-borne pathogens Listeria
monocytogenes, Campylobacter jejuni
and/or Penicillium nordicum (see tables
13-17 for details).
Seven strains demonstrated antimicrobial
activity against multiple pathogens (tables
13 and 14). No protective and probiotic

strains have been identified to date which
demonstrate in-vitro antimicrobial activity
against the pathogens Escherichia coli or
Saccharomyces cerevisiae.
Note: strains from the genus Enterococcus
were not selected for further investigation/
feasibility trials in feed or food applications
within the PathogenCombat project.
Although some strains of Enterococcus
faecium show a long history of apparent
safe use in food or feed, Enterococci are
among the leading causes of community
and hospital-acquired infections in humans. The safety concerns related to
Table 13. PathogenCombat strains which demonstrated in-vitro antimicrobial activity

against multiple pathogens (Listeria monocytogenes and Campylobacter jejuni).
Strain
Origin
Leuconostoc
pseudomesenteroides
PCK 18
PCD 71
Maasai milk
(Kenya)
Sausage
Listeria
monocytogenes
antimicrobial activity
Very strong
Campylobacter
jejuni antimicrobial
activity
Moderate
Strong
Moderate
Table 14. PathogenCombat strains which demonstrated in-vitro antimicrobial activity

against multiple pathogens (Campylobacter jejuni and Penicillium nordicum).
Strain
Origin
Lactobacillus pentosus
PCA 227
PCA 236
PCA 263
PCA 275
PCS 20
Unknown
Moderate
Confirmed
Kasseri cheese Moderate
Confirmed
Xynotyri cheese Moderate
Confirmed
Feta cheese
Moderate
Confirmed
Cheese
Moderate
Confirmed
83
these bacteria are further heightened by

the intrinsic resistance of these bacteria
to a variety of antibiotics. In addition,
some species, such as Enterococcus durans and Enterococcus hirae, have been
associated with infections in chickens.
As a result of these safety concerns and
the lack of information on safety, the
European Food Safety Authority (EFSA)
has decided that no members of the
genus Enterococcus can be proposed for
QPS (Qualified Presumption of Safety)
status. This means that the safety of
each Enterococcus spp. strain must be
assessed on a case-by-case basis.
Some protective and probiotic strains
from the PathogenCombat Culture
Collection have been selected for feasibility trials at laboratory scale in specific
feed and food applications. If the laboratory trials are successful, the strains
will be tested at industrial scale in the
final feed or food application at Institutional Partners or food-producing SMEs/

Industrial Partners.
The utilisation of protective and probiotic
cultures from the PathogenCombat
Culture Collection could be implemented
on a broader scale for greater impact in
reducing the incidence of food-borne
pathogens within the food chain and improving food safety within the European
Community.
PathogenCombat protective
and probiotic strains with invitro antimicrobial activity
against select pathogens
Salmonella
This pathogen was not targeted for investigation within the scope of the PathogenCombat project.
Table 15. PathogenCombat protective and probiotic strains which demonstrated invitro antimicrobial activity against Campylobacter jejuni.
Strain
Leuconostoc PCK 73
Bifidobacterium longum PCB 133
Lactobacillus pentosus PCA 227
Enterococcus faecium PCD 71
Lactobacillus plantarum PCA 236
Lactobacillus plantarum PCS 20
Lactobacillus delbrueckii PCK 103
Leuconostoc
pseudomesenteroides PCK 18
Bifidobacterium longum
biovar suis PCD 733B
Enterococcus durans PCD 103
Origin
Coffee fermentation (Ethiopia) Strong
New-born infant
Strong
Unknown
Moderate
Sausage
Moderate
Kasseri cheese
Moderate
Xynotyri cheese
Moderate
Feta cheese
Moderate
Cheese
Moderate
Salgam (Turkey)
Moderate
Maasai milk (Kenya)
Moderate
Pig (International Culture
Collection)
Sausage (Austria)
Moderate
Moderate
84
Campylobacter
A total of twelve strains were identified
which demonstrated in-vitro antimicrobial
activity against Campylobacter jejuni
(table 15).
Bifidobacterium longum PCB 133
Introduction
Isolated from a new-born infant, the protective and probiotic strain Bifidobacterium
longum PCB 133 demonstrated strong activity in vitro against Campylobacter jejuni.
Although resistant to high temperature,
this strain is sensitive to low pH which may
cause survival difficulties during processing
or in the final feed or food application.
Industrial production for
feasibility trials in final applications
The strain Bifidobacterium longum PCB
133 has been successfully produced in
freeze-dried form at industrial scale and
microencapsulated for improved survival
in specific final applications.
Application in poultry feed
The use of probiotic strains could have
application as an additive in feed for livestock poultry against intestinal pathogens in order to reduce the use of
antibiotics and contamination of the
meat.
A study was conducted at the University
of Bologna to evaluate the capacity of
two different orally administered probiotics (Lactobacillus plantarum PCS 20 and
Bifidobacterium longum PCB 133) to colonise the intestinal tract of broiler chickens and to assess their effect on the
Campylobacter jejuni population.
Each probiotic group was administered a

daily dosage of 1-10 million of viable cells
for 15 consecutive days. Results of this experiment demonstrate that only the probiotic strain Bifidobacterium longum PCB
133 colonised the intestinal tract of the
broiler chickens and was detected in the faeces of the treatment group; Lactobacillus
plantarum PCS 20 was not recovered.
Feed studies with the microencapsulated
probiotic culture of Bifidobacterium
longum PCB 133 in poultry are currently
in progress at the University of Bologna.
In addition, another trial is in the planning
stages to evaluate the use of this microencapsulated strain (Bifidobacterium
longum PCB 133) as an animal probiotic
to reduce Campylobacter jejuni contamination in turkey meat products in
Germany.
Application in poultry meat
products
The industrial, freeze-dried probiotic culture of Bifidobacterium longum PCB 133
was sent to the University of Burgos in
Spain for preliminary laboratory trials in
specific poultry meat applications. Upon
successful results of these tests, the strain
will be sent to the Spanish food-producing SME for industrial feasibility trials at
the Cooperativa Avicola y Ganadera de
Burgos.
Application in a functional food (probiotic dairy product)
In collaboration with the dairy SME Pittas
in Cyprus, trials were conducted to develop
a probiotic sheep milk yogurt with the use
of the protective and probiotic strain
Bifidobacterium longum PCB 133.
Although prototypes of the probiotic yo-
85
gurt were successfully produced at laboratory scale, the stability of the strain in the
final product was not acceptable, possibly
related to the strains sensitivity to low pH
as identified in WP10. Efforts are now concentrated on the use of another protective
and probiotic strain (Lactobacillus plantarum PCS 20) in the yogurt product.
Upon successful development and stability trials, production will be scaled up to

evaluate the feasibility of the probiotic yogurt process at industrial scale. Focus
groups will then be conducted to obtain
consumer feedback on the probiotic
sheep milk yogurt produced in these
trials.
Introduction
Introduction
Isolated from cheese, the protective and

probiotic strain Lactobacillus plantarum
PCS 20 showed in-vitro antimicrobial activity against the mould Penicillium nordicum and moderate activity against
Campylobacter jejuni. It is tolerant or
semi-resistant to low pH and high temperature which may facilitate its survival during processing and in the final food or
feed application.
Isolated from cheese, the protective and

probiotic strain Lactobacillus plantarum
PCA 236 is active against Penicillium nordicum and Campylobacter jejuni. In addition, the strain produces a bacteriocin
(Plantaricin EF) and survives well in low pH
and high temperature environments.
Based upon preliminary results, it also inhibits Mycobacterium avium subsp. paratuberculosis (MAP), a resistant pathogen
encountered in milk which can cause diseases in farm animals and has also been
shown to infect humans.
Application in a functional food (probiotic dairy product)

Trials were conducted to develop a probiotic sheep milk yogurt with the use of
Lactobacillus plantarum PCS 20. Pilot batches of yogurt were successfully produced
with this strain co-grown with the starter
culture of Streptococcus thermophilus
(YO8). In addition, the desired results in
terms of stability were achieved as the viability of the strain PCS 20 in the final yogurt product was demonstrated after 35
days at +5 C.
Another development trial is planned to assess the feasibility of producing the probiotic yogurt with Pittas sheep milk and
Lactobacillus plantarum PCS 20 co-grown
with a starter culture of Streptococcus thermophilus in combination with Lactobacillus
bulgaricus.
Application in goat feed

The strain was successfully produced in
freeze-dried form and sent to the
Agricultural University of Athens for a
probiotic feed trial in goats in collaboration with SME Pittas in Cyprus. The industrial produced strain Lactobacillis plantarum PCA 236 survived gastrointestinal
transit in the goat GI tract. In addition, the
population levels of harmful bacteria
(Clostridia) were reduced and lactic acid
bacteria populations (beneficial bacteria)
increased in the goats which were fed the
PCA 236 strain. A statistically significant
increase (13%) in milk production was
also reported in the probiotic group compared to the control. Analysis of the milk
86
(for antioxidant activity) and blood (for

antibodies and antioxidant activity) from
goats treated with the animal probiotic
PCA 236 is pending; results are expected
in July 2009.
Listeria
A total of eight strains were identified
activity against Listeria (table 16).
cessing or in the final feed or food application.

Application in lamb and beef meat
products
The strain Leuconostoc pseudomesenteroides PCK 18 has been successfully produced in freeze-dried form at Laboratory
pilot scale and sent to the University of
Burgos in Spain for preliminary trials in
specific beef and lamb food applications.
Table 16. PathogenCombat protective and probiotic strains which demonstrated invitro antimicrobial activity against Listeria monocytogenes.
Strain
Origin
Leuconostoc pseudomesenteroides
PCK 18
Enterococcus faecium PCK 38
Enterococcus faecium PCD 71
Lactobacillus pentosus PCD 101
Leuconostoc mesenteroides PCD 119
Pediococcus pentosaceus PCD 215
Pediococcus pentosaceus PCD 237
Maasai milk (Kenya)
Listeria
monocytogenes
Very strong
Maasai milk (Kenya)

Maasai milk (Kenya)
Sausage
Sausage (Austria)
Pasta filled with mince meat
Ham
Sausage
Very strong
Very strong
Strong
Strong
Strong
Strong
Strong
Leuconostoc
pseudomesenteroides PCK 18
Introduction
Isolated from maasai milk, the protective
and probiotic strain Leuconostoc pseudo-
Upon successful laboratory trials, the

strain(s) will be produced at industrial
scale and sent to Spanish food-producing
SMEs for industrial feasibility trials in lamb
products at Colear Castilla and/or in beef
products at Martinez Loriente, S.A.
mesenteroides PCK 18 demonstrated very
Lactobacillus pentosus PCD 101
strong in-vitro antimicrobial activity
Introduction
against Listeria and moderate activity

against Campylobacter jejuni. However,
the strain is sensitive to low pH which
may cause survival difficulties during pro-
Isolated from sausage, the protective and

probiotic strain Lactobacillus pentosus
PCD 101 showed strong antimicrobial activity against Listeria. Although resistant
87
to high temperature, this strain is sensitive to low pH which may cause survival
difficulties during processing or in the
final feed or food application.
Application in beef and lamb meat
products
The strain Lactobacillus pentosus PCD
101 has been successfully produced in
freeze-dried form at laboratory pilot
scale and sent to the University of
Burgos in Spain for preliminary trials in
specific beef and lamb food applications. Upon successful laboratory trials,
the strain(s) will be produced at industrial scale and sent to Spanish food-producing SMEs for industrial feasibility
trials in lamb products at Colear Castilla
and/or in beef products at Martinez
Loriente, S.A.
A total of ten strains were identified
activity against Penicillium nordicum (table
17).
Escherichia coli
No protective and probiotic strains have
been identified to date which demonstrate in-vitro antimicrobial activity against
the pathogen Escherichia coli.
Glossary
Disease: any condition caused by the presence of an invading organism or a toxic
component (i.e. pathogen) which causes
damage to the host.
Pathogen: organisms, frequently microorganisms or components of these organisms which cause disease or illness.
Microbial pathogens include various species of bacteria (e.g. the bacterium
Mycobacterium tuberculosis causes tuberculosis), viruses (e.g. pathogenic viruses
cause smallpox, influenza, mumps, measles, chickenpox and rubella), protozoa
(e.g. malaria), and fungi (e.g. infections
due to Candida and Cryptococcus).
Probiotic: live micro-organisms which
when administered in adequate amounts
confer a health benefit on the host.
Table 17. PathogenCombat protective and probiotic strains which demonstrated invitro antimicrobial activity against Penicillium nordicum.
Strain
Lactobacillus pentosus PCA 227
Lactobacillus gasseri PCA 185
Lactobacillus fermentum PCA 144
Lactobacillus fermentum PCK 129
Origin
Unknown
Kasseri cheese
Xynotyri cheese
Feta cheese
Cheese
Cheese
Feta cheese
Kasseri cheese
Kunun-zaki (Nigeria)
Maasai milk (Kenya)
88
Probiotic culture: culture of probiotic

micro-organisms which confers a beneficial
effect upon the host, either a farm animal
through probiotic animal feed or the final
consumer by a probiotic food product.
lected microorganisms referred to EFSA, Opinion

of the Scientific Committee.The EFSA Journal
2007; 587:1-16.
Protective culture: culture of micro-organisms which reduces or controls the growth

of pathogenic micro-organisms in the farm
environment and/or the final food product.
Lan PT, Binh LT, Benno Y. Impact of two probiotic

Lactobacillus strains feeding on fecal lactobacilli
and weight gains in chicken. J. Gen. Appl.
Microbiol. 2003; 49:29-36.
Zoonosis: also called zoonose or zoonotic

disease, this term refers to a disease
which can be transmitted from animals
(wild or domestic) to humans.
Lawson AJ, Shafi MS, Pathak K, Stanley J.

Detection of Campylobacter in gastroenteritis:
comparison of direct PCR assay of faecal samples with selective culture. Epidemiology and
Infection, 1998; 121:547-53.
Acknowledgements
We grately acknowledge funding from financial participation by the European
Community under the Sixth Framework
Programme for research, technological development and demonstration activities for
the Integrated Project PathogenCombat
Food-CT-2005-007081.
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The application of PathogenCombat

research results in practice
F. Gagga, B. Biavati
The overall objective of Pathogen

Combat is to provide new essential information and methods to the food industry and public authorities on how to
reduce the prevalence of new and emerging food borne pathogens
The aim of the Work Package 13
Application in the Food Chain is related
to the application of the knowledge and
tools produced within the project and to
the development of support measures to
food industries. In particular, research activities and results obtained from different
Partners in several WPs (WP 4, 5, 6, 10,
11, 12, 14 and 15) are made available to
be disseminated and tested into SMEs
and industrial partners. The dissemination
and transfer into practical application in
this WP comprise the food processing
SME and Industrial partners of Pathogen
Combat, belonging to dairy, ruminant,
poultry and pork food chain (table 1).
Considering food safety, the combat of
pathogens is of outmost importance to
assure quality products to the consumer.
To approach the omnipresent threats from

new and emerging pathogens, the
European project PathogenCombat,
aims, first of all, at increasing knowledge
on the factors, which enable the viability,
persistence and virulence of pathogens in
the food chain, in order to reduce their incidence.
Despite significant investment, the incidence of food derived disease is still too
high in the EU. Besides being of fundamental importance to the consumer, fighting pathogens is also of imperative significance to the food industry and
economy, as impact on trade and competitiveness is substantial.
The project deals with eight pathogens:
Listeria monocytogenes, Mycobacterium
avium subsp. paratuberculosis, Campylobacter
jejuni and shiga-like toxin producing
Escherichia coli (STEC), invasive variants
of Saccharomyces cerevisia, ochratoxin A
producing filamentous fungus (Penicillium
nordicum), hepatitis E virus (HEV) and
tickborne encephalitis virus (TBEV); pre-
Table 1. List of SMEs and Industrial partner participating to Pathogen Combat.

Cheese and Milk
Partner 27 GRAN-I
Partner 28 PITTAS-G
Partner 31 BERG-D
Poultry
Partner 29 CAGB-E
Partner 33 ZIEGLER-D
Pork
Partner 29 CAGB-E
Partner 34 JAMSA-E
Beef and Lamb

Partner 46 COLEAR-E
Partner 47 MARLOSA-E
90
diction and detection of Staphylococcus

aureus enterotoxins are also included.
SMEs and Industrial partners involved in
the project, along the last three years of
project, aim to apply and improve:
New detection methods and prediction
of the occurrence and virulence of pathogens in the food chain and at time of
consumption with molecular biology
based on culture independent techniques and microarrays.
New processing technologies and systems, new hygienic design, protective
and probiotic cultures and new information on host-pathogen interaction to
prevent pathogen transmission along
the food chain.
New mathematical models for pathogen control throughout the food
chain and at time of consumption.
Their Food Safety Management System
preventing microbial food borne diseases.
In this paper, we will describe some important applications that have been developed within the project, concerning
new culture-independent detection methods based on molecular biology, and applied in practice inside SMEs and Industrial
partners plants.
The quality control and monitoring along
the food chains is necessary to ensure
safe end products; mainly food industry
rely on international procedures based
on culturing methods to estimate the
presence, the viability and enumeration
of pathogens throughout the food chain
taking into account the large amount of
time required to achieve the final results.
Since producers, processing industry and

consumers increasingly demand higher
quality for starting material and food
products, there is a rising interest for
rapid, sensitive and specific diagnostic
methods.
The aim of WP4 within PathogenCombat
is the development and the optimization
of culture independent methods (e.g FISH
technique, PCR-DGGE, qPCR and RT-qPCR)
for the identification of microbial populations in food systems without the need of
traditional isolation and plating counts, taking into consideration both the definition
of the microbial ecology of foodstuffs and
the detection of food-borne pathogens.
The Fluorescence in Situ Hybridization
(FISH) technique has been optimized in
WP4 in order to produce experimental protocols to be used in WP13 for a direct profiling of the microbial populations present
in a specific food ecosystem or in samples
from critical points along the food chain.
The developed protocols were applied on
samples collected from project partners in
particular from a dairy company for the detection of viable bacteria and the presence
of different pathogenic bacteria, like
Listeria monocytogenes, or hygiene indicator microorganisms, like coliform bacteria and yeasts. Mainly process water, biofilms and cheese samples were sent and
analysed. The results obtained allowed to
point out that, by using the adapted FISH
protocols on process samples, useful information for the producers in relation to the
bacterial community composition (e.g.
high speed detection of pathogens) and
on the hygienic status of the production
process could be obtained.
The application of PathogenCombat research results in practice
91
Furthermore, the hands-on time for the

analyses requires only few minutes and
results are available within 3 hours (only
a short pre-enrichment is required) leading to a time-saving of 3 to 5 days when
compared with conventional methods.
The WP4 is interested also in the development of quantitative PCR (qPCR), for detection, quantification and typing of pathogens such as L. monocytogenes, E. coli
STEC and C. jejuni in food matrices.
Of the identified hazards in Pathogen
Combat relevant to dairy industry operations, Listeria monocytogenes has the greatest attention and placed surveillance; it
can survive and multiply under conditions
frequently used for food preservation; this
makes L. monocytogenes problematic to
the food industry. The University of Torino
(Italy) has optimized a protocol for the detection and quantification of L. monocytogenes that has been applied in a dairy
company within the project. A total of 150
samples (cheese during and at the end of
the production, swabs and brines) were collected for three consecutive weeks; the
analyses were carried out by molecular and
traditional methods. Some cheese and environmental samples were found to be positive for L. monocytogenes; in some samples a slight discrepancy in the results
obtained with the two methods has been
detected (17%). Following these results an
inspection and check were performed on
the continuous flow pasteurizer (and its
cleaning system) of liquid brines: a failure
was detected and then a corrective action
was performed.
A deeper look and investigation on the
applicability of the new methods is obviously necessary to obtain repeatability,
reproducibility and a validated methodology; however this kind of application represents a real advantage for the food
producing companies, because in a relatively short time (about 18-24 h) they can
obtain information on the presence of
pathogens in their plants and take appropriate preventing measures. The evaluation of the risk, its prevention and/or its
solving could be deal within a reasonable
time offering to the personnel precious
days to act and take decisions.
Some specific works and studies were initiated also from two Spanish Companies,
partners of Pathogen Combat, to approach and develop new culture-independent techniques in order to analyze different steps of the poultry, pork and
ruminants food chain.
Important results have been obtained in
a Spanish SME; samples of pork loin
cured were analysed after a treatment
with High Pressure. The analyses were
performed both by conventional microbiology using the ISO procedures for the
detection of L. monocytogenes (ISO
11290-1:1996) and culture independent
method, specifically by Real Time PCR
(with a protocol developed within the
Pathogen Combat research).
A good correspondence between the two
methods was detected and also in this
case a reduction in the analyses time (1
day against 5 days with traditional methods) was observed.
In addition to the important applications
described above, the analysis of the water
supply systems of two German SMEs and
two Spanish SMEs has been completed
using molecular biology, and non-culturing methods (DNA extraction, conven-
92
tional and seminested PCR and TaqMan

q-PCR), for the direct detection of pathogenic microorganisms.
Molecular biological methods (PCR-DGGE)
for the analysis of the bacterial population
composition and stability at the different
water sampling points of the food industry
facilities have been also applied. The protocols have been optimised for the detection
of the following pathogens: L. monocytogenes, M. avium subsp. paratuberculosis,
C. jejuni, Enterococcus ssp, Salmonella ssp,
E.coli and P. aeruginosa. Several litres of
water from each sampling point were taken
and the original present bacteria were concentrated by filtration, and directly used for
molecular biology analyses. Furthermore a
questionnaire on drinking water distribution has been prepared for the SMEs in
order to achieve a complete evaluation of
the water supply system inside these SMEs.
Finally partners of WP5 are working in
the development and validation of robust
microarrays to monitor the existence and
expression of the involved genetic factors
for virulence and pathogenicity for
Listeria, Campylobacter and E.coli. The
basic procedure include: a) samples collection and concentration; b) nucleic
acid purification and labelling; c) hybridization to array; d) data capture and interpretation.
The arrays will be test and used in collaboration with the SME and industrial food
producers in WP 13 on cheese and meat
samples from the processing lines.
The steps to develop rapid and reliable detection methods based on culture independent techniques require time and efforts
(optimization of the primers and probes,
nucleic acid extraction) and should be cost
effective, ensuring high repeatability, reproducibility and a good training of the personnel performing the test. Moreover, the
protocols need to be optimized to meet
every requirements taking into account the
context, specific for each company.
Therefore a strict collaboration between
partners, taking into charge the development of the new protocols, and the companies participating to the Project has been
achieved. In that sense PathogenCombat
created a direct line among partners for the
rapid information exchange, assistance and
dissemination of the knowledge.
It is a good opportunity for SMEs and
Industrial partner to get in touch with a
new scientific approach made available by
universities or private companies with the
necessary equipments and laboratories facilities; a valid contribution to the improvement of Food Safety has been performing with the aim to transfer and
disseminate the new knowledge in the
European context.
Bribliography
Van Amerongen A, Barug D, Lauwaars M.
Rapid Methods. Wageningen Academic
publishers. 2005.
Annual Report PathogenCombat. 2006.
Roberts A, Cai S, Wiedmann M. The contribution of actA to virulence differences
among Listeria monocytogenes strains.
103rd Annual Meeting of the American
Society for Microbiology, Washington,
DC. 2003.
EHEDG: mtodo para la comprobacin de

la evaluacin de la limpieza in situ de los
equipos para el procesado de los alimentos
M. I. Llorca
Introduccin
La inocuidad alimentaria es un objetivo
comn al sector agroalimentario y una
clara exigencia y preocupacin de los consumidores. Para conseguirlo la industria
alimentaria debe cumplir con una serie de
medidas y controles que garanticen la obtencin de alimentos inocuos.
El mantenimiento de un elevado nivel de
limpieza de los equipos y de las instalaciones y del entorno de trabajo, afecta de
forma directa sobre la inocuidad del producto final. Para conseguirlo no slo
deben ser regularmente limpiados y desinfectados sino que su diseo inicial debe
facilitar la realizacin de estas operaciones
eficazmente as como garantizar que
tanto las instalaciones como los equipos
por su diseo no se conviertan en foco de
contaminacin de los alimentos.
El diseo de un equipo o instalacin se considera higinico si incorpora, con carcter
preventivo, caractersticas que reducen o
eliminan el riesgo de constituir una fuente
de contaminacin para los alimentos, tanto
de forma directa como indirecta.
Por ejemplo, gracias a un adecuado diseo
higinico es posible garantizar que un
equipo o instalacin determinada no transfiere ningn cuerpo extrao, sustancia qu-
mica, ni microorganismo. Para ello, en el

mbito del diseo higinico de equipos e
instalaciones se consideran factores tales
como los materiales de construccin, superficies de contacto, drenabilidad, hermeticidad, accesibilidad, entre otros muchos.
Errores o deficiencias en el diseo higinico de instalaciones y de equipos pueden
hacer fracasar o dificultar la obtencin de
alimentos inocuos, aumentando y/o dificultando las tareas de mantenimiento,
limpieza, desinfeccin, control de plagas
y control del proceso fundamentales y necesarias para asegurar unas condiciones
de produccin adecuadas.
Por lo tanto, para conseguir equipos e instalaciones higinicas stas deben disearse
y construirse cumpliendo requisitos higinicos. Pero aun as en algunos casos por razones funcionales no es posible cumplir totalmente con estos requisitos y se hace
necesario disponer de mtodos o ensayos
que nos permitan comprobar que aun as
el equipo o instalacin es limpiable o esterilizable, segn exigencias de uso.
En el apartado 3 del presente artculo se
describe el mtodo de EHEDG para evaluar la limpieza CIP de equipos pequeos.
En esta sentido AINIA, centro tecnolgico
desde su creacin, viene desarrollando actuaciones de formacin, investigacin,
94
anlisis y apoyo tcnico al sector en el mbito de la inocuidad alimentaria.

Entre estas actividades sealamos las siguientes lneas de actuacin:
Desarrollo de nuevas tecnologas para
la mejora y control de la seguridad alimentaria en las industrias del sector.
Investigacin y mejora de la higiene as
como del impacto medio ambiental asociado. Estudio de tcnicas de limpieza y
desinfeccin.
Servicios analticos acreditados por
ENAC que permitan la deteccin de
contaminantes en los alimentos.
Diseo higinico y limipiabilidad (diseo
y puesta a punto de mtodos).
AINIA es miembro y sede regional del
Grupo Europeo de Ingeniera y Diseo
Higinico, EHEDG, en Espaa.
EHEDG es el referente europeo en diseo
higinico, en el siguiente apartado del presente artculo se procede a su descripcin.
AINIA, como sede regional de EHEDG, desarrolla diversas acciones para fomentar en
Espaa la adopcin de criterios de higiene
en la proyeccin, ampliacin y reformas de
las industrias alimentarias, as como en el
diseo de maquinaria y equipos.
EHEDG tiene vnculos fuertes con varias

organizaciones internacionales y tiene
entre sus planes buscar relaciones globales adicionales.
Como la seguridad alimentaria no termina en los lmites de Europa, EHEDG
promueve activamente la armonizacin
de las diferentes guas y normas. Las organizaciones americanas NSF y 3-A, estn
de acuerdo en cooperar con EHEDG en el
desarrollo de sus guas y, por su parte,
EHEDG colabora en el desarrollo de las
normas 3-A y NSF.
EHEDG proporciona asesoramiento en aspectos de ingeniera higinica para la elaboracin de alimentos inocuos.
Organizacin interna
Presidente
Comit ejecutivo
Fundacin
Miembros
Subgrupos
Secciones
Regionales
Organismo
Certificacin
Qu es EHEDG
EHEDG (European Hygienic Engineering
and Design Group) es un consorcio europeo de fabricantes de equipos, industrias alimentarias, institutos de investigacin y autoridades pblicas, fundado en
1989 con el objeto de promover la higiene durante el procesado y envasado de
alimentos.
El Presidente es elegido por el Grupo

Principal para un periodo de tres aos. Su
funcin es mantener la organizacin
unida, representar formalmente a EHEDG,
y presidir la Asamblea General.
El Grupo Principal, compuesto por el
Comit Ejecutivo y miembros en general,
dirige EHEDG y revisa y aprueba las guas
EHEDG: mtodo para la comprobacin de la evaluacin de la limpieza in situ de los equipos
95
previo a su publicacin. Se espera que en

el conjunto de miembros haya una representacin equilibrada de fabricantes de
equipos, industria agroalimentaria, organizaciones de investigacin y legisladores. En el Grupo Principal hay representantes de 3-A y de NSF International,
por lo que existe una cooperacin
formal. Adems, el Grupo Principal est
representado en 3-A y NSF International.
Cuando es oportuno, se invita a representantes de otras organizaciones con
objetivos similares.
El Comit Ejecutivo ejecuta las decisiones del Grupo Principal y se hace
cargo de las tareas cotidianas de
EHEDG. Est formado por el Presidente,
el Secretario General, el Tesorero y los
miembros. Los miembros del Comit
Ejecutivo tienen funciones de coordinacin especficas y tareas relacionadas:
coordinacin de subgrupos, coordinacin de las Secciones Regionales, coordinacin de estndares, coordinacin de
los cuerpos gubernamentales, coordinacin de certificacin y coordinacin de
eventos. Los coordinadores de los subgrupos hacen un seguimiento e informan sobre el progreso de los trabajos
de cada subgrupo. El Comit Ejecutivo
se rene cuatro veces al ao.
Los miembros de EHEDG trabajan principalmente para:
Incrementar los conocimientos sobre higiene de los alimentos.
Ayudar en la prevencin de problemas
de seguridad alimentaria.
Sostener de esta manera la imagen de
la industria alimentaria entre los consumidores.
Las principales tareas de la Fundacin son

salvaguardar el uso del logo EHEDG y supervisar el trabajo de las organizaciones
autorizadas para realizar las pruebas de
evaluacin. Publican las guas y materiales
de formacin, controlan la pgina web,
organizan reuniones y conferencias.
Las principales funciones de las Secciones
Regionales son la traduccin de las guas
al idioma nacional, informar en su mbito
de influencia de las actividades internacionales de EHEDG, realizacin de seminarios, talleres sobre diseo higinico, fomentar la transferencia de conocimiento
entre los miembros, promulgar el diseo
higinico.
Todos los miembros y patrocinadores
estn invitados a participar en la
Asamblea General durante la conferencia
que se realiza una vez cada dos aos.
Objetivos
Proporcionar asesoramiento en aspectos
de ingeniera higinica para la elaboracin de alimentos inocuos.
Ofrecer un forum o punto de encuentro
a los fabricantes de equipos, la industria
alimentaria, usuarios y reguladores para
tratar aspectos de diseo higinico y fomentar la obtencin de alimentos seguros.
Proporcionar documentos gua sobre
las prcticas y normas esenciales de diseo higinico, basadas en ciencia y
tecnologa, y revisarlas peridicamente. Estas guas ofrecen asesoramiento a fabricantes de equipos y
usuarios sobre cmo cumplir con la legislacin nacional e internacional.
96
Desarrollar mtodos de comprobacin

que puedan ser utilizados por terceras
partes para la evaluacin del diseo higinico conforme a la legislacin.
Asegurar que el uso del nombre y logo
de EHEDG se controlan convenientemente.
Identificar reas donde el conocimiento
del diseo higinico es insuficiente y fomentar la investigacin y el desarrollo
en estas reas.
mbito de trabajo
El mbito principal de trabajo de EHEDG
se centra en la divulgacin de la ingeniera
higinica en el sector agroalimentario y
fabricantes de equipos. Para ello trabaja
Grupo de trabajo
Equipos y componentes.
Contact: J. Kastelein
Principios.
Contact: J. Hofmann
Procesado y servicios/instalaciones auxiliares.

Contact: M. Stringer
Formacin y educacin.
Contact: K. Lorenzen
en el establecimiento de requisitos tcnicos de diseo higinicos, as como en el

asesoramiento y divulgacin del diseo higinico y limpiabilidad de equipos e instalaciones agroalimentarias.
Para ello en la actualidad existen 25 subgrupos de trabajo que se organizan en los
siguientes cuatro grupos de trabajo:
Adems de las actividades de formacin
y divulgacin de la ingeniera higinica a
continuacin se describen otras actividades que realiza EHEDG:
Elaboracin, actualizacin y publicacin
de guas: EHEDG promueve la prevencin
a travs de la elaboracin de guas basadas en la combinacin de los conocimientos de sus miembros y los datos cien-
Alcance
Refrigeracin y enfriamiento
Equipos para el procesado de carne/pescado
Juntas mecnicas
Envasadoras
Tubera y conexiones
Bombas
Sensores
Vlvulas
Diseo de instalaciones
Principios de diseo higinico
Manipulado de alimentos secos o deshidratados
Integracin sistemas de higiene
Materiales de construccin
Mtodo de ensayos/certificacin
Soldaduras
Manejo del aire
Instalaciones elctricas
Tratamientos de calor
Lubricantes
Agua de procesor
Facilitador
Toolbox
Formacin
97
tficos disponibles. En la actualidad existen

32 guas.
Certificacin de equipos: se dispone de un
procedimiento tcnico para la certificacin
del diseo higinico de equipos. El esquema de certificacin de equipos de
EHEDG consiste en una evaluacin tcnica
del diseo higinico del equipo seguido
de la comprobacin de la facilidad de limpieza, limpiabilidad. Para ello los expertos primero realizan una comprobacin
del cumplimiento de los requisitos de diseo higinico. Una vez superada esta
evaluacin se procede a la realizacin de
los ensayos oportunos para comprobar la
facilidad de su limpieza. Estos ensayos slo
pueden realizarse por los organismos autorizados por EHEDG (TNO, DTI, CCFRA,
Universidad de Munich).
Ventajas de convertirse en miembro
EHEDG
Las empresas miembros de EHEDG se
comprometen con los ms altos estndares de seguridad alimentaria en sus empresas y se esfuerzan en mejorar la
imagen global de la industria que los consumidores tienen. El uso del logo de
EHEDG como patrocinador es una
muestra de este compromiso.
A travs de la red de nuestra organizacin,
las empresas miembro pueden promocionar su visin, dado que apoyan los objetivos EHEDG. Las empresas miembro
pueden marcar tendencias y obtener reconocimiento internacional de sus esfuerzos.
La contribucin anual de cada empresa
miembro depende de su facturacin relacionada con el negocio alimentario. Todos
los miembros (individuales y empresas)
estn invitados a la Asamblea General

que se realiza cada dos aos. Las contribuciones se basan en la facturacin anual
de la empresa relacionada con el negocio
de la alimentacin y oscilan entre 500 y
10.000 . En el caso de miembros individuales la cuota es de 100 .
Otras ventajas:
Uso del logo de EHEDG registrado para
empresas en la documentacin de la empresa, tras la firma de un contrato, en el
que se especifican las condiciones de uso.
Por ejemplo, las empresas fabricantes de
equipos no pueden usar ese logo para indicar que un producto est certificado.
Emplear el logo o nombre de EHEDG en
programas de cursos, folletos, etc.
Obtener una copia gratuita de las guas
en CD-ROM para empresas miembros y
con un 50% de descuento para miembros individuales.
Ser componentes de hasta dos subgrupos de trabajo.
Enlace desde la pgina web de EHEDG.
Mtodo EHEDG para

comprobar la limpiabilidad CIP
de equipos para el procesado
de los alimentos
En el presente apartado se describe el mtodo de EHEDG para comprobar la limpiabilidad CIP de equipos moderadamente
pequeos descrito en el documento o
gua n 2 de EHEDG.
EHEDG dispone de un esquema de certificacin del diseo higinico de
equipos para equipos cerrados que re-
98
quieran limpieza CIP y sobre los que

existan dudas o no pueda demostrarse
el cumplimiento total de los requisitos
de higiene por un experto autorizado.
Se debe someter a dicho ensayo de
comprobacin de la limpiabilidad.
El procedimiento de ensayo est diseado
para indicar las zonas con un diseo higinico deficiente en los equipos y est basado
en una comparacin de la limpiabilidad de
un elemento sometido a ensayo con la de
un trozo de tubera recto, o tubo de referencia. El grado de limpieza se basa en la
eliminacin de una solucin ensuciadora
que contiene bacterias y se valora evaluando el crecimiento de las bacterias que
quedan despus de la limpieza.
El ensayo est pensado, por lo tanto,
como ensayo bsico de comprobacin del
diseo de equipos higinicos y no es indicativo del comportamiento en situaciones
de limpieza industrial.
Mtodo de ensayo
El equipo o elemento que se va a ensayar
con objeto de comprobar su limpiabilidad
y por tanto su diseo higinico, junto con
la tubera de referencia se someten a un
proceso de ensuciamiento y posteriormente a su limpieza.
Para su ensuciamiento se utiliza leche
agria inoculada con GeoBacillus stearothermophilus ya que es de crecimiento rpido, tiene esporas que son resistentes a
la solucin detergente que se usa en el
procedimiento de ensayo y produce reacciones de color bien definidas en el medio
de crecimiento utilizado.
El agar utilizado es MSHA Shapton y
Hindes modificado, que con la presencia
del Geobacillus provoca un cambio en el
pH y el agar vira de color morado a color

amarillo siendo muy fcil su deteccin.
El equipo a ensayar se llena con la solucin ensuciadora y se presuriza tres veces
a 5 bares durante 2 minutos. Mientras
estn sometidos a la presin, las partes
mviles que existan se accionan para simular las condiciones de empleo (por
ejemplo, se abren y se cierran las vlvulas).
A continuacin se vaca y se seca con aire
seco filtrado, se requiere un valor final de
HR < 5% a 15-25 C.
La seccin de ensayo ensuciada se monta
en un banco de pruebas CIP construido a
propsito (ver esquema), para proceder a
su limpieza:
Aclarar con agua fra (10-20 C) durante
un tiempo igual al tiempo medio de residencia (t) y no inferior a 1 minuto.
Hacer circular una solucin detergente al
1,0% (p/v) a 63 C 2 C durante 10 minutos (el volumen de solucin detergente
usado debe ser al menos 20 veces el volumen interior del elemento sometido a
ensayo).
Aclarar con agua fra (10-20 C) durante
un tiempo igual al tiempo medio de residencia (t) y no inferior a 1 minuto.
Las soluciones de limpieza se hacen circular
a una velocidad de flujo media de 1,5 m
s-1 0,1 m s-1 dentro del tubo de referencia
y se debe mantener una presin hidrosttica positiva en las tuberas de retorno para
evitar burbujas de aire en el sistema.
Tras el proceso de limpieza se retira el
equipo y la tubera de referencia del banco
de pruebas y se procede a la evaluacin de
su limpiabilidad para ello se procede a rellara tanto el equipo como la tubera con
99
TEST
ITEM
vlvula de
derivacin
tubo de referencia
TI
agua de
aclarado
solucin
detergente
FI
vlvula de
regulacin
de caudal
PI
bomba
centrfuga
FI indicador de caudal
PI indicador de presin
TI indicador de temperatura
Esquema del banco de pruebas CIP para el ensayo de limpiabilidad.
agar MSHA Despus de que el agar se haya

solidificado completamente, se coloca casi
vertical en una estufa de cultivo a 58C durante 16-24 horas.
Para facilitar la diferenciacin entre el

color amarillo y el prpura y estandarizar
los resultados de ensayo realizados por diferentes operarios, se dispone de un disco
comparador de colores.
Evaluacin de resultados
El porcentaje de zona amarilla 5-30%

sirve como control de la variabilidad tanto
intra como interlaboratorios.
Despus de la incubacin, el equipo de ensayo y del tubo de referencia se examinan

para buscar la presencia de zonas de coloracin amarilla en el MSHA prpura.
El tubo de referencia se abre y se extrae
el agar solidificado. Para ello, con una herramienta especial, se extrae el ncleo
central del agar. Luego se abre el tubo de
agar que se ha formado convirtindolo en
una lmina plana y se coloca sobre una
cuadrcula de recuento transparente.
Es preferible utilizar una cuadrcula que
tenga divisiones de 5 mm x 5 mm, lo
que facilitar la evaluacin de las zonas
amarillas.
Por lo general, y asumiendo que existe

una pequea cantidad de color amarillo
en el tubo de referencia, son posibles tres
resultados para el equipo o elemento sometido a ensayo:
Presencia de residuos de leche: si hay presencia de residuos visibles de leche
cuando se observa/desmonta el elemento
sometido a ensayo antes de que se
aplique el agar, no es necesario realizar
ningn examen microbiolgico, ya que
las esporas estn claramente presentes
por lo que presenta graves defectos en el
diseo. Sin embargo, se requiere un an-
100
lisis microbiolgico del tubo de referencia

para confirmar que el ensayo fue adecuado. Se debe repetir el ensayo.
ms limpiable que la tuberia de referencia, el equipo sometido a ensayo es

apto para certificar su diseo higinico.
En pruebas comparativas de equipos, la
superficie relativa de zonas amarillas que
permanece en cada una de las piezas del
equipo ensayado se puede usar como
medida de su limpiabilidad. La interpretacin de las comparaciones de limpiabilidad es slo aproximada, ya que el grado
de color amarillo puede estar influido por
el espesor del agar sobre la superficie del
elemento sometido a ensayo.
Presencia de zonas y/o colonias amarillas:

se debe repetir el procedimiento de ensayo hasta un mximo de cinco veces. La
presencia de suciedad retenida en la
misma zona del elemento sometido a ensayo en tres ocasiones distintas es indicativa de zonas que son de difcil limpieza
y, por consiguiente, zonas en las que se
deben considerar mejoras en el diseo higinico. La limpiabilidad del elemento sometido a ensayo se compara con la del
tubo de referencia evaluando las superficies porcentuales correspondientes de
zonas amarillas:
Si la superficie porcentual de zonas
amarillas en el elemento sometido a ensayo es similar a la del tubo de referencia, el grado de limpiabilidad es, por
consiguiente, similar.
Si la superficie porcentual de zonas amarillas en el elemento sometido a ensayo
es inferior o superior a la del tubo de referencia, el elemento sometido a ensayo
es, respectivamente, ms o menos fcil
de limpiar. En el caso de que sea igual o
Sin zonas/colonias amarillas: si estas condiciones se dan en tres ocasiones sucesivas, no es necesario repetir ms el ensayo y el elemento sometido a ensayo se
puede describir como especialmente fcil
de limpiar o limpiable.
En ocasiones algunos materiales empleados
en juntas de estanquidad pueden tener
propiedades antibacterianas que impidan
que las esporas presentes en su superficie
germinen y/o crezcan. As, zonas con diseo higinico deficiente pueden no aparecer como zonas amarillas, lo que da lugar
a resultados falsamente negativos.
Para conocerlo, se deben comprobar las
propiedades antibacterianas de todas las
juntas tricas y juntas de estanquidad, para
ello se inocula con esporas un volumen
adecuado de MSHA (aproximadamente 102
ml-1 de agar). A continuacin se colocan las
juntas de estanquidad/juntas tricas estriles en placas petri adecuadas, se cubren
con el MSHA fundido y se incuban a 58 C
durante 16-24 horas. Si las juntas de estanquidad/juntas tricas tienen propiedades
antibacterianas, se ve una zona prpura a
su alrededor.
101
Centros autorizados por EHEDG para

la realizacin del test de limpiablidad
cidos por EHEDG para convertirse en

centro autorizado:
En la actualidad EHEDG dispone de un

total de cinco centros autorizados para la
realizacin de las pruebas de limpiabilidad
y para la emisin de informes favorables
necesarios para la certificacin del diseo
higinico de equipos:
Ser miembro del subgrupo de trabajo

de EHEDG de Mtodos de Ensayo, y
participar en las reuniones de trabajo.
Dinamarca: DTI.
Alemania: Universidad de Munich.
Holanda: TNO.
Reino Unido: Campden BRI.
EE.UU.: Universidad de Pardue.
Es objetivo de AINIA convertirse en el
sexto centro autorizado. Actualmente
AINIA cumple con los requisitos establecidos internamente por EHEDG y slo
tiene pendiente de conseguir la acreditacin ISO 17025 del mtodo. Est previsto
conseguir esta acreditacin en el segundo
semestre de 2009.
A continuacin y a modo de resumen se
indican los principales requisitos estable-
Disponer de un plan de negocio con el

compromiso formal de actuar como instituto autorizado (disponer de instalaciones, cualificacin del personal, mantenimiento preventivo de instalaciones
y equipos).
Disponer de personal con formacin y
experiencia demostrable en el rea de
higiene.
El personal responsable del mtodo
debe realzar el curso avanzado de diseo higinico de EHEDG.
Recibir formacin y experiencia previa
de la mano de otro centro o experto autorizado.
Obtener la acreditacin ISO 17025 del
mtodo de ensayo.
Participar en cualquier actividad propuesta por el grupo de trabajo as como
en la pruebas interlaboratorio.
New cleaning and disinfection methods

and summary of methods applied for
verification of their efficiency
H. Lje, A. Friis, L. Gram, B. Carpentier, J. Verran
Introduction
Cleaning and disinfection procedures
have to be considered as an integral part
of food production. If the cleaning is not
effective, micro-organisms and product
residues will remains at concentrations
that may affect the quality and safety of
the food product (Gibson et al, 1999).
The attachment of bacteria, with possible
subsequent development of biofilm in
food processing environments, is a potential source of contamination of finished
product that may shorten the shelf-life or
encourage transmission of diseases
(Sharma and Anand, 2002).
Biofilm are communities of microorganisms
on surfaces, typically encased in some
polymer matrix that has been produced by
the microorganism themselves. Microor-ganisms attached as in biofilms exhibit different properties to microorganisms that float
around in liquid: they are more resistant to
antibiotics and biocides and are more difficult to remove from the surface. In the food
processing environment, particularly within
closed systems, conditions favour attachment and biofilm formation, i.e. flowing
water, suitable attachment surfaces, ample
nutrients and raw material (Gibson et al,
1999). Furthermore the formation of biofilm protects bacteria from hostile conditions
and thus these bacteria are much more resistant to detergents and disinfectants on
open surfaces (Notermanns, 1994). The
choice of cleaning and disinfection product
is also highly dependent on the food matrix
in which the organism is embedded (Gram
et al, 2007; Verran et al, 2008).
Cleaning should both remove soil and reduce the number of microorganisms present. The disinfection should further reduce
the surface population of viable microorganisms via removal or destruction, and/or to
prevent surface microbial growth during
the inter-production period (Holah, 2003).
In 2003 Holah gave a review of new cleaning agents and cleaning methods available
at that time. This chapter is a further description of the new cleaning agents and
methods. In addition the newest results
from WP11 partners in PathogenCombat
are included.
Cleaning and disinfection:

quantitative assessment
Methods for differentiating soil and
microorganisms
Methods for differentiating soil (e.g. meat
or dairy soil) and microorganisms in open
systems have been developed by
104
Manchester Metropolitan University (MMU)

(Verran et al, 2008). The aim of this work
was to recommend a specific and simple
method for fouling and cleanability assays
to industry, particularly SMEs, that is relevant to their product, and whose results are
supported by more sophisticated analytical
techniques.
A large experiment involved soiling of surfaces with key components of dairy and
meat industry food soils (eg oils, carbohydrates, proteins) at different concentrations,
and then using a range of analytical and
detection methods to compare ease of use,
sensitivity, and relationship with one
another. Two commercially available kits,
UV light detection method and ATP
Bioluminescence, were included in the
study. Briefly, UV light detection method (eg
Bactoforce) provides a convenient, macroscopic indication of poor hygiene (general
fouling), but does not differentiate between
cells and soil. Some problems were encountered with the use of ATP hygiene testing,
in terms of the efficiency of removal of
soil/cells from the surface, but this method
generally provided an indication of the presence of microorganisms. Viable counts
from swabs suffered the same problem in
terms of poor recovery. Fluorescent staining
of cells and soil on the surface provided the
most rapid and simple method, although
the area imaged is microscopic, therefore
small, and the technology is perhaps not
easily accessible by SMEs (Whitehead et al,
2008). The combination of cells and soil
components increased complexity for
analysis, but staining and subsequent differential analysis of the coverage of microscopic fields of the surface by microorganisms and cells will enable useful evaluation
of the effectiveness of given soil/cell/clea-
ning and disinfection regime/industry combination, and separation of the different removal kinetics of cells and soil.
Combinations of different stains enable discrimination between cells and food soil;
DAPI and Rhodamine; DAPI and fluorescein
and DAPI and acridine orange.
Quantification of attached cells
Quantification of attached bacteria is a key
parameter in assessing persistence and effect of different soils and cleaning and disinfecting agents. Work comparing different quantification methods (indirect
conductance, real-time PCR, sonication/ colony counting, direct microscopy) have
shown that several methods can be used
to quantify bacteria on surfaces. Direct microscopy is well suited at medium bacterial
densities but cannot be used when very
high or low numbers are present. Hence
the method is not suitable when measuring
reducing effects of disinfectants. During the
PathogenCombat work, the partners have
primarily used the methodology developed
by AFSSA (Assr et al, 2008) where bacteria are removed by high energy ultrasound (28 kHz, 150 W) for 4 min and subsequently quantified (Kastbjerg and Gram,
2009; Marouani-Gadri et al, 2009a and
2009b). When only culturable cells are
quantified after cleaning and disinfection,
it is not possible to distinguish the part due
to detachment from the part due to killing
in the reduction of the bacterial numbers.
Real-time PCR was thus used to quantify
total cells which reduction indicates detachment (Marouani-Gadri et al, 2009c).
Ethidium monoazide real-time PCR (Novga
et al, 2003; Rudi et al, 2005) was also used
to quantified viable cells, i.e. the culturable
cells and the viable-but-non-culturable ones
New cleaning and disinfection methods and summary of methods applied for verification...
105
which numbers may be 2 to 3 log greater

than culturable cells counts (Peneau et al,
2007; Marouani-Gadri et al, 2009c).

cleaning agents
Enzymes
Several studies have reported on the positive cleaning effects of enzymes on ultrafiltration membranes fouled with proteinbased residue from milk or meat processing
environments (Allie et al, 2003; Argello et
al, 2003; Muoz-Aguado et al, 1996).
Enzyme based cleaning will find practical
application in bioprocess operations only if
no residual activity remains on the surface
post-cleaning. After the cleaning process,
equipment is often sanitized/sterilized by
exposure to live steam or boiling water.
These steps will almost certainly inactivate
any residual enzymes remaining on the test
surface. Enzymes were able to remove soil
and the cleaning efficacy was increased by
incorporation of a detergent (Turner et al,
2005).
Initial issues with respect to the use of
enzymes to clean dairy equipment included
high costs and low cleaning efficiency
(Grasshoff, 1997). However, with increasing
environmental concern, enzymatic cleaners
are a promising alternative to traditional
chemicals (Grasshoff, 2002). The industry
for textile detergents has employed such
methods, resulting in reduction in the chemicals required and reduced heating, hence
energy saving. Enzymes have been successively used for the cleaning of cold milk processing equipment (Potthoff et al, 1997)
and membrane cleaning. A number of investigations on the use of enzymes to clean
milk heaters have been reported (Grasshoff,

1997).
Enzyme detergents have also proved to
be effective in disrupting the extracellular
polymers which form the biofilm matrix
and thus helped in removal of biofilms
(Potthoff et al, 1997). A mixture of
enzyme activities may be necessary for a
sufficient degradation of bacterial biofilm
due to the heterogeneity of the extracellular polysaccharides in the biofilm
(Johansen et al, 1997). A complex
mixture of polysaccharide-hydrolyzing
enzymes was able to remove bacterial
biofilm from steel and polypropylene
substrata but did not have a significant
antibacterial activity. Combining oxidoreductases with polysaccharide-hydrolyzing
enzymes resulted in bactericidal activity
and removal of the biofilm (Johansen et
al, 1997). The use of enzymes for removal of bacterial biofilm is still limited
due to the very low prices of chemicals in
use.
Enzymes and detergents have also been
used as synergists, to boost disinfectant
efficacy (Johansen et al, 1997). The specific mode of action makes it difficult however to find enzymes that are effective
against all different types of biofilm
(Meyer, 2003).

new mechanical actions for
cleaning
Ice-pigging
Ice-pigging is a novel and innovative new
pigging technique that has significant advantages over conventional solid pigs. The
method was developed by Professor Joe
106
Quarini and his team at the Department

of Mechanical Engineering, University of
Bristol (Quarini, 2002). The ice pig plug is
formed from a thermodynamically stable
ice slurry combined with freezing point
depressant which is capable of cleaning a
product from ductwork and/or separating
products in different phases of the production cycle. Ice pigging employs dense
suspensions of ice to purge products from
process lines. These softsolid plugs can
be pumped safely through a range of
pipe fittings and process units, achieving
very good degrees of sweeping.
Noteworthy features of the technique include its benign chemical nature, safety,
small fluid inventory, and relief of blockage (by melting). Ice pigging must however, be accompanied by clean in place
(CIP) or other methods in order to make
reliably clean surfaces.
The cleaning efficiency of ice-pigging indicates that the ice pig could easily and efficiently remove soft fouling. The fouling
material tested included jam and fats (food
industry), tooth paste and fine silt and sand
(Quarini, 2002). The advantages of this
technique include low environmental impact and the ability to separate and recover
products. Results show a significant improvement in cleaning behaviour compared
with water at 20C; however, the results
were not compared with cleaning with chemicals. It is also not yet clear to what extent
pigging removes very thin layers of deposit,
but the method may significantly reduce
rinsing times.
Two beneficial advantages of the ice pig
are that it is environmental very friendly
and that it never gets stuck in pipework
for very long. Over time, at normal temperature it will melt to water (Quarini,
2002).Thus ice pigging with its ability to

cope with difficult geometrics appears to
offer an innovative defouling methodology (Shire et al, 2005).
Ultrasound
Ultrasound is defined as acoustic energy or
sound waves with frequencies above 20
kHz. Ultrasound provides an effective
means of cleaning surfaces because of the
physical effects on the cavitation phenomenon. The destruction and removal of microbial cells by ultrasound can be achieved
by choosing combinations of ultrasonic
power, exposure time, temperature and distance from the contaminated surface.
Dismantled or small items of equipment
can be effectively cleaned in minutes.
Ultrasonication was found to be a suitable
cleaning method for both cheese moulds
and transportation crates (Salo and
Wirtanen, 2007). To ensure the effectiveness of cleaning, the quality of cleaning
liquid should be measured frequently and
threshold limits for changing the cleaning
liquid should be set.
Mott et al, (1998) found that ultrasound
(30 sec. pulses 20-150-350 KHz) was capable of removing bacterial biofilm along
50 cm lengths of glass tubing, and this
length might be the maximum.
Ultrasound could also be propagated
along lengths of water-filled tubing and
a study using 20 or 150 kHz transducers
showed that Proteus mirabilis biofilms
could be effectively removed from 50 cm
tubes by cavitational activity (Mott et al,
1998). Muthukumaran et al, (2004)
found that ultrasound was good on the
cleaning of whey-fouled membranes. The
use of surfactants in combination with ul-
107
trasonic cleaning showed a synergistic effect providing a better efficiency than

either cleaning process alone.
Ultrasound in combination with heat, pressure or non-foaming detergents appears to
be more effective than any of the treatments alone in cleaning or decontamination of foods (Guerroro et al, 2001).
Ultrasonic treatment in conjunction with a
water temperature of 60 C has shown to
reduce the microbial contamination of crate
surfaces and there the microbial hazards associated with transporting live poultry to
the processing plant (Allen et al, 2008).
Tolvanen et al, (2007) found that logarithmic reduction of L. monocytogenes was
significantly greater in stainless steel than it
was in plastic materials. The difference in
cleaning efficacy for the various material
tested can partly be explained by the hardness of the material. Ultrasonic cleaning is
more efficient on hard surfaces than it is on
soft materials (Tolvanen et al, 2007).
Nevertheless it was found that ultrasonic
cleaning was an effective method of detaching L. monocytogenes from conveyor
materials. Short ultrasonic washing treatment may provide a new possibility in cleaning conveyor belts that are difficult to
clean with conventional methods. It was
found that cleaning time could be as short
as 30s without impairing the cleaning results. Combinations of ultrasound and
steam efficiently killed bacteria on stainless
steel and flooring surfaces in a dose dependant manner, however, it did cause a minor
spread of bacterial cells to surrounding
areas (Vogel et al, 2009).
Pulsing Flow
Pulsing flows have been investigated as a
way of controlling, or preventing, fouling.
Flow pulsing involves imposing a velocity

pulse on a steady flow, increasing the
local shear rate and pressure at the deposit/liquid interface. The dynamics and
applications of pulsating flows in pipes
were reviewed by Edwards and Wilkinson
(1971).
Flow pulsing is an attractive technology
for systems where the liquid is too viscous
to achieve turbulent flow or the inventory
of fluid is to be minimized (Bode et al,
2007). Shamel et al, (1999) found that
both the pulse frequency and amplitude
were important parameters to improve
pulsing flow in cleaning.
Flow pulsing has been used as a method
for enhancing the rate of cleaning of whey
protein deposits in CIP systems. Gillham et
al, (2000) investigated the enhancement
of alkali-based CIP of whey protein deposits by flow pulsing using low frequence
(< 2Hz) fluid pulses. Enhancement of cleaning rates varied up to 250% and depended on the initial amount of deposit
present, pulsing frequency and pulse amplitude. Flow pulsing strongly affected the
cleaning behaviour after the initial deposits swelling stage. The pulsing has the effect of raising the maximum shear stress,
at the pipe wall and thus increasing the cleaning rate (Gillham et al, 2000).
Wall shear stress influences the rate of removal and the amount of soil removed
from the surface exposed to a moving
fluid. Higher velocities tend to break up
the fouling layer due to an increase in
shear stresses acting on the surface. A
pulsating flow could create momentary,
large accelerations of the liquid flow
around the fouling layer, thus resulting in
an increase in removal of the layer (Bode
108
et al, 2007). Jensen et al, (2006) studied

cleaning of an upstand with a pulsating
flow rate. The results showed that a pulsation only has minor effect on the removal of the soil used. Increasing the flow
rate increased velocity generally, but only
small increases were seen in areas, which
are always difficult to clean.
Bode et al, (2007) found that cleaning
time using pulsing flow of whey protein
fouling layer could be shortened by two
and a half times, which correspond to the
results by Gillham et al, (2000).
Two phase flow (water/air)
In 1959 Jennings found that effectiveness
of cleaning was increased by permitting
air to leak into the vacuum side of the
system. Using two phase flow (air/water)
reduces the water required for circulation,
increases flow velocities and enhances
mechanical cleaning action (Reinemann,
1996).
To remove membrane fouling, there are
many means and methods available in
practice to either detach biomass during
membrane cleaning or inactivated microorganisms during biocide dosing.
However chemical cleaning alone is
usually not enough to control biofouling,
the biomass has also to be physically removed (Cornelisssen et al, 2007). In order
to inactivate biomass from membrane elements, Cornelissen et al, (2007) investigated air/water cleaning and daily copper
sulphate dosing. It was found that both
air/water cleaning and daily copper sulphate dosing proved to be very effective
methods in reducing membrane fouling
due to feed spacer fouling. The daily
air/water cleaning was somewhat less ef-
fective in control of bio-fouling than the

copper sulphate dosing but better than
the reference (no cleaning). The cost of
operation may be reduced strongly by
applying this cleaning method. In the
study by Cornelissen et al, (2007), biofouling is connected with membranes applied for the production of drinking water,
process water and for desalination of seawater. Copper sulphate is not a common
disinfecting agent in food industry.
A new disinfecting process:

plasma jet
A new disinfection mechanism using atmospheric plasmas has been investigated
(Lee et al, 2003; Araya et al, 2007). Gases
such as nitrogen, argon and a mixture of
nitrogen and oxygen can be used as
plasma gas. Araya et al, (2007) found that
paint on plastic surfaces was removed by
using dry air as plasma gas in an atmospheric non-thermal plasmas jet. High pressure cold plasma jet has been used to destroy biofilm, and the results indicated the
potential of plasmas as an alternative way
for biofilm removal (Abramzon et al,
2006).
Cleaning and disinfection

products: application method
Mist, foam, fogging and gel
techniques
The main difference between mist, foam
and gel techniques is their ability to maintain a detergent/soil/surface contact time.
Mist spraying is undertaken using small
hand-pumped containers sprayers or pressure washing systems at low pressure.
Misting will only wet vertical smooth sur-
109
faces. Foams can be generated and applied by the entrapment of air in highpressure equipment or by the addition of
compressed air in low-pressure systems.
Gels are thixotropic chemicals which are
fluid at high and low concentrations but
become thick and gelatinous at concentrations of approximately 5-10%. Gels are
easily applied through high and low pressure systems or from specific portable
electric pumped units and physically adhere to the surface (Holah, 2003).
Fogging disinfection implies dispersal of
finely disposed droplets of a disinfectant
within a room. The intention of fogging
is to ensure that all regions and equipment in the room receive an adequate application of the disinfectant. Fogging systems are costly, but could be cost efficient
and also result in improved hygiene if
used appropriately (Bore and Langsrud,
2005). Only few studies have investigated
the use of fogging. Bagge-Ravn et al,
(2003) compared the efficacy of peracetic
acid-based fogging with hypochlorite
based foam in a salmon smokehouse. The
results indicated that the procedure based
on fogging gave similar or better reduction in micro-organism than the foambased method.
Russell (1999) found that fogging is successful for the disinfection of horizontal,
upward facing surfaces but is ineffective
for disinfecting vertical surfaces, undersides and dismantled equipment. These
need spraying. It was also pointed out
that fogging must be regarded as an additional safeguard only and must not replace traditional cleaning and disinfection
routines. Unwanted processing material
left on surfaces prevents the fog reaching
the micro-organisms and can deactivate

certain chemicals.
Holah (2003) has in his review given at
status of the foams, mist, gels and fogging systems and compared them.
Readers are referred to read more in
Holahs review (Holah, 2003).
Disinfectants
Chlorine, a strong oxidant, and chlorine
releasing compounds have been widely
used as disinfectants in the food industry
(Holah, 2003) for example for washing
fresh produce and sodium hypochlorite
has been used in several CIP systems
(Birks, 2003).
Chlorine has been shown to have the ability to depolymerise the polymeric matrix
of biofilm so that it can also detach bacterial cells. Carpentier (unpublished results) found that chlorinated alkaline detergents were the most effective in
removal of Pseudomonas fluorescens biofilm cells from stainless steel. Acidic and
neutral detergents detached no more cells
than water.
Chlorine compounds can be corrosive to
equipment and hazardous to health, and
should always be handled with care and
in the appropriate concentrations (Holah,
2003). Chlorine and chlorine-derived
compounds are now considered undesirable due to the health concerns based
on toxicity and ecological concerns and
are gradually being withdrawn from use.
Ozonated cold water/ozone. Ozone is a
powerful antimicrobial substance due to
its potential oxidizing capacity and it is
widely used as a disinfectant in a variety
of applications. Ozone (O3) is formed by
110
addition of an oxygen atom to molecular

diatomic oxygen. The triatomic oxygen
is highly unstable and rapidly degrades
by releasing the third oxygen atom. The
tri-oxygen molecule of ozone is the second most powerful oxidizer after fluorine. There are many advantages of
ozone in the food industry such as food
surface hygiene, sanitation of food plant
equipment, treatment of food plant
waste and reuse of waste water (GuzelSeydim et al, 2004). Guzel-Seydim et al,
(2000) found that on ozonated water
pretreatment was better than pre-treatment with hot water. However the difference was slight. The investigations have
been done in steady-state systems with
the only movement coming from the
bubbles of ozone and the corresponding
bubbles of air for the hot water cleaning.
Ozone treatment was cold (10 C) and
hot water was at 40 C.
Ozone can be applied both as a gas and
in ozonated water. When used in combination with water it provides a strong
sterilising agent several times more powerful than chlorine, suitable for equipment and production area washdown,
thus eliminating the need for hot water
or hazardous chemicals (Birks, 2003).
Some recent studies have examined new
methods of applying ozone by fogging
ozonated water and charging it electrostatically to increase the effectiveness of
applicationon vertical surfaces (Birks,
2003).
Pascual et al, (2007) have in a review
described the use of ozone as a disinfectant agent, as well as it uses to clean and
disinfect surfaces and equipment, and
the environmental impact of cleaning
and disinfection using ozone. A table is
presented which summarize findings on

efficacy of ozone on various surfaces and
micro-organisms. Moderate doses of
ozone (between 0.5 ppm and 3.5 ppm)
both in gas form and as ozonated water
were sufficient to achieve significant microbial reductions. When ozone is applied as a gas, the necessary exposure
times are considerably longer (1-4 hours)
than for application in ozonated water
(1-10 minutes) (Pascual et al, 2007).
Haibara et al, (2005) found that in order
to increase the capability of organic
matter removal, a high temperature and
a high concentration of dissolved ozone
in ozonated water was most effective.
Guzel-Seydim et al, (2004) reviewed the
use of ozone in the food industry and
concluded that there are sound advantages of ozone applications in food industry. Ozone does not leave any residue
due to its quick decomposition. However
restrictions should be applied to human
exposure to ozone. In humans, ozone
primarily affects the respiratory tract.
Dosti et al, (2005) found that the killing
effect for microorganisms on surfaces
was better with ozone than heat and chlorine. Microorganisms in biofilm have
the same resistance to ozone and chlorine.
Sanitising agents based on chlorine are
now considered undesirable due to health concerns. The interest in ozone as
an alternative to chlorine and other chemical disinfectants in cleaning and disinfection operations is based on its high
biocide efficacy, wide antimicrobial spectrum, absence of by-products that are
detrimental to health, and the ability to
be generated it on demand, in situ, wit-
111
hout a storage requirement. It also has

the significant advantage of being environmental friendly that reduces the companys environmental cost (Pascual et al,
2007).
Electrolysed strong acid water (ESAW) &
Electrolysed weak acid water (EWAW)
also known as activated water. ESAW
and EWAW have a high oxidation reduction potential. These solutions can be
prepared using salt and tap water, and
lose their oxidative and acidic properties
when exposed to the environment. The
process of generation of electrolyzed
water is simple: electrolyzed water is generated by the electrolysis of a 0.1%
concentration of NaCl solution in deionized water (Mahmoud, 2007). ESAW
has a pH value between 2.3 and 2.7
while EWAW has a pH value between
5.0 and 6.0. Mahmoud (2007) has reviewed electrolysed water and its use for
food decontamination.
Urata et al, (2003) investigate the disinfection of endoscope and found that
ESAW and EWAW were effective after
mechanical cleaning of upper gastro-intestinal endoscopes, and could, therefore, be used in the endoscopy unit.
Electrolysed water is more effective for
killing than ozonated water (for microorganisms found on medical equipment). However it also has a corrosive effect on the steel.
Electrolyzed oxidizing water is created
when electric current flowing through
two electrodes immersed in a weak
salt solution and separated by a membrane produces an alkaline and an
acidic solution. Research results have
showed that with 7.5-10 min the elec-
trolyzed oxidizing water was as effective

as conventional treatments in removing
organic matter from the milk pipes.
Other claims have been more obscure or
less transparent (Napper, 2007).
Additional considerations
To compare sensitivity of different bacterial
strains to disinfectants, it is very important
to standardize the assays used with respect
to biomass. Also, the system must be calibrated to allow a measurable reduction, i.e.
some bacterial cells must remain after the
activity. DTU Aqua has developed such methodology for planktonic bacteria, attached
bacteria and biofilm bacteria (Kastbjerg and
Gram 2009). The set-up uses, deliberately,
very low concentrations of disinfectants to
achieve measurable reductions that can be
statistically compared. We hypothesised
that the cause of persistence could be the
appearance of persister cells within a L. monocytogenes population. This has been studied in collaboration with KU-Life and we
have not found indications of such persister
sub-populations using FRIM to measure intracellular pH of surface attached L. monocytogenes (Kastbjerg et al, 2009a).
Since processing equipment is the most
common source of L. monocytogenes as
product contamination, the influence of
disinfectants on phenotypic behaviour is
important. Especially, PathogenCombat
has in collaboration with Professor Hanne
Ingmer, KU-Life determined if sub-lethal
concentrations of disinfectants has any effect on the virulence of L. monocytogenes. Our data, currently being prepared
for publication, indicate that indeed some
disinfectants may either upregulate or
down regulate the central virluence regulator, prfA (Kastbjerg et al, 2009b).
112
Comparison of persistent and nonpersistent strains (e.g Listeria monocytogenes) with respect to their
sensitivity to disinfections (Incimaxx)
A collection of persistent and non-persistent Listeria monocytogenes strains has
been tested for sensitivity to a common
acidic disinfectant (Incimaxx). DTU Aqua
has not been able to demonstrate a systematic difference in tolerance between
these two groups. A sub-group of these
has been tested against one other disinfectants (Triquart). As with Incimaxx, no
systematic strain difference was detected.
This has been published in Kastbjerg and
Gram (2009).
The effect of simple food
preservation parameters (e.g. NaCl)
on disinfectants
The effect of simple food preservation parameters has been studied. DTU Aqua has
demonstrated that when grown with 3-5%
NaCl, most L. monocytogenes strains autoaggregate and stick to plastic surfaces. In
PathogenCombat, we have for the first
time demonstrated that the adhesion to
stainless steel is also enhanced by presence
of moderate levels of NaCl and 1/2-1 log
higher cell counts are seen on steel surfaces
if L. monocytogenes has been grown with
moderate levels of salt than if grown in
media with 1/2-1% NaCl. We have found
that NaCl protects planktonic cells of L. monocytogenes against the acidic disinfectant,
Incimaxx. This protective effect is, however,
not seen for surface spotted bacteria.
Measurment of intracellular pH (collaboration with WP1) has confirmed that NaCl
protects that planktonic cells against the
stress encountered when exposed to the di-
sinfectant. These results were presented at

IAFP summer 2008 and are being published
in Kastbjerg and Gram (2009). Using measurement of intracellular pH by FRIM
(Kastbjerg et al, 2009a), it has also been clearly demonstrated that bacteria grown with
moderate NaCl levels are more tolerant to
disinfectants as measured by a higher intracellular pH.
Conclusion
Within the last few years several new
approaches for cleaning agents and cleaning methods in the food industry have
been described. Many of these are physical/mechanical methods rather than
chemical agents. One of the most promising procedures seems to be ultrasound, which can be used in critical
places in closed equipment and for
pieces of open equipment. Ozonated
water has potential as a disinfectant,
lacking some of the toxicity/environmental issues associated with some
more conventional biocides.
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Aplicacin de nuevas tcnicas eco

eficientes de limpieza y desinfeccin en
sistemas CIP, basadas en el uso del ozono.
Proyecto OZONECIP
V. Martnez
Problemtica de limpieza y
desinfeccin y ventajas del
uso del ozono
neracin de subproductos peligrosos de

la desinfeccin.
Las operaciones de limpieza y desinfeccin

son imprescindibles para la industria agroalimentaria ya que aseguran el mantenimiento de los niveles de higiene exigibles y
necesarios para producir con unos niveles
de calidad adecuados. Sin embargo, estas
operaciones llevan asociado un elevado impacto medio ambiental en lo que se refiere
a consumos de agua, productos qumicos
y energa, as como en la generacin de
aguas residuales y residuos slidos. En la
mayor parte de los casos estos impactos
son los ms relevantes que se producen en
las instalaciones agroalimentarias.
En lo que respecta al consumo de agua, las

instalaciones de procesado de alimentos
consumen diariamente grandes cantidades
de agua (por ejemplo, una industria lctea
puede consumir entre 1 y 5 m3 por Tm de
leche procesada, una cervecera puede consumir entre 4 y 8 Hl por Hl de cerveza elaborado). Normalmente, las operaciones de
limpieza son las que mayor consumo de
agua conllevan en la industria alimentaria.
La limpieza de depsitos y redes de tuberas son las actividades que mayor consumo
de agua implican. Normalmente este tipo
de elementos se limpian mediante sistemas
CIP (clean in place).
Habitualmente, los procedimientos de

limpieza se disean atendiendo nicamente a su eficacia higinica, olvidando
el impacto global que dichas actuaciones
tienen sobre el medio ambiente. No es extrao encontrar en las industrias agroalimentarias un consumo desmesurado de
agua y productos qumicos, elevada produccin de residuos y residuos de envases, alto consumo de energa trmica y
elctrica, etc. Otro aspecto medio ambiental que debera considerarse es la ge-
En cuanto a las aguas residuales generadas,

se debe tener en cuenta que en la industria
alimentaria, casi toda el agua utilizada en
las distintas actividades se convierte en un
efluente de agua residual (por ejemplo, en
el sector lcteo se generan 3-5 m3 por Tm
de leche procesada, en el sector cervecero
2,5-7,2 hl/hl de cerveza elaborada). En el
caso del sector lcteo, no se precisa agua
para la formulacin del producto final, pero
se utiliza una gran cantidad de agua y se
genera gran cantidad de aguas residuales
118
en las actividades de limpieza y desinfeccin de conducciones, depsitos, filtros, etc.

Habitualmente esta agua residual contiene
N, P, COT y cloruros como principales elementos contaminantes.
El proyecto OZONECIP que se describe en
el siguiente apartado experimenta con
tcnicas de limpieza y desinfeccin ms
limpias que conjuguen la variable higinica y la variable ambiental manteniendo
la viabilidad econmica de estos procesos.
De esta forma se asegurarn los rendimientos higinicos y adems se alcanzar
un menor impacto medio ambiental. Este
planteamiento coincide con la filosofa de
las Mejores Tcnicas Disponibles (MTDs).
Es conveniente recordar que el cloro, es un
desinfectante con importantes ventajas que
lo han hecho prcticamente universal en las
instalaciones agroalimentarias. Sin embargo, hay que tener en cuenta algunas
desventajas relacionadas con la generacin
de derivados del cloro, especialmente trihalometanos, cloraminas o clorofenoles, que
han sido relacionados con enfermedades
como el cncer y vinculados a otros efectos
adversos, y por ello se buscan alternativas,
siendo el ozono una de las ms prometedoras con la ventaja adicional de su mejor
resultado medio ambiental. A continuacin
se destacan las principales caractersticas
funcionales por las cuales el ozono resulta
de gran inters para las operaciones de limpieza y desinfeccin:
El ozono tiene un mayor oxidante que el
resto de desinfectantes qumicos, con lo
que se ahorra agua y tiempo. La accin
antimicrobiana de O3 est basada en su
alto potencial de oxidacin (2,07 V),
mucho mayor que los de otros com-
puestos qumicos como H2O2 (1,78 V),

HOCl (1,49 V), Cl2 (1,36 V), ClO2 (1,27 V)
o I2 (0,54 V). El ozono destruye los microorganismos mediante la oxidacin de
las paredes celulares de los mismos, que
con su ataque quedan desintegradas
(lisis). Este es un mecanismo muy diferente al del cloro, el cual se difunde a
travs de la pared celular haciendo la
pared susceptible del ataque enzimtico.
Ahorro de consumo de agua: el ozono
no genera residuos, por lo que no es
necesario el uso de aguas de aclarado.
El agua ozonizada que ha sido usada en
la desinfeccin puede ser reutilizada
nuevamente en las etapas de limpieza
previas, directamente o despus de una
nueva ozonizacin.
Proyecto OZONECIP
Introduccin
El proyecto OZONECIP (LIFE 05 ENV/
E/000251) es un proyecto cofinanciado por
la Comisin Europea a travs del programa
Life-Environment cuyo objetivo fue contribuir a la reduccin del impacto ambiental
de las operaciones de limpieza y desinfeccin a travs de una tcnica innovadora,
ms eficiente en trminos medio ambientales, consistente en el empleo de ozono
como agente desinfectante alternativo a los
agentes qumicos de desinfeccin tradicionales en el mbito de la limpieza y desinfeccin de equipos cerrados mediante sistemas CIP (Clean in Place).
El proyecto se circunscribe a los sectores
lcteo, vincola y cervecero como usuarios intensivos de los sistemas tipo CIP y
suficientemente representativos para
Aplicacin de nuevas tcnicas eco eficientes de limpieza y desinfeccin en sistemas CIP...
119
otros tipos de subsectores interesados:

zumos, bebidas refrescantes, etc. El proyecto tuvo una duracin de 36 meses,
inicindose el 1 de diciembre de 2005.
Para su realizacin, adems de AINIA
centro tecnolgico como coordinador
de proyecto, han participado otros centros europeos: Bionord en Alemania y la
Universidad de Gdansk en Polonia, as
como socios industriales: Bodegas
Domecq en Espaa, la cervecera Inbev y
la lctea MG Lang en Alemania.
vocado actualmente por las operaciones

de limpieza y desinfeccin de depsitos y
equipos cerrados.
Durante el primer ao se realizaron una

serie de revisiones del estado del arte de
las tecnologas implicadas: CIP y ozonizacin y lograr su adecuada integracin. Por
otro lado se revisaron otros aspectos no
tecnolgicos que pudieran afectar la viabilidad del sistema propuesto.
La siguiente tabla muestra los resultados

obtenidos por AINIA en diferentes empresas lcteas en los procesos convencionales de limpieza y desinfeccin tipo CIP
de depsitos de almacenamiento, fermentadores y pasteurizadores.
Paralelamente, durante el primer ao se

realizaron una serie de trabajos de campo
en industrias colaboradoras para obtener
datos reales del impacto ambiental pro-
Como ejemplo de resultados obtenidos

en el sector lcteo, destacar que el consumo de agua est principalmente ligado
a las operaciones de limpieza y desinfeccin representando de forma general alrededor del 80%. Por otro lado, el
arrastre de producto en las aguas de lavado supone un incremento considerable
de la carga orgnica del vertido.
Con la informacin obtenida a travs de

las actividades del primer ao, el segundo ao se dise y construy un
prototipo piloto que permite simular
procesos de lavado CIP convencionales
Tabla 1. Muestreo de aguas residuales en sector lcteo.
Leche
Yogur
pH
(unidades)
6,66
4,15
Conductividad
(mS/cm)
5,25
155
DQO
(mg/L)
160,500
184,500
Nitrgeno
(mg/L)
590
370
PO4-P
(mg/L)
1.680
980
Tabla 2. Aguas residuales de limpieza de equipos lcteos.
1 Enjuagado con agua

Lavado alcalino
Lavado cido
ltimo enjuague
pH (unidades)
Conductividad (S/cm)
DQO (mg O2/L)
8,1-11,61
12,8-13,11
8,63-13,22
2,49-2,50
2,65-4,97
7,00-8,00
430-1.700
13,280-39.200
485-18.760
4,840-9.920
1,170-6.940
412-1.040
28-1.465
196-568
32-1.190
428-958
31-428
30-60
120
de cualquier tipo y ensayar ciclos alternativos basados en el empleo de agua

ozonizada de forma que fuera posible
monitorizar ambas opciones y obtener
datos comparativos en relacin a su respectivo impacto ambiental en trminos
de agua consumida y caractersticas del
vertido generado.
el sistema ozono y un PLC principal que gobierna el sistema en su conjunto. La planta

se opera desde un SCADA que permite la
comunicacin con el PLC principal y modificar las secuencias y condiciones de operacin de los diversos ensayos a efectuar. Las
imgenes a continuacin muestran el aspecto del CIP y del depsito objetivo.
La planta piloto se divide en tres sistemas:

sistema generacin de ozono, sistema CIP y
sistema objetivo a limpiar y desinfectar (depsito de acero inoxidable 316 de 500 litros).
El conjunto cuenta con un PLC que gobierna
El tercer y ltimo ao de proyecto se realizaron ensayos comparativos de ciclos de

limpieza habituales en la industria y ciclos
alternativos basados en el empleo del
ozono, que se describen a continuacin.
Entrada
de agua
Water in
Drenaje
Drain
Prototipo OZONECIP.
11Agua.
Water
Alkali solution
22Solucin
alcalina.
Acid solution
33Solucin
cida.
Ozonated
water
44Agua
ozonizada.
Disinfectant
injection
55Inyeccin
de desinfectante.
6 Ozonation system:
6 Sistema de ozonizacin
formed por
by injector,
formado
inyector,
ozonegenerators
33generadores
de ozono
5 PSA units
yand
5 unidades
PSA.
Target diana.
vessel
77Tanque
121
OZONECIP ensayos comparativos:

materiales y mtodo
Con objeto de disponer de datos concretos del impacto ambiental de las operaciones de limpieza y desinfeccin, se
realizaron ensayos en planta piloto, ejecutando ciclos de limpieza convencionales y ciclos basados en el empleo de
agua ozonizada con objeto de obtener
datos comparativos de la eficiencia higinica y del impacto ambiental de
ambas metodologas.
Como ejemplo se describen seguidamente los ciclos empleados en los ensayos
realizados con leche:
En los ensayos se emple un depsito de

acero inoxidable de 500 litros que para las
diversas pruebas fue ensuciado con:
Vino en rama y con vino inoculado con
cepas de Brettanomyces y de bacterias
acticas.
Cerveza inoculada con Lactobacilllus brevi,
Pectinatus cerevisiphiluy y Saccharomyces
cerevisiae.
Leche inoculada con Bacillus pumilis,
Listeria inocua y Saccharomyces cerevisiae.
En cada ensayo se control la eficiencia
de la limpieza y desinfeccin mediante
placas RODAC, analizndose la carga microbiana en la superficie interior del de-
Tabla 3. Protocolos de limpieza y desinfeccin convencionales.

DCIP1
Enjuague con agua
Lavado con hidrxido sdico
Enjuague con agua
Lavado con cido ntrico
Enjuague con agua
Desinfeccin con cido peractico
Enjuague con agua
tiempo
2
5
2
2
2
5
2
DCIP2
conc
2%
1,5%
0,5%
tiempo
2
5
2
2
2
5
2
conc
0,5%
1,5%
0,5%
Tabla 4. Protocolos de limpieza y desinfeccin alternativas con ozono.

O3CIP4
O3CIP5
tiempo
2
conc
1 ppm
tiempo
2
conc
1 ppm
Lavado con hidrxido sdico
2%
0,50%
Enjuague con O3 a vertido
1 ppm
1 ppm
Lavado con cido ntrico
1,50%
1,50%
1 ppm
1 ppm
Enjuague con O3 en recirculacin
1 ppm
1 ppm
122
psito antes y despus de la limpieza y la

calidad de las ltimas aguas de enjuague.
Paralelamente se control el volumen de
agua consumida (por tanto el volumen de
vertido) y se determin la carga orgnica
del vertido generado.
Resultados del proyecto
De los ensayos realizados de limpieza y
desinfeccin CIP con vino, cerveza y leche
se obtuvieron resultados de eficacia de
desinfeccin de superficies y aguas de enjuagado y de calidad ambiental de las
aguas residuales generadas.
Seguidamente se describen como ejemplo
los resultados obtenidos en las pruebas
realizadas con leche contaminadas con
una mezcla de microorganismos: Bacillus
pumilis, Listeria inocua y Saccharomyces
cerevisiae a una concentracin media por
microorganismo de 105 unidades formadoras de colonia/100 ml.

La tabla 5 muestra los resultados de la eficacia de desinfeccin. Los resultados
dieron valores aceptables en las superficies del depsito muestreadas, no identificndose diferencias significativas entre
los tratamientos con cido peractico y
ozono. De igual forma los resultados microbiolgicos del agua del ltimo enjuague tambin dieron valores aceptables.
La tabla 7 muestra los resultados ambientales de muestras integradas de las aguas
residuales generadas en las pruebas de
limpieza y desinfeccin. Como principales
resultados, destacar el menor consumo de
agua obtenido en la pruebas con ozono
debido a que no es necesario la realizacin de un enjuague final, junto con la reduccin del COD y la carga orgnica en
Tabla 5.
Superficie sucia Superficie limpia ltimo enjuague Superficie Superficie
ufc/25 cm2
ufc/25 cm2
ufc/100 ml
sucia ATP limpia ATP
Bacillus pumilis
CIP1
> 100
<1
31
9.500
128
CIP2
> 100
<1
23
9.200
90
O3CIP4
> 100
<1
20
7.900
140
O3CIP5
> 100
<1
34
13.525
85
Listeria inocua
CIP1
CIP2
O3CIP4
> 100
> 100
> 100
<1
<1
<1
<1
<1
1
9.500
9.200
7.900
128
90
140
O3CIP5
> 100
<1
13.525
85
Saccharomyces cerevisiae
CIP1
> 100
CIP2
> 100
O3CIP4
> 100
<1
8
<1
<1
<1
5
9.500
9.200
7.900
128
90
140
O3CIP5
<1
<1
13.525
85
> 100
123
trminos de reduccin de gramos de COD

(ms del 75%) cuando se usa el ozono
como agente desinfectante.
Consideraciones
econmicas
Coste del equipamiento
Un equipo completo de ozono para la
aplicacin en un sistema CIP est compuesto principalmente por un generador
de ozono, un sistema de alimentacin
de gas (aire u oxgeno concentrado), inyector, tanque de reaccin y destructor
de ozono gas, equipos de medida de
concentracin de ozono gas en alimentacin, ozono disuelto y ozono ambiental, unidad de control y bomba de
recirculacin.
Para un sistema CIP convencional las necesidades de higienizacin se deter-
minan generalmente mediante el tamao y la potencia de la bomba de circulacin, o por la capacidad de generacin de ozono medido en g O 3/h. Una
vez definidos estos trminos es cuando
se determinan las especificaciones de los
componentes del sistema. Un ejemplo
orientativo de coste de un sistema de
ozono para una unidad convencional
CIP, se especfica en la siguiente tabla:
La cuantificacin incluye: el generador
de ozono, inyector, destructor de ozono
residual, medidor de concentracin de
ozono disuelto, unidad de control y estructura de soporte. Considerando que
el prototipo utilizado en el proyecto
OZONECIP, utilizaba principalmente
para las pruebas un generador de 30 g
O 3/h, dicho prototipo pude utilizarse
para la higienizacin de equipos a nivel
industrial.
Tabla 6. Calidad ambiental del agua del ltimo enjuague.

pH
unidades
Conductividad
S/cm
DQO
mg O2/l
CIP1
CIP2
O3CIP4
7,71
7,81
8,06
1.068
1.044
1.057
4,1
7,1
5,5
O3CIP5
8,1
1.074
5,6
Tabla 7. Calidad ambiental de las aguas residuales generadas.

pH
unidades
Conductividad
S/cm
DQO
mg O2/l
Volumen
l
Carga
g COD
CIP1
CIP2
O3CIP4
9,81
4,85
12,03
2.860
1.800
2.900
1.425
1.320
795
1.401
1.399
551
1.996
1.847
438
O3CIP5
3,2
2.680
740
563
417
124
Tabla 8. Cuantificacin de diferentes equipos de ozono, conforme con Air-Tree Europe

GMBH (precios de 2006).
Flujo de agua
Produccin de ozono
Coste de la inversin en
2 m3/h
10 g/h
20.000
5 m /h
20 g/h
25.000
10 m /h
40 g/h
35.000
20 m /h
100 g/h
40.000
3
3
Coste de uso
Cuando se evala el coste de explotacin
del uso del ozono en sistema CIP, al igual
que en un CIP convencional hay que tener
en cuenta los siguientes aspectos:
Consumo de energa.
Consumo de agua.
Consumo de productos qumicos.
Tabla 9. Consumo medio de energa en

funcin de la produccin de ozono.
Produccin
de ozono
4 g O3/h
Consumo medio
de energa
80-180 W
10 g O3/h
200-1.000 W
40 g O3/h
Alrededor de 1.500 W
Costes de mantenimiento.
Consumo de energa
En funcin de la concentracin de ozono
producida por el generador el consumo
puede variar entre 80 W y 1.500 W. En la
siguiente tabla se describe el consumo
medio de energa en funcin de la produccin de ozono. El rango de consumo
puede variar en funcin del fabricante,
tipo de sistema de generacin de ozono,
electrodos, etc.
ozonizada era recirculada en circuito cerrado para su posterior uso. En las

pruebas de limpieza y desinfeccin con
ozono OZONECIP se redujo alrededor de
un 50% el consumo de agua.
Consumo de productos qumicos
Cuando se utiliza agua ozonizada como
agente desinfectante no se requieren productos qumicos adicionales.
El consumo de energa para la generacin

de ozono es relativamente baja comparada con la requerida para calentar las soluciones detergentes y desinfectantes en
sistemas CIP convencionales.
Costes de mantenimiento
Consumo de agua
Destacar que los costes de mantenimiento

del sistema de ozonizacin son relativamente bajos puesto que ninguna de las
El consumo de agua se vio claramente reducido cuando se utiliz ozono y el agua
En los clculos de mantenimiento esperados para un sistema CIP basado en el

uso del ozono, hay que considerar de
forma adicional los generados por el sistema de ozonizacin.
125
partes del mismo requiere un cambio peridico. Entre los costes del sistema ms significativos se incluyen los relacionados con
la calibracin de los equipos de medicin.
Conclusiones
Con los datos medio ambientales obtenidos en el proyecto OZONECIP se confirma la importancia del impacto generado en las operaciones de limpieza y
desinfeccin tal como recoga cualitativamente el BREF y lo complementan cuantitativamente con valores referidos a operaciones particulares, con los siguientes
comentarios generales:
En torno al 80% del agua total consumida en los sectores estudiados se consume en operaciones de limpieza y desinfeccin (valor an mayor en el caso
de bodegas elaboradoras de vino).
La operacin manual de los sistemas CIP
es una prctica an muy extendida en
la industria. La automatizacin y la monitorizacin de los sistemas conducira
a una mayor reproducibilidad de las
operaciones y a ahorros en los actuales
niveles de consumo de agua de enjuague u agentes de limpieza.
Los primeros enjuagues y las soluciones
con desinfectante son generalmente
vertidas.
Valores para diferentes operaciones de
limpieza han sido obtenidos. Tales valores
constituyen slo una referencia del orden
de magnitud de la carga contaminante,
los rangos son muy amplios dado que la
operacin manual causa una gran variabilidad en funcin de la toma o ausencia
de buenas prcticas y de la propia pericia

de los operadores. As, se observan picos
de pH (pH=12 y pH=3) y de conductividad. La carga orgnica es muy variable.
La segregacin de las primeras aguas de
enjuague extremadamente cargadas es
un factor clave para mejorar significativamente la calidad del vertido. Se debe extremar la precaucin en no sobredosificar
detergentes ni desinfectantes puesto que
si se sobredosifica se requieren ingentes
cantidades de agua para eliminar espumas y asegurarse de que no quedan
restos de productos qumicos en los
equipos. Adems puede conferir toxicidad a las aguas residuales.
En los tres productos utilizados en las
pruebas (vino, leche y cerveza) se ha
conseguido una reduccin del 50% en
el consumo de agua cuando se aplicaba
agua ozonizada como agente desinfectante.
La reduccin de la carga orgnica en
trminos de gramos de COD fue de un
50% cuando se us agua ozonizada
como agente desinfectante.
En todas las pruebas se obtuvieron valores de eficacia de desinfeccin de las
superficies del depsito higienizado
con agua ozonizada similares a los obtenidos con el cido peractico. En lo
que concierne a los resultados microbiolgicos de las aguas de enjuague,
cabe destacar que algunas muestras
con tratamiento con ozono presentaron valores ms elevados que los obtenidos con el cido peractico. Esto
puede ser debido a que el ozono con
126
el paso del tiempo no alcanz una

concentracin residual aceptable en el
medio al transformarse en oxgeno.
La revisin tecnolgica realizada en
torno a la integracin de las tecnologas del ozono en los sistemas CIP
muestra que sera relativamente sencillo de implantar en sistemas CIP ya
existentes, adoptando ciertas medidas
adicionales. As, cabe revisar la compatibilidad de los materiales que entraran en contacto con el ozono, en
general las instalaciones son de acero
inoxidable por lo que slo habra que
revisar componentes en vlvulas,
codos, sondas, etc. Por otro lado, se
produce un pase de ozono desde la
fase acuosa a la fase gaseosa en los
equipos, tales como depsitos, por lo
que podra ser conveniente, segn
equipos y productos, el forzar la salida
del aire rico en ozono empujndolo
con un gas inerte: nitrgeno o con
aire y destruir el ozono residual con ultravioletas. Por ltimo, no debe olvidarse que el ozono es un gas txico y
debe prevenirse la exposicin de los
trabajadores al mismo. El actual valor
VLA-ED (valor de exposicin diaria)
ante el ozono es 0,1 ppm. El Instituto
Nacional de Seguridad e Higiene en el
Trabajo adems indica los siguientes

valores en funcin de la actividad:
As, los sistemas deben incorporar sensores de nivel de ozono en ambiente que
detengan el sistema en caso de detectar
fugas y acten en la renovacin rpida del
aire ambiente del rea afectada.
Trabajo pesado
Trabajo moderado
Trabajo ligero
Trabajo pesado,
moderado
ligero ( 2 horas)
ppm
0,05
0,08
0,1
0,2
mg/m3
0,1
0,16
0,2
0,4
TLV para concentracin de ozono en ambientes de

trabajo en Espaa.
En definitiva, como consecuencia del proyecto OZONECIP puede apuntarse que la

incorporacin de las tecnologas del
ozono en nuevos protocolos de lavado y
desinfeccin en sistemas CIP nuevos y
existentes es factible, adoptando ciertas
medidas ya apuntadas anteriormente, obteniendo una eficacia en la higiene y desinfeccin igual o superior que el sistema
tradicional basado en el uso exclusivo de
agentes qumicos pero con un potencial
gran ahorro en la cantidad de agua necesaria para efectuar la operacin y una mejora en la calidad del vertido de aguas residuales generado.

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Cmo puedo aumentar la seguridad alimentaria de mis productos?

How to improve the food safety of my products?

Cmo puedo aumentar la seguridad

INSTITUTO TOMS PASCUAL SANZ

Cmo puedo aumentar la seguridad

Cmo puedo aumentar la seguridad

Obstacles and solutions for the knowledge transfer between

La seguridad alimentaria en el siglo XXI

Tools to support the self assessment of the performance of Food

Prevention of biofilm formation and foodborne infections by control of moisture management

How can the food industry contribute to decrease the risk of

New processing technologies that can reduce the presence of

Reducing foodborne pathogens in the food chain by the use of

The application of PathogenCombat research results in practice

EHEDG: mtodo para la comprobacin de la evaluacin de la

New cleaning and disinfection methods and summary of methods

Aplicacin de nuevas tcnicas eco eficientes de limpieza y

PathogenCombat es la abreviatura del proyecto Control y prevencin a niveles

Table 1. Overall, the objetives of PathogenCombat can be describeb as follows:

Cmo puedo aumentar la seguridad alimentaria de mis productos?

External Advisory Board

Project Management Group

Leader:Clare Hall, SAC

Application and management

Leader: Susanne Braun, STUTT

Control and prevention

Leader: Susanne Braun, STUTT

Figure 1. Organizational chart of the PathogenCombat-project.

Cmo puedo aumentar la seguridad alimentaria de mis productos?

Table 2. Achievements: examples.

Obstacles and solutions for the knowledge

technology changes, shorter product life

Cmo puedo aumentar la seguridad alimentaria de mis productos?

In this context, the distribution of knowledge transfer has become an important

Figure 1. Classification of enterprises based on

For a successful knowledge transfer from

Pulp and paper

Food and drink

All NACE branches

The importance of technology transfer is

Figure 2. Research activity as percentage of added

prises regarding their research potential.

Figure 3. Business expenditure on research by food

The focus of knowledge transfer to SMEs

The cooperation of public research bodies

Cmo puedo aumentar la seguridad alimentaria de mis productos?

The formerly low levels of knowledge distribution of universities were attributed to

Employees with higher education

Manufacture of pulp paper

Manufactur of textiles and

Products of wood and cork;

Motor vehicles, trailers and

Manufacture of chemicals and

Computer services and

Scientific information is easily available,

Electrical and optical

Another important problem arises when

of the process of knowledge transfer, as

knowledge transfer process should have

For a successful knowledge transfer, information has to be processed into a form

Cmo puedo aumentar la seguridad alimentaria de mis productos?

workshops or meetings are either written

and associations related to the food

With more than 40%, the Pathogen