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¿Cómo puedo aumentar la seguridad alimentaria de mis productos?

How to improve the food safety of my products?

Coordinación editorial:

Alberto Alcocer, 13, 1º D. 28036 MadridTel.: 91 353 33 70. Fax: 91 353 33 73. [email protected]

Reservados todos los derechos. Ninguna parte de esta publicación puede ser reprodu-cida, transmitida en ninguna forma o medio alguno, electrónico o mecánico, incluyendolas fotocopias, grabaciones o cualquier sistema de recuperación de almacenaje de in-formación, sin permiso escrito del titular del copyright.

ISBN: 978-84-7867-054-3ISBN: 978-84-92681-13-6Depósito Legal: M-20018-2010

© Universidad de BurgosHospital del Rey s/n. 09001 Burgos

© INSTITUTO TOMÁS PASCUAL SANZpara la nutrición y la salud

Pº de la Castellana 178 - 3º Dcha. 28046 MadridTel.: 91 703 04 97. Fax: 91 350 92 [email protected] • www.institutotomaspascual.es

¿Cómo puedo aumentar la seguridadalimentaria de mis productos?


Autores/Authors

B. BiavatiUniversity of Bologna. Italy.

S. BraunDepartment of Economic Policy. University of Stuttgart. Germany.

N. CarliniProbiotical S.p.A., Novara, Italy.

B. CarpentierAFSSA, Research Laboratory on Food Quality and Processes. Maisons-Alfort Cedex, France.

R. CockerCocker Consulting Ltd. Ireland.

F. DevlieghereDepartment 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. Gaggìa

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

Department of Food Safety and Food Quality, Laboratory of Food Preservationand Food Microbiology, Faculty of Bioscience Engineering, Ghent University. Belgium.

V. JassonDepartment of Food Safety and Food Quality, Laboratory of Food Preservation

and Food Microbiology, Faculty of Bioscience Engineering, Ghent University. Belgium.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. KussagaProduct Design and Quality Management Group, Department of Agrotechnology and Food Sciences,

Wageningen University. The Netherlands.

H. LøjeNational Food Institute, Technical University of Denmark. Lyngby, Denmark.

PA. LuningProduct Design and Quality Management Group, Department of Agrotechnology and Food Sciences,

Wageningen University. The Netherlands.M.ª I. Llorca

AINIA Centro Tecnológico. Spain.WJ. Marcelis

Management Studies Group, Department of Social Sciences, Wageningen University. The Netherlands.V. Martínez

AINIA Centro Tecnológico. Spain.A. Martínez-López

Instituto de Agroquímica y Tecnología de Alimentos, CSIC. Burjassot, Valencia. Spain.L. Mogna

Probiotical S.p.A., Novara, Italy.S. Osés

Department of Biotechnology and Food Science. University of Burgos. Spain.I. Pavlik

Veterinary Research Institute, Brno, Czech Republic.MC. Pina-Pérez

Instituto de Agroquímica y Tecnología de Alimentos, CSIC. Burjassot, Valencia. Spain.A. Rajkovic

Laboratory of Food Microbiology and Food Preservation, Department of Food Safety and Food Quality, Faculty ofBioscience Engineering, Ghent University, Ghent, Belgium.

D. RodrigoInstituto de Agroquímica y Tecnología de Alimentos, CSIC. Burjassot, Valencia. Spain.

J. RoviraDepartment of Biotechnology and Food Science. University of Burgos. Spain.

AB. Silva-AnguloInstituto de Agroquímica y Tecnología de Alimentos, CSIC. Burjassot, Valencia. Spain.

N. SmigicLaboratory of Food Microbiology and Food Preservation, Department of Food Safety and Food Quality, Faculty of

Bioscience Engineering, Ghent University, Ghent, Belgium.GP. Strozzi

Probiotical S.p.A., Novara, Italy.M. Uyttendaele

Laboratory of Food Microbiology and Food Preservation, Department of Food Safety and Food Quality, Faculty ofBioscience Engineering, Ghent University, Ghent, Belgium.

M. Van der SpiegelProduct Design and Quality Management Group, Department of Agrotechnology and Food Sciences,

Wageningen University. The Netherlands.J. Verran

Manchester Metropolitan University, School of Biology, Chemistry and Health Science. United Kingdom.

¿Cómo puedo aumentar la seguridadalimentaria de mis productos?


Índice/Index7 Prólogo

D. Ricardo Martí Fluxá

9 Prologue

Prof. Mogens Jakobsen

15 Obstacles and solutions for the knowledge transfer betweenscience and industry

S. Braun, K. Hadwiger

23 La seguridad alimentaria en el siglo XXI

MC. Pina-Pérez, AB. Silva-Angulo, D. Rodrigo, A. Martínez-López

31 Tools to support the self assessment of the performance of FoodSafety Management Systems

PA. Luning, L. Jacxsens, V. Jasson, WJ. Marcelis, J. Kussaga,M. Van der Spiegel, M. Kousta, S. Osés, J. Rovira,F. Devlieghere, M. Uyttendaele

45 Prevention of biofilm formation and foodborne infections by con-trol of moisture management

R. Cocker

55 How can the food industry contribute to decrease the risk ofcontamination by mycobacteria: a hypothetical case discussion

K. Hruska, I. Pavlik, RA. Juste

63 New processing technologies that can reduce the presence ofpathogens in foods

A. Rajkovic, M. Uyttendaele, N. Smigic, F. Devlieghere

73 Reducing foodborne pathogens in the food chain by the use ofprotective and probiotic cultures

N. Carlini, L. Mogna, GP. Strozzi

89 The application of PathogenCombat research results in practice

F. Gaggìa, B. Biavati

93 EHEDG: método para la comprobación de la evaluación de lalimpieza in situ de los equipos para el procesado de los alimentos

M.ª I. Llorca

103 New cleaning and disinfection methods and summary of methodsapplied for verification of their efficiency

H. Løje, A. Friis, L. Gram, B. Carpentier, J. Verran

117 Aplicación de nuevas técnicas eco eficientes de limpieza ydesinfección en sistemas CIP, basadas en el uso del ozono.Proyecto OZONECIP

V. Martínez

PathogenCombat es la abreviatura del proyecto “Control y prevención a nivelescelulares y moleculares de patógenos emergentes y futuros dentro de la cadenaalimentaria”. El título es bien expresivo y demuestra la importancia que la UniónEuropea concede a la seguridad alimentaria.

Las nuevas técnicas de procesado y los envases con atmósfera modificada pro-curan conservar al máximo las propiedades sensoriales y nutritivas de los ali-mentos pero, al mismo tiempo, posibilitan la generación de nuevos nichos eco-lógicos donde especies que antes no eran deterioradoras o patógenas van aserlo en esas circunstancias. La ciencia debe abrir caminos a la industria ali-mentaria, describiendo estos fenómenos y enseñando como detectarlos y con-trolarlos.

Esta publicación recoge las actas del Seminario ¿Cómo puedo aumentar la se-guridad alimentaria de mis productos?, una de las actividades académicas pro-gramadas en la Cátedra Tomás Pascual Sanz, Universidad de Burgos, aprove-chando la reunión en Burgos de científicos participantes en el proyecto queexpusieron sus investigaciones y resultados. Organizado por la Universidad deBurgos y dirigido por su Vicerrector de Investigación, el Dr. Jordi Rovira, en esteseminario se trataron temas relacionados con la desinfección y limpieza deplantas, mejora del nivel de calidad de los alimentos y equipos agroalimenta-rios y sistemas de gestión de la seguridad alimentaria, todo ello desde un en-foque multidisciplinar y colaborativo entre los centros europeos que formanparte del Proyecto PathogenCombat.

Quizás sorprenda al lector que publiquemos este libro en el idioma inglés. Perola naturaleza científica del proyecto y lo plural de sus participantes nos acon-sejan esta decisión. Este es un libro para todos los participantes del proyectoy su trascendencia va más allá del ámbito nacional.

Para nuestro Instituto es un orgullo contribuir a la difusión de los resultadosde este proyecto, interesante para la ciencia y para la empresa, abriendo ca-minos a la ciencia española y en particular a los equipos de la Universidad deBurgos, cuyo trabajo merece todo nuestro apoyo.

D. Ricardo Martí FluxáPresidente Instituto Tomás Pascual Sanzpara la nutrición y la salud

Prólogo

OverviewThe “PathogenCombat”-project is an integrated project under the 6th FrameworkProgram of the European Union. It started in 2005 and will run till April 2010. The ove-rall objectives are summarised in table 1.

The “Pathogen Combat”–project deal with the issue of food safety, which becomesmore and more important for consumers and industry. With the media coverage of foodborn diseases and consistently occurring food scandals, the public becomes more awareand interested in these problems. So, while the investment in research in this broad spec-trum is rising, the problem still doesn’t seem to fade. The “PathogenCombat”–projectincluding the problem of food pathogens by a multidisciplinary approach, dealing withknown and upcoming food borne pathogens making use of a variety of methods throug-hout the entire food chain.

The project developed a several of platforms to investigate the occurrence, virulence andsurvival of pathogens in the food chain, from primary production to the consumer. Theplatforms rest upon fluorescent ratio imaging microscopy (FRIM), laser tweezers, phagedisplay, functional cell models, functional genomics and microarrays. Emerging food

Prologue

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 andvirulence of pathogens.

3. Detection and prediction of the occurrence and virulence of pathogens in the food chainwith molecular biology based culture independent techniques and microarrays.

4. Determination of host-pathogen interaction with functional cell model replacing the use ofexperimental animals.

5. Prevention of pathogen transmission along the food chain by new processing technologiesand 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.


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borne bacteria, yeast, fungi and viruses are targeted for milk and dairy products, rumi-nants, poultry and pigs and their meat products.

The pillars of RTD (Research, Technology and Development) and NTC (Network, Trainingand Consumer) are the two supporting legs of the project.

In overal terms the RTD Pillars are dealing with basic research, scientific developmentand 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 in-vestigate the factors that enable a pathogen to establish itself in food and on feed ma-trices and food, feed and water contact surfaces. Microbial surface molecules respon-sible for microbial adhesion to matrices and surfaces were determined and subsequentlyused as target for prevention of adhesion. PCR techniques and microarrays were deve-loped for determination and prediction of microbial expression of virulence, expressionof stress and for expression of genes responsible for adhesion. Microarrays for gene ex-pressions have been developed to study the behaviour of pathogens in the food chainat the molecular level i.e. pork, poultry and ruminants and enable host pathogen inte-

Coordinator

Consortium meetings

External Advisory Board

RTD team

Effiue Tskalidou AUA

Larry Beuchat, USAGrahame Gould, UKKarel Hruska, CZWihelm Holzapfel, DENiels Skovgaard, DKServé Notermans, NL

Francois Lefvre, INRAJørn D. Mikkelsen, DANISCOLuca Cocolín, UNIUDBruno Biavati, UNIBO

NTC teamSusanne Braun, STUITTClare HALL, SAC

Mogens Jakobsen, LIFE

Knowledgemanagement& IPR

Administration

Finance

Reporting& communication

Knowledgemanagement& IPR

Project Management GroupMember of External Advisory Board (Larry Beuchat)

Coordinator (Mogens Jakobsen)

Deputy Coordinator

RTD Pillars

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Figure 1. Organizational chart of the “PathogenCombat”-project.

Prologue

11

ractions to be studied at the molecular level. Functional cell models were established forpork, 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 rapidand specific methods methods for detecting new and emerging pathogens. This is im-portant in relation to developments in the food industry in which raw materials and in-gredients originate from many different countries and different production systems. Suchexternal sources of new and emerging pathogens are also a likely source of pathogenswith unknown patterns of resistance towards control methods and with unknown viru-lence traits. Stress adaptation and new virulence traits may develop within the particularfood chain and in both situations call for appropriate control and detection methods tobe applicable throughout the chain.

Based upon the functional genomics studies, culture independent techniques to detectnew and emerging pathogens were developed. DNA-arrays for detection of pathogenswere developed to predict the occurrence of pathogens as well as their virulence at agiven time in the food chain and at the time of consumption. The methods developednot only report numbers of pathogens, but most importantly they give information ontheir virulence and relevance in the food chain.

RTD III Control and Prevention

Attachment and establishment of viable and virulent pathogens in or on solid matricesis considered the most important route of transmission along the food chain. Detachmente.g. by cleaning or exposure to various food processing stresses leading to inactivationas well as loss of virulence will break this transmission.

Studies to determine and improve the hygienic design and close the present gap bet-ween hygiene and technology were conducted. The optimal characteristics of food con-tact surfaces and efficient cleaning and desinfection procedures have been addressed.Protective and probiotic bacteria, which can prevent attachment by competitive exclu-sion or inactive pathogens in the food chain including the intestinal tract of pigs, chickenand ruminants as estimated by use of the functional cell model, were identified. Finallyinactivation of pathogens by mild processing techniques, e.g. intense UV light pulsesand hydro-static pressure was included.

RTD IV Application and Management

The food processing SME Partners and the food chain they belong to were the targetsand collaborators in application of the results obtained in RTD I, II and III and for the de-velopment of the generic and specific Food Safety Management Systems. This is the tran-sition of research and development into practical application in the food industry fullyintegrated with networking of SMEs and the training activities described in NTC I andNTC 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 es-timation played a major role in the development of the Food Safety Management Systemby identification of CCPs of the HACCP based system developed.

The NTC Pillars are dealing with the tasks of transferring the new information and techno-logies to the users. This is done by creating a network of enterprises that would profit fromthese findings, providing, processing and distributing knowledge in appropriate form, offe-ring 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 be-ginning and the continuous exchange of knowledge and experiences gained are essen-tial for the project.

Through contacts to SMEs having experience from participating in EU projects in depthinformation are obtained on the optimal method to integrate SMEs in PathogenCombat.The network is operational through a net-based system of communication. The func-tion of the network is supported by the training activities and the dissemination of Projectresults 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 appli-cation. Transition from research to practical application is carried out to allow a Europeanwide production of food with no or acceptably low level of pathogens. This training is or-ganized in three levels. Level one addresses the food producing SME Partners ofPathogenCombat. At Level two SMEs outside PathogenCombat are included in several re-gional workshops to teach the participants how to apply the deliverables ofPathogenCombat including the Food Safety management System developed. Level threeis pan-European and based upon the SME network created in NTC I including national andEuropean SME organizations as well as regulatory agencies.

NTC III Consumer Awareness

This activity involves the participation of consumers and consumer groups and organiza-tions. It establishes methods for effectively communicating accurate information regardingfood safety issues to European consumers. It seeks to strengthen our understanding of therole of food safety in consumer behavior and thereby develop best practice in communica-ting with consumers to enhance their awareness of food safety issues. It is an objective ofthis activity to raise awareness of food safety issues throughout Europe and to strengthenthe 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 platformsNovel approaches to analyse the interactions at cellular and molecular level between pathogensand food and feed matrices and contact surfaces in the food chain including the intestinal tractof farm animals. To understand the mechanisms, by which pathogens enter, adapt, persist andexpress 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 pathogensListeria 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 methodsMicroarrays and molecular biology culture-independent techniques, have been developed forpathogens included in PathogenCombat. The methods will not only report numbers, but includea new approach to estimate viability and virulence of pathogens throughout the food chain.

IV. Virulence expression in matricesA 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 modelsModels developed for pigs, chicken and ruminants. Applied in host-pathogen interactions andselection of protective and probiotic cultures. A new opportunity has been made available fordeterminations of dose-response and risk assessment.

VII. Food Safety ManagementDiagnostic instruments and tools have been developed for SMEs for identification of technologicaland managerial interventions which can improve food safety management systems (FSMS) andlead to integration of the new knowledge and methods developed in PathogenCombat. Conceptof web-based FSMS support systems for SMEs has been developed.

VIII. Interaction with food producing SMEsFor new knowledge obtained and methods and tools developed, validation and testing ofapplicability in SMEs are in progress.

IX. The consumer, the food industry and the regulatory bodies in EuropeThe consumers’ understanding for key food safety issues has been evaluated and the most effectiveways of communicating results is being assessed. A strategy for interaction and exchange ofinformation with the food industry and regulatory agencies has been established.

Prof. Mogens JakobsenCoordinator of PathogenCombat. Faculty of Life Sciences, University of Copenhagen, Denmark

Obstacles and solutions for the knowledgetransfer between science and industry S. Braun, K. Hadwiger

Overview

Knowledge transfer

Knowledge transfer is defined as the pro-blem of transferring knowledge betweendifferent organizations or from one partof an organization to another part of thesame organization. In other words, know-ledge transfer is the gateway for thetransfer of knowledge between donatingand receiving entities. In most cases, thedonor is a public organization, like a uni-versity or research institute, while the re-ceiving entity, which often has interest incommercializing the knowledge, is a com-pany. Furthermore, knowledge transfercan be seen as a more general term, des-cribing the distribution of knowledge th-rough human beings. This approach fo-cuses on characteristics of communicationbetween experts and the person that re-ceive the knowledge.

Despite the different views, knowledgetransfer is often hindered by a variety ofissues. Nowadays, removal of these ba-rriers and the improvement of knowledgetransfer are seen as a main driver of eco-nomic growth. The present society hasbeen defined as a knowledge society. Dueto increasing importance of knowledge,a close relationship between science andtechnology became more important.Generally, the increased competition on aglobal scale led to an increasing rate of

technology changes, shorter product lifecycles and increasing product quality. Thefinding and utilization of advanced know-ledge and new technologies is more thanever substantial for the survival and pros-perity of any firm.

Many large enterprises have their ownscientific department. Therefore, the im-portance of distribution of knowledgecreated by public bodies has becamemore important, especially, for small andmedium enterprises (SMEs) that cannotafford the cost of research and are depen-dent on external knowledge to improvetheir production processes. Although it isan important advantage for large enter-prises that they are able to conduct rese-arch, it has become increasingly difficultto rely solely on internal knowledge pro-duction.

Companies are classified as small and me-dium-sized enterprises (SMEs) by theEuropean Union if the number of theiremployees does not exceed 250 and ifthey are independent from large compa-nies. In addition, their annual turnovermay not be beyond € 50 million, or theirannual balance sheet beyond € 43 mi-llion. SMEs are divided into three subca-tegories: micro-enterprises with 10 or lessemployees, small enterprises with 10 to49 employees and medium enterpriseswith 50 to 249 employees.


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The importance of technology transfer isobvious when one takes into account that98.8% of all European enterprises are infact SMEs and they provide 67% of alljobs in the private sector. Moreover,91.5% of SMEs are in fact micro-enter-prises with less than 10 employees.

For a successful knowledge transfer fromany knowledge donor to a SME, it is notsufficient to provide knowledge or tech-nology, considering their limited possibi-lities, also the financial support of theSME is necessary to make the implemen-tation of new technology possible.

The different industrial branches dependon this external knowledge on a varyingdegree. The food industry in the EuropeanUnion, which consists mainly of SMEswith 10 or less employees, is heavily de-pendent on this knowledge due to a lackof resources to carry out any research anddevelopment activity. It is estimated that70% of all SMEs of the food sector donot carry out any kind of research activity.In addition to that, this industry has todeal with the demand for safer productsand higher quality, as well as a high com-petition. To cope with these challenges,new technologies have to be introduced.

In this context, the distribution of know-ledge transfer has become an importantsubject of scientific research, as well as ineconomic and public policy. An importantrole of the PathogenCombat project is thesupport of SMEs of the food sector in thisprocess and helps with the implementa-tion of new technologies.

The food sector is the largest manufactu-ring sector in the European Union, withan annual turnover of € 910 Billions in2008. Its importance is also shown in itshigh level of legal regulation. Many na-tional and international regulations deter-mine the form of production and foodhandling as well as food quality itself.New regulations are added at a regularbasis and the demand is still increasing.SMEs have to survive in an overregulatedmarked, with high requirement andstrong competition. The implementationof new knowledge in the food industry isnot only optional, but necessary for allcompanies. Its assistance should be con-sidered as highest priority.

The task of supporting innovation in foodproducing SMEs becomes even more im-portant when taking into consideration

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Obstacles and solutions for the knowledge transfer between science and industry

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that exports of the food industry have sh-runken in the last years. The market shareof world food and drink exports of theEuropean Union diminished from 24.8%in 1998 to 19.2% in 2008. New innova-tions in the food sector could help aver-ting a further decline and improve com-petitiveness on the world market.

The focus of knowledge transfer to SMEsis on the distribution of knowledge cre-ated at universities, which is mostly des-cribed as being largely underexploited.However, recent studies indicate a posi-tive trend in university-industry interac-tion. The reasons for this improvementare changes in public policies and institu-tional environment and a strong encou-ragement of commercialization of scien-tific discoveries. Prior to this improvement,scientific discoveries arose directly fromthe “ivory tower” of science, and therewas no specific aim to utilize and com-mercialize them. At present, academic re-search is more focused on future indus-trialization and exploitation of theirdiscoveries.

Universities and other public research ins-titutions are not comparable with enter-

prises regarding their research potential.Contrary to the knowledge transfer bet-ween two enterprises, knowledge createdby universities is widely available.Moreover, there are many advantages forenterprises and universities to collaborate.The enterprise can benefit from the highlytrained staff of a public institution, andcan also improve their corporate imagedue to this joint venture. The universitycan benefit from additional fund for rese-arch, the opportunity to research close tothe market and an increased opportunityof employment for students and gra-duates.

The cooperation of public research bodieslike universities and private enterprisescan work in a variety of ways. The mostcommon way is the support of research,mostly by financial contributions. This fun-ding was often unspecific and unres-tricted in the past, but nowadays it isbound to specific research areas and ac-tivities that presumably will benefit the fi-nancer by bringing in new knowledgeand technology. Another possibility of co-operation is cooperative research. Thispostulates a close teamwork in the fieldof research. The research teams, consti-tuted by scientists of both partners, haveto work together on a topic to maximizeefficiency and prevent interference of re-search fields.

There is also a possibility of direct know-ledge or technology transfer in both di-rections. With this method both teamsneed also to have a broad understandingof the particular research field and full in-sight in each other’s work. However, theimplementation of new technology has tobe conducted with full support of the do-nating partner.

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The formerly low levels of knowledge dis-tribution of universities were attributed toa lack of knowledge structures at the en-terprise side and a lack of stimulant gra-tification for the distribution of scientificknowledge by the academic side.Different approaches have been tried toeliminate this problems. Universities haveaccumulated a lot of experience in the lastyears on transfer of technologies, and pu-blic organizations which focus on the dis-tribution and transfer of knowledge bet-ween science and industry have beenfounded.

PathogenCombat tries to contribute tothe knowledge transfer in the area offood safety. Later in this article, the pro-blems concerning knowledge transferbetween science and industry will be des-cribed, and the role of PathogenCombatwill be presented in the interaction bet-ween science and industry and its contri-bution to technology and knowledgetransfer.

Obstacles in knowledge transfer

Some problems concerning knowledgetransfer have already been mentioned inthis article (see overview). The mostcommon problems are:

• The lack of willingness to share know-ledge by the donor, due to:

– Mistrust.

– Assumed benefits of possessingknowledge exclusively.

– Absence of ability to transfer know-ledge to a non-specialist.

– Language and culture barriers.

– Lacking of rewards for knowledgetransfer.

• The lack of willingness to accept know-ledge by the receiving entity, due to:

– Lack of structures for knowledge pro-cessing.

– Missing contacts to knowledge pro-ducers.

– Lack of knowledge concerning theknow-how transfer process.

– Lack of ability to understand and im-plement knowledge.

– Language and culture barriers.

Knowledge transfer is hampered by a va-riety of ways. Both donors and receivingentities of knowledge can be the reasonfor stopping the transfer and implemen-tation of knowledge and technology.

Firstly, the knowledge donor should havethe willingness to share his knowledge toa third party. Knowledge, as a property ofsome worth, is nothing to give away ea-sily because it is often perceived as earnedby hard work and years of research. Ascientist who publishes his findings inscientific papers will clearly receive a re-ward in form of reputation. In the processof technology transfer, the benefits canbe less evident. Whichever form of coo-peration is chosen, important research re-sults have to be given away, often, beforepublishing. As a consequence, it is veryimportant to provide an appropriate re-ward for sharing knowledge. It has to bevery clear that the receiving entity and theknowledge donor will equally benefitfrom this process. It is important that bothpartners build up their relationship basedon trust. Knowledge has a considerablevalue and, unlike other goods, it can begiven away quite easily, even unkno-wingly. Consequently, the partners in a


19

knowledge transfer process should havea high degree of reliance.

Another important problem arises whenboth partners are on a different level ofinsight to the matter in hand.

Scientific information is easily available,often for free. While some scientists payto the scientific journals for publishingtheir scientific articles, the author him-self provides these papers willingly forfree. Despite this, research results willnot find its way to the enterprises thatmay need them; especially small andmedium enterprises (SMEs) that cannotafford to conduct research on their own.The reason for this is the barrier bet-ween entrepreneur and specialists. Let’stake a look at the food sector, forexample, which consists mainly of SMEsof five to ten employees. Most of entre-preneurs and managers of these SMEsdo not have an academic degree andmost of them would not have theknowledge on how to obtain scientificinformation. Even if they know how toobtain new scientific findings, either byscientific articles or personal communi-cation, it is unlikely that a non-specialistcan understand and digest this informa-tion - partly due to the fact that scien-tific language is very hard to compre-hend without appropriate training. Inaddition, to comprehend a scientific ar-ticle, good background knowledge is re-quired that only a specialist in this fieldof activity can have.

While many large enterprises have dedi-cated structures that deal with knowledgetransfer, like scientific trained employeesor research departments, most SMEs lackemployees which have an understanding

of the process of knowledge transfer, aswell as knowledge processing. In fact, en-terprises relying mostly on external know-ledge have many problems receiving andprocessing it. This disadvantage can becounteracted by choosing the right pre-sentation form.

For a successful knowledge transfer, in-formation has to be processed into a formthat is understandable for anyone. It hasto be easily readable and understandableand, at best, easy to implement into theexisting production process. Nevertheless,it still has to contain all the critical infor-mation needed for the full and successfulknow-how transfer. While this is easilymanageable with small efforts in someareas, this can be almost impossible inother fields of science with state of theart developments or very specialized rese-arch. Here, the only possibility is theknowledge transfer through extensiveworkshops and intensive assistance to thereceiving partner.

An additional problem is the language ba-rrier, which often represents the main ba-rrier of knowledge transfer. Currently,English is the language for science andbusiness, and most of scientific articles,

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20

workshops or meetings are either writtenor hold in English. The English languageis widespread across the world; however,only people with a relatively high level ofeducation are able to read and fluentlycommunicate in English. This ability is al-most absent in less well educated people,except for those with English is theirmother tongue. The first step to ease andstimulate the knowledge transfer processwould be the translation of the Englishworks into the local language. This wouldgive the possibility for many SMEs withemployees of lower educational back-ground to obtain critical knowledge.

Both partners share the responsibility fora successful knowledge transfer. On onehand, the donor entity has to process theknowledge to make it understandable fornon-specialists. On the other hand, the re-ceiving entity has to be willing to acceptto work with a new process, to be opento new circumstances, and to have acommitment to the project.

The contribution ofPathogenCombat to theknowledge transferPathogenCombat has made a variety ofcontributions to the transfer of knowledgeand technology to SMEs throughout theEuropean Union and beyond its borderline.The implementation and transfer of newscientific findings during the project hasbeen a priority task from the beginning.The distribution of knowledge was accom-plished through different approaches.

A widespread network of contacts th-roughout the European Union was esta-blished, consisting of SMEs, industry, re-search institutions, government agencies

and associations related to the foodsector. A database with several thousandsof addresses is created, maintained, andconstantly extended during the course ofthe project.

The PathogenCombat-Newsflash was cre-ated and published throughout theNetwork as a source of up-to-date infor-mation for the food industry. This includesresults of state of the art research, currentnational and Pan-European developmentsand upcoming events like workshops andmeetings.

In addition, a web portal was establishedas contact point for SMEs. The webpageoffers a wide range of useful information.Amongst others, visitors can find the latestresearch results and publications in thefield of food quality and safety from thePathogenCombat consortium members,information about upcoming workshopsas well as outcomes of the previous ones,and information about PathogenCombatproject and its partners.

Workshops organized by PathogenCombatare greatly contributing to the process ofknowledge transfer. These are open toSMEs, scientists and organizations inte-rested in the topics of food safety, foodquality and food processing. They allow forpersonal contact and communication bet-ween knowledge donors and receiving en-tities. Special attention was paid to the pro-blem of the language barrier and thespecific requirements of SMEs in the foodsector. The collaboration with local part-ners and associations provided valuable in-sight in the ‘hot’ topics for the local SMEsand their expectations. Workshops heldentirely in the local language added to asuccessful transfer of research.


21

With more than 40%, the PathogenCombat project consortium has a very highproportion of members that are SMEs.Though not all results are immediately ap-plicable for the industry, especially at thebeginning of the project where funda-mental research has been conducted, theproject SMEs were able to transform manynew findings into countable results.

A good example of a successful knowledgetransfer to a PathogenCombat SME is PittasDairy Industries, based in Cyprus. The com-pany was founded in 1939 and producesover 100 different dairy products, fromcheese to yogurt. Pittas is a small but inter-national operating company, and theyknow that innovation is an important factorif one wants to stay ahead of competitors.Therefore, when the Agricultural Universityof Athens contacted Pittas and asked fortheir participation in the project, they didnot hesitate to join it. The role of Pittas,along with other SMEs, was to apply newresearch results in their production process.This included developing a predictive modelfor the shelf life of yoghurt, using probioticstrains in animal feed, developing new pro-ducts with protective cultures and compa-ring their own food safety systems againstnational and international standards.

For Andreas Hadjipetrou of Pittas, the coo-peration was a full success. In the five yearsof participation in the project, the companywas able to gain new insight in the processof food production. Additionally, they wereable to meet experts from different fields ofexpertise, like microbiology, food safety andnutritional science. In cooperation withPathogenCombat, Pittas was able to in-crease food quality and safety, while laun-ching new products and gaining new cus-tomers European-wide.

Further information about knowledgetransfer and PathogenCombat can be ob-tained by visiting www.pathogen-combat.com

BibliographyBaardseth 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 aca-demic technology transfer organizations in im-proving industry science links; Research Policy,Number of Employees with higher educationper sector (Source: Hollanders & Arundel2005; 34 (3):321-42.

European Commission, SME Success Storiesin the area of Food Agriculture and Fisheriesand Biotechnology (EUR 23612EN), 2008;14-5.

Feller I. Universities as engines of R&D basedeconomic growth: they think they can,Research Policy, 1990; 19:349-55.

Kaufmann A, Tödling F. Science-industry inte-raction in the process of innovation: the im-portance of boundary-crossing between sys-tems. Research Policy; 30:791-04.

Kingsley G, Bozeman B, Coker K. Technologytransfer and absorption: an R&D value map-ping approach. Research Policy, 1996;25:967-95.

Mansfield E, Lee JY. The modern university:contributor to industrial innovation and reci-pient of industrial R&D support. ResearchPolicy, 1996; 25:1047-58.

Mansfield E. Academic research and industrialinnovations, Research Policy, 1991; 26:773-6.

Meyer-Krahmer F, Schmoch U. Science-based technologies: university–industry in-teractions in four fields. Research Policy,1998; 27:835-51.

Mowery DC, Sampat BN. Universities in na-tional innovation systems, The OxfordHandbook of Innovation, Oxford UniversityPress, Oxford, 2005; 209-39.


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Paula E. Stephan. Educational Implications ofUniversity-Industry Technology Transfer. TheJournal 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 techno-logy centrality in industry-university interactions,Research Policy, 2002; 31:1163-80.

Traill W, Meulenberg M. Innovation in the foodindustry. Agribusiness, 2002; 18 (1):1-21.

Von Hippel E. The Sources of Innovation; OxfordUniversity Press, Oxford. 1988.

IntroducciónDada la magnitud y el alcance de la ca-dena de suministro de alimentos en lassociedades modernas, es muy complejogarantizar que todos los alimentos dis-tribuidos y consumidos estarán alejadosde fuentes de contaminación poten-ciales. En su lugar, la seguridad alimen-taria se debe incrementar concentrandolos esfuerzos de forma sistemática demanera que se minimicen las oportuni-dades de contaminación en cada puntode la cadena de producción, procesadoy distribución hasta su preparación yconsumo. A pesar de que ha habido im-portantes avances en la conservación ycontrol de alimentos, las enfermedadestransmitidas por los mismos perma-necen como la principal causa de enfer-medad y mortalidad en el mundo. Enconsecuencia, las políticas de la UniónEuropea y de los Estados miembro,debe enfocarse para alcanzar el nivelmás alto de seguridad alimentaria conobjeto de reducir la incidencia de lasenfermedades producidas por el con-sumo de alimentos. Para alcanzar esteobjetivo, el análisis de riesgos debe serel pilar en el cual se base la política deseguridad alimentaria, complemen-tando esta herramienta con la creaciónde la Autoridad Europea para laSeguridad Alimentaria y las distintasagencias nacionales de los Estadosmiembro (AESAN, por ejemplo). Todas

estas estructuras deben trabajar deforma cercana de manera que el es-fuerzo vaya en la misma dirección y seaeficaz en el control de los alimentos yen reducir la incidencia de enferme-dades transmitidas por los mismos.

Contaminación microbianaLos alimentos, dada su naturaleza bioló-gica, proporcionan a los consumidores losnutrientes necesarios para cubrir sus ne-cesidades; a la vez, son sustratos en losque el crecimiento de microorganismos esposible en un amplio rango de condi-ciones. Desde que un alimento se pro-duce (agrícolas, ganaderos, pesqueros,etc.) o fabrica (cualquier alimento manu-facturado: pan, queso, entre otros), tieneriesgos de contaminarse.

La presencia de microorganismos pató-genos potenciales en nuestro ambiente,la habilidad de algunos de ellos para so-brevivir, crear resistencias o proliferar encondiciones de refrigeración y a bajasconcentraciones de oxígeno, y en ciertoscasos, las bajas concentraciones necesa-rias para producir enfermedad, agravan elproblema al que nos enfrentamos encuanto a seguridad alimentaria (de Swarteand Donker, 2005).

El proceso de infección por patógenos ali-mentarios se produce de acuerdo a la re-lación alimento-patógeno-consumo:

La seguridad alimentaria en el siglo XXIMC. Pina-Pérez, AB. Silva-Angulo, D. Rodrigo, A. Martínez-López


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Las enfermedades de origen alimentarioprovocadas por los microorganismos sonde tres tipos:

• Intoxicaciones.

• Infecciones.

• Toxicoinfecciones.

Entre los principales microorganismos pa-tógenos presentes en alimentos, destacanpor la gravedad de los síntomas y altastasas de incidencia, los siguientes:

Salmonella spp

Salmonella spp es una de las causas máscomunes de enfermedades transmitidaspor los alimentos en los seres humanos, laduplicación de la incidencia de los casos desalmonelosis en las dos últimas décadas haacompañado la modernización de las in-dustrias alimentarias (Altekruse et al,1997).

Este grupo se puede dividir en dos grandescategorías: los que causan la fiebre ti-foidea y los que no: S. typhy, S. paratyphiy S. enteritidis, ésta última como causantede gastroenteritis por consumo de huevos,leche, alimentos que contienen huevoscrudos, carne, aves de corral y productosfrescos (Acheson, 2003).

Campylobacter jejuni

Campylobacter jejuni es un patógenonuevo transmitido por los alimentos, ac-

tualmente se considera la principal causade ETA en Estados Unidos. Hay dos prin-cipales especies de Campylobacter queson responsables de la mayoría de las en-fermedades en seres humanos. C. jejunirepresenta a la mayoría (90%) y C. coli re-presenta sólo el 10%, estos pertenecen ala flora natural de los intestinos de mu-chos animales, incluyendo aves y animalesdomésticos. La mayoría de estas infec-ciones están asociadas con el consumo deaves de corral mal preparadas o en malestado, el consumo de leche cruda y aguano clorada.

Escherichia coli O157:H7

E. coli O157:H7 es una de las principalescausas de diarrea sanguinolenta en todoel mundo, además es la causa del sín-drome urémico hemolítico, la principalcausa de insuficiencia renal aguda enniños de Estados Unidos con una tasa demuerte del 5%. Este microorganismo seha asociado con el consumo de carne deres molida, lechuga, leche cruda y aguano tratada, y también se han documen-tado casos de transmisión persona a per-sona (McCabe and Beattie, 2004).

Listeria monocytogenes

L. monocytogenes es un microorganismocomún. Se sabe que entre 1 a 10% dela población mundial son portadores deL. monocytogenes. Los alimentos dondefrecuentemente se encuentra incluyenleches contaminadas después de su pas-teurización, paté, quesos, vegetalescrudos y carne mal cocida. Aunque es unmicroorganismo que fácilmente muerecon el calor, la recontaminación de ali-mentos es muy frecuente, además de so-portar y ser capaz de crecer a tempera-

Microorganismo Consumo

Alimento

La seguridad alimentaria en el siglo XXI

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turas de refrigeración. Las mujeres em-barazadas son un grupo altamente sus-ceptible a este microorganismo, debidoa que tiene la capacidad de llegar al feto,provocando aborto espontáneo, naci-miento prematuro, sepsis neonatal y me-ningitis. Además, la listeriosis puedecausar muertes, meningitis o sepsis enpersonas inmunocomprometidas; te-niendo una tasa de letalidad del 40%.

Clostridium perfringensy Bacillus cereus

Estos dos microorganismos son las princi-pales causas de brotes alimentarios enbanquetes, debido a su fácil proliferacióncuando los alimentos se mantienen a unatemperatura ambiente por un largo pe-riodo de tiempo, causando gastroenteritismuy leve. Además, comparten caracterís-ticas comunes: formación de esporas,producción de enterotoxinas que causangastroenteritis, están presentes en ali-mentos previamente sometidos a trata-mientos térmicos como salsas y arroz.

Los alimentos más susceptibles de estarimplicados en los brotes con B. cereus in-cluyen carnes y verduras cocidas, pro-ductos lácteos pasteurizados, cereales yarroz cocido.

Yersinia spp

De los tres miembros de este genero, Y.enterocolitica y Y. pseudotuberculosis sonconsiderados como patógenos transmi-tidos por alimentos. No es un microorga-nismo comúnmente causante de enfer-medades transmitidas por los alimentoscomparado con Salmonella spp oCampylobacter spp; sin embargo, se sabeque es transmitido por los alimentos a los

seres humanos en los que causa unagrave enfermedad gastrointestinal; el ali-mento comúnmente implicado en estosbrotes es el cerdo. La leche es otra fuentefrecuentemente asociada con este micro-organismo.

Control y reducción del riesgo El control y reducción del riesgo causadopor el consumo de alimentos contami-nados puede llevarse a cabo actuandosobre tres factores: el patógeno, el con-sumidor, o el vehículo de transmisión, eneste caso, los alimentos en los cuales vivee interactúa (Havelaar et al, 2002).

Estos tres factores son necesarios paraque ocurra la enfermedad transmitida porlos alimentos y existen interacciones com-plejas entre ellos. Cambios producidos enestos tres factores pueden reducir la pro-babilidad de adquirir una enfermedad porconsumo de alimentos, por ejemplo la es-terilización comercial de un alimento des-truye a los microorganismos patógenoseliminando el factor exposición, lo quehace al alimento más seguro. Por el con-trario, los cambios en estos tres factorespueden favorecer que emerjan nuevospatógenos, este es el caso de individuosinmuno-comprometidos por haber sufridoun transplante. Microorganismos que ha-bitualmente no producen enfermedadespueden colonizar el tracto intestinal de losbebes prematuros, personas mayores, etc.Se pueden producir cambios de virulenciacomo consecuencia de los tratamientosde conservación, en este caso el patógenoadquiere características que le ayudan ainvadir el cuerpo humano. Estos tres fac-tores son una pieza clave en la reducciónde la enfermedad de origen alimentario.


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Como se verá más adelante, la industriay agricultura han introducido una seriede medidas de control efectivas enfo-cadas a limitar alguno de los factoresclaves de la enfermedad por consumo dealimentos.

En 1896, el U.S. Public Health Service(USDA/FSIS, 1996) comenzó a investigarla calidad sanitaria de la leche, y medianteestudios epidemiológicos sobre enferme-dades humanas relacionadas con la po-blación consumidora de leche, establecióel primer criterio específico para pasteu-rización de leche a 161 ºF y 15 s, con elobjetivo de eliminar los microorganismosmás resistentes al calor, no formadores deesporas.

También en la década de los 90 se esta-blece una regulación en lo referente a con-servas de productos de baja acidez, exi-giendo en las conserveras el alcance de laesterilidad comercial con el objetivo de des-truir Clostridium botulinum. La esterilidadcomercial puede ser definida como la“aplicación de un tratamiento térmico su-ficientemente intenso como para que elproducto esterilizado esté libre de micro-organismos capaces de reproducirse en lascondiciones normales de almacenamientosin refrigeración y libre de microorganismosincluyendo esporas, que puedan compro-meter la salud del consumidor”.

Actualmente existe una amplia gama deopciones para prevenir y controlar lasenfermedades transmitidas por ali-mentos; a nivel de producción, lasBuenas Prácticas Agroalimentarias (BPA)pueden aplicarse y reforzarse, mejo-rando la explotación y el saneamiento,la adopción del sistema “HazardAnalysis and Critical Control Points”

(HACCP) durante toda la cadena de pro-ducción y el control de la contaminacióny temperatura durante el transporte y al-macenamiento deben ser capaces degarantizar la inocuidad de los alimentos;a nivel de los manipuladores de ali-mentos se deben mantener las condi-ciones de higiene adecuadas, BuenasPrácticas Higiénicas (BPH). Por otraparte, las tecnologías de control micro-biológico como la pasteurización y tec-nologías emergentes se ha confirmadoque pueden evitar muchas enferme-dades. Su objetivo final es claro: ayudara producir alimentos más seguros y enconsecuencia, reducir el número debrotes de las enfermedades transmitidaspor ellos (Tauxe, 2002; Panisello, 2000;Pina, 2006).

El sistema y concepto del HACCP se de-sarrolló hace 30 años pero sólo hace unadécada que se ha incluido en el programade control de los alimentos de las autori-dades de Salud Pública, en la UniónEuropea el término HACCP se presentópor primera vez en la Directiva 93/43/CE(1993); reconociéndose como un métodode referencia para el aseguramiento de lainocuidad de los alimentos y un sistemade regulación en los sistemas de controlde los alimentos. El sistema HACCP sebasa en siete principios: 1) análisis deriesgos, identificar los peligros y especi-ficar las medidas de control; 2) identificarlos puntos críticos de control (PCC); 3) es-tablecer límites críticos; 4) establecer pro-cedimientos de vigilancia; 5) establecermedidas correctivas; 6) establecer los pro-cedimientos de verificación y; 7) docu-mentar los procedimientos (Motarjemi,1996; Motarjemi, 1999).


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Control dentro de la UniónEuropeaDe acuerdo con el Libro Blanco para la se-guridad alimentaria (Comisión of theEuropean Communities 2000), los consu-midores deben disponer de una oferta am-plia de alimentos seguros y de alta calidadprocedentes de los Estados miembro. Estoes esencial en el mercado interno de laUnión Europea y significa que cada Estadomiembro tiene una responsabilidad no sólohacia sus propios ciudadanos sino tambiénhacia todos los ciudadanos de la Unión yterceros países por los alimentos producidosen su territorio. Las acciones estratégicas atomar para reducir los peligros deben porlo tanto comenzar a nivel nacional en cadaEstado miembro incluyendo una implica-ción a nivel regional, local e internacional.

Para garantizar la seguridad de los ali-mentos procesados se debe aplicar el aná-lisis de riesgos, cuya misión es mejorar losactuales sistemas de control de alimentos(HACCP).

El análisis de riesgos es un proceso com-plejo, exige un enfoque multidisciplinar eincluye tres componentes básicos: la eva-luación, la gestión y la comunicación delriesgo (Hugas et al, 2007). La evaluaciónde riesgos es una evaluación científica delos efectos adversos, potenciales o efec-tivos, resultantes de la exposición a unconsumo de alimentos en los que se en-cuentran presentes agentes patógenos; lagestión es el proceso que comprende lavaloración de las posibles alternativas paraminimizar o reducir el riesgo o mejorar lasalternativas en aplicación; y la comunica-ción es el proceso interactivo de inter-cambio de información y opinión entreasesores y evaluadores del riesgo.

Durante la pasada década, el análisis deriesgos en seguridad alimentaria fue mejo-rado, llevado a cabo en gran cantidad de es-tudios (EFSA, 2009) e integrado dentro delproceso de vigilancia de la seguridad a nivelnacional, internacional y comunitario, comouna herramienta basada en el conocimientocientífico, herramienta en la que se apoyael proceso de toma de decisión, para me-jorar los sistemas de control de alimentos, ypara fijar prioridades sobre los diferentesproblemas en seguridad alimentaria.

Para asegurar la salud del consumidor, enel proceso de análisis se proponen unaserie de objetivos o criterios de seguridad,así aparece el concepto de “nivel de pro-tección apropiado” (ALOP), el conceptode “objetivo de seguridad o Food SafetyObjective” (FSO). Adicionalmente, elComité de Higiene Alimentaria (CodexAlimentarius Commission, 2003b) definecriterios microbiológicos específicos,desde el procesado de alimentos hasta elmomento del consumo, como sigue:

• El “nivel apropiado de protección” (ALOP)puede ser definido como la expresióncuantitativa de que se produzca un efectoadverso sobre la salud de los consumi-dores o la probabilidad de incidencia deenfermedad.

• “Objetivos de seguridad” (FSO): la fre-cuencia o concentración máxima de lapresencia de un riesgo en un producto enel momento de consumo que contribuyea establecer un adecuado nivel de protec-ción (Appropiate Level of Protection,ALOP).

• “Objetivo de consecución” (Perfor-manceObjective, PO): la frecuencia máxima y/oconcentración de un riesgo en un pro-ducto en un momento específico de la


28

cadena de producción antes del mo-mento de consumo y que contribuye alestablecimiento de un FSO, ALOP comoaplicable.

• “Criterio de realización” (Perfor-manceCriteria, PC): el efecto en frecuencia y/oconcentración de un riesgo en un ali-mento que debe ser conseguido por laaplicación de una o más medidas decontrol para asegurar o contribuir a unPO o a un FSO.

• “Criterio microbiológico” (Microbiolo-gical Criteria, MC): define la aceptabilidadde un producto o un lote de productos,basada en la ausencia o presencia, o nú-mero de microorganismos, incluidos porunidad de masa, volumen o lote.

En ciertos casos, y para microorganismosde gran virulencia, como el caso de E. coli0157:H7 se establecen criterios de “tole-rancia zero”. En 1996, el FISIS (USDA/FISIS,1996) desarrolló un criterio estándar parala carne de vaca cruda, a consecuencia deun brote por E. coli 0157:H7 en el que sedieron 700 casos de enfermedad y 4muertes, por consumo de comida rápidaen un restaurante (Bell et al, 1994). Estecriterio es único y reconoce la práctica fre-cuente de consumidores de este tipo deproducto de cocinar insuficientemente elproducto crudo no destruyendo así los pa-tógenos presentes. Esta práctica aplicatemperaturas que son inadecuadas paradestruir los microorganismos patógenosdel interior del producto, siendo el calen-tamiento superficial, y por ello puedecausar enfermedad, colitis hemorrágica ysíndrome urémico hemolítico, por lo queFISIS estableció el nivel “zero” de toleranciaen 1994.

ConclusionesSe aplicarán criterios de control de micro-organismos en la fuente y en la selecciónde la materia prima, a continuación en eldiseño y ejecución de proceso, mediante laaplicación de buenas prácticas higiénicas, yla mejora de los sistemas de análisis y con-trol de puntos críticos a lo largo de la ca-dena de producción/consumo (FAO/WHO,2001; ICMSF, 2002).

Las autoridades de Salud Pública debenpromover la seguridad alimentaria en lasociedad y, en particular, entre los con-sumidores, para que éstos no sóloadopten prácticas seguras de manipula-ción de los alimentos en sus hogares,sino que también sean capaces de de-mandar Buenas Prácticas Higiénicas(BPH) y aprecien los esfuerzos de las em-presas alimentarias por ofrecer ali-mentos inocuos, siendo estos esfuerzosinnovadores y continuos por parte delas industrias alimentarias de todo elmundo (Motarjemi, 1999). Al mismotiempo deben impulsar el desarrollo delas agencias o autoridades de seguridadalimentaria con la misión de llevar acavo una evaluación de riesgos que per-mita a las autoridades sanitarias llevar acabo políticas de seguridad alimentariaeficaces en España y en los distintosEstados miembro de la Unión Europea.

BibliografíaAltekruse S, Cohen M, Swerdlow D. Perspectives.Emerging foodborne diseases. Centers for di-sease control and prevention, 3. 1997.

Bell BP, Goldoft M, Griffin PM, Davis MA, GordonDC, Tarr PI, Bartleson CA, Lewis JH, Barrett TJ,Wells JG, et al. A multistate outbreak ofEscherichia coli O157:H7 associated with bloodydiarrhea and hemolytic uremic syndrome. 1994.


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Bertolini M, Rizzi A, Bevilacqua M. An alterna-tive approach to HACCP system implementa-tion. Journal of Food Engineering, 2007;79:1322-8.

Codex Alimentarius Commission. Report of the35th session of the Codex Committee on foodhygiene. ALINORM 03/13A, Secretariat of theJoint FAO/WHO Food Standards Programme,FAO, Rome. Available from ftp://ftp.fao.org/codex/alinorm03/ al0313ae.pdf (2003b).

De Swarte C, Donker RA. Towards an FSO/ALOPbased food safety policy. Food Control, 2005;16:825-30.

European Food Safety Authority. Quantitativeestimation of the impact of setting a new targetfor the reduction of Salmonella in breeding hensof Gallus gallus 1. Scientific Opinion of the Panelon Biological Hazards (Question No EFSA-Q-2008-291) 2009.

European Food Safety Authority. Assessment ofthe Public Health significance of meticillin resis-tant Staphylococcus aureus (MRSA) in animalsand foods1. Scientific Opinion of the Panel onBiological Hazards (Question No EFSA-Q-2008-300) 2009.

FAO/WHO. Principles for the establishmentand application of microbiological criteria forfoods. General Requirements. CAC/GL 21-1997. Joint FAO/WHO Food StandardsProgramme, Codex Alimentarius Commission.Food and Agriculture Organization of theUnited Nations/World Health Organization,Rome, Italy. 2001.

Havelaar A, Takumi K, Teunis P, de Jonge R,Garsen J. Modeling the interactions betweenpathogens, their hosts and their environment.Dose-response modelling. Maryland. ArieHavellar. 2002.

Hugas M, Tsigarida E, Robinson T, Calistri P. Riskassessment of biological hazards in the

European Union. International Journal of FoodMicrobiology, 2007; 120:131-5.

ICMSF. “Microorganisms in Foods 7”.International Commission on MicrobiologicalSpecifications for Foods. Kluwer Academic/Plenum Publishers, New York, N.Y. 2002.

Motarjemi Y, Käferstein F, Moy G, Miyagawa S,Miyagishima K. Importance of HACCP for pu-blic Elath and development. The role of theWorld Health Organization. Food Control, 1996;7:77-85.

Motarjemi Y, Käferstein F. Food safety, Hazardanalisis and critical control point and the increasein foodborne diseases: a paradox? Food Control,1999; 10:325-33.

Panisello P, Rooney R, Quantick P, Smith R.Application of foodborne diasease outbreakdata in the development and maintenance ofHACCP systems. International Journal of FoodMicrobiology, 2000; 59:221-34.

Pina M, Ferrer C, Rodrigo M, Klein G, MartínezA. Organization of risk analysis in the EU and thenew EU food regulation. 2006; XX,113:401-32.

Tauxe R. Surveillance and investigation of food-borne diseases; roles for public health in mee-ting objetives for food safety. Food Control,2002; 13:363-9.

Stringer M. Summary report: Food safety objec-tives-role in microbiological food safety mana-gement. Food Control, 2005; 16:775-94.

USDA/FISIS. Pathogen reduction; hazard analysisand critical control point (HACCP) systems. FinalRule. Fed. Reg. 1996; 61(144): 38805-989.

Walls I, Buchanan RL. Use of food safety objec-tives as a tool for reducing foodborne listeriosis.Food Control, 2005; 16:795-9.

Zwietering M. Practical considerations onfood safety objectives. Food Control, 2005;16:817-23.

IntroductionChanges in food supply chains, health anddemographic situations, lifestyle and socialsituations, environmental conditions, and in-creased legislative requirements have led tosignificant efforts in the development ofquality and safety management systems inagribusiness and food industry worldwide(Ropkins and Beck, 2000; Efstratiadis, Karirti,and Arvanitoyannis, 2000; Jacxsens, et al,2009a, Luning and Marcelis, 2009a).Nowadays, companies have implementedvarious quality assurance (QA) guidelinesand standards, such as GMP and HACCPguidelines (like General Principles of foodhygiene (Codex Alimentarius 2003), GFSIguidance document (GFSI (2007), and qua-lity assurance standards (like ISO 9001:2008(2008), ISO22000:2005 (2005), BRC (2008),and IFS (2007) into their company own foodsafety management system. The perfor-mance of such systems in practice is, ho-wever, still variable. Moreover, the conti-nuous pressure on food safety managementsystem (FSMS) performance and the dy-namic environment wherein the systemsoperate (such as emerging pathogens,changing consumer demands, develop-ments in preservation techniques) requirethat 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 theEuropean project entitled ‘PathogenCombat-EU FOOD-CT-2005-007081’ various toolshave been developed to support food com-panies and establishments in systematicallyanalysing and judging their food safety ma-nagement system and its microbiologicalperformance as basis for strategic choiceson interventions to improve the FSMS per-formance. This chapter describes brieflyprinciples of the major tools that have beendeveloped and some others, which are stillunder still under construction.

Quality assurance evaluationgridsThe wide range of quality assurance stan-dards and guidelines commonly leads to dif-ficulties for small and medium enterprises(SME) to select and implement them intotheir company specific Quality ManagementSystem (QMS) and or Food SafetyManagement System (FSMS). It is often hardfor SME’s to understand the detailed diffe-rences between various QA standards andguidelines and to judge the possible conse-quences of implementation, because they

Tools to support the self assessment of theperformance of Food Safety ManagementSystemsPA. Luning, L. Jacxsens, V. Jasson, WJ. Marcelis, J. Kussaga, M. Van der Spiegel,M. Kousta, S. Osés, J. Rovira, F. Devlieghere, M. Uyttendaele


32

not always have the necessary expertise, ex-perience, and resources (e.g. financial, staf-fing capabilities) (Yapp and Fairman, 2006;Aggelogiannopoulos et al, 2007; Karipidis,et al, 2009). Therefore, quality assuranceevaluation grids have been developed,which show the major differences betweenacknowledged QA standards and guidelineson distinct features. The QA evaluation gridsmay support companies in the agri-foodchain to balance the benefits of implemen-ting certain QA standards (and guidelines)against the efforts that are required.Moreover, it might serve as a compact over-view of possibilities and consequences ofimplementing QA standards and guidelineswhen supporting companies to improvetheir own FSMS. The features that havebeen included in the grids are in table 1

summarised. For details the reader is refe-rred to Kussaga and co-authors (2009).

Food Safety ManagementSystem Diagnostic Instrument(FSMS-DI)

Stakeholders (like government, branch or-ganisations, customers, retail, etc) put de-mands on the design of a company’s FSMSby requiring the implementation of certain(sets) of quality assurance (QA) standardsand or guidelines. However, each companyor establishment has a unique company-specific FSMS depending on how standardsand guidelines have been translated intothe own situation (Jacxsens, et al, 2009a;Luning and Marcelis, 2009a). Recently, a

Table 1. Features on which acknowledged QA standards and guidelines have beenevaluated (modified from Kussaga et al, 2009b).

Features related to position of QA Standards and GuidelinesFocus 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 wholeregion/continent like Europe, Middle East), or worldwide recognized.

Features related to type of requirements of QA Standards and Guidelines

Comprehensiveness Comprehensiveness refers to the extent of detail of the requirements,requirements and to how they are in the document formatted.

Extent validity Extent of validity requirements refers to what degree demands are putrequirements on assuring that the system is really effective in practice.Degree of Degree of organisational demands refers to what extent QA standardorganisational and guideline set requirements on typical organisational issuesdemands (like training of personnel, setting procedures).

Tools to support the self assessment of the performance of Food Safety Management Systems

33

diagnostic tool has been developed usinga techno-managerial research approach toconsider both technological factors andpeople behaviour in the performance offood safety management systems (Luningand Marcelis, 2006, 2007, 2009b). The toolis called “food safety management systemdiagnostic instrument” (FSMS-DI). TheFSMS-DI is a tool that enables a systematicanalysis and assessment of a company‘sunique food safety management system in-dependent of the QA standards and or gui-delines that have been implemented(Luning et al, 2008, 2009a, b, c; Jacxsenset al, 2009c). The instrument consists ofcomprehensive lists with sets of indicatorsto analyse respectively which core controland core assurance activities are addressedin the company specific FSMS, which majorcontextual factors could affect FSMS per-formance, and to analyse the microbiolo-gical safety performance of the system.Moreover, the FSMS-DI encompasses gridsto assess respectively levels of control andassurance activities (i.e. more or less ad-vanced), contextual situations (i.e. more orless ‘risky’) wherein the FSMS has to ope-rate, and the microbiological safety level.For each indicator, to assess core control orassurance activities or food safety perfor-mance, four different levels have been des-cribed (i.e. 0, 1, 2, and 3 representing a low,

basic, average, and advanced level respec-tively). Similarly, for each indicator, to assesscontextual factors, three different risk levelshave been described (i.e. 1, 2, and 3 repre-senting low, moderate and high-risk con-text respectively).

The elements of the FSMS-DI are summa-rised in figure 1, it starts with introductoryquestions followed by defining a represen-tative production unit for which a QA ma-nager can do the self assessment (part I).Part II includes the indicators and grids toassess the major contextual factors ‘productcharacteristics’, ‘process characteristics’, ‘or-ganisational characteristics’, and ‘chain en-vironment characteristics’. Part III is for as-sessment of the core control activities‘design of preventive measures’, ‘design ofintervention measures’, ‘design of monito-ring systems’, and ‘actual operation of con-trol measures’, whereas the core assuranceactivities ‘setting system requirements’, ‘va-lidation’, ‘verification’, and ‘documentationand record-keeping are covered in part IV.Part V includes the indicators (called theFood Safety Performance Indicators FSPI)and grids to assess ‘internal’ and ‘externalfood safety performance’ (Jacxsens et al,2009c). The assumption behind the FSMS-DI is that companies operating in a high-risk context (due to highly risky product and

Table 1. Features on which acknowledged QA standards and guidelines have beenevaluated (modified from Kussaga et al, 2009b) (continuation).

Features related to Certification of QA standardsScope 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 waycertification certification requirements can be fulfilled.Frequency Frequency of certification refers to how often certification audits must

be by third parties carried out.


34

processes, less supporting organisationalconditions, highly vulnerable and dependchain position) need to have an advancedFSMS (i.e. based on precise information,scientifically underpinned, criticallyanalysed, procedure-based, systematic, andindependent) to realise a predictable andcontrollable food safety performance. In amoderate-risk context an average FSMS isexpected to be sufficient to realise a goodFS performance, while in a low-risk contexteven 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 mea-

sured by experiments using the microbial

Assessment Scheme (MAS) of Jacxsens and

co-authors (2009b) (Section 4).

Figure 1. Overview of elements of food safety management system diagnostic instrument.

PART I: introductory section for Food Safety Management System (FSMS)A. Introduction questions (1 -11)B. Selection of Representative Production Unit (RPU) for self-assessment (12-20)

PART II: assessment of contextual factorsA. Assessment of product characteristics (A1-3)B. Assessment of process characteristics (B4-6)C. Assessment of organisation characteristics (C7-13)D. Assessment of chain environment characteristics (D14-17)

PART III: assessment of core safety control activitiesE. Assessment of preventive measures design (E18-23)F. Assessment of intervention processes design (F24-27)G. Assessment monitoring system design (G28-34)H. Assessment of operation of preventive measures, intervention process and (H35-41)

monitoring systems

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

assurance

PART V: assessment of food safety performanceM. EXTERNAL Food Safety Performance (M51-54)N. INTERNAL Food Safety Performance (N55-57)


35

The FSMS-DI has been tested and vali-dated in pre-tests and a pilot study with15 food producing companies in the areaof dairy, pork, beef & lamb, and poultryproducts. Moreover, the instrument hasbeen slightly adapted and applied in thecatering sector (50 food service establish-ments) in Spain (Chinchilla, 2009).Recently, the ‘paper based’ instrumenthas been transformed to a ‘web based’application, i.e. the FSMS self assess-ment tool. This self assessment tool isnow used for a quantitative study to as-sess food safety management systems indairy, pork, beef & lamb, and poultrycompanies in Europe. For details aboutthe diagnostic tool, the reader is referredto Luning and co-authors (2008, 2009 a,b, c), Jacxsens and co-authors (2009c),and the Pathogen Combat website(www.pathogencombat.com).

Microbiological AssessmentScheme (MAS)

As previously stated, the actual microbiolo-gical performance of FSMS in practice is stillvariable (e.g. Cormier et al, 2007; Manninget al, 2006; Tsalo et al, 2007). In fact, atten-tion has been shifted from implementingQA standards to better understanding theperformance of an FSMS (Doménech et al,2008; Luning et al, 2008; Stringer and Hall,2007) and various audit tools have beendeveloped to determine performance to-wards certain QA standards (e.g. Wallaceet al, 2005; CIES, 2007; Cormier et al,2007). However, these audit tools basicallycheck on compliance to the set require-ments, for instance, during internal or ex-ternal auditing (Van der Spiegel et al, 2005),whereas the FSMS-DI focuses on crucialcontrol and assurance activities (not linkedto specific QA standards). Although, theFSMS-DI can give an indication about themicrobiological safety performance, it givesrestricted insight in the actual microbiolo-gical performance.

In practice, food processing companiescommonly use microbial testing of finalproducts to assess if their products meetfood safety criteria (e.g. ICMSF, 2002;Legan, 2001). These criteria are set by dif-ferent stakeholders or regulatory bodies(like EU and/or country regulations and/orcustomers’ requirements), but can also beused to guide the evaluation of a manu-facturing process to define preventive ac-tions (Kvenberg and Schwalm, 2000;Martins and Germano, 2008). However,no procedure to systematically evaluatethe microbiological performance of aFSMS was yet available. Therefore, theMicrobial Assessment Scheme (MAS)

Figure 2. Principle assumption behind the researchwork of tools to measure the performance of FoodSafety Management Systems.

FS

3

2

1

Context FSMS

3=bestperformance

3=mostdangerous

3=highestlevel

3

2

1

3

1

2


36

tool has been developed to support a sys-tematic analysis of microbial counts to as-sess the current microbial performance ofan implemented FSMS.

The MAS tool is a procedure that definesthe identification of critical sampling loca-tions (CSL), the selection of microbiologicalparameters, the assessment of sampling fre-quency, the selection of sampling methodand method of analysis, and finally dataprocessing and interpretation (figure 3).

Based on the MAS assessment, microbialsafety level profiles can be derived, indi-cating which microorganisms and to whatextent they contribute to microbiologicalsafety for a specific food processing com-pany. A microbial safety level can be clas-sified from 1 to 3, where level 3 reflects a

good performance (legal criteria or guide-lines are respected, no improvements areneeded –current level of FSMS is highenough to cover this hazard), level 2 co-rresponds with a moderate performance(legal criteria or guidelines are exceeded,improvements need to be made on asingle control activity of the FSMS) andlevel 1 represents a poor performance(legal criteria or guidelines are exceeded,improvements need to be made on mul-tiple control activities of the FSMS). Thesum of the levels is resulting in the micro-bial food safety level profile. The principlebehind the MAS tool is that low numbersof microorganisms and small variations inmicrobiological counts imply a well func-tioning FSMS (Jacxsens et al, 2009b).

Identification CriticalSampling locations CSL)

Locations provide info about microbial performance of product flow(e.g. raw materials, intermediated food products and final foodproducts) and core control strategies (e.g. contact surfaces, hands ofpersonnel, 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, 3times (days), 3 times a day

Product sampling & surface sampling per CSLUse of ISO methods for sampling & analysis (reliable, accurate,robust) or via acknowledged and validated alternative methods

Show variability in raw dataUse legal (available) criteria to judge outcome Development food safety level profiles

Selection microbiologicalparameters

Assessment sampling frequency

Selection sampling method& method of analysis

Data processing& interpretation of data

Figure 3. Steps of the MAS scheme (modified from Jacxsens et al, 2009b).


37

A MAS scheme will differ depending on theproduction processes and food type thatare addressed in the specific company.MAS-schemes have been specified for mi-crobiological analyses in respectively poultry,dairy, beef & lamb, and pork companies.Depending on the product and productionprocesses, specific microorganisms havebeen selected to indicate respectively safety(e.g. Listeria monocytogenes, Salmonellaspp, Campylobacter), hygiene indicators(e.g. E. coli and Enterobacteriaceae, Staphy-loccocus aureus), and overall performance(total aerobic count). Data provided in-depth insight in microbiological counts inproduct flows (both raw materials, interme-diate, and final products), contact surfaceareas (like at critical cutting areas, knives,conveyor belts, etc), and people (hands andgloves).

The detailed MAS data provide insight inwhich indicator microorganisms exceed li-mits and at which critical locations, but alsoreveals the extent of variation in microbio-logical counts. The microbial safety levelprofiles give an immediate insight in theroom o improvement and or which micro-biological parameters. These profiles canalso be used to compare the microbiolo-gical performance of different companieswith the same type of production processesand food products as benchmarking tool.As such, microbiological problems in asector can be identified, independent of thetype of company. The food safety perfor-mance indicators (FSPI) have been analysedon their indicative value by comparing datawith the extensive MAS data for nineEuropean companies. (Jacxsens et al,2009c). The food safety performance diag-nosis can be a useful tool to have a first in-dication about the microbiological perfor-

mance of an operational food safety ma-nagement system.

For more details the reader is referred toJacxsens and co-authors (2009a, 2009c),and Sampers and co-authors (2009), andKussaga and co-authors (2009b).

MAS analysis methodselection toolDifferent authors recommended the use ofmicrobial testing to evaluate critical controlpoints (e.g.), to evaluate procedures forGood Hygienic practices (GHPs) andStandard Operating Procedures (SOPs) (e.g.Brown et al, 2000; Swanson andAnderson, 2000; Kvenberg and Schwalm,2000; Gonzalez-Miret et al, 2001; Cormieret al, 2007; Martins and Germano, 2008).The MAS-scheme can support food safetyexperts in systematically designing a tai-lored scheme to asses the microbiologicalperformance of implement ted food safetymanagement systems (Jacxsens et al,2009b). According to the MAS protocolappropriate methods for sampling andanalyses of pathogens and other micro-or-ganisms (to indicate hygiene or total per-formance) need to be selected. In the cu-rrent MAS protocol, the authors refer tothe use of internationally acknowledgedsampling and analysis methods accordingto ISO standards. However, nowadays awide range of methods to sample and oranalyse micro-organisms (and specificallypathogens) are existing or have been re-cently developed. Each method has its ownspecific characteristics, which may affectthe choice of a certain method.

Therefore a MAS analysis method selec-tion tool has been developed, which canaid in the process of decision-making regar-


38

ding selection of microbial analysing met-hods in specific situations. A comprehen-sive review of literature regarding differentenumeration and detection method wasperformed. Based on this review specificmethod characteristics were determinedthat have used as a parameter in the selec-tion tool. The major characteristics onwhich the methods have been analysed areshown in table 2. Moreover, a decision treewas made that allows classifying the MA

Regarding the selection tool it should benoticed that no method exist that is a100% sensitive, 100% specific, that can beperformed in real-time and that is comple-tely without costs. All methods have advan-tages and disadvantages. The challenge isto select the method that fulfils the most ofthe characteristics of the ideal method in aspecific situation. Advantages of a methodshould be optimally exploited and the di-sadvantages should be recognized. The se-

that needs to be performed. The decisiontree is based on a techno-managerial pointof view.

lection tool can aid in finding the most ap-propriate method for a specific situation inneed of microbial analysis.

Table 2. Some examples of characteristics on which microbiological methods have beenevaluated as basis of the MAS method analysis selection tool (modified from Jasson etal, 2009).

Characteristics Brief descriptionAlternative method An alterative method is a method of analysis that demonstrates or

estimates, for a given category of products, the same analyte as ismeasured by using the corresponding reference method. Thisalternative method can be proprietary or non-commercial and coversan entire analysis procedure, that is, from the preparation of samplesto the test results either as such or may include references to otherprocedures in order to be complete. The alternative method exhibitsattributes appropriate to the user’ needs, e.g.: speed of analysis and/orresponse, ease of execution and/or automation, analytical properties,miniaturisation or reduction of cost

Time Total time to result is the time needed from sample until countingresults or presence/absence result (confirmation not included)

Matrix Food matrix. A method should be applicable for the food matrix ofinterest

Validation Users of commercially available kits (proprietary methods) needcertificate guarantees regarding the performance of these kits. Validation of

alternative methods is a process that determines if an alternativemethod can obtain the same analyte as is measured by using thecorresponding reference method

Type of microbial Multi-functionality of the method regarding different microorganismsparameters


39

For details about the MAS analysismethod selection tool, the reader is refe-rred to Jasson et al, (2009).

Improvement roadmap forFSMSAfter companies have analysed theirsystem by using the self assessment toolFSMS-DI alone or in combination withMAS, they have detailed insight in the le-vels at which they execute their core con-trol and assurance activities (ranging fromabsent, low, medium to high (= level 3).They also have an idea about the typicalcontextual situation wherein their systemhas to operate (ranging from highly, to res-tricted and not vulnerable, ambiguous anduncertain (situation 3-1) moreover, theyhave an indication about the (actual) mi-crobiological performance. As previously,stated the principle behind the diagnosis isthat companies that operate in a morerisky context (i.e. more vulnerable, ambi-guous and uncertainly) require a more ad-vanced (high level) FSMS to be able to re-alise and ensure safety requirements(Luning et al, 2009b). If the assessmentdata reveal food safety levels below 3(Jacxsens et al, 2009b,c) and this is per-ceived as a problem, then a companycould first consider those core control andassurance activities that are at level 1 (is as-sociated with aspect, like not scientificallyunderpinned, general, not structured, in-complete, not independent) or at level 0(absent, not used, unknown), to considerpossible interventions in the FSMS to im-prove the performance. However, one canalso consider those contextual factors thatare allocated in situation 3, to identify pos-sible interventions in the contextual situa-

tion, which are commonly long-term inter-ventions (like changing production process,increasing competence level of operators,improving information system, enhancingsupplier relationships, etc) (Luning andMarcelis, 2009a; Luning et al, 2009c).

To support the improvement process, ge-neric roadmaps have been made showinghow to go through the different steps ofan improvement process The systematic ap-proach is based on the principles of thefood quality relationship model (food qua-lity = f (food behaviour, human behaviour),the food quality management decisionsgrid, and the principles of improvementprocesses (Luning and Marcelis, 2006,2007, 2009a, b). The basic steps of an im-provement cycle are: 1) map problem area,i.e. collecting information and documenta-tion, 2) analyse problem area: i.e. identifi-cation of causes and effects, and 3) rede-sign: i.e. development and implementationof solutions as depicted in Figure 4.

Improvement processes are characterisedby a gradual nature, it is a step-by-step on-going process. Depending on the startingsituation, improvements can vary fromsimple measures to reduce variation in pro-ducts and decision-making on the shortterm, to changes in the infrastructure onthe long-term. Using the food quality rela-tionship we have defined three levels of in-creasing improvement efforts, i.e. a)changes in product and people behaviour,b) changes in technological and decision-making process conditions, and c) changesin the technological and organisational in-frastructure. After each improvement cyclethe new situation should be reassessed inorder to judge the effect of the improve-ment. Subsequently, the new situationmust be assured (Luning and Marcelis,


40

2009). Using above approach, an exampleof a roadmap has been elaborated indica-ting typical activities that could be done byfood companies when they want to im-prove problems with aw materials, table 3shows typical activities in the three impro-vement steps for the different levels of im-provement. The activities are a selection ofinformation gathering, analyses methods,and improvement measures addressingboth technological and managerial issuesto demonstrate how food companies cansystematically improve their FSMS. Compa-nies have to select themselves which tools,techniques, and methods are most suitablefor their own situation.

The principle of generic roadmaps for im-provement will be further in the near fu-ture (Luning et al, 2009c).

Additional supporting toolsData from the pre-tests and pilot studies in-dicated that validation and verification ac-tivities but also design of sampling plans are

still not yet well worked out in practice.Protocols have been developed to supportcompanies in improving their validation andverifications activities, and a protocol to im-prove design of sampling plans.

To support companies in improving theirFSMS they need to have access to informa-tion, knowledge, and experience aboutthese tools. In this perspective, a food sa-fety management support system hasbeen developed to provide in a systematicway information about control and assu-rance principles, supporting tools (like newenumeration, detection and monitoringtechniques for pathogens, new interven-tion techniques and methods, protocolsand procedures on sanitation, validation,verification, microbial assessment, etc), prin-ciples and structure of acknowledged qua-lity assurance standards and guidelines, andlegislative requirements.

The FSMS support system is available viathe Pathogen Combat website (www.pat-hogencombat.com).

Level of improvemenet efforts

1. Map problemsituation

2. Analyseproblem

Roadmap1

2

3

1

2

3

1

2

33. Redesign

a. Change productand people behaviour

b. Change technologicaland decision-makingprocess conditions

c. Change technologicaland organisationalinfrastructure

Figure 4. Generic approach to develop roadmaps.


41

Final considerationsIt is evident that an implemented FSMS ina company in the agri-food chain must beseen as a dynamic system, which needs tobe frequently analysed, judged, improved,and tailored to the actual and changing si-tuation with respect to the control and as-surance activities and the contextual factorsaffecting the performance of the company’sunique FSMS. The FSMS self assessmenttool in combination with the FSMS supportsystem (including all relevant tools deve-

loped in PathogenCombat, useful guide-lines, legislative requirements, scientificknowledge) can be used to search forknowledge, information and tools toanalyse, judge, and improve an imple-mented FSMS.

BibliographyAggelogiannopoulos D, Drosinos EH, Athanaso-poulos P. Implementation of a quality manage-ment system according to the ISO 9000 familyin a Greek small-sized winery: A case study. FoodControl; 18 (9):1077-85.

Table 3. Typical activities in the improvement steps for problems with ‘Raw materials’

a. Change product andpeople behaviour

b. Change technologicaland decision-makingprocess conditions

c. Change technologicaland organisationalinfrastructure

1. Mapproblemsituation:gatheringinformation

Gather materialsinformation, likerejections, incidencereports, complaints,microbial loadproducts, % realisedinspections

Gather process conditioninformation, like storagetemperatures, complaints,actual availability of andcompliance to procedures,actual availability ofmaterials and supplierinformation

Gather information onstorage facilities andsuppliers, like microbialload walls and floors,supplier performance,communicationproblems, quality systemperformance

2. Analyseproblems:methodsand tools

Use basic statistictools, andbrainstorming foranalysing structuraldeviations

Use CCP analysis, riskanalysis, Total ProductiveMaintenance, and literatureanalysis

Use CCP analysis,hygienic designmethods/principles, riskanalysis, predictivemodelling, and literatureanalysis

3. Redesign:improvement options

Possible measures forimprovement: changecorrective actions,change inspectionfrequency, changefrequency of recordingdata, changeinstructions, intensifysupervision

Possible measures forimprovement: changeincoming materialinspection, change storagetemperature control,change corrective measures,change procedures,training, intensify supportquality department,intensify information supply

Possible measures forimprovement: buildingconditioned storagerooms, change suppliers,intensify supply chainrequirements, changesupplier agreements,change organisationalresponsibilities


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BRC. Global standard for food safety (issue 5).www.brc.org.uk. 2008.

Chinchilla K. Assessment of food safety in food-service establishments. PhD thesis BurgosUniversity Spain (in preparation). 2009.

CIES. Global Food Safety Initiative Guidance do-cument 5th edition, [email protected],2007; 41 pp. Accessed 8th of July 2008.

Cormier RJ, Mallet M, Chiasson S, MagnússonH, Valdimarsson G. Effectiveness and perfor-mance of HACCP-based programs. FoodControl, 2007; 18:665-71.

Doménech E, Escriche I, Martorell S. Assessingthe effectiveness of critical control points to gua-rantee food safety. Food Control, 2008; 19(6):557-65.

Efstratiadis MM, Karirti AC, ArvanitoyannisIS. Implementation of ISO 9000 to the foodindustry: an overview. International Journalof Food Sciences and Nutrition, 2000; 51(6):459-73.

González-Miret ML, Coello MT, Alonso S,Heredia FJ. Validation of parameters in HACCPverification using univariate and multivariatestatistics. Application to the final phases ofpoultry meat production. Food Control, 2001;12:261-8.

GFSI. Global Food Safety Initiative version 5.2007. www.ciesnet.com

IFS. International Featured Standards. 2007;http://www.food-care.info

ISO 22000: 2005. Food safety management sys-tems. Requirements for any organization in thefood chain, 2005; http://www.iso.org/iso

ISO 9000. Quality management systems.Fundamentals and vocabulary. 2005.http://www.iso.org/iso

Jacxsens L, Devlieghere F, Uyttendaele M.Quality Management Systems in the FoodIndustry. Book in the framework of Erasmus,2009a.

Jacxsens L, Kussaga J, Luning PA, Van derSpiegel M, DeVlieghere F, Uyttendale M. A mi-crobial assessment scheme to support microbialperformance measurements of food safety ma-nagement systems. International Journal of food

Microbiology. doi: 10.1016/j.ijfoodmicro.2009.02.018. 2009b.

Jacxsens L, Uyttendaele M, Devlieghere F,Luning PA. Food safety performance indicatorsto benchmark food safety output of food sa-fety management systems. Submitted inSpecial Issue International journal of foodMicrobiology.

Jasson V, Jacxsens L, Luning PA, Uyttendaele M.A selection tool for alternative rapid microbial met-hods in the framework of food safety manage-ment systems performance (in preparation). 2009.

Karipidis P, Athanassiadis K, Aggelopoulos S,Giompliakis E. Factors affecting the adoption ofquality assurance systems in small food enter-prises. Food control, 2009; 20 (2):93-8.

Kussaga J, Jacxsens L, Luning PA, Kousta M,Drosinos EH, Uyttendaele M. Quality assu-rance grids to support agribusiness and foodcompanies in selecting QA standards andguidelines. Submitted in Food Control.2009a.

Kussaga J, Luning PA, Jacxsens L, Van BoekelMAJS, Uyttendaele M, Marcelis WJ.Interpreting results of FSMS diagnosis and mi-crobiological assessment as a basis for stra-tegic decisions on interventions to improveFSMS. (in preparation). 2009b.

Kvenberg JE, Schwalm DJ. Use of microbialdata for HACCP- food and drug administra-tive perspective. Journal of Food Protection,2000; 63:810-4.

Legan JD, Vandeven M, Dahms S, Cole M.Determining the concentration of microorga-nisms controlled by attributes sampling plans.Food Control, 2001; 12:137-47.

Luning PA, Marcelis WJ. A techno-managerialapproach to food quality management. Trendsin Food Science and Technology, 2006;17:378-85.

Luning PA, Marcelis WJ. A Food QualityManagement functions model from a techno-managerial perspective. Trends in Food Science& Technology, 2007; 18 (3):159-66.

Luning PA, Marcelis W. Food QualityManagement: technological and managerialprinciples and practices. Wageningen


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Academic Publishers: Wageningen, TheNetherlands. ISBN: 987-90-8686-116-3. 2009a.

Luning PA, Marcelis WJ. A food quality mana-gement research methodology. Integrating tech-nological and managerial theories. Trends inFood Science & Technology 2009b; 20:35-44.

Luning PA, Bango L, Kussaga J, Rovira J,Marcelis WJ. Comprehensive analysis and dif-ferentiated assessment of food safety controlsystems: a diagnostic instrument. Trends inFood Science & Technology, 2008; 19:522-34.

Luning PA, Marcelis WJ, Rovira J, Van der SpiegelM, Uyttendaele M, Jacxsens L. Systematic assess-ment of core assurance activities in companyspecific food safety management systems.Trends in Food Science & Technology, 2009a; 20:300-12.

Luning PA, Marcelis WJ, Rovira J, Van Boekel,MAJS, Uyttendaele M, Jacxsens L. Assessmentof context factors on riskiness within the FoodSafety Management System-DiagnosticInstrument (FSMS-DI). Submitted in SpecialIssue International Journal of FoodMicrobiology. 2009b.

Luning PA, Jacxsens L, Kussaga J, Kousta M,Uyttendaele M, Drosinos E, Marcelis WJ.Assessing food safety management systemperformance: a critical evaluation of microbio-logical safety performance against requiredfood safety control and assurance activitiesSubmitted in Special issue International JournalFood Microbiology. 2009c.

Manning L, Baines RN, Chadd SA. Food safetymanagement in broiler meat production.British Food Journal, 2006; 08 (8):605-21.

Martins EA, Germano PML. Microbiological in-dicators for the assessment of performance inthe hazard analysis and critical control points(HACCP) system in meat lasagne production.Food Control, 2008; 19:764-71.

Kvenberg JE, Schwalm DJ. Use of microbialdata for HACCP- food and drug administra-tive perspective. Journal of Food Protection,2000; 63:810-4.

Ropkins K, Beck AJ. Evaluation of worldwideapproaches to the use of HACCP to controlfood safety. Trends in Food Science &Technology, 2000; 11 (1):10-21.

Sampers I, Jacxsens L, Luning PA, MarcelisWJ, Dumoulin A, Uyttendaele M. Relationbetween Campylobacter contamination andperformance of Food Safety ManagementSystems in poultry meat industries.Submitted in Journal of Food Protection.2009.

Swanson KMJ, Anderson JE. Industry perspec-tives on the use of microbial data for hazardanalysis and critical control point validationand verification. Journal of Food Protection,2000; 63:815-8.

Tsalo E, Drosinos EH, Zoiopoulos P. Impact ofpoultry slaughter house modernisation andupdating of food safety management sys-tems on the microbiological quality and sa-fety of products. Food control, 2007;19:423-31.

Van der Spiegel M, Luning PA, de Boer WJ,Ziggers GW, Jongen WMF. How to improvefood quality management in the bakery sector.NJAS-Wageningen Journal of Life Sciences,2005; 53 (2):131-50.

Van der Spiegel M, Luning PA, Boer de WJ,Ziggers GW, Jongen WMF. Measuring effecti-veness of food quality management in the ba-kery sector. Total Quality Management andBusiness Excellence, 2006; 17 (6):1-19.

Wallace CA, Powell SC, Holyoak L.Development of methods for standardisedHACCP assessment. British Food Journal,2005; 107 (10):723-42.

Yapp C, Fairman R. Factors affecting food sa-fety compliance within small and medium-sized enterprises: implications for regulatoryand enforcement strategies. Food Control,2006; 17 (1):42-51.

Introduction

This article is based on a presentationgiven at the PathogenCombat/EHEDGSME Workshop at the University ofBurgos, Spain, on 18th June 2009. Duringthe PathogenCombat project, CockerConsulting Ltd had the objective of wor-king with SMEs on hygienic engineeringand design, with a secondary responsibi-lity in the objective of minimising the for-mation of biofilms and assisting in the de-velopment of practical advice on how toremove biofilms once they had formed.The advice contained in the presentationat the PathogenCombat workshops inLjubljana, Copenhagen, Burgos and in fu-ture in Copenhagen and Budapest wasbased on my background as first, a gra-duate microbiologist, followed by a doc-torate and extensive practical learning inthe field of aseptic and hygienic designand engineering. This work included visitsin Spain and Ireland to SMEs in the meat,poultry and dairy sectors and visits tothese sectors, plus fish processing, on be-half of Tesco and many other clients, bothSMEs and those with world famousbrands.

This article concentrates mainly on issuesapplying to open equipment, which isused extensively in meat, poultry, fish anddairy processing.

Conclusions • To a greater or lesser extent, food-proces-

sing organisations operate with hazardsand risks to food safety. Risk is the pro-duct of the magnitude of a hazard and itsprobability. In my extensive visits and au-dits, many food-processing organisationsdid not have the insight in hygienic de-sign and engineering to assign a proba-bility to the various hazards. They unkno-wingly accepted an excessive number andmagnitude of hygiene risks because theoccurrence had also been zero. This wasdespite that fact that a future occurrenceof any one of these hazards could wrecktheir business and seriously injure or killconsumers.

• The importance of good moisture ma-nagement and hygienic design was notunderstood. The following exampleswere repeated (in large organisationsand SMEs alike:

– Regulators and auditors demandingwashing of a dry process area.

– An emphasis on performing the ritualof cleaning, rather than on preventionby design.

– Cleaning that was not working pro-perly and leaving a biofilm. Some ofthese biofilms were not visible innormal light, but they were visibleusing U.V. light.

Prevention of biofilm formationand foodborne infections by controlof moisture managementR. Cocker


46

– Equipment left wet overnight or for awhole weekend after cleaning.

– Footwear poorly cleaned on exitingproduction areas and on re-entering.

– Poor drainage, poor access for inspec-tion and cleaning, wet films, conden-sation and aerosols.

– There was often a mistaken confidencein the effectiveness of cleaning-mostopen equipment had many unrea-chable crevices, formed by unsealedjoints, tack welds and threaded fittingsyet owners felt that their cleaning waseffective.

– Many suppliers, inspectors, veterina-rians and auditors lacked hygieneknowledge.

– The users were not very aware either.There were both product safety andoccupational hazards, stemming frompoor control of moisture.

• The poor moisture control and poor hy-gienic design was associated with exces-sive environmental and cleaning costs.

• Most new, CE-marked food processingequipment (estimate: over 70%) did notcomply with the hygiene provisions of theMachinery Directive, 98/37/EC (“TheDirective”).

• The user organisations did not knowthis and did not understand their rightsto have equipment and instructions thatcould allow them to produce safe food.

• Claims related to alleged deficiencies inhygienic design which Cocker ConsultingLtd and fellow consultants had been in-volved with were settled out-of-court,with confidentiality conditions. Thismeant that not only were the hygiene

provisions of 98/37/EC not policed, therewas also a lack of publicity to warn otherequipment suppliers of the size of the riskto their businesses. Note: The Directive,98/37/EC will be replaced by Directive2006/42/EC from December 2009.

• Aggressive fluxes of energy, thermal tre-atments and chemicals were thoughtunavoidable. However, hygienic buil-dings and equipment were needed inorder to realise the benefits of “ecolo-gical” cleaning methods, longer processtimes, increased safety and lower costs.

• A common target was “visually clean”.This could be misleading.

• Enforcement and customer demandsoften suggested poor knowledge.

• Inspectors, regulators and auditors ne-eded better training in hygienic design.

• Operators, fitters, quality assurance per-sonnel, engineers and designers also ne-eded better training in hygienic design.

• Apart from the knowledge of the designprinciple, engineers, designers and othersneeded an outline of the functional pro-perties of microbes that were relevant tothe design principles, together with theknowledge of how these functional pro-perties relate to hygienic design. This ap-plied not just in the specific area of equip-ment design, but also to training and tothe whole food production systems, or-ganisations and procedures.

• The reasons and evidence for the designsin EN 1672-2 are not explained. Moreknowledge was needed in the industry ofthe hygiene provisions of The MachineryDirective and EN 1672-2, the standardthat was written to explain how to meetthese provisions.

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

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• It was concluded that at best the engi-neers and designers at equipment ma-nufacturers lack the microbiologicalknowledge mentioned above, whichmay make the hygienic design principlesin EN 1672-2 credible to them and alsothe skills in food technology necessaryto carry out a hygiene risk assessment,as required by The Directive.

• A scientific basis for designs and for thevalidation of equipment, such as that ofthe European Hygienic Engineering andDesign Group (EHEDG), was needed.

Explanations • The Role of Moisture in the establish-

ment of Biofilms:

– Many microbes, including bacteria,can either swim or grow via films ofliquid. Stagnant liquid provides aready breeding-ground for microbesand cleaning sprays may redistributepathogens from such pools.Unwittingly, manufacturers may assistthis by promoting a wet environment.Even the distinctions of product andnon productcontact surfaces, onwhich the designs may rely for theirhygienic performance, can becomemeaningless under such conditions.

In case further motivation is needed, the re-port of the United Kingdom Health andSafety Executive established that 30% of allmajor injuries were slips and that 90% ofthese slips were caused by wet floors. 95%resulted in broken bones and 1,000,000days were lost per annum, at an averagecompensation cost of STG 4,000 per acci-dent.

• The transport of microbes through theair:

– Because of electrostatic attraction, mi-crobes in the air are inevitably atta-ched to other particles. They have noactive means of flying, but are insteadalways passengers, either in liquid orsolid aerosols, or in sprays.

• Dry conditions and products:

These do not support the growth orpropagation of microbes.

• Moisture Management:

Water is a major component of most foodproducts and processing often involvestransfer of this water from the food.

In cleaning, water is the dominant carrierand 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 ofa preventive strategy, embodied in thebasic principles of hygienic design (Seethe free-to-download Document #8www.ehedg.org). The key point is thatequipment and its surroundings need toshed liquid quickly and to retain it onlyon demand, for example by closure ofa drain valve or by turning a vesselupright. There was poor recognition ofthe ability of horizontal surfaces to re-tain liquids, especially downwards-fa-cing surfaces. Many of these down-wards-facing surfaces were not easilyaccessible for inspection or cleaning.This adaptation of a schematic from theEHEDG Trainers’ Toolbox illustrates thepoint for framework.

The image shows the effect under-neath a ventilator and underneathadjacent cylindrical and square-sec-tion piping:

And below are such areas immediatelyabove a conveyer belt:

Active moisture removal usually con-sumes energy, for example ventilation,extraction, heating and the use of ab-sorbent materials, rubber blades, and,for closed systems, vacuum. It is oftennecessary after wet cleaning.

– Protection of microorganisms by soilResidues of food products and biofilmson equipment have been shown toprotect microorganisms from respecti-vely thermal and chemical treatmentsdesigned to kill them.


49

It has also been demonstrated that thedeeper the crevice, the greater the pro-tection that is afforded by the soil.

There are plenty of examples of suchcrevices in recentlypurchased equip-ment, such as this at an SME dairy:

The dairy in question was consideringpurchase of a further scraped surfaceheat exchanger, so advice was providedon improved designs.

The “Phoenix” • A principle difference between micro-

bial contaminants and other contami-nants such as chemicals and foreign bo-dies is that microbes are capable ofre-growing after any setback. Dilution

is perfectly effective for controlling che-mical contaminants, for example, butusually offers only temporary relief,where microbes are concerned.

• A typical sequence is that crevices andhidden surfaces collect proteins, fats andmicrobes, then escape effective cleaningand detection, even though 99.99% ofthe rest of the surfaces are very well cle-aned and disinfected. The managers in-volved conclude on the basis of visual ins-pection and possibly, point-sampling ofaccessible surfaces, that they have cleanequipment. Each crevice is then a readylocus of contamination, which can leadto dissemination and biofilm formation,and then in turn to more frequent conta-mination events and to increased conta-mination-levels. This is especially so if theequipment does not have dry surfaces.

It is worth ensuring that all concerned un-derstand the importance and mechanicsof biofilm consolidation, which is mea-sured in hours and days and is characte-rised by physiological and metabolicchanges that lead to increased resistanceto lethal agents, increased adhesion andan ability to survive in the presence of lownutrient concentrations. As we all knowfrom the biofilms that form on our teeth,mechanical force is needed to removebiofilms, once consolidation has takenplace, and because of the kinetics of bio-film consolidation, the frequency of cle-aning is crucial to maintaining control.

In the presence of nutrients, thegrowth-rate of microbes can vary fromexponential down to apparent stasis,depending on nutrient sufficiency. Thegreen curves in the following schema-tics represent a hygienic design, versusa non-hygienic design (yellow).


50

This can allow significant advantages tobe gained from increasing levels of hy-gienic design, until ultimately for asepticsystems, the absence of contaminatingmicrobes means that there is no growthof contaminants, and extremely longprocessing times are possible, withoutthe need for cleaning or sterilization.

• Aerosol Formation

An appreciation of necessary hygienic de-sign considerations for open systems re-quires some understanding of aerobio-logy, or the mechanisms by whichmicroorganisms survive and spread whilstin the air. Even a drop of liquid fallingfrom a pipette onto a lab bench gene-rates a large quantity of micro-droplets.(Dimmick & Akers, 1969). The rate andintensity is proportional to the mechanicalenergy-flux (Dimmick factor). In a food-processing environment, examples in-clude high-pressure jets from cleaning,high-speed fans and high-speed slicers.Direct and reflected spray can carry mi-croorganisms and nutrients a few feetaway, but the associated aerosol can becopious and can spread much further.The finest aerosols have neutral buoyancyin the air and hang in the air for a verylong time. This is why, for example, jetcleaning should never be performed inrooms where food or clean equipment iswithin reach of the aerosol cloud. It is alsowhy high-speed, water-lubricated, slicer-blades can give problems like those atMaple Foods in Ontario. The spray andaerosol from the disc disseminates mois-ture, microbes and food, and there isample time between cleaning shifts formultiplication. There were 22 deathsfrom Listeria monocytogenes in this out-break.


51

High-pressure sprayers have been shownto become contaminated and to back-contaminate the water supply in foodplants (Gagniere S, Auvray F, Carpentier,B. Spread of a green fluorescent protein-tagged Pseudomonas putida in a waterpipe following airborne contamination. JFood Prot 2006; 69:2692-6).

This phenomenon is supported by thefinding that water-cooled high-speeddentist drills also suffer the same problem(Appl. Env. Microbiol. 66:6.636).

EHEDG Document #13, Open Equipment,draws attention to the positioning ofcommon sources of high energy, such asmotor drives and fans, highvoltage insecttraps and static components such as pi-ping and cable trays (which can carry li-quid to a point where it can contact sucha device). The following image is takenfrom the EHEDG Trainers’ Toolbox, whichwas developed using EU funding withinthe HYFOMA project.

The use of high-pressure sprays, espe-cially those targeted near the floor ornear drains with the intention of trans-porting food materials to the drain. It iseasy to see how even the coarse, visible

mist can spread contamination, letalone the invisible fine aerosol.

During PathogenCombat visits toSpanish food processors, it was foundthat for the ripening rooms of jamoniberico, both veterinarians and cus-tomer representatives (auditors) had de-manded wet cleaning of the floor.


52

To prepare for this, the processor hadto remove all the hams from the (verylarge) ripening rooms and then a ride-on floor scrubber/dryer was used.Unfortunately, this type of machinesucks the remaining fluid from the floorand exhausts it as an aerosol.

We recommended against using thefloor scrubber or wet cleaning. The ri-pening-rooms have an ambient relativehumidity of around 40%, so it wascounterproductive to add water.Instead, we recommended using asheet of material under each row ofham frames, rolling this up when it wastime to clean. It could be recyclable,reusable or disposable. If reusable, itcould be cleaned elsewhere, withoutthe need to move all the hams out ofthe room and then move them backagain.

For moisture-management, it is recom-mended to implement a “dry floor” po-licy. This means:

– Removing waste at source and spiri-ting liquid waste straight to drain.

– NO rubber boots or aprons.

– Normal safety shoes.

– NO boot-washers at production.

– Hoses & mops locked during production.

– Rubber blades with scoops and binsonly, for the removal of waste thatfalls to the floor.

– Good ventilation.

– Controlled wet cleaning where neces-sary, for example, use of impregnatedwet wipes.

Whilst this discussion is focused on li-quid aerosols, it would be misleading todraw attention away from dust andsolid aerosols, for example from buil-ding work or other sources of dust.

• In the meat, poultry, fish and dairy in-dustries, poorly-controlled sources ofmoisture 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 en-gines (battery versions OK).

– Traffic.

– Drafts.

– Boot-washers at entrances.

A significant moisture-source was themovement of lift-trucks between areas.This should be avoided as far as possible,by use of roller- and belt-conveyers, pas-sing via “windows” from one room toanother, and split at the door, so that therooms and conveyers can be isolated forcleaning and when product is not beingtransported.


53

Note that the conveyer above is also co-vered, effectively making it a “closed”part of the system, unaffected by thesurroundings until opened.

• The role of correct integration:

Humidity control and many other fac-tors important for food safety oftenbroke down once the layout and flowof products, people, equipment, mate-rials and waste products became dis-rupted. This could have happened be-cause of poor initial planning and/orbecause of badly planned expansions ofproduction. There should be a syste-matic, sequential flow of product in onedirection, with air, waste and peoplerunning counter to this. In thePathogenCombat visits, it was almostalways the case that we were led th-rough the process from beginning toend, dirty to clean. Many other visitshave been the same.

The slide below is a schematic of thecorrect flows. The example is illustratedwith respect to a poultry plant, as anexample where the beginning of the

process is very contaminated, and thevisitors (yellow group) have just passedthough from the live bird intake, viaslaughter and evisceration.

The last Integration topic in the Burgospresentation was to re-emphasise thatfood safety requires a holistic approach.A forgotten aspect in many plants wasthe Maintenance Department, whereequipment, clothing and people thatfind their way into processing areas(note the hairnets) were not handled ina hygienic fashion.

AbstractA case study is presented as a contributionto the knowledge dissemination from thePathogenCombat project. A general admi-nistrative regulation for Mycobacteriumavium subsp. paratuberculosis in milk andMycobacterium spp in drinking water is notyet available. Dairies and dairy food produ-cers should be interested in the paratuber-culosis herd prevalence in the cluster oftheir milk suppliers. Their classification as li-kely MAP free should be based on regularanalysis of bulk tank milk or milk filters byreal time quantitative PCR. Farmer shouldbe encouraged to control paratuberculosisin their herds and mycobacteria-free milkshould be used for baby foods production.

The hypothesisThere are some infections that cause insome individuals an altered immune res-ponse that leads to persistent local inflam-mation at the environment-organism in-terface. They have protracted incubationperiods since they are caused by low viru-lence organisms replicating at very lowrates. Also some cell wall components ofbacteria are able to trigger chronic humanillnesses. People in risk are mainly new-born babies and people with a specificmutation of one gene.

Let researchers assess this hypothesis astrue or false. However, unless the riskwill not be credibly excluded, it shouldbe taken as a real one and measures forconsumer protection should be applied.The published data support sufficientlyneeds to develop an efficient systemhow to eliminate or at least how to de-crease this risk. The following case studyis an example, how food industry, fromsmall to global enterprises, can contri-bute to the consumer protection. Thebasic premise is that a solution does notconsist either in a simple elimination ofa risky foods and tap water from humanconsumption, or in pasteurization or boi-ling. Cattle is the largest producer ofMycobacterium avium subspecies paratu-berculosis (MAP), but milk and beef anddairy products cannot be generally ex-cluded from human diet.

The implications and the possible economicimpacts on milk demand were analysed(Groenendaal and Zagmutt, 2008). Threescenarios were developed based on the ef-fectiveness of possible risk-mitigation stra-tegies. In the first scenario, it was assumedthat an effective strategy exists; therefore,a negligible demand decrease in the con-sumption of dairy products was expected.In the second scenario, it was assumed thatnew risk mitigation would need to be im-

How can the food industry contribute todecrease the risk of contamination bymycobacteria: a hypothetical case discussionK. Hruska, I. Pavlik, RA. Juste


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plemented to minimize the health hazardfor humans. In this case, a small milk de-mand decrease was expected, but largerdemand decreases were also possible. Thethird scenario assumed that no fully effec-tive risk mitigation was available, and thisresulted in a considerable demand decreaseand a potential reduction in milk supply asa result of regulatory measures. A milk de-mand reduction of 1 or 5% resulted in a re-duction in consumer surplus of $600 mi-llion and $2.9 billion, and a reduction indairy farm income of $270 million and $1.3billion, respectively. A decrease in milksupply would cause a slight increase in totallosses, but would cause the greatest lossesto test-positive dairy farms. Given the cu-rrent scientific knowledge about MAP andCrohn’s disease, the authors conclude thatif a link were established, it is most likelythat the first or second scenario wouldoccur. Thus, consumer response and eco-nomic consequences to the discovery ofsuch a link are expected to be limited, butcould be large if the consumer's perceptionof risk is large or if risk-mitigation strategieswere ineffective (Groenendaal andZagmutt, 2008).

The basic termsThe basic terms have to be defined in thefirst place before any decisions should bedone. In each item should be known ifknowledge is scientifically confirmed. Adoubtful definition has to be explicitlymarked. In this case study the importantterms are as follows:

Crohn’s disease

CD is a devastating illness in search of acause and a cure. More than 800 000 pe-

ople in North America suffer from CD, agastrointestinal disorder characterized bysevere abdominal pain, diarrhoea, blee-ding, bowel obstruction, and a variety ofsystemic symptoms that can impede theability to lead a normal life during chronicepisodes that span months to years.Researchers and clinicians agree thatonset of CD requires a series of events;implicated are certain inherited genetictraits, an environmental stimulus, and anoverzealous immune and inflammatoryresponse. The combination of these fac-tors contributes to a disease whosecourse is variable among patients andwhose symptoms range from mild to de-vastating on any given day. The economicand social impact of this disease is subs-tantial for the patient, the family, thecommunity, and the healthcare system(Nacy and Buckley, 2008).

Paratuberculosis (Johne’s disease)

Paratuberculosis, also known as Johne’s di-sease, is a bacterial infection of the gas-trointestinal tract and is characterised by ch-ronic diarrhoea, persistent weight loss,decreased milk production and eventuallydeath (Davies et al, 2009). The disease isnot treatable and vaccinations avoids cli-nical disease but do not completely preventinfection; therefore, economic loss is subs-tantial in both the dairy and beef industries.Conflicting opinions have been publishedthat indicate a potential link between thecausative agent (Mycobacterium aviumsubspecies paratuberculosis) and Crohn’sdisease in humans, via the consumption ofinfected dairy products (Chiodini andRossiter, 1996; Bakker et al, 2000). In theUnited States, economic losses from the di-sease have been estimated to exceed

How can the food industry contribute to decrease the risk of contamination by mycobacteria…

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$1,500,000,000 per year, mainly from theeffects of reduced milk production (Okaforet al, 2008).

Mycobacteria (http://en.wikipedia.org/wiki/mycobacterium)

Mycobacterium is the only genus in the fa-mily Mycobacteriaceae belonging to orderActinomycetales, phyllum Actinobacteria.The genus includes pathogens known tocause serious diseases in mammals, inclu-ding tuberculosis and leprosy. All mycobac-terial species share a characteristic cell wall,thicker than in many other bacteria, whichis hydrophobic, waxy, and rich in mycolicacids (mycolates). The cell wall consists ofthe hydrophobic mycolic layer and a pepti-doglycan layer held together by a polysac-charide, arabinogalactan. The cell wallmakes a substantial contribution to the har-diness of this genus. Many mycobacterialspecies adapt readily to growth on verysimple substrates, using ammonia or aminoacids as nitrogen sources and glycerol as acarbon source in the presence of mineralsalts. Optimal growth temperatures vary wi-dely according to the species and rangefrom 25 °C to over 50 °C.

With regards to food safety the most im-portant species are Mycobacterium bovisbecause of its possible presence in unpas-teurized milk and cheese and Mycobacte-rium avium subspecies paratuberculosis be-cause of the huge amounts shed by cowsirregularly in their faeces (1012 cells pergram) and that might contaminate foodsand environment. (Anon, 2000). AmericanMicrobiology Academy in its report (Nacyand Buckley, 2008) quotes a recent studyusing culture-dependent methods detectedviable MAP in 2.8% of homogenized milkcartons sampled from supermarket shelves

in the U.S. The prevalence of 19.7% IS900PCR-positive bulk-milk samples shows awide distribution of subclinical MAP-infec-tions in dairy stock in Switzerland. The pre-valence, however, in the different regionsof Switzerland shows significant differencesand ranged from 1.7% to 49.2% (Cortiand Stephan, 2002). Other studies detectedMAP in samples of cheese (Ikonomopouloset al, 2005; Stephan et al, 2007). Overall,different levels of MAP contamination ofmilk, meat and food products have beennoted in the U.S. and a variety of countriesaround the world (Bosshard et al, 2006;Clark et al, 2006; Slana et al, 2008). MAPhas also been identified in environmentalsources, including river water and municipalwater (Pickup et al, 2006). MAP has beendetected using IS900 PCR in 49.0 % ofbaby foods. These results correspond to theepidemiological situation in Europe and arenot unexpected (Hruska et al, 2005). Acomprehensive review of mycobacteria inthe environment and their impact on ani-mal’s and human’s health has been pu-blished recently (Kazda et al, 2009). Equally,the demonstration of frequent presence ofMAP in muscle of clinically and subclinicallyinfected cattle opens a hitherto overlookedroute of exposure of humans through food.Since consumption of raw cattle meat isvery common in some cultures, this routecould be even more relevant that the milk,since in the case of raw beef there is noteven a significant thermal treatment.

The beef industry also needs to both requirecertification of reduction or eradication ofMAP from its suppliers and to establish acontrol of the paratuberculosis status of theindividuals entering its commercial chain. Ageneral administrative regulation is not ex-pected from several reasons. The herd pre-


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valence of paratuberculosis is estimated insome countries as high as 50% to 85%.The identification of shedders is difficult andtheir culling contributes to the disease era-dication but does not exclude shedding ofMAP by another animal next week or nextmonths. A substitution of culled animalsfrom another herd is not secure because noherd is absolutely paratuberculosis free. Theeradication certification of herds as MAPfree will be a long and expensive procedure.Use of vaccines is a common practice insome countries, it is inexpensive and hasshown a high efficacy in reducing rates ofbacterial excretion for which it could makea good immediate mitigation strategy.However, even though it is a practice cu-rrently in the increase, especially in small ru-minants, still, due to prejudices regardingto interference with TB control programs,it remains largely unused for cattle in mostcountries. The consequence is that very littleis being done in paratuberculosis control.

Slow infections and microbialtriggers

The term was first used by Bjorn Sigurdsonin 1954 (Sigurdsson, 1954) referring to thesheep diseases that spread through someregions of Iceland in the thirties as a conse-quence of an import of apparently healthyrams that had even been submitted to longquarantine periods. This concept has notbeen widely used, but fits nicely with a setof chronic diseases usually not lethal bythemselves if proper care is given. Since theagents are difficult to grow or detect, areubiquitous and do not act directly, but byway of the own host response, they are verydifficult to causally associate to a series ofdiseases for which an infectious cause has

long been suspected but, that do not fullycomply with classical causation postulates.

In the report of American Academy ofMicrobiology (Carbone et al, 2005), a mi-crobial trigger is defined broadly to meanany organism that sets in motion or expe-dites a disease process. Hence, a micro-bial trigger can bring on disease in any ofa number of ways, including persistenceas a chronic infection and the inductionof destructive host immune responses.The priming effect can be mediated alsoby the components of bacterial cells, e.g.peptidoglycan and its minimal motif mu-ramyldipeptide.

In 1974, muramyldipeptide (MDP) wasdiscovered as the minimal structure res-ponsible for the improved reaction to my-cobacteria in Freund’s complete adjuvant.Numerous reports suggest that MDP andother muropeptides directly induce cyto-kines, thus activating and modulating im-mune responses and inflammation (Traubet al, 2006). Hence not only the life bac-teria, containing MDP, but also dead cellsand their components participate in de-velopment of some diseases. Timing oftheir ingestion or inhalation, the way ofentering, the effective mass and host’s ge-netic disposition are without a doubt theimportant factors in disease pathogenesis.

Food borne diseases

At: http://www.cdc.gov/ncidod/dbmd/di-seaseinfo/files/foodborne_illness_FAQ.pdfa simple but detailed description of foodborne diseases is available. According tothe U.S. Centers for Disease Control andPrevention a foodborne disease is causedby consuming contaminated foods or be-verages. Many different disease causing


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microbes, or pathogens, can contaminatefoods, so there are many different foodborne infections. In addition, poisonouschemicals, or other harmful substancescan cause food borne diseases if they arepresent in food. More than 250 differentfood 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 byharmful toxins or chemicals that havecontaminated the food, for example, poi-sonous mushrooms or pesticide residues.

Autoimmune and autoinflammatorydiseases (McGonagle and McDermott,2006)

Triggered by ingested bacterial compo-nents can be classified as specific foodborne diseases. The evident differencefrom current food borne infections is se-veral or dozens years of latent period andabsence of apparent clusters of diseasedpersons usual in food borne infectionsoutbreaks. Direct proving or disprovingthe origin of clinical forms of chronic di-seases is impossible and Koch’s postulatescannot be applied. Bacterial cells and theircomponents should be better describes asharmful contaminants, closer to toxic xe-nobiotics or pollutants than to pathogens.

A hypothetical case discussion

It is not possible to take the assignment to-gether as a quite different approach has tobe applied on different mycobacteria con-sidered as risky from the food safety pointof view. Mycobacterium bovis andMycobacterium tuberculosis can appear in

milk and meat from animals suffering frombovine or human tuberculosis if the veteri-nary inspection fails. The problem can arisewhen bioproducts can be sold from thefarm directly to consumers. The food in-dustry enterprises in the European Unionare responsible for using the traceable fe-edstock from official sources under inspec-tion. Milk and meat primary contaminationthus should be avoided by the veterinaryinspection and food producers have to fo-llow only the possibility of secondary con-tamination from people suffering from tu-berculosis in open form. An apparentmanifestation of the disease, mainly coug-hing, should not be overlooked.

Mycobacterium avium subspecies paratu-berculosis represents another problem, notyet suitably treated by legislative norms.Therefore the following recommendationsare not obligatory and should be appliedvoluntarily to the consumer’s benefit. Therecommendations are addressed (1) to far-mers (how to decrease economic lossesfrom paratuberculosis and keep their herdsunder control, (2) to water companies (howto decrease the mycobacterial contamina-tion of water in the pipe systems), (3) toconsumers (how to decrease the risk of my-cobacteria ingestion namely is there ishigher risk of their disposition to Crohn’s di-sease), (4) to the research and developmentsector (how to apply the semiquantitativeanalyses of mycobacterial contamination ofwater, milk and meat and how to improvethe control of paratuberculosis), and (5) toadministration (how the financial supportto the agriculture, environmental a healthsectors should be directed and the con-sumer protection legislatively treated). Inagreement with the aim of this workshop


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the recommendation addressed to the foodindustry are described in more detail.

Possible case treatmentAll dairies and dairy food producers shouldbe interested in the paratuberculosis herdprevalence in the cluster of their milk sup-pliers. Their classification as likely MAP freeshould be based on quarterly analysis ofbulk tank milk or milk filters by real timequantitative PCR as long as the system ofherd certification would not be used.Having the results steps should be directedtowards the consumer protection. Entirelyin the enterprise management is securingthe use of tap water free of mycobacteria.It is necessary to collaborate with the labo-ratory, specialized in mycobacteria determi-nation both in milk and water analysis. Theappropriate methods are not used in everyroutine microbiological or molecular biolo-gical laboratory and ready-to-use kits arenot yet available.

The enterprises producing baby food, na-mely for newborns, pre-term borne andyoungest babies should follow the possibi-lity to introduce the MAP free formulas assoon as possible. Again, the input check ofmilk and selection of suppliers should bebased on interest of food industry itself asthe farmers need not to produce MAP freemilk yet. Similarly, the food industry needsnot to warrant MAP free product but thecomparative advantage can be used in mar-keting. However, once the product is de-clared as MAP free, the consumer protec-tion organizations will check the retailproducts certainly. A great responsibility ison the managements of producers of bot-tled water, designed as suitable for the ba-bies. Beside the currently required quality

the mycobacteria should be tested.Moreover, the consumers should be well in-formed about the risk of mycobacteria gro-wing on the inner surface of botles.

Paratuberculosis should be a notifiable di-sease and the national system for certifica-tion of MAP free herds or with infection re-duction strategies should be opened. Aslong as the certification is not available allherds have to be assumed as infected. Forcommercial purposes the customer (dairy)could require periodical evaluation of bulktank milk or milk filters on its own accountand for its own production managementand HACCP system, which has to considermycobacteria as a risk.

The quick, reliable, robust and accredi-table semiquantitative or quantitativetests for MAP DNA in milk or meat andfor mycobacteria DNA in water are avai-lable. Nevertheless, a development ofnew formats and principles of tests canbe expected.

In summary, consumer protection from therisk of mycobacteria contamination of foodis at present a voluntary task for the foodindustry. The changes of regulations are ex-pected, but both for farmers and state ad-ministration they will very difficult, expen-sive and long time required assignment.Nevertheless, if only the prevalence ofCrohn’s disease would decrease by 5%only, at least 100 000 consumers could beprotected in the USA, Canada and Europe.

BibliographyAlonso-Hearn M, Molina E, Geijo M, Vázquez P,Sevilla I, Garrido JM, Juste RA. Isolation ofMycobacterium avium subsp. paratuberculosisfrom Muscle Tissue of Naturally Infected Cattle.Foodborne Pathogens and Disease, 2009; DOI:10.1089=fpd.2008.0226.


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Anon. Possible links between Crohn's diseaseand Paratuberculosis. Report of the ScientificCommittee on Animal Health and AnimalWelfare Adopted 21 March 2000(SANCO/B3/R16/2000); 1-76.

Bakker D, Willemsen PTJ, van Zijderveld FG.Paratuberculosis recognized as a problem at last:A review. Veterinary Quarterly, 200; 22:200-4.

Bosshard C, Stephan R, Tasara T. Application ofan F57 sequence-based real-time PCR assay forMycobacterium paratuberculosis detection inbulk tank raw milk and slaughtered healthy dairycows. Journal of Food Protection, 2006;69:1662-7.

Carbone KM, Luftig RB, Buckley MR. Microbialtriggers of chronic human ilness. AmericanAcademy of Microbiology Colloquium, 2005;1-14.

Chiodini RJ, Rossiter CA. Paratuberculosis: A po-tential zoonosis? Veterinary Clinics of NorthAmerica-Food Animal Practice, 1996; 12:457-67.

Clark DL, Anderson JL, Koziczkowski JJ, EllingsonJLE. Detection of Mycobacterium avium subspe-cies paratuberculosis genetic components in re-tail cheese curds purchased in Wisconsin andMinnesota by PCR. Molecular and CellularProbes, 2006; 20:197-202.

Corti S, Stephan R. Detection of Mycobacteriumavium subspecies paratuberculosis specific IS900insertion sequences in bulk-tank milk samplesobtained from different regions throughoutSwitzerland. BMC Microbiol, (26-6-2002); 2:15.

Davies G, Genini S, Bishop SC, Giuffra E. An as-sessment of opportunities to dissect host geneticvariation in resistance to infectious diseases in li-vestock. Animal, 2009; 3:415-36.

Groenendaal H, Zagmutt FJ. Scenario analysis ofchanges in consumption of dairy productscaused by a hypothetical causal link betweenMycobacterium avium subspecies paratubercu-losis and Crohn's disease. Journal of DairyScience, 2008; 91:3245-58.

Hruska K, Bartos M, Kralik P, Pavlik I. Mycobac-terium avium subsp paratuberculosis in pow-dered infant milk: paratuberculosis in cattle -thepublic health problem to be solved. VeterinarniMedicina, 2005; 50:327-35.

Ikonomopoulos J, Pavlik I, Bartos M, Svastova P,Ayele WY, Roubal P, Lukas J, Cook N, GazouliM. Detection of Mycobacterium avium subspparatuberculosis in retail cheeses from Greeceand the Czech republic. Applied andEnvironmental Microbiology, 2005; 71:8934-6.

Kazda J, Pavlik I, Falkinham III JO, Hruska K. Theecology of mycobacteria: Impact on animal's andhuman's health. Eds Springer, 2009; 560 pp.

McGonagle D, McDermott MF. A proposed clas-sification of the immunological diseases. PlosMedicine, 2006; 3:1242-8.

Nacy C, Buckley M. Mycobacterium avium pa-ratuberculosis: Infrequent human pathogen orpublic health threat? A report from the AmericanAcademy of Microbiology, 2008; 1-37.

Okafor C, Grooms D, Alocilja E, Bolin S.Fabrication of a novel conductometric biosensorfor detecting Mycobacterium avium subsp pa-ratuberculosis antibodies. Sensors, 2008;8:6015-25.

Pickup RW, Rhodes G, Bull TJ, Arnott S, Sidi-Boumedine K, Hurley M, Hermon-Taylor J.Mycobacterium avium subsp paratuberculosis inlake catchments, in river water abstracted fordomestic use, and in effluent from domestic se-wage treatment works: diverse opportunities forenvironmental cycling and human exposure.Applied and Environmental Microbiology, 2006;72:4067-77.

Sigurdsson B. Maedi, a slow progressive pneu-monia of sheep: an epizootiological and patho-logical study. British Veterinary Journal, 1954;110:225-70.

Slana I, Paolicchi F, Janstova B, Navratilova P,Pavlik I. Detection methods for Mycobacteriumavium subsp paratuberculosis in milk and milkproducts: a review. Veterinarni Medicina, 2008;53:283-306.

Stephan R, Schumacher S, Tasara T, Grant IR.Prevalence of Mycobacterium avium subspeciesparatuberculosis in swiss raw milk cheeses co-llected at the retail level. Journal of Dairy Science,2007; 90:3590-5.

Traub S, von Aulock S, Hartung T, Hermann C.MDP and other muropeptides - direct and syner-gistic effects on the immune system. Journal ofEndotoxin Research, 2006; 12:69-85.

IntroductionNumber of alternative methods and tech-nologies rose up to replace historicallyproven heat treatments in the attempt tosatisfy modern trends in food consumption.These new trends were induced by thechange in the consumers’ perception offood quality and nutrition. The modern con-sumer seeks fresh looking, convenient andnutritionally healthy food, which requiresfrom industry to adopt new strategies insafe food production. The main change interms of microbial food safety is that steri-lization and pasteurization as we knewthem are in great extent replaced by verymild heat treatment, high pressure proces-sing, pulsed electric fields, intense lightpulses, application of organic and naturalpreservatives etc. The ability of these tech-niques, alone and even more in combina-tion, to inactivate and suppress recoveryand growth of surviving microorganisms atlow temperatures is beneficial for applica-tions of heat sensitive foods and ingredientsand to minimize adverse effects on the sen-sory characteristics of food products. Manyof these novel technologies have been al-ready subject of extensive research, but be-fore actual commercial application numberof technical, economical, and regulatory is-sues are to be solved. Among the factorsthat will determine the success of certainnovel technology is the consumers’ accep-

tance, which influences their purchase in-tent. Although many consumers prefer thenon-thermal food processing technologiesto manufacture higher quality, value-addedfoods that feature higher vitamin and nu-trient retention, and improved sensory at-tributes the lack of knowledge may pose anobstacle in their buying behavior (table 1).

Table 1. Percent of (n = 198) respondentsthat were “very” or “extremely”concerned with foods processed bynovel food processing techniques(adopted from Wright et al, 2007).

Food % very or processing extremelymethod concerned % uncertain

Genetically modified 54 17 Irradiation 49 17 Radio frequency 40 21sterilizationHigh pressure treatment 20 18 Microwave processing 18 12 Thermal processing 18 14 Heat pasteurization 13 6

The ideal processing technique would bethe one that meets following demands(Raso et al, 2005):

• Improvement of shelf life and safety byinactivating enzymes and spoilage andpathogenic microorganisms.

New processing technologies that canreduce the presence of pathogens in foodsA. Rajkovic, M. Uyttendaele, N. Smigic, F. Devlieghere

.


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• No changes in organoleptic and nutri-tional attributes.

• No residues left on food.

• Convenient to apply.

• Cheap.

• No objections from consumers and le-gislators.

Pulsed electric field (PEF)Pulsed electric field (PEF) technology is anon-thermal inactivation technology basedon the use of electric fields instead of heat.Next to microbial inactivation PEF maintainsquality attributes such as sensory qualityand nutritional value. Successful applicationof PEF technology to liquid products suchas fruit juices (Aguilo-Aguayo et al, 2009;Elez-Martínez et al, 2004; Elez-Martínez etal, 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 laboratoryand pilot plant levels suggests the potentialof this technology as a substitute for tradi-tional thermal pasteurization. While for thereduction of wine and must spoilage floraan optimum treatment of 186 kJ/kg at 29kV/cm has been established (Puertolas et al,2009), opposite was found for PEF applica-tion in beer production (Evrendilek et al,2004) due to significant degradation inflavor and mouth feel.

The exact mechanism by which PEF inacti-vate microorganisms is not yet completelyunderstood; however, much of the rese-arch in the field points toward damage ofthe cell membrane as the principal factor

responsible for microbial inactivation.Other effects resulting from the applicationof high-intensity pulsed electric fields, suchas DNA damage and generation of toxiccompounds, have been suggested, alt-hough some of the later studies rejectedthese hypotheses (Barbosa-Canovas andSepulveda, 2005). Although the currentknowledge does not provide ultimateanswer on the antimicrobial mechanism(s)of PEF it seems that there is reasonabledoubt if the membrane integrity is the onlyfactor to be considered.

Numbers of factors play a role in the de-termination of the effectiveness of pulsedelectric field technology as a microbial-inactivation process. Among those thekey role can be attributed to the type ofthe equipment used, setting of the treat-ment parameters, the type of media/foodprocessed, and the target microorganism(Aronsson et al, 2005; Barbosa-Canovasand Sepulveda, 2005; de Azeredo et al,2008; Jin et al, 2009; Wouters et al,2001). The relationship between thesefactors and their overall contribution tothe measured effectives of the PEF still re-quires further investigation.

High pressure processingHistorically seen food processing withhigh pressure goes long back to the endof 19th century. In 1899 (Heinz and Knorr,2005 and references therein), Hite sub-jected milk to high hydrostatic pressuretreatment as an alternative to classic heatsterilization in an attempt to avoid thesensory shortcomings of heat sterilizedmilk, while maintaining its microbiologicalsafety and quality. Hite achieved a 4-logreduction in microbial count in milk with

New processing technologies that can reduce the presence of pathogens in foods

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a 10-min treatment at approximately 700MPa at room temperature. Below 200MPa, the lethal effect of pressure wasfound to be significantly reduced. The ef-fect on spores was already than noticedto be much less effective (Heinz andKnorr, 2005 and references therein).Therefore, an increasing interest exists inproducing shelf-stable pressure-treatedfoods using HPP in conjunction with heat(initial temperatures around 80-90 °C,and compression heating temperatures> 121 °C) to kill resistant spores. Highpressure technology (100-1000 MPa, i.e.1000-10000 bar) is in general of increa-sing interest to biological and food sys-tems primarily because it permits micro-bial inactivation at low or moderatetemperature. The commercial productionof pressurized foods has become a realityin Japan, France, Spain, the USA andmany other countries. This is in a greatdeal results of extensive scientific rese-arch, technological and technical ad-vances in equipment production and de-crease in the processing costs (Chefteland Culioli, 1997). Nowadays, commer-cial application of high hydrostatic pres-sure has been found its place in the pro-duction of juices, smoothies, ready-to-eatmeat products, guacamole, oysters etc(Hjelmqwist, 2005; Patterson et al, 2007).

A typical high pressure system for food pro-cessing consists of a pressure vessel inwhich food packages are loaded and intowhich the pressure medium, usually water,is pumped, and a pressure-generating de-vice. In the case of liquids, such as fruitjuices, the vessel is filled with the juice,which acts as the pressure transmissionfluid. Once the desired pressure is reached,the pressure can be maintained without

further need for energy input. A funda-mental principle underlying HPP is the isos-tatic process allowing that all regions of thefood are rapidly exposed to a uniform pres-sure. The work of compression during HPPtreatment also increases the temperatureof foods through a process known as adia-batic heating, and the extent of the tempe-rature increase varies with the compositionof the food (normally 3-9 °C/100 MPa). HPPis traditionally a batch process. A series ofthese vessels can work in a staggered se-quence for an overall system that is semi-continuous.

HPP treatment is generally considered to acton bacterial cell membranes and impairtheir permeability and ion exchange(Cheftel, 1995; Hoover et al, 1989;Yaldagard et al, 2008). Microorganisms varywidely 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 atpressures around 300-400 MPa at ambienttemperature or higher temperatures. In re-cent study of Alpas et al. (2003) differentjuices were inoculated with Alicyclobacillusacidoterrestris cells to 6 log CFU/ml andwere pressurized at 350 MPa at 50 °C for20 min. More than 4 log cycle reductionwas achieved in all juices studied immedia-tely after pressurization. The inactivation ofspores by HPP is less efficient and requireshigher pressures and higher temperatures.Bacterial spores were found to survive upto 1200 MPa at room temperature (SanMartin et al, 2002; Zhang and Mittal, 2008and references therein) Furthermore, a de-tailed review of Zhang et al. (2008) com-piled much of the published data showingthat there can be significant variations inthe requirements of high pressure and tem-


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perature among different bacterial sporesspecies and also among strains of the samespecies. In general spores are unlikely to be(sufficiently) inactivated by HPP at roomtemperature. Therefore, optimization of theHPP conditions or combination with othertreatments and agents may be needed fora successful inactivation of spores. Most ofthe time the inactivation of spores is a two-step process: first, germination of thespores, and second, subsequent inactiva-tion of the germinated spores. Germinationis the process by which a stimulus is appliedto induce the dormant spores to convert toa metabolically active vegetative state. Heatshock is probably the most common sti-mulus. In principle, if all of the spores pre-sent in a food material could be induced togerminate, the food material could then besterilized by a subsequent preservation tre-atment that would be milder than the tre-atment needed to inactivate ungerminatedspores (Murad et al, 2007).

Not only bacterial inherent characteristicsand treatment parameters play an impor-tant role in effectiveness of HPP, but alsothe environment in which bacteria arefound. Patterson et al. (1995) reported thattreating E. coli O157:H7 under the sameconditions of 700 MPa for 30 min at 20 °Cresulted in a 6 log reduction in phosphate-buffered saline, a 4 log reduction in poultrymeat, and a < 2 log reduction in UHT milk.This reason is probably in the protective roleof certain food constituents. However, thepH and water activity (aw) of foods can alsosignificantly affect the inactivation of micro-organisms by HPP. Most microorganismstend to be more susceptible to pressure inlower pH environments, and pressure-da-maged cells are less likely to survive in acidic

environments (Patterson, Linton, andDoona, 2007).

Regarding inactivation of foodborne virusesdifferences in HPP effect were noticed bet-ween different viruses, different treatmentparameters and different foods/media werereported (Baert et al, 2009; Kingsley et al,2007). In review of Baert et al. (2009) it wasreported that exposure of hepatitis A virusto pressures of 375 MPa at 21 °C for 5 mininduced reduction of respectively 4.3 and4.7 log in strawberry puree and on slicedgreen onions. HPP treatment of oysters witha pressure of 400 MPa for 1 min (9.0 °C) in-duced 3 log reduction of HAV whereasMNV-1 was reduced by 4 log (5 °C). On theother hand, Aichivirus and coxsackievirusB5 remained fully infectious if 600 MPa wasapplied for 5 min at ambient temperaturewhereas coxsackievirus A9 was reduced by7.6 log under the same conditions. Similarlypoliovirus was found to be resistant to 600MPa for 1 h. It can be concluded that thesensitivity towards HPP does not agree bet-ween genetically related taxonomic groupsor even strains. A possible explanation couldbe the difference in protein sequence andstructure (Baert et al, 2009).

Intense light pulsesIntense light pulse (ILP) is one of the emer-ging non-thermal techniques investigatedas an alternative to traditional thermal tre-atment. ILP is a technique to decontami-nate surfaces by killing microorganismsusing short time pulses of an intense broadspectrum, rich in UV-C light (the portion ofthe electromagnetic spectrum correspon-ding to the band between 200 and 280nm), which has been proven to be effectivefor microbial inactivation on food surfaces


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and food packages. The mode of action ofthe pulsed light process is attributed to theeffect of the high peak power and the UVcomponent of the broad spectrum of theflash. Inactivation occurs by several mecha-nisms, including chemical modification andcleavage of DNA, protein denaturation andother cellular materials alteration (Turtoi andNicolau, 2007 and Barbosa-Canovas et al,2004 therein).

The following units are commonly usedto characterize the ILP treatment (Gómez-López et al, 2007):

• Fluence rate: is measured in Watt/ meter2

(W/m2) and is the energy received fromthe lamp by the sample per unit area persecond.

• Fluence: is measured in Joule/meter2

(J/m2) and is the energy received fromthe lamp by the sample per unit areaduring the treatment.

• Dose: used sometimes as a synonym offluence.

• Exposure time: length in time (seconds)of the treatment.

• Pulse width: time interval (fractions of se-conds) during which energy is delivered.

• Pulse-repetition-rate (prr): number ofpulses per second (Hertz [Hz]) or com-monly expressed as pps (pulses per se-cond).

• Peak power: is measured in Watt (W)and is pulse energy divided by the pulseduration.

The trend of susceptibility of different mi-croorganisms towards ILP has been re-viewed by Gómez-López (Gómez-López etal, 2007). The contradiction in reporteddata can be based on the experimental se-

tups and technology (equipment) used.Namely, contradictory findings were notedbetween Gómez-López et al. (2005) whodid not observe any sensitivity patternamong different groups of microorganisms,after studying 27 bacterial, yeast and mouldspecies and decreasing order of sensitivityobserved by Anderson et al. (2000): Gram-negative bacteria, Gram-positive bacteriaand fungal spores.

Gómez-López et al. (2005) described thatfor an industrial implementation: the posi-tion and orientation of strobes in an unitwill determine the lethality, that productsto be treated should be flashed as soon aspossible after contamination occurs, that acooling system should be used for heat-sensitive products and that flashed productsshould be light protected. However, it is im-portant to note that latest results haveshown that repetitive cycles of inactivationwith ILP can result in increased resistance,but will not affect growth characteristics ofresistance cells (Rajkovic et al, Resistance ofListeria monocytogenes, Escherichia coliO157:H7 and Campylobacter jejuni afterexposure to repetitive cycles of mild bacte-ricidal treatments Food Microbiology –ac-cepted for publication–).

Food preservation bycombined processes (hurdletechnology)For almost all treatments that do notcause complete inactivation of microor-ganisms it is characteristic to induce su-blethal injury to the present bacterial cells.Depending on the type of the injury, typeof the organism and surrounding environ-ment these injured microbial cells havethe potential to resuscitate and resume


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growth under favorable conditions. Theseconditions are commonly found in foodsand the safety of foods treated with alter-native, non-thermal and less-than sterili-zing technologies requires to be wellthought-out. In addition to the inactiva-tion technologies applied to foods, bothmicrobial growth and survival can be in-fluenced by different intrinsic factors ofthe food. This means that intrinsic factors,alone or combined with the extrinsic fac-tors, can enhance or inhibit recovery andgrowth of microbial cells. Therefore, thesafety and stability of food can be im-proved by a combination of several fac-tors that will prevent surviving and injuredcells to proliferate. These multiple intrinsicfactors are part of a dynamic system thatchanges from the moment of applicationto the moment of consumption. Duringthis process, each factor plays a role ofdifferent magnitude and such magnitudechanges over time (Raso, Pagan, andCondon, 2005 and references therein).

Food preservation by combined processes(aka hurdle technology) supports the com-bination of existing and novel preservationtechniques to establish a series of preserva-tion factors (hurdles) that no microorganism(of concern) present should be able to over-come (Leistner, 1992; Leistner, 1994;Leistner and Gorris, 1995; Raso andBarbosa-Canovas, 2003; Raso, Pagan, andCondon, 2005; Ritz et al, 2002; Shin et al,2006). This is especially true for the hardymicroorganisms and bacterial spores thatare very resistant to processing treatments.To apply principles of food preservation bycombined processes correctly, an appro-priate understanding of the mechanisms ofaction of the individual factors alone and incombination is needed. This understanding

allows justified and well balanced combi-nation of hurdles to achieve desired level ofsafety and quality, avoiding the need toapply only one factor at such high intensitythat it causes severe changes in the food’squality (Raso, Pagan, and Condon, 2005).Instead, using combined hurdles one caninterfere with the microbial homeostasisand extend the effect of sublethal injury bybreaking the homeostatic mechanisms andrendering cells incapable of responding tothe stresses produced by the preservationfactors. This can not only results in growthinhibition, but can also impair survival pos-sibilities leading the death of injured micro-bial cells. As said before several studies haveindicated that HPP and PEF inflict sublethalinjury. Also for other technologies similarfindings were reported. Van Houteghem etal. (2008) studied the effects of carbon dio-xide in modified atmospheres on the resus-citation of Listeria monocytogenes cells in-jured by intense light pulses (ILP), chlorinedioxide (ClO2), lactic acid (LA) and mild heatmild bactericidal treatments during storageat 7 °C were examined. The results indi-cated additional bactericidal effects of CO2

on cultures treated with LA, ClO2 and ILP,with additional reductions in viable L. mo-nocytogenes of 0.5-1.0 log cfu/ml. Lagphase duration was significantly differentbetween the different treatments, withnon-treated cells having the shortest lagphase, followed by that of heat, intenselight pulses, lactic acid and finally ClO2 tre-ated cells. The authors have found rela-tionship between the amount of sub-let-hally damaged cells after a mild inactivationtreatment and the lag phase duration in theCO2 environment. Similarly, Rajkovic et al.(2009) reported on the effect of partialinactivation of Listeria monocytogenes with


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LA, liquid ClO2 and ILP on injury and post-treatment growth under increased NaClconcentration and reduced pH values. Theresults showed that the inactivation levelsand the percentage of sub-lethal were de-pendent upon strain and type of inactiva-tion technique used. The biggest effect onthe growth retardation was at every pH ob-served for the cultures treated with ClO2,followed by LA and ILP. Under increasedNaCl concentration LA treated cells sufferedhardest growth retardation, followed byClO2 and ILP, respectively. Recovery of ILPtreated cultures was not always differentfrom untreated cultures. In general, da-maged microorganisms become moreexacting in growth requirements and aremore sensitive to other preservation factorslike low pH, antimicrobial components, etc.(Raso, Pagan, and Condon, 2005 andMackey, 2000 therein).

In foods preserved by combined methodsthe microbial homeostasis is threatened ondifferent multiple sites asking for a complexand energy demanding microbial response(Raso, Pagan, and Condon, 2005 andMontville and Matthews, 2001 therein).This fact enables the obtaining of safe andstable foods by balancing different factorsand strategies. Particularly under mildlylethal stress, the ultimate cause of inactiva-tion is subject of cellular response to addi-tional regulation that integrates informa-tion about the global state of the cell andits environment (Aertsen and Michiels,2004). It is therefore an art of combiningdifferent suboptimal factors that will pushmicrobial cell over the thin line betweenbacterial growth and inactivation. The ex-tended post-treatment effect based on thegrowth retardation or inhibition of injuredcells under sub-optimal conditions can be

utilized as an important tool in conditioningof microbial food safety.

The risks to be consideredThe European and World food industryaims increasingly at applying novel andmild preservation techniques for the pro-duction of food products that will meetdemands of a modern consumer. Hence,their product range is shifting more andmore towards refrigerated, microbiologi-cally unstable food products, which re-sults in an increased complexity to controlthe microbial safety of their products. Asseen in principles of food preservation bycombined processes for the production ofmildly preserved food products, often aninactivation step is applied as a first step.Most often, a mild heat treatment is ap-plied for this purpose. However, the in-dustry resorts more and more to non-thermal alternatives such as highhydrostatic pressure, decontaminationwith organic acids or other decontamina-tion agents (e.g. chlorine dioxide in thegas phase), electrical inactivation and in-tense light pulses. When this type of noveland mild inactivation techniques are ap-plied, incomplete inactivation and su-blethal damage of the target organismsis often obtained, but by the applicationof suboptimal inhibiting factors, growthduring preservation is prevented orfurther inactivation is promoted.

In the scientific community, but also amongfood processors and legislators there is aconcern about the fact that the applicationof sublethal stress factors could induce(cross) resistance mechanisms in the survi-ving population and change their virulencecharacteristics (Rajkovic et al, Resistance of


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Listeria monocytogenes, Escherichia coliO157:H7 and Campylobacter jejuni afterexposure to repetitive cycles of mild bacte-ricidal treatments Food Microbiology ac-cepted for publication; Abee and Wouters,1999; Hill et al, 2002; Lou and Yousef,1997; Rowan, 1999). This increased resis-tance stems from the fact that bacteria, asliving organisms, can respond to and har-ness themselves against stresses to whichthey are exposed. A quantification of theseresponses as well as understanding the mo-lecular and cellular mechanisms of thisadaptive response is of great interest, be-cause it may lead to improved strategies forcombating microbes, not only in foods, butalso in disease.

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AbstractWith a significant impact on trade andcompetitiveness, food safety is of funda-mental importance to the European con-sumer, food industry and economy. Butdespite significant investment in the areaof food safety, the incidence of food-borne disease is still on the rise in theEuropean Union.

The use of protective and probiotic culturesmay be a useful and effective strategy toprevent or reduce the incidence of patho-gens in the food chain, improve food sa-fety and enhance consumer health.

The benefits of these protective and pro-biotic cultures can be effected at any levelof the food process chain, from the farmanimals to the final food product (“Farmto Fork” approach). More specifically,these beneficial bacteria can be usedeither as protective cultures (which reduceor control the growth of pathogens in thefarm environment or in the final food pro-duct) or as probiotic cultures (which confera beneficial effect upon the host, either afarm animal through probiotic animal feedor the final consumer through a probioticfood product).

Specific research objectives were identi-fied within the PathogenCombat project

to identify and characterise a collection ofprotective and probiotic strains which de-monstrated the following multifunctionalproperties:

• Inhibit a variety of pathogenic orga-nisms common in the food industry.

• Capable of survival in food processingconditions or the gastric system of ani-mals and humans.

• Safe for consumption by farm animalsand humans.

Of the 1087 strains screened to date, 70 de-monstrated inhibitory activity against spe-cific pathogens. Selected strains were thentested for survival in simulated food proces-sing conditions (high temperatures, pre-sence of salt, lack of nutrients) and animaland human gastrointestinal tract conditions(low pH and presence of bile salts).

As a result of this research, a total of 23promising protective and probiotic strainshave been identified which inhibit in-vitropathogenic organisms found in the foodindustry and are capable of surviving foodprocessing conditions and the gastricsystem of animals and humans.

Some of these protective and probioticcultures were selected for further testingin specific food chain applications.

Reducing foodborne pathogens in the foodchain by the use of protective andprobiotic culturesN. Carlini, L. Mogna, GP. Strozzi


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Preliminary results are encouraging and po-sitive benefits have been demonstrated todate in specific poultry and dairy applications.

Additional testing with PathogenCombatprotective and probiotic strains in otherapplications is recommended to furtherunderstand the impact of these findingsand the potential of these strains to im-prove food safety.

Introduction to pathogensand diseaseA disease is any condition caused by thepresence of an invading organism or a toxiccomponent (i.e. pathogen) which causesdamage to the host. In humans, diseasescan be caused by the growth of micro-or-ganisms such as bacteria, viruses, protozoa,and fungi. However, bacterial growth is notmandatory to cause disease.

Not all pathogens cause diseases whichhave the same severity of symptoms. Forexample, an infection with the influenzavirus can cause the short term aches andfever that are hallmarks of the flu, or it cancause more serious symptoms, dependingon the type of virus which causes the in-fection. Bacteria also vary in the damagethey cause. For example, ingestion of foodcontaminated with Salmonella enteriticacauses intestinal upset. But consumptionof Escherichia coli O157:H7 can cause a se-rious disease which can permanently da-mage the kidneys and even be fatal.

Pathogens can be spread from person toperson in a number of ways and not allpathogens use all available routes. Forexample, the influenza virus is transmittedfrom person to person through the air, ty-pically via sneezing or coughing. But thevirus is not transmitted via water. In con-

trast, Escherichia coli is readily transmittedvia water, food, and blood, but is not re-adily transmitted via air or insect bite.

Zoonoses are infections or diseases whichare transmitted from animals to humans.The infection can be acquired directlyfrom animals or by ingestion of contami-nated foodstuffs. The severity of these di-seases can vary from mild symptoms tolife-threatening conditions.

Pathogens and zoonoticdisease in the EuropeanCommunity

In order to prevent the occurrence of zoo-noses and protect human health, it is im-portant to identify which animals and fo-odstuffs are the main sources of infectionor disease. For this reason, information iscollected every year from all EuropeanUnion Member States and analysed by theEuropean Food Safety Authority (EFSA) incollaboration with the European Centre forDisease Prevention and Control (ECDC).

An annual Community Zoonoses SummaryReport is published, which reports the oc-currence of infectious diseases transmittedfrom animals to humans. The recently pu-blished report for 2007 highlights thatmany bacteria are still being transmittedfrom animals to our food.

Campylobacter

Campylobacteriosis is an infectious diseasecaused by bacteria of the genusCampylobacter found in animals such aspoultry, cattle, pigs, wild birds and wildmammals. Most human illness is caused byone species, Campylobacter jejuni. This spe-cies is well adapted to birds, whose body

temperature (approximately 42 °C) allowsfor optimal growth of the bacteria. Birds cancarry these bacteria without becoming ill.

Most people who become ill with campy-lobacteriosis experience diarrhoea, cram-ping, abdominal pain, and fever withintwo to five days after exposure to the or-ganism. The diarrhoea may be bloody andcan be accompanied by nausea and vo-miting. The illness typically lasts oneweek. In individuals with compromisedimmune systems, Campylobacter occasio-nally spreads to the bloodstream andcauses a serious life-threatening infection.

Campylobacter infection has also been as-sociated with complications such as laterjoint inflammation and, on rare occasions,Guillain-Barré syndrome, a temporary butsevere paralysis which may be total.

In 2007, infections from Campylobacterwere again the most frequently reportedzoonotic disease in humans across theEuropean Union with a total of 200,507cases reported. This represents an increaseof 14.2% from 175,561 in the previousyear.

Reducing foodborne pathogens in the food chain by the use of protective and probiotic cultures

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Table 1. Campylobacter in fresh broiler meat.

Samples tested % Positive Sampling point Samples tested % Positive in EU in EU in Spain in Spain7598 26.0%

At slaughter 147 55.8%

At processing plant 168 29.0%

At retail 208 30.8%

Table 2. Campylobacter in other foods.

Food Samples % Positive Total samples % Positive tested in EU in EU tested in Spain in Spain

Pig meat 537 0.9% 36 0%

Bovine meat 695 1.2% 0 Not applicable

Cow’s milk 4,158 0.5% 0 Not applicable

Dairy products 520 1.1% 96 0%

Table 3. Campylobacter in animals.

Animal Total % Positive Samples % Positive tested in EU in EU tested in Spain in Spain

Broiler flocks 10,260 25.2% 89 46.1%

Pigs 1,102 56.1% 230 71.3%

Cattle 12,539 5.9% 163 46.0%


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In foodstuffs, Campylobacter was mostlyfound in raw poultry meat with an averageof 26% of samples showing contamina-tion. In live animals, Campylobacter wasfound in poultry, pigs and cattle.

Salmonella

Salmonellosis is caused by bacteria belon-ging to the genus Salmonella. This genusconsists of more than 2,500 serotypes butthe two serotypes Salmonella typhimuriumand Salmonella enteritidis account for themajority (70-80%) of Salmonellosis casesreported in Europe.

The infections may occur in small, con-tained outbreaks in the general popula-

tion or in large outbreaks, particularly inhospitals, restaurants, or institutions hou-sing children or the elderly.

Salmonellosis is generally contracted bythe consumption of contaminated foodof animal origin that has not been pro-perly heat-treated or is eaten raw (mainlymeat, poultry, eggs and milk). Many otherfoods, including green vegetables conta-minated from manure, have also been im-plicated in its transmission.

Food prepared on surfaces previously incontact with raw meat or meat productscan, in turn, become contaminated withthe bacteria. This is known as cross-con-tamination.

Table 4. Salmonella in fresh broiler meat.

Samples % Positive Sampling point Samples % Positive tested in EU in EU tested in Spain in Spain28,012 5.5% Slaughter 184 22.3%

Processing 144 2.8%/cutting plantRetail 206 10.2%

Table 5. Salmonella in table eggs.

Samples % Positive Sampling point Samples % Positive tested in EU in EU tested in Spain in Spain16,626 0.8% Packing centre 1,653 2.8%

Retail 98 1.0%Not specified 41 2.8%

Table 6. Salmonella in fresh pig meat.

Samples % Positive Sampling point Samples % Positive tested in EU in EU tested in Spain in Spain81,131 1.1% Slaughter 315 4.8%

Processing 63 7.9%/cutting plantRetail 66 6.1%

Most people infected with Salmonelladevelop diarrhoea, fever, and abdominalcramps 12 to 72 hours after infection.The illness usually lasts 4 to 7 days, andmost recover without treatment.However, in some cases, the diarrhoeamay be so severe that the patient needsto be hospitalized. In these patients, theSalmonella infection may spread fromthe intestines to the blood stream, andthen to other body sites and can causedeath unless the person is treatedpromptly with antibiotics. The elderly, in-fants, and those with impaired immunesystems are more likely to have a severeillness.

Although the number of cases showed adecrease for the fourth successive year, atotal of 151,995 people in the EuropeanCommunity were still affected by the bac-terium Salmonella in 2007 (compared to164,011 cases reported in 2006).

Poultry and pig meat were reported asthe foods most frequently associatedwith Salmonella, and an average of5.5% of all fresh poultry meat sampleswithin the European Union was foundto be contaminated. Eggs and egg pro-ducts were also found to be contami-nated, while the bacterium was only ra-rely detected in raw dairy products,vegetables and fruits.

In animal populations, Salmonella wasmost frequently detected in poultry flocks.In 2007, the European Commission laun-ched a new control programme againstSalmonella in breeding poultry flocks. Atthe end of 2007, 15 Member States hadalready met the target of 1% which mustbe achieved by the end of 2009.

Listeria

The bacterium Listeria monocytogenes,commonly referred to as listeria, is foundin soil, vegetation, sewage, water and thefeces of animals and humans. Listeria canalso be found in unpasteurised dairy pro-ducts, raw vegetables and meats and pro-cessed foods including deli meats and hotdogs.

Listeriosis is a rare but potentially lethalfood-borne infection caused by Listeria mo-nocytogenes. Infected pregnant womenmay experience only a mild, flu-like illness.However, infections during pregnancy canlead to miscarriage or stillbirth, prematuredelivery, or infection of the newborn.

In addition, the elderly and individuals suf-fering from immuno-compromising dise-ases such as cancer or HIV are particularlyvulnerable to listeriosis. In these cases, lis-teriosis may lead to meningitis, brain in-fection, and severe blood infection.

In 2007, the number of Listeria infectionsin humans in the European Union remainedat the same level as 2006 with 1,554 con-firmed cases. Although less frequent thanCampylobacter and Salmonella, infectionsfrom Listeria are quite dangerous due totheir high mortality rate (20%).

Cases of Listeria above the legal safetylimit were found in ready-to-eat foods,most often in smoked fish and other fis-hery products, followed by meat productsand cheese.

Escherichia coli

Escherichia coli or E. coli is a bacteriumfrom the family Enterobacteriaceaewhich is usually found in the digestivesystem of healthy humans and animals.


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There are hundreds of known E. colistrains. Many are harmless but somestrains, such as E. coli O157:H7 and E.coli O121:H19, belong to a group ofEscherichia coli known as verotoxigenicE. coli (VTEC) or shiga-like toxin produ-cing E. coli (STEC).

VTEC organisms have several characteris-tics which make them so dangerous. Theyproduce one or more verocytotoxins (VT)which cause severe damage to the intes-tinal tract lining of those infected. VTEC or-ganisms also have a very low infectious

dose, which means that only a relativelysmall number of bacteria are needed “toset-up housekeeping” in the intestinal tractand cause infection. Finally, they are quitehardy and can survive for quite some time,depending on the environmental condi-tions. Some organisms (e.g. E. coliO157:H7) can survive at low temperaturesand in acidic conditions which makes it dif-ficult to eradicate them in nature.

The acute disease associated with E. coliO157:H7 is named hemorrhagic colitis.Symptoms characteristic of this disease in-

Table 7. Listeria in food.

Food Samples % Positive Samples % Positivetested in EU in EU tested in Spain in Spain

Bovine meat 932 1.8% No data No dataPig meat 21,245 2.2% 418 4.1%Red, mixed or 2,644 2.5% No data No dataunspecified meatPoultry meat 2,581 2.6% 31 6.5%Cheeses from 4,879 0.1% No data No datacow milkCheeses from sheep 1,064 1.0% No data No dataand goat milkFish 2,629 18.3% No data No dataCrustaceans, shellfish or 2,328 2.5% 653 5.2%unspecified fishery products

Table 8. Listeria in animals.

Animal Total % Positive Total tested % Positivetested in EU in EU in Spain in Spain

Gallus gallus (fowl) 4,860 0.1% No data No dataTurkeys and ducks 140 0% No data No dataPigs 5,834 0.1% No data No dataCattle (bovine animals) 76,376 0.2% 68,311 0%Goats 689 3.8% No data No dataSheep 2,973 2.4% No data No data

clude watery and/or bloody diarrhea,fever, nausea, severe abdominal cram-ping, and vomiting; these symptoms mayappear within hours or up to several daysafter ingestion of the bacteria. Treatmentis usually not necessary as most people re-cover from the illness on their own aftera duration of 5-10 days.

However, particularly virulent strains of E.coli can cause serious illness or death in theelderly or those with weakened immunesystems. Some individuals may develop he-molytic uremic syndrome (HUS). In the veryyoung, this disorder may cause renal failure,hemolytic anemia (destruction of red bloodcells), or even permanent loss of kidneyfunction. In the elderly, these symptoms aswell as thrombotic thrombocytopenic pur-pura (TTP) can occur and the mortality ratedue to TTP can be as high as 50% in thispopulation.

Transmission of E. coli usually occurs byconsumption of contaminated food orwater or by contact with an infected animalor person.

Categories of foodstuffs where VTEC repre-sents a hazard to public health have beenidentified and include: raw or undercookedbeef and possibly meat from other rumi-nants; minced and/or fermented beef andproducts thereof; raw milk and raw milkproducts; fresh produce, in particularsprouted seeds, and unpasteurised fruit andvegetable juices; and water.

In the European Union, VTEC accounted fora total of 2,905 human infections in 2007.Among animals and foodstuffs, VTEC wasmost often reported in cattle and bovinemeat and very rarely in vegetables.

The utilisation of protectiveand probiotic cultures as astrategy to prevent or reducepathogen transmission alongthe food chainThe utilisation of protective and pro-biotic cultures may be a useful and ef-fective strategy to prevent or reduce theincidence of food-borne pathogens in


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Table 9. VTEC in fresh bovine meat.

Total samples % Positive Sampling point Samples % Positive tested in EU in EU tested in Spain in Spain14,115 0.3%

At slaughter, cutting 201 1.8%/processing plantAt retail 69 1.4%Not specified No data No data

Table 10. VTEC in cattle.

Animal Total tested % Positive Total tested % Positive in EU in EU in Spain in Spain

Total animal 5,154 3.6% 312 17.0%Total herd/holding 559 8.1% No data No data


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the food chain, improve food productsafety and enhance consumer health.

The benefits of these protective and pro-biotic cultures can be effected at anylevel of the food process chain, fromfarm animals to the final food product(“Farm to Fork” approach). More speci-fically, these beneficial bacteria can beused in the food process chain as protec-tive cultures which reduce or control thegrowth of pathogens in the farm envi-ronment and in the final food product oras probiotic cultures which confer a be-neficial effect upon the host, either afarm animal through probiotic animalfeed or the final consumer through aprobiotic food product.

Specific objectives of the scientific researchwithin WP 10 of the PathogenCombat pro-ject were to identify and characterise a co-llection of bacterial strains which demons-trated the following multifunctionalproperties:

• Inhibit a variety of pathogenic organisms

common in the food industry (pathogens

targeted for investigation within the pro-

ject are identified in table 11).

• Capable of survival in food processing con-

ditions 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 thePathogenCombat project.

Pathogen Category PathogenGram-positive bacteria Listeria monocytogenes

Mycobacterium avium subsp.paratuberculosis (MAP)

Gram-negative bacteria Campylobacter jejuni, Escherichia coliYeast Saccharomyces cerevisiaeOchratoxin A producing filamentous fungus Penicillium nordicumViruses Hepatatis E virus (HEV)

Tickborne encephalitis virus (TBEV)

Table 12. Resistance of PathogenCombat strains to stress conditions (low pH and hightemperature).

Strain Origin Survival at low pH Survival at high(2.5 for 3 hours) temperature

(55 °C for 15 minutes)Lactobacillus Unknown Medium survival Inconclusive resultspentosus PCA 227Enterococcus faecium Sausage Low survival High survivalPCD 71

Lactobacillus pentosus Sausage Low survival High survivalPCD 101 (Austria)


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Table 12. Resistance of PathogenCombat strains to stress conditions (low pH and hightemperature) (continuation).

Lactobacillus plantarum Kasseri cheese Inconclusive results Inconclusive resultsPCA 236Lactobacillus plantarum Xynotyri cheese Medium survival Inconclusive resultsPCA 263Lactobacillus plantarum Feta cheese Low–medium survival Medium-high survivalPCA 275Lactobacillus plantarum Cheese Medium-high survival Medium-high survivalPCS 20Lactobacillus plantarum Cheese Medium-high survival Low survivalPCS 22Lactobacillus gasseri Feta cheese High survival High survivalPCA 185Lactobacillus fermentum Kasseri cheese High survival Medium-high survivalPCA 144Lactobacillus fermentum Kunun-zaki High survival Medium-high survivalPCK 129 (Nigeria)Lactobacillus delbrueckii Salgam Low–medium survival High survivalPCK 103 (Turkey)Leuconostoc Maasai milk Low survival Inconclusive resultspseudomesenteroides (Kenya)PCK 18Leuconostoc mesenteroides Pasta filled Low survival High survivalPCD 119 with mince

meatLeuconostoc PCK 73 Coffee Low survival Medium-high survival

fermentation(Ethiopia)

Pediococcus pentosaceus Ham Medium survival High survivalPCD 215Pediococcus pentosaceus Sausage Medium-high survival High survivalPCD 237Enterococcus faecium Maasai milk Low survival Medium-high survivalPCK 38 (Kenya)Enterococcus faecium Maasai milk Inconclusive results Medium-high survivalPCK 45 (Kenya)Enterococcus faecium Maasai milk Low survival High survivalPCK 49 (Kenya)Bifidobacterium longum New-born Low survival High survivalPCB 133 infantBifidobacterium Pig Low survival High survivallongum biovar suis (International PCD 733B Culture

Collection)Enterococcus durans Sausage Low survival High survivalPCD 103 (Austria)


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PathogenCombat protectiveand probiotic strains with in-vitro activity against food-borne pathogensAs a result of the scientific research con-ducted in WP 10 of the PathogenCombatproject, twenty-three protective and pro-biotic strains have been identified whichdemonstrate in-vitro antimicrobial activityagainst the food-borne pathogens Listeriamonocytogenes, Campylobacter jejuniand/or Penicillium nordicum (see tables13-17 for details).

Seven strains demonstrated antimicrobialactivity against multiple pathogens (tables

13 and 14). No protective and probioticstrains have been identified to date whichdemonstrate in-vitro antimicrobial activityagainst the pathogens Escherichia coli orSaccharomyces cerevisiae.

Note: strains from the genus Enterococcuswere not selected for further investigation/feasibility trials in feed or food applicationswithin the PathogenCombat project.

Although some strains of Enterococcusfaecium show a long history of apparentsafe use in food or feed, Enterococci areamong the leading causes of communityand hospital-acquired infections in hu-mans. The safety concerns related to

Table 13. PathogenCombat strains which demonstrated in-vitro antimicrobial activityagainst multiple pathogens (Listeria monocytogenes and Campylobacter jejuni).

Strain Origin Listeria Campylobacter monocytogenes jejuni antimicrobial antimicrobial activity activity

Leuconostoc Maasai milk Very strong Moderatepseudomesenteroides (Kenya)PCK 18Enterococcus faecium Sausage Strong ModeratePCD 71

Table 14. PathogenCombat strains which demonstrated in-vitro antimicrobial activityagainst multiple pathogens (Campylobacter jejuni and Penicillium nordicum).

Strain Origin Campylobacter jejuni Penicillium nordicum antimicrobial activity antimicrobial activity

Lactobacillus pentosus Unknown Moderate ConfirmedPCA 227Lactobacillus plantarum Kasseri cheese Moderate ConfirmedPCA 236Lactobacillus plantarum Xynotyri cheese Moderate ConfirmedPCA 263Lactobacillus plantarum Feta cheese Moderate ConfirmedPCA 275Lactobacillus plantarum Cheese Moderate ConfirmedPCS 20

these bacteria are further heightened bythe intrinsic resistance of these bacteriato a variety of antibiotics. In addition,some species, such as Enterococcus du-rans and Enterococcus hirae, have beenassociated with infections in chickens.

As a result of these safety concerns andthe lack of information on safety, theEuropean Food Safety Authority (EFSA)has decided that no members of thegenus Enterococcus can be proposed forQPS (Qualified Presumption of Safety)status. This means that the safety ofeach Enterococcus spp. strain must beassessed on a case-by-case basis.

Some protective and probiotic strainsfrom the PathogenCombat CultureCollection have been selected for feasi-bility trials at laboratory scale in specificfeed and food applications. If the labo-ratory trials are successful, the strainswill be tested at industrial scale in the

final feed or food application at Institu-tional Partners or food-producing SMEs/Industrial Partners.

The utilisation of protective and probioticcultures from the PathogenCombatCulture Collection could be implementedon a broader scale for greater impact inreducing the incidence of food-bornepathogens within the food chain and im-proving food safety within the EuropeanCommunity.

PathogenCombat protectiveand probiotic strains with in-vitro antimicrobial activityagainst select pathogens

Salmonella

This pathogen was not targeted for inves-tigation within the scope of the Patho-genCombat project.


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Table 15. PathogenCombat protective and probiotic strains which demonstrated in-vitro antimicrobial activity against Campylobacter jejuni.

Strain Origin Campylobacter jejuniantimicrobial activity

Leuconostoc PCK 73 Coffee fermentation (Ethiopia) StrongBifidobacterium longum PCB 133 New-born infant StrongLactobacillus pentosus PCA 227 Unknown ModerateEnterococcus faecium PCD 71 Sausage ModerateLactobacillus plantarum PCA 236 Kasseri cheese ModerateLactobacillus plantarum PCA 263 Xynotyri cheese ModerateLactobacillus plantarum PCA 275 Feta cheese ModerateLactobacillus plantarum PCS 20 Cheese ModerateLactobacillus delbrueckii PCK 103 Salgam (Turkey) ModerateLeuconostoc Maasai milk (Kenya) Moderatepseudomesenteroides PCK 18Bifidobacterium longum Pig (International Culture Moderatebiovar suis PCD 733B Collection)Enterococcus durans PCD 103 Sausage (Austria) Moderate


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Campylobacter

A total of twelve strains were identifiedwhich demonstrated in-vitro antimicrobialactivity against Campylobacter jejuni(table 15).

Bifidobacterium longum PCB 133

Introduction

Isolated from a new-born infant, the pro-tective and probiotic strain Bifidobacteriumlongum PCB 133 demonstrated strong ac-tivity in vitro against Campylobacter jejuni.Although resistant to high temperature,this strain is sensitive to low pH which maycause survival difficulties during processingor in the final feed or food application.

Industrial production forfeasibility trials in final applications

The strain Bifidobacterium longum PCB133 has been successfully produced infreeze-dried form at industrial scale andmicroencapsulated for improved survivalin specific final applications.

Application in poultry feed

The use of probiotic strains could haveapplication as an additive in feed for li-vestock poultry against intestinal pat-hogens in order to reduce the use ofantibiotics and contamination of themeat.

A study was conducted at the Universityof Bologna to evaluate the capacity oftwo different orally administered probio-tics (Lactobacillus plantarum PCS 20 andBifidobacterium longum PCB 133) to co-lonise the intestinal tract of broiler chic-kens and to assess their effect on theCampylobacter jejuni population.

Each probiotic group was administered adaily dosage of 1-10 million of viable cellsfor 15 consecutive days. Results of this ex-periment demonstrate that only the pro-biotic strain Bifidobacterium longum PCB133 colonised the intestinal tract of thebroiler chickens and was detected in the fa-eces of the treatment group; Lactobacillusplantarum PCS 20 was not recovered.

Feed studies with the microencapsulatedprobiotic culture of Bifidobacteriumlongum PCB 133 in poultry are currentlyin progress at the University of Bologna.

In addition, another trial is in the planningstages to evaluate the use of this micro-encapsulated strain (Bifidobacteriumlongum PCB 133) as an animal probioticto reduce Campylobacter jejuni contami-nation in turkey meat products inGermany.

Application in poultry meatproducts

The industrial, freeze-dried probiotic cul-ture of Bifidobacterium longum PCB 133was sent to the University of Burgos inSpain for preliminary laboratory trials inspecific poultry meat applications. Uponsuccessful results of these tests, the strainwill be sent to the Spanish food-produ-cing SME for industrial feasibility trials atthe Cooperativa Avicola y Ganadera deBurgos.

Application in a functional food (pro-biotic dairy product)

In collaboration with the dairy SME Pittasin Cyprus, trials were conducted to developa probiotic sheep milk yogurt with the useof the protective and probiotic strainBifidobacterium longum PCB 133.Although prototypes of the probiotic yo-

gurt were successfully produced at labora-tory scale, the stability of the strain in thefinal product was not acceptable, possiblyrelated to the strain’s sensitivity to low pHas identified in WP10. Efforts are now con-centrated on the use of another protectiveand probiotic strain (Lactobacillus plan-tarum PCS 20) in the yogurt product.

Lactobacillus plantarum PCS 20

Introduction

Isolated from cheese, the protective andprobiotic strain Lactobacillus plantarumPCS 20 showed in-vitro antimicrobial ac-tivity against the mould Penicillium nor-dicum and moderate activity againstCampylobacter jejuni. It is tolerant orsemi-resistant to low pH and high tempe-rature which may facilitate its survival du-ring processing and in the final food orfeed application.

Application in a functional food (pro-biotic dairy product)

Trials were conducted to develop a pro-biotic sheep milk yogurt with the use ofLactobacillus plantarum PCS 20. Pilot bat-ches of yogurt were successfully producedwith this strain co-grown with the starterculture of Streptococcus thermophilus(YO8). In addition, the desired results interms of stability were achieved as the via-bility of the strain PCS 20 in the final yo-gurt product was demonstrated after 35days at +5 °C.

Another development trial is planned to as-sess the feasibility of producing the pro-biotic yogurt with Pittas sheep milk andLactobacillus plantarum PCS 20 co-grownwith a starter culture of Streptococcus ther-mophilus in combination with Lactobacillusbulgaricus.

Upon successful development and stabi-lity trials, production will be scaled up toevaluate the feasibility of the probiotic yo-gurt process at industrial scale. Focusgroups will then be conducted to obtainconsumer feedback on the probioticsheep milk yogurt produced in thesetrials.

Lactobacillus plantarum PCA 236

Introduction

Isolated from cheese, the protective andprobiotic strain Lactobacillus plantarumPCA 236 is active against Penicillium nor-dicum and Campylobacter jejuni. In addi-tion, the strain produces a bacteriocin(Plantaricin EF) and survives well in low pHand high temperature environments.Based upon preliminary results, it also in-hibits Mycobacterium avium subsp. para-tuberculosis (MAP), a resistant pathogenencountered in milk which can cause di-seases in farm animals and has also beenshown to infect humans.

Application in goat feed

The strain was successfully produced infreeze-dried form and sent to theAgricultural University of Athens for aprobiotic feed trial in goats in collabora-tion with SME Pittas in Cyprus. The indus-trial produced strain Lactobacillis plan-tarum PCA 236 survived gastrointestinaltransit in the goat GI tract. In addition, thepopulation levels of harmful bacteria(Clostridia) were reduced and lactic acidbacteria populations (beneficial bacteria)increased in the goats which were fed thePCA 236 strain. A statistically significantincrease (13%) in milk production wasalso reported in the probiotic group com-pared to the control. Analysis of the milk


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(for antioxidant activity) and blood (forantibodies and antioxidant activity) fromgoats treated with the animal probioticPCA 236 is pending; results are expectedin July 2009.

Listeria

A total of eight strains were identifiedwhich demonstrated in-vitro antimicrobialactivity against Listeria (table 16).

cessing or in the final feed or food appli-cation.

Application in lamb and beef meatproducts

The strain Leuconostoc pseudomesente-roides PCK 18 has been successfully pro-duced in freeze-dried form at Laboratorypilot scale and sent to the University ofBurgos in Spain for preliminary trials inspecific beef and lamb food applications.

Upon successful laboratory trials, thestrain(s) will be produced at industrialscale and sent to Spanish food-producingSMEs for industrial feasibility trials in lambproducts at Colear Castilla and/or in beefproducts at Martinez Loriente, S.A.

Lactobacillus pentosus PCD 101

Introduction

Isolated from sausage, the protective andprobiotic strain Lactobacillus pentosusPCD 101 showed strong antimicrobial ac-tivity against Listeria. Although resistant

Leuconostoc

pseudomesenteroides PCK 18

Introduction

Isolated from maasai milk, the protective

and probiotic strain Leuconostoc pseudo-

mesenteroides PCK 18 demonstrated very

strong in-vitro antimicrobial activity

against Listeria and moderate activity

against Campylobacter jejuni. However,

the strain is sensitive to low pH which

may cause survival difficulties during pro-

Table 16. PathogenCombat protective and probiotic strains which demonstrated in-vitro antimicrobial activity against Listeria monocytogenes.

Strain Origin Listeriamonocytogenesantimicrobial activity

Leuconostoc pseudomesenteroides Maasai milk (Kenya) Very strongPCK 18Enterococcus faecium PCK 38 Maasai milk (Kenya) Very strongEnterococcus faecium PCK 45 Maasai milk (Kenya) Very strongEnterococcus faecium PCD 71 Sausage StrongLactobacillus pentosus PCD 101 Sausage (Austria) StrongLeuconostoc mesenteroides PCD 119 Pasta filled with mince meat StrongPediococcus pentosaceus PCD 215 Ham StrongPediococcus pentosaceus PCD 237 Sausage Strong

to high temperature, this strain is sensi-tive to low pH which may cause survivaldifficulties during processing or in thefinal feed or food application.

Application in beef and lamb meatproducts

The strain Lactobacillus pentosus PCD101 has been successfully produced infreeze-dried form at laboratory pilotscale and sent to the University ofBurgos in Spain for preliminary trials inspecific beef and lamb food applica-tions. Upon successful laboratory trials,the strain(s) will be produced at indus-trial scale and sent to Spanish food-pro-ducing SMEs for industrial feasibilitytrials in lamb products at Colear Castillaand/or in beef products at MartinezLoriente, S.A.

Penicillium nordicum

A total of ten strains were identifiedwhich demonstrated in-vitro antimicrobialactivity against Penicillium nordicum (table17).

Escherichia coli

No protective and probiotic strains havebeen identified to date which demons-trate in-vitro antimicrobial activity againstthe pathogen Escherichia coli.

GlossaryDisease: any condition caused by the pre-sence of an invading organism or a toxiccomponent (i.e. pathogen) which causesdamage to the host.

Pathogen: organisms, frequently micro-organisms or components of these orga-nisms which cause disease or illness.Microbial pathogens include various spe-cies of bacteria (e.g. the bacteriumMycobacterium tuberculosis causes tuber-culosis), viruses (e.g. pathogenic virusescause smallpox, influenza, mumps, me-asles, chickenpox and rubella), protozoa(e.g. malaria), and fungi (e.g. infectionsdue to Candida and Cryptococcus).

Probiotic: live micro-organisms whichwhen administered in adequate amountsconfer a health benefit on the host.


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Table 17. PathogenCombat protective and probiotic strains which demonstrated in-vitro antimicrobial activity against Penicillium nordicum.

Strain OriginLactobacillus pentosus PCA 227 UnknownLactobacillus plantarum PCA 236 Kasseri cheeseLactobacillus plantarum PCA 263 Xynotyri cheeseLactobacillus plantarum PCA 275 Feta cheeseLactobacillus plantarum PCS 20 CheeseLactobacillus plantarum PCS 22 CheeseLactobacillus gasseri PCA 185 Feta cheeseLactobacillus fermentum PCA 144 Kasseri cheeseLactobacillus fermentum PCK 129 Kunun-zaki (Nigeria)Enterococcus faecium PCK 49 Maasai milk (Kenya)


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Probiotic culture: culture of probioticmicro-organisms which confers a beneficialeffect upon the host, either a farm animalthrough probiotic animal feed or the finalconsumer by a probiotic food product.

Protective culture: culture of micro-orga-nisms which reduces or controls the growthof pathogenic micro-organisms in the farmenvironment and/or the final food product.

Zoonosis: also called zoonose or zoonoticdisease, this term refers to a diseasewhich can be transmitted from animals(wild or domestic) to humans.

AcknowledgementsWe grately acknowledge funding from fi-nancial participation by the EuropeanCommunity under the Sixth FrameworkProgramme for research, technological de-velopment and demonstration activities forthe Integrated Project PathogenCombatFood-CT-2005-007081.

BibliographyAltekruse SF, Stern NJ, Fields PI, Swerdlow DL.Campylobacter jejuni-An Emerging FoodbornePathogen Emerg Infect Dis. Jan-Mar, 1999; 5(1):28-35. Annex 3: Assessment of Gram-Positive Non-Sporulating (GPNS) bacteria with respect to aQualified Presumption of Safety. The EFSAJournal. 2007; 587:1-16.Castillo M, Martín-Orué SM, Manzanilla EG,Badiola I, Martín M, Gasa J. Quantification oftotal bacteria, enterobacteria and lactobacilli po-pulations in pig digesta by real-time PCR.Veterinary Microbiology, 2006; 114:165-70.Davies AR, Capell C, Jehanno D, Nychas GJE,Kirby RM. Incidence of foodborne pathogens onEuropean fish. Food Control. 2001; 12(2):67-71. Inglis GD, Kalischuk LD, HW, Kastelic JP. Coloni-zation of cattle intestines by Campylobacter jejuniand Campylobacter lanienae. Appl EnvironMicrobiol. Sept 2005; 71 (9):5.145-53.Introduction of a Qualified Presumption ofSafety (QPS) approach for assessment of se-

lected microorganisms referred to EFSA, Opinionof the Scientific Committee.The EFSA Journal2007; 587:1-16.

Jemmi T, Stephan R. Listeria monocytogenes:food-borne pathogen and hygiene indicator.Rev. Sci. Tech. Aug 2006; 25 (2):571-80.

Lan PT, Binh LT, Benno Y. Impact of two probioticLactobacillus strains feeding on fecal lactobacilliand weight gains in chicken. J. Gen. Appl.Microbiol. 2003; 49:29-36.

Lawson AJ, Shafi MS, Pathak K, Stanley J.Detection of Campylobacter in gastroenteritis:comparison of direct PCR assay of faecal sam-ples with selective culture. Epidemiology andInfection, 1998; 121:547-53.

Nauta M, Hill A, Rosenquist H, Brynestad S,Fetsch A, van der Logt P, Fazil A, Christensen B,Katsma E, Borck B, Havelaar A. A comparison ofrisk assessments on Campylobacter in broilermeat.Int J of Food Microb. 2009; 129:107-23.

Patterson JA, Burkholder KM. Application ofPrebiotics and Probiotics in Poultry Production.Poultry Science, 2003; 82:627-31.

Rhoades JR, Duffy G, Koutsoumanis K.Prevalence and concentration of verocytotoxi-genic Escherichia coli, Salmonella enterica andListeria monocytogenes in the beef productionchain: a review. Food Microbiol. Jun 2009; 26(4):357-76. Epub 2008, Nov 11.

Saleha AA, Mead GC, Ibrahim AL.Campylobacter jejuni in poultry production andprocessing in relation to public health. J WorldPoultry Sci. 1998; 54:49-58.

Sandberg M, Nygård K, Meldal H, Valle PS,Kruse H, Skjerve E. Incidence trend and risk fac-tors for campylobacter infections in humans inNorway. BMC Public Health, 2006; 6:179.

Skånseng B, Trosvik P, Zimonja M, Johnsen G,Bjerrum L, Pedersen K, Wallin N, Rudi K. Co-in-fection dynamics of a major food-borne zoo-notic pathogen in chicken. PLoS Pathog.November 2007; 3 (11):e175.

The Community Summary Report on trends andsources of zoonotic agents in the EuropeanUnion in 2007. The EFSA Journal (2009), 223.

Swaminathan B, Gerner-Smidt P. The epidemio-logy of human listeriosis. Microbes Infect. Aug2007; 9 (10):1236-43. Epub 2007, May 7.

The overall objective of “PathogenCombat” is to provide new essential in-formation and methods to the food in-dustry and public authorities on how toreduce the prevalence of new and emer-ging food borne pathogens

The aim of the Work Package 13“Application in the Food Chain” is relatedto the application of the knowledge andtools produced within the project and tothe development of support measures tofood industries. In particular, research ac-tivities and results obtained from differentPartners in several WPs (WP 4, 5, 6, 10,11, 12, 14 and 15) are made available tobe disseminated and tested into SMEsand industrial partners. The disseminationand transfer into practical application inthis WP comprise the food processingSME and Industrial partners of “PathogenCombat”, belonging to dairy, ruminant,poultry and pork food chain (table 1).

Considering food safety, the combat ofpathogens is of outmost importance toassure quality products to the consumer.

To approach the omnipresent threats fromnew and emerging pathogens, theEuropean project “PathogenCombat”,aims, first of all, at increasing knowledgeon the factors, which enable the viability,persistence and virulence of pathogens inthe food chain, in order to reduce their in-cidence.

Despite significant investment, the inci-dence of food derived disease is still toohigh in the EU. Besides being of funda-mental importance to the consumer, figh-ting pathogens is also of imperative sig-nificance to the food industry andeconomy, as impact on trade and compe-titiveness is substantial.

The project deals with eight pathogens:Listeria monocytogenes, Mycobacteriumavium subsp. paratuberculosis, Campylobacterjejuni and shiga-like toxin producingEscherichia coli (STEC), invasive variantsof Saccharomyces cerevisia, ochratoxin Aproducing filamentous fungus (Penicilliumnordicum), hepatitis E virus (HEV) andtickborne encephalitis virus (TBEV); pre-

The application of PathogenCombatresearch results in practiceF. Gaggìa, B. Biavati

Table 1. List of SMEs and Industrial partner participating to “Pathogen Combat”.

Cheese and Milk Poultry Pork Beef and LambPartner 27 GRAN-I Partner 29 CAGB-E Partner 29 CAGB-E Partner 46 COLEAR-EPartner 28 PITTAS-G Partner 33 ZIEGLER-D Partner 34 JAMSA-E Partner 47 MARLOSA-EPartner 31 BERG-D


90

diction and detection of Staphylococcusaureus enterotoxins are also included.

SMEs and Industrial partners involved inthe project, along the last three years ofproject, aim to apply and improve:

• New detection methods and predictionof the occurrence and virulence of pat-hogens in the food chain and at time ofconsumption with molecular biologybased on culture independent techni-ques and microarrays.

• New processing technologies and sys-tems, new hygienic design, protectiveand probiotic cultures and new informa-tion on host-pathogen interaction toprevent pathogen transmission alongthe food chain.

• New mathematical models for pat-hogen control throughout the foodchain and at time of consumption.

• Their Food Safety Management Systempreventing microbial food borne dise-ases.

In this paper, we will describe some im-portant applications that have been de-veloped within the project, concerningnew culture-independent detection met-hods based on molecular biology, and ap-plied in practice inside SMEs and Industrialpartners plants.

The quality control and monitoring alongthe food chains is necessary to ensuresafe end products; mainly food industryrely on international procedures basedon culturing methods to estimate thepresence, the viability and enumerationof pathogens throughout the food chaintaking into account the large amount oftime required to achieve the final results.

Since producers, processing industry andconsumers increasingly demand higherquality for starting material and foodproducts, there is a rising interest forrapid, sensitive and specific diagnosticmethods.

The aim of WP4 within PathogenCombatis the development and the optimizationof culture independent methods (e.g FISHtechnique, PCR-DGGE, qPCR and RT-qPCR)for the identification of microbial popula-tions in food systems without the need oftraditional isolation and plating counts, ta-king into consideration both the definitionof the microbial ecology of foodstuffs andthe detection of food-borne pathogens.

The Fluorescence in Situ Hybridization(FISH) technique has been optimized inWP4 in order to produce experimental pro-tocols to be used in WP13 for a direct pro-filing of the microbial populations presentin a specific food ecosystem or in samplesfrom critical points along the food chain.

The developed protocols were applied onsamples collected from project partners inparticular from a dairy company for the de-tection of viable bacteria and the presenceof different pathogenic bacteria, likeListeria monocytogenes, or hygiene indi-cator microorganisms, like coliform bac-teria and yeasts. Mainly process water, bio-films and cheese samples were sent andanalysed. The results obtained allowed topoint out that, by using the adapted FISHprotocols on process samples, useful infor-mation for the producers in relation to thebacterial community composition (e.g.high speed detection of pathogens) andon the hygienic status of the productionprocess could be obtained.

Furthermore, the hands-on time for theanalyses requires only few minutes andresults are available within 3 hours (onlya short pre-enrichment is required) lea-ding to a time-saving of 3 to 5 days whencompared with conventional methods.

The WP4 is interested also in the develop-ment of quantitative PCR (qPCR), for de-tection, quantification and typing of pat-hogens such as L. monocytogenes, E. coliSTEC and C. jejuni in food matrices.

Of the identified hazards in PathogenCombat relevant to dairy industry opera-tions, Listeria monocytogenes has the gre-atest attention and placed surveillance; itcan survive and multiply under conditionsfrequently used for food preservation; thismakes L. monocytogenes problematic tothe food industry. The University of Torino(Italy) has optimized a protocol for the de-tection and quantification of L. monocyto-genes that has been applied in a dairycompany within the project. A total of 150samples (cheese during and at the end ofthe production, swabs and brines) were co-llected for three consecutive weeks; theanalyses were carried out by molecular andtraditional methods. Some cheese and en-vironmental samples were found to be po-sitive for L. monocytogenes; in some sam-ples a slight discrepancy in the resultsobtained with the two methods has beendetected (17%). Following these results aninspection and check were performed onthe continuous flow pasteurizer (and itscleaning system) of liquid brines: a failurewas detected and then a corrective actionwas performed.

A deeper look and investigation on theapplicability of the new methods is ob-viously necessary to obtain repeatability,

reproducibility and a validated methodo-logy; however this kind of application re-presents a real advantage for the foodproducing companies, because in a rela-tively short time (about 18-24 h) they canobtain information on the presence ofpathogens in their plants and take appro-priate preventing measures. The evalua-tion of the risk, its prevention and/or itssolving could be deal within a reasonabletime offering to the personnel preciousdays to act and take decisions.

Some specific works and studies were ini-tiated also from two Spanish Companies,partners of Pathogen Combat, to ap-proach and develop new culture-indepen-dent techniques in order to analyze diffe-rent steps of the poultry, pork andruminants food chain.

Important results have been obtained ina Spanish SME; samples of pork loincured were analysed after a treatmentwith High Pressure. The analyses wereperformed both by conventional micro-biology using the ISO procedures for thedetection of L. monocytogenes (ISO11290-1:1996) and culture independentmethod, specifically by Real Time PCR(with a protocol developed within thePathogen Combat research).

A good correspondence between the twomethods was detected and also in thiscase a reduction in the analyses time (1day against 5 days with traditional met-hods) was observed.

In addition to the important applicationsdescribed above, the analysis of the watersupply systems of two German SMEs andtwo Spanish SMEs has been completedusing molecular biology, and non-cultu-ring methods (DNA extraction, conven-

The application of PathogenCombat research results in practice

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tional and seminested PCR and TaqManq-PCR), for the direct detection of patho-genic microorganisms.

Molecular biological methods (PCR-DGGE)for the analysis of the bacterial populationcomposition and stability at the differentwater sampling points of the food industryfacilities have been also applied. The proto-cols have been optimised for the detectionof the following pathogens: L. monocyto-genes, M. avium subsp. paratuberculosis,C. jejuni, Enterococcus ssp, Salmonella ssp,E.coli and P. aeruginosa. Several litres ofwater from each sampling point were takenand the original present bacteria were con-centrated by filtration, and directly used formolecular biology analyses. Furthermore aquestionnaire on drinking water distribu-tion has been prepared for the SMEs inorder to achieve a complete evaluation ofthe water supply system inside these SMEs.

Finally partners of WP5 are working inthe development and validation of robustmicroarrays to monitor the existence andexpression of the involved genetic factorsfor virulence and pathogenicity forListeria, Campylobacter and E.coli. Thebasic procedure include: a) samples co-llection and concentration; b) nucleicacid purification and labelling; c) hybridi-zation to array; d) data capture and in-terpretation.

The arrays will be test and used in colla-boration with the SME and industrial foodproducers in WP 13 on cheese and meatsamples from the processing lines.

The steps to develop rapid and reliable de-tection methods based on culture indepen-dent techniques require time and efforts(optimization of the primers and probes,nucleic acid extraction) and should be cost

effective, ensuring high repeatability, repro-ducibility and a good training of the per-sonnel performing the test. Moreover, theprotocols need to be optimized to meetevery requirements taking into account thecontext, specific for each company.

Therefore a strict collaboration betweenpartners, taking into charge the develop-ment of the new protocols, and the com-panies participating to the Project has beenachieved. In that sense PathogenCombatcreated a direct line among partners for therapid information exchange, assistance anddissemination of the knowledge.

It is a good opportunity for SMEs andIndustrial partner to get in touch with anew scientific approach made available byuniversities or private companies with thenecessary equipments and laboratories fa-cilities; a valid contribution to the impro-vement of Food Safety has been perfor-ming with the aim to transfer anddisseminate the new knowledge in theEuropean context.

Bribliography

Van Amerongen A, Barug D, Lauwaars M.Rapid Methods. Wageningen Academicpublishers. 2005.

Annual Report “PathogenCombat”. 2006.



Roberts A, Cai S, Wiedmann M. The con-tribution of actA to virulence differencesamong Listeria monocytogenes strains.103rd Annual Meeting of the AmericanSociety for Microbiology, Washington,DC. 2003.

Introducción La inocuidad alimentaria es un objetivocomún al sector agroalimentario y unaclara exigencia y preocupación de los con-sumidores. Para conseguirlo la industriaalimentaria debe cumplir con una serie demedidas y controles que garanticen la ob-tención de alimentos inocuos.

El mantenimiento de un elevado nivel delimpieza de los equipos y de las instala-ciones y del entorno de trabajo, afecta deforma directa sobre la inocuidad del pro-ducto final. Para conseguirlo no sólodeben ser regularmente limpiados y de-sinfectados sino que su diseño inicial debefacilitar la realización de estas operacioneseficazmente así como garantizar quetanto las instalaciones como los equipospor su diseño no se conviertan en foco decontaminación de los alimentos.

El diseño de un equipo o instalación se con-sidera “higiénico” si incorpora, con carácterpreventivo, características que reducen oeliminan el riesgo de constituir una fuentede contaminación para los alimentos, tantode forma directa como indirecta.

Por ejemplo, gracias a un adecuado diseñohigiénico es posible garantizar que unequipo o instalación determinada no trans-fiere ningún cuerpo extraño, sustancia quí-

mica, ni microorganismo. Para ello, en elámbito del diseño higiénico de equipos einstalaciones se consideran factores talescomo los materiales de construcción, super-ficies de contacto, drenabilidad, hermeti-cidad, accesibilidad, entre otros muchos.

Errores o deficiencias en el diseño higié-nico de instalaciones y de equipos puedenhacer fracasar o dificultar la obtención dealimentos inocuos, aumentando y/o difi-cultando las tareas de mantenimiento,limpieza, desinfección, control de plagasy control del proceso fundamentales y ne-cesarias para asegurar unas condicionesde producción adecuadas.

Por lo tanto, para conseguir equipos e ins-talaciones higiénicas éstas deben diseñarsey construirse cumpliendo requisitos higié-nicos. Pero aun así en algunos casos por ra-zones funcionales no es posible cumplir to-talmente con estos requisitos y se hacenecesario disponer de métodos o ensayosque nos permitan comprobar que aun asíel equipo o instalación es limpiable o este-rilizable, según exigencias de uso.

En el apartado 3 del presente artículo sedescribe el método de EHEDG para eva-luar la limpieza CIP de equipos pequeños.

En esta sentido AINIA, centro tecnológicodesde su creación, viene desarrollando ac-tuaciones de formación, investigación,

EHEDG: método para la comprobación dela evaluación de la limpieza in situ de losequipos para el procesado de los alimentosM.ª I. Llorca


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análisis y apoyo técnico al sector en el ám-bito de la inocuidad alimentaria.

Entre estas actividades señalamos las si-guientes líneas de actuación:

• Desarrollo de nuevas tecnologías parala mejora y control de la seguridad ali-mentaria en las industrias del sector.

• Investigación y mejora de la higiene asícomo del impacto medio ambiental aso-ciado. Estudio de técnicas de limpieza ydesinfección.

• Servicios analíticos acreditados porENAC que permitan la detección decontaminantes en los alimentos.

• Diseño higiénico y limipiabilidad (diseñoy puesta a punto de métodos).

AINIA es miembro y sede regional delGrupo Europeo de Ingeniería y DiseñoHigiénico, EHEDG, en España.

EHEDG es el referente europeo en diseñohigiénico, en el siguiente apartado del pre-sente artículo se procede a su descripción.

AINIA, como sede regional de EHEDG, de-sarrolla diversas acciones para fomentar enEspaña la adopción de criterios de higieneen la proyección, ampliación y reformas delas industrias alimentarias, así como en eldiseño de maquinaria y equipos.

Qué es EHEDG EHEDG (European Hygienic Engineeringand Design Group) es un consorcio eu-ropeo de fabricantes de equipos, indus-trias alimentarias, institutos de investiga-ción y autoridades públicas, fundado en1989 con el objeto de promover la hi-giene durante el procesado y envasado dealimentos.

EHEDG tiene vínculos fuertes con variasorganizaciones internacionales y tieneentre sus planes buscar relaciones glo-bales adicionales.

Como la seguridad alimentaria no ter-mina en los límites de Europa, EHEDGpromueve activamente la armonizaciónde las diferentes guías y normas. Las or-ganizaciones americanas NSF y 3-A, estánde acuerdo en cooperar con EHEDG en eldesarrollo de sus guías y, por su parte,EHEDG colabora en el desarrollo de lasnormas 3-A y NSF.

EHEDG proporciona asesoramiento en as-pectos de ingeniería higiénica para la ela-boración de alimentos inocuos.

Organización interna

El Presidente es elegido por el GrupoPrincipal para un periodo de tres años. Sufunción es mantener la organizaciónunida, representar formalmente a EHEDG,y presidir la Asamblea General.

El Grupo Principal, compuesto por elComité Ejecutivo y miembros en general,dirige EHEDG y revisa y aprueba las guías

Presidente

Miembros

Comité ejecutivo Fundación

OrganismoCertificación

SeccionesRegionalesSubgrupos

EHEDG: método para la comprobación de la evaluación de la limpieza in situ de los equipos…

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previo a su publicación. Se espera que enel conjunto de miembros haya una repre-sentación equilibrada de fabricantes deequipos, industria agroalimentaria, orga-nizaciones de investigación y legisla-dores. En el Grupo Principal hay repre-sentantes de 3-A y de NSF International,por lo que existe una cooperaciónformal. Además, el Grupo Principal estárepresentado en 3-A y NSF International.Cuando es oportuno, se invita a repre-sentantes de otras organizaciones conobjetivos similares.

El Comité Ejecutivo ejecuta las deci-siones del Grupo Principal y se hacecargo de las tareas cotidianas deEHEDG. Está formado por el Presidente,el Secretario General, el Tesorero y losmiembros. Los miembros del ComitéEjecutivo tienen funciones de coordina-ción específicas y tareas relacionadas:coordinación de subgrupos, coordina-ción de las Secciones Regionales, coor-dinación de estándares, coordinación delos cuerpos gubernamentales, coordina-ción de certificación y coordinación deeventos. Los coordinadores de los sub-grupos hacen un seguimiento e in-forman sobre el progreso de los trabajosde cada subgrupo. El Comité Ejecutivose reúne cuatro veces al año.

Los miembros de EHEDG trabajan princi-palmente para:

• Incrementar los conocimientos sobre hi-giene de los alimentos.

• Ayudar en la prevención de problemasde seguridad alimentaria.

• Sostener de esta manera la imagen dela industria alimentaria entre los consu-midores.

Las principales tareas de la Fundación sonsalvaguardar el uso del logo EHEDG y su-pervisar el trabajo de las organizacionesautorizadas para realizar las pruebas deevaluación. Publican las guías y materialesde formación, controlan la página web,organizan reuniones y conferencias.

Las principales funciones de las SeccionesRegionales son la traducción de las guíasal idioma nacional, informar en su ámbitode influencia de las actividades interna-cionales de EHEDG, realización de semi-narios, talleres sobre diseño higiénico, fo-mentar la transferencia de conocimientoentre los miembros, promulgar el diseñohigiénico.

Todos los miembros y patrocinadoresestán invitados a participar en laAsamblea General durante la conferenciaque se realiza una vez cada dos años.

Objetivos

• Proporcionar asesoramiento en aspectosde ingeniería higiénica para la elabora-ción de alimentos inocuos.

• Ofrecer un forum o punto de encuentroa los fabricantes de equipos, la industriaalimentaria, usuarios y reguladores paratratar aspectos de diseño higiénico y fo-mentar la obtención de alimentos se-guros.

• Proporcionar documentos guía sobrelas prácticas y normas esenciales de di-seño higiénico, basadas en ciencia ytecnología, y revisarlas periódica-mente. Estas guías ofrecen asesora-miento a fabricantes de equipos yusuarios sobre cómo cumplir con la le-gislación nacional e internacional.


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• Desarrollar métodos de comprobaciónque puedan ser utilizados por terceraspartes para la evaluación del diseño hi-giénico conforme a la legislación.

• Asegurar que el uso del nombre y logode EHEDG se controlan conveniente-mente.

• Identificar áreas donde el conocimientodel diseño higiénico es insuficiente y fo-mentar la investigación y el desarrolloen estas áreas.

Ámbito de trabajo

El ámbito principal de trabajo de EHEDGse centra en la divulgación de la ingenieríahigiénica en el sector agroalimentario yfabricantes de equipos. Para ello trabaja

en el establecimiento de requisitos téc-nicos de diseño higiénicos, así como en elasesoramiento y divulgación del diseño hi-giénico y limpiabilidad de equipos e ins-talaciones agroalimentarias.

Para ello en la actualidad existen 25 sub-grupos de trabajo que se organizan en lossiguientes cuatro grupos de trabajo:

Además de las actividades de formacióny divulgación de la ingeniería higiénica acontinuación se describen otras activi-dades que realiza EHEDG:

• Elaboración, actualización y publicación

de guías: EHEDG promueve la prevención

a través de la elaboración de guías ba-

sadas en la combinación de los conoci-

mientos de sus miembros y los datos cien-

Grupo de trabajo Alcance Equipos y componentes. • Refrigeración y enfriamientoContact: J. Kastelein • Equipos para el procesado de carne/pescado

• Juntas mecánicas• Envasadoras• Tubería y conexiones• Bombas• Sensores• Válvulas

Principios. • Diseño de instalacionesContact: J. Hofmann • Principios de diseño higiénico

• Manipulado de alimentos secos o deshidratados• Integración sistemas de higiene• Materiales de construcción• Método de ensayos/certificación• Soldaduras

Procesado y servicios/instalaciones auxiliares. • Manejo del aireContact: M. Stringer • Instalaciones eléctricas

• Tratamientos de calor• Lubricantes• Agua de procesor

Formación y educación. • Facilitador Contact: K. Lorenzen • Toolbox

• Formación


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tíficos disponibles. En la actualidad existen32 guías.

• Certificación de equipos: se dispone de unprocedimiento técnico para la certificacióndel diseño higiénico de equipos. El es-quema de certificación de equipos deEHEDG consiste en una evaluación técnicadel diseño higiénico del equipo seguidode la comprobación de la facilidad de lim-pieza, “limpiabilidad”. Para ello los ex-pertos primero realizan una comprobacióndel cumplimiento de los requisitos de di-seño higiénico. Una vez superada estaevaluación se procede a la realización delos ensayos oportunos para comprobar lafacilidad de su limpieza. Estos ensayos sólopueden realizarse por los organismos au-torizados por EHEDG (TNO, DTI, CCFRA,Universidad de Munich).

Ventajas de convertirse en miembroEHEDG

Las empresas miembros de EHEDG secomprometen con los más altos están-dares de seguridad alimentaria en sus em-presas y se esfuerzan en mejorar laimagen global de la industria que los con-sumidores tienen. El uso del logo deEHEDG como patrocinador es unamuestra de este compromiso.

A través de la red de nuestra organización,las empresas miembro pueden promo-cionar su visión, dado que apoyan los ob-jetivos EHEDG. Las empresas miembropueden marcar tendencias y obtener reco-nocimiento internacional de sus esfuerzos.

La contribución anual de cada empresamiembro depende de su facturación rela-cionada con el negocio alimentario. Todoslos miembros (individuales y empresas)

están invitados a la Asamblea Generalque se realiza cada dos años. Las contri-buciones se basan en la facturación anualde la empresa relacionada con el negociode la alimentación y oscilan entre 500 y10.000 €. En el caso de miembros indivi-duales la cuota es de 100 €.

Otras ventajas:

• Uso del logo de EHEDG registrado paraempresas en la documentación de la em-presa, tras la firma de un contrato, en elque se especifican las condiciones de uso.Por ejemplo, las empresas fabricantes deequipos no pueden usar ese logo para in-dicar que un producto está certificado.

• Emplear el logo o nombre de EHEDG enprogramas de cursos, folletos, etc.

• Obtener una copia gratuita de las guíasen CD-ROM para empresas miembros ycon un 50% de descuento para miem-bros individuales.

• Ser componentes de hasta dos sub-grupos de trabajo.

• Enlace desde la página web de EHEDG.

Método EHEDG paracomprobar la limpiabilidad CIPde equipos para el procesadode los alimentosEn el presente apartado se describe el mé-todo de EHEDG para comprobar la limpia-bilidad CIP de equipos moderadamentepequeños descrito en el documento oguía nº 2 de EHEDG.

EHEDG dispone de un esquema de cer-tificación del diseño higiénico deequipos para equipos cerrados que re-


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quieran limpieza CIP y sobre los queexistan dudas o no pueda demostrarseel cumplimiento total de los requisitosde higiene por un experto autorizado.Se debe someter a dicho ensayo decomprobación de la limpiabilidad.

El procedimiento de ensayo está diseñadopara indicar las zonas con un diseño higié-nico deficiente en los equipos y está basadoen una comparación de la limpiabilidad deun elemento sometido a ensayo con la deun trozo de tubería recto, o tubo de refe-rencia. El grado de limpieza se basa en laeliminación de una solución ensuciadoraque contiene bacterias y se valora eva-luando el crecimiento de las bacterias quequedan después de la limpieza.

El ensayo está pensado, por lo tanto,como ensayo básico de comprobación deldiseño de equipos higiénicos y no es indi-cativo del comportamiento en situacionesde limpieza industrial.

Método de ensayo

El equipo o elemento que se va a ensayarcon objeto de comprobar su limpiabilidady por tanto su diseño higiénico, junto conla tubería de referencia se someten a unproceso de ensuciamiento y posterior-mente a su limpieza.

Para su ensuciamiento se utiliza lecheagria inoculada con GeoBacillus stearot-hermophilus ya que es de crecimiento rá-pido, tiene esporas que son resistentes ala solución detergente que se usa en elprocedimiento de ensayo y produce reac-ciones de color bien definidas en el mediode crecimiento utilizado.

El agar utilizado es MSHA Shapton yHindes modificado, que con la presenciadel Geobacillus provoca un cambio en el

pH y el agar vira de color morado a coloramarillo siendo muy fácil su detección.

El equipo a ensayar se llena con la solu-ción ensuciadora y se presuriza tres vecesa 5 bares durante 2 minutos. Mientrasestán sometidos a la presión, las partesmóviles que existan se accionan para si-mular las condiciones de empleo (porejemplo, se abren y se cierran las válvulas).A continuación se vacía y se seca con aireseco filtrado, se requiere un valor final deHR < 5% a 15-25 ºC.

La sección de ensayo ensuciada se montaen un banco de pruebas CIP construido apropósito (ver esquema), para proceder asu limpieza:

• Aclarar con agua fría (10-20 ºC) duranteun tiempo igual al tiempo medio de re-sidencia (t) y no inferior a 1 minuto.

• Hacer circular una solución detergente al1,0% (p/v) a 63 ºC ± 2 ºC durante 10 mi-nutos (el volumen de solución detergenteusado debe ser al menos 20 veces el vo-lumen interior del elemento sometido aensayo).

• Aclarar con agua fría (10-20 ºC) duranteun tiempo igual al tiempo medio de re-sidencia (t) y no inferior a 1 minuto.

Las soluciones de limpieza se hacen circulara una velocidad de flujo media de 1,5 ms-1± 0,1 m s-1 dentro del tubo de referenciay se debe mantener una presión hidrostá-tica positiva en las tuberías de retorno paraevitar burbujas de aire en el sistema.

Tras el proceso de limpieza se retira elequipo y la tubería de referencia del bancode pruebas y se procede a la evaluación desu limpiabilidad para ello se procede a re-llara tanto el equipo como la tubería con


99

agar MSHA Después de que el agar se hayasolidificado completamente, se coloca casivertical en una estufa de cultivo a 58ºC du-rante 16-24 horas.

Evaluación de resultados

Después de la incubación, el equipo de en-sayo y del tubo de referencia se examinanpara buscar la presencia de zonas de colo-ración amarilla en el MSHA púrpura.

El tubo de referencia se abre y se extraeel agar solidificado. Para ello, con una he-rramienta especial, se extrae el núcleocentral del agar. Luego se abre el tubo deagar que se ha formado convirtiéndolo enuna lámina plana y se coloca sobre unacuadrícula de recuento transparente.

Es preferible utilizar una cuadrícula quetenga divisiones de 5 mm x 5 mm, loque facilitará la evaluación de las zonasamarillas.

Para facilitar la diferenciación entre elcolor amarillo y el púrpura y estandarizarlos resultados de ensayo realizados por di-ferentes operarios, se dispone de un discocomparador de colores.

El porcentaje de zona amarilla 5-30%sirve como control de la variabilidad tantointra como interlaboratorios.

Por lo general, y asumiendo que existeuna pequeña cantidad de color amarilloen el tubo de referencia, son posibles tresresultados para el equipo o elemento so-metido a ensayo:

• Presencia de residuos de leche: si hay pre-sencia de residuos visibles de lechecuando se observa/desmonta el elementosometido a ensayo antes de que seaplique el agar, no es necesario realizarningún examen microbiológico, ya quelas esporas están claramente presentespor lo que presenta graves defectos en eldiseño. Sin embargo, se requiere un aná-

solucióndetergente

tubo de referenciaTI

PI

FI

válvula deregulaciónde caudal

válvula dederivación

bombacentrífuga

FI indicador de caudalPI indicador de presiónTI indicador de temperatura

TESTITEM

agua deaclarado

Esquema del banco de pruebas CIP para el ensayo de limpiabilidad.


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lisis microbiológico del tubo de referenciapara confirmar que el ensayo fue ade-cuado. Se debe repetir el ensayo.

• Presencia de zonas y/o colonias amarillas:se debe repetir el procedimiento de en-sayo hasta un máximo de cinco veces. Lapresencia de suciedad retenida en lamisma zona del elemento sometido a en-sayo en tres ocasiones distintas es indica-tiva de zonas que son de difícil limpiezay, por consiguiente, zonas en las que sedeben considerar mejoras en el diseño hi-giénico. La limpiabilidad del elemento so-metido a ensayo se compara con la deltubo de referencia evaluando las superfi-cies porcentuales correspondientes dezonas amarillas:

– Si la superficie porcentual de zonasamarillas en el elemento sometido a en-sayo es similar a la del tubo de refe-rencia, el grado de limpiabilidad es, porconsiguiente, similar.

– Si la superficie porcentual de zonas ama-rillas en el elemento sometido a ensayoes inferior o superior a la del tubo de re-ferencia, el elemento sometido a ensayoes, respectivamente, más o menos fácilde limpiar. En el caso de que sea igual o

más limpiable que la tuberia de refe-rencia, el equipo sometido a ensayo esapto para certificar su diseño higiénico.

En pruebas comparativas de equipos, lasuperficie relativa de zonas amarillas quepermanece en cada una de las piezas delequipo ensayado se puede usar comomedida de su limpiabilidad. La interpre-tación de las comparaciones de limpiabi-lidad es sólo aproximada, ya que el gradode color amarillo puede estar influido porel espesor del agar sobre la superficie delelemento sometido a ensayo.

• Sin zonas/colonias amarillas: si estas con-diciones se dan en tres ocasiones suce-sivas, no es necesario repetir más el en-sayo y el elemento sometido a ensayo sepuede describir como especialmente fácilde limpiar o limpiable.

En ocasiones algunos materiales empleadosen juntas de estanquidad pueden tenerpropiedades antibacterianas que impidanque las esporas presentes en su superficiegerminen y/o crezcan. Así, zonas con di-seño higiénico deficiente pueden no apa-recer como zonas amarillas, lo que da lugara resultados falsamente negativos.

Para conocerlo, se deben comprobar laspropiedades antibacterianas de todas lasjuntas tóricas y juntas de estanquidad, paraello se inocula con esporas un volumenadecuado de MSHA (aproximadamente 102

ml-1 de agar). A continuación se colocan lasjuntas de estanquidad/juntas tóricas esté-riles en placas petri adecuadas, se cubrencon el MSHA fundido y se incuban a 58 ºCdurante 16-24 horas. Si las juntas de estan-quidad/juntas tóricas tienen propiedadesantibacterianas, se ve una zona púrpura asu alrededor.


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Centros autorizados por EHEDG para

la realización del test de limpiablidad

En la actualidad EHEDG dispone de untotal de cinco centros autorizados para larealización de las pruebas de limpiabilidady para la emisión de informes favorablesnecesarios para la certificación del diseñohigiénico de equipos:

• Dinamarca: DTI.

• Alemania: Universidad de Munich.

• Holanda: TNO.

• Reino Unido: Campden BRI.

• EE.UU.: Universidad de Pardue.

Es objetivo de AINIA convertirse en elsexto centro autorizado. ActualmenteAINIA cumple con los requisitos estable-cidos internamente por EHEDG y sólotiene pendiente de conseguir la acredita-ción ISO 17025 del método. Está previstoconseguir esta acreditación en el segundosemestre de 2009.

A continuación y a modo de resumen seindican los principales requisitos estable-

cidos por EHEDG para convertirse encentro autorizado:

• Ser miembro del subgrupo de trabajode EHEDG de Métodos de Ensayo, yparticipar en las reuniones de trabajo.

• Disponer de un plan de negocio con elcompromiso formal de actuar como ins-tituto autorizado (disponer de instala-ciones, cualificación del personal, man-tenimiento preventivo de instalacionesy equipos…).

• Disponer de personal con formación yexperiencia demostrable en el área dehigiene.

• El personal responsable del métododebe realzar el curso avanzado de di-seño higiénico de EHEDG.

• Recibir formación y experiencia previade la mano de otro centro o experto au-torizado.

• Obtener la acreditación ISO 17025 delmétodo de ensayo.

• Participar en cualquier actividad pro-puesta por el grupo de trabajo así comoen la pruebas interlaboratorio.

IntroductionCleaning and disinfection procedureshave to be considered as an integral partof food production. If the cleaning is noteffective, micro-organisms and productresidues will remains at concentrationsthat may affect the quality and safety ofthe food product (Gibson et al, 1999).

The attachment of bacteria, with possiblesubsequent development of biofilm infood processing environments, is a poten-tial source of contamination of finishedproduct that may shorten the shelf-life orencourage transmission of diseases(Sharma and Anand, 2002).

Biofilm are communities of microorganismson surfaces, typically encased in somepolymer matrix that has been produced bythe microorganism themselves. Microor-ga-nisms attached as in biofilms exhibit diffe-rent properties to microorganisms that floataround in liquid: they are more resistant toantibiotics and biocides and are more diffi-cult to remove from the surface. In the foodprocessing environment, particularly withinclosed systems, conditions favour attach-ment and biofilm formation, i.e. flowingwater, suitable attachment surfaces, amplenutrients and raw material (Gibson et al,1999). Furthermore the formation of bio-film protects bacteria from hostile conditions

and thus these bacteria are much more re-sistant to detergents and disinfectants onopen surfaces (Notermanns, 1994). Thechoice of cleaning and disinfection productis also highly dependent on the food matrixin which the organism is embedded (Gramet al, 2007; Verran et al, 2008).

Cleaning should both remove soil and re-duce the number of microorganisms pre-sent. The disinfection should further reducethe surface population of viable microorga-nisms via removal or destruction, and/or toprevent surface microbial growth duringthe inter-production period (Holah, 2003).

In 2003 Holah gave a review of new clea-ning agents and cleaning methods availableat that time. This chapter is a further des-cription of the new cleaning agents andmethods. In addition the newest resultsfrom WP11 partners in PathogenCombatare included.

Cleaning and disinfection:quantitative assessment

Methods for differentiating soil andmicroorganisms

Methods for differentiating soil (e.g. meator dairy soil) and microorganisms in opensystems have been developed by

New cleaning and disinfection methodsand summary of methods applied forverification of their efficiency H. Løje, A. Friis, L. Gram, B. Carpentier, J. Verran


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Manchester Metropolitan University (MMU)(Verran et al, 2008). The aim of this workwas to recommend a specific and simplemethod for fouling and cleanability assaysto industry, particularly SMEs, that is rele-vant to their product, and whose results aresupported by more sophisticated analyticaltechniques.

A large experiment involved soiling of sur-faces with key components of dairy andmeat industry food soils (eg oils, carbohy-drates, proteins) at different concentrations,and then using a range of analytical anddetection methods to compare ease of use,sensitivity, and relationship with oneanother. Two commercially available kits,UV light detection method and ATPBioluminescence, were included in thestudy. Briefly, UV light detection method (egBactoforce) provides a convenient, macros-copic indication of poor hygiene (generalfouling), but does not differentiate betweencells and soil. Some problems were encoun-tered with the use of ATP hygiene testing,in terms of the efficiency of removal ofsoil/cells from the surface, but this methodgenerally provided an indication of the pre-sence of microorganisms. Viable countsfrom swabs suffered the same problem interms of poor recovery. Fluorescent stainingof cells and soil on the surface provided themost rapid and simple method, althoughthe area imaged is microscopic, thereforesmall, and the technology is perhaps noteasily accessible by SMEs (Whitehead et al,2008). The combination of cells and soilcomponents increased complexity foranalysis, but staining and subsequent dif-ferential analysis of the coverage of micros-copic fields of the surface by microorga-nisms and cells will enable useful evaluationof the effectiveness of given soil/cell/clea-

ning and disinfection regime/industry com-bination, and separation of the different re-moval kinetics of cells and soil.

Combinations of different stains enable dis-crimination between cells and food soil;DAPI and Rhodamine; DAPI and fluoresceinand DAPI and acridine orange.

Quantification of attached cells

Quantification of attached bacteria is a keyparameter in assessing persistence and ef-fect of different soils and cleaning and di-sinfecting agents. Work comparing diffe-rent quantification methods (indirectconductance, real-time PCR, sonication/ co-lony counting, direct microscopy) haveshown that several methods can be usedto quantify bacteria on surfaces. Direct mi-croscopy is well suited at medium bacterialdensities but cannot be used when veryhigh or low numbers are present. Hencethe method is not suitable when measuringreducing effects of disinfectants. During thePathogenCombat work, the partners haveprimarily used the methodology developedby AFSSA (Asséré et al, 2008) where bac-teria are removed by high energy ultra-sound (28 kHz, 150 W) for 4 min and sub-sequently quantified (Kastbjerg and Gram,2009; Marouani-Gadri et al, 2009a and2009b). When only culturable cells arequantified after cleaning and disinfection,it is not possible to distinguish the part dueto detachment from the part due to killingin the reduction of the bacterial numbers.Real-time PCR was thus used to quantifytotal cells which reduction indicates detach-ment (Marouani-Gadri et al, 2009c).Ethidium monoazide real-time PCR (Novgaet al, 2003; Rudi et al, 2005) was also usedto quantified viable cells, i.e. the culturablecells and the viable-but-non-culturable ones

which numbers may be 2 to 3 log greaterthan culturable cells counts (Peneau et al,2007; Marouani-Gadri et al, 2009c).

Cleaning and disinfection:cleaning agents

Enzymes

Several studies have reported on the posi-tive cleaning effects of enzymes on ultrafil-tration membranes fouled with protein-based residue from milk or meat processingenvironments (Allie et al, 2003; Argüello etal, 2003; Muñoz-Aguado et al, 1996).Enzyme based cleaning will find practicalapplication in bioprocess operations only ifno residual activity remains on the surfacepost-cleaning. After the cleaning process,equipment is often sanitized/sterilized byexposure to live steam or boiling water.These steps will almost certainly inactivateany residual enzymes remaining on the testsurface. Enzymes were able to remove soiland the cleaning efficacy was increased byincorporation of a detergent (Turner et al,2005).

Initial issues with respect to the use ofenzymes to clean dairy equipment includedhigh costs and low cleaning efficiency(Grasshoff, 1997). However, with increasingenvironmental concern, enzymatic cleanersare a promising alternative to traditionalchemicals (Grasshoff, 2002). The industryfor textile detergents has employed suchmethods, resulting in reduction in the che-micals required and reduced heating, henceenergy saving. Enzymes have been succes-sively used for the cleaning of cold milk pro-cessing equipment (Potthoff et al, 1997)and membrane cleaning. A number of in-vestigations on the use of enzymes to clean

milk heaters have been reported (Grasshoff,1997).

Enzyme detergents have also proved tobe effective in disrupting the extracellularpolymers which form the biofilm matrixand thus helped in removal of biofilms(Potthoff et al, 1997). A mixture ofenzyme activities may be necessary for asufficient degradation of bacterial biofilmdue to the heterogeneity of the extrace-llular polysaccharides in the biofilm(Johansen et al, 1997). A complex mixture of polysaccharide-hydrolyzingenzymes was able to remove bacterialbiofilm from steel and polypropylenesubstrata but did not have a significantantibacterial activity. Combining oxidore-ductases with polysaccharide-hydrolyzingenzymes resulted in bactericidal activityand removal of the biofilm (Johansen etal, 1997). The use of enzymes for re-moval of bacterial biofilm is still limiteddue to the very low prices of chemicals inuse.

Enzymes and detergents have also beenused as synergists, to boost disinfectantefficacy (Johansen et al, 1997). The spe-cific mode of action makes it difficult ho-wever to find enzymes that are effectiveagainst all different types of biofilm(Meyer, 2003).

Cleaning and disinfection:new mechanical actions forcleaning

Ice-pigging

Ice-pigging is a novel and innovative newpigging technique that has significant ad-vantages over conventional solid pigs. Themethod was developed by Professor Joe

New cleaning and disinfection methods and summary of methods applied for verification...

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Quarini and his team at the Departmentof Mechanical Engineering, University ofBristol (Quarini, 2002). The ice pig plug isformed from a thermodynamically stableice slurry combined with freezing pointdepressant which is capable of cleaning aproduct from ductwork and/or separatingproducts in different phases of the pro-duction cycle. Ice pigging employs densesuspensions of ice to purge products fromprocess lines. These soft–solid plugs canbe pumped safely through a range ofpipe fittings and process units, achievingvery good degrees of sweeping.Noteworthy features of the technique in-clude its benign chemical nature, safety,small fluid inventory, and relief of bloc-kage (by melting). Ice pigging must ho-wever, be accompanied by clean in place(CIP) or other methods in order to makereliably clean surfaces.

The cleaning efficiency of ice-pigging indi-cates that the ice pig could easily and effi-ciently remove “soft” fouling. The foulingmaterial tested included jam and fats (foodindustry), tooth paste and fine silt and sand(Quarini, 2002). The advantages of thistechnique include low environmental im-pact and the ability to separate and recoverproducts. Results show a significant impro-vement in cleaning behaviour comparedwith water at 20°C; however, the resultswere not compared with cleaning with che-micals. It is also not yet clear to what extentpigging removes very thin layers of deposit,but the method may significantly reducerinsing times.

Two beneficial advantages of the ice pigare that it is environmental very friendlyand that it never gets stuck in pipeworkfor very long. Over time, at normal tem-perature it will melt to water (Quarini,

2002).Thus ice pigging with its ability tocope with difficult geometrics appears tooffer an innovative defouling methodo-logy (Shire et al, 2005).

Ultrasound

Ultrasound is defined as acoustic energy orsound waves with frequencies above 20kHz. Ultrasound provides an effectivemeans of cleaning surfaces because of thephysical effects on the cavitation pheno-menon. The destruction and removal of mi-crobial cells by ultrasound can be achievedby choosing combinations of ultrasonicpower, exposure time, temperature and dis-tance from the contaminated surface.Dismantled or small items of equipmentcan be effectively cleaned in minutes.

Ultrasonication was found to be a suitablecleaning method for both cheese mouldsand transportation crates (Salo andWirtanen, 2007). To ensure the effective-ness of cleaning, the quality of cleaningliquid should be measured frequently andthreshold limits for changing the cleaningliquid should be set.

Mott et al, (1998) found that ultrasound(30 sec. pulses 20-150-350 KHz) was ca-pable of removing bacterial biofilm along50 cm lengths of glass tubing, and thislength might be the maximum.Ultrasound could also be propagatedalong lengths of water-filled tubing anda study using 20 or 150 kHz transducersshowed that Proteus mirabilis biofilmscould be effectively removed from 50 cmtubes by cavitational activity (Mott et al,1998). Muthukumaran et al, (2004)found that ultrasound was good on thecleaning of whey-fouled membranes. Theuse of surfactants in combination with ul-

trasonic cleaning showed a synergistic ef-fect providing a better efficiency thaneither cleaning process alone.

Ultrasound in combination with heat, pres-sure or non-foaming detergents appears tobe more effective than any of the treat-ments alone in cleaning or decontamina-tion of foods (Guerroro et al, 2001).Ultrasonic treatment in conjunction with awater temperature of 60 °C has shown toreduce the microbial contamination of cratesurfaces and there the microbial hazards as-sociated with transporting live poultry tothe processing plant (Allen et al, 2008).Tolvanen et al, (2007) found that loga-rithmic reduction of L. monocytogenes wassignificantly greater in stainless steel than itwas in plastic materials. The difference incleaning efficacy for the various materialtested can partly be explained by the hard-ness of the material. Ultrasonic cleaning ismore efficient on hard surfaces than it is onsoft materials (Tolvanen et al, 2007).Nevertheless it was found that ultrasoniccleaning was an effective method of deta-ching L. monocytogenes from conveyormaterials. Short ultrasonic washing treat-ment may provide a new possibility in cle-aning conveyor belts that are difficult toclean with conventional methods. It wasfound that cleaning time could be as shortas 30s without impairing the cleaning re-sults. Combinations of ultrasound andsteam efficiently killed bacteria on stainlesssteel and flooring surfaces in a dose depen-dant manner, however, it did cause a minorspread of bacterial cells to surroundingareas (Vogel et al, 2009).

Pulsing Flow

Pulsing flows have been investigated as away of controlling, or preventing, fouling.

Flow pulsing involves imposing a velocitypulse on a steady flow, increasing thelocal shear rate and pressure at the de-posit/liquid interface. The dynamics andapplications of pulsating flows in pipeswere reviewed by Edwards and Wilkinson(1971).

Flow pulsing is an attractive technologyfor systems where the liquid is too viscousto achieve turbulent flow or the inventoryof fluid is to be minimized (Bode et al,2007). Shamel et al, (1999) found thatboth the pulse frequency and amplitudewere important parameters to improvepulsing flow in cleaning.

Flow pulsing has been used as a methodfor enhancing the rate of cleaning of wheyprotein deposits in CIP systems. Gillham etal, (2000) investigated the enhancementof alkali-based CIP of whey protein depo-sits by flow pulsing using low frequence(< 2Hz) fluid pulses. Enhancement of clea-ning rates varied up to 250% and de-pended on the initial amount of depositpresent, pulsing frequency and pulse am-plitude. Flow pulsing strongly affected thecleaning behaviour after the initial depo-sits swelling stage. The pulsing has the ef-fect of raising the maximum shear stress,at the pipe wall and thus increasing the cle-aning rate (Gillham et al, 2000).

Wall shear stress influences the rate of re-moval and the amount of soil removedfrom the surface exposed to a movingfluid. Higher velocities tend to break upthe fouling layer due to an increase inshear stresses acting on the surface. Apulsating flow could create momentary,large accelerations of the liquid flowaround the fouling layer, thus resulting inan increase in removal of the layer (Bode


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et al, 2007). Jensen et al, (2006) studiedcleaning of an upstand with a pulsatingflow rate. The results showed that a pul-sation only has minor effect on the re-moval of the soil used. Increasing the flowrate increased velocity generally, but onlysmall increases were seen in areas, whichare always difficult to clean.

Bode et al, (2007) found that cleaningtime using pulsing flow of whey proteinfouling layer could be shortened by twoand a half times, which correspond to theresults by Gillham et al, (2000).

Two phase flow (water/air)

In 1959 Jennings found that effectivenessof cleaning was increased by permittingair to leak into the vacuum side of thesystem. Using two phase flow (air/water)reduces the water required for circulation,increases flow velocities and enhancesmechanical cleaning action (Reinemann,1996).

To remove membrane fouling, there aremany means and methods available inpractice to either detach biomass duringmembrane cleaning or inactivated micro-organisms during biocide dosing.However chemical cleaning alone isusually not enough to control biofouling,the biomass has also to be physically re-moved (Cornelisssen et al, 2007). In orderto inactivate biomass from membrane ele-ments, Cornelissen et al, (2007) investi-gated air/water cleaning and daily coppersulphate dosing. It was found that bothair/water cleaning and daily copper sulp-hate dosing proved to be very effectivemethods in reducing membrane foulingdue to feed spacer fouling. The dailyair/water cleaning was somewhat less ef-

fective in control of bio-fouling than thecopper sulphate dosing but better thanthe reference (no cleaning). The cost ofoperation may be reduced strongly byapplying this cleaning method. In thestudy by Cornelissen et al, (2007), biofou-ling is connected with membranes ap-plied for the production of drinking water,process water and for desalination of se-awater. Copper sulphate is not a commondisinfecting agent in food industry.

A new disinfecting process:plasma jetA new disinfection mechanism using at-mospheric plasmas has been investigated(Lee et al, 2003; Araya et al, 2007). Gasessuch as nitrogen, argon and a mixture ofnitrogen and oxygen can be used asplasma gas. Araya et al, (2007) found thatpaint on plastic surfaces was removed byusing dry air as plasma gas in an atmosp-heric non-thermal plasmas jet. High pres-sure cold plasma jet has been used to des-troy biofilm, and the results indicated thepotential of plasmas as an alternative wayfor biofilm removal (Abramzon et al,2006).

Cleaning and disinfectionproducts: application method

Mist, foam, fogging and geltechniques

The main difference between mist, foamand gel techniques is their ability to main-tain a detergent/soil/surface contact time.Mist spraying is undertaken using smallhand-pumped containers sprayers or pres-sure washing systems at low pressure.Misting will only wet vertical smooth sur-

faces. Foams can be generated and ap-plied by the entrapment of air in high-pressure equipment or by the addition ofcompressed air in low-pressure systems.Gels are thixotropic chemicals which arefluid at high and low concentrations butbecome thick and gelatinous at concen-trations of approximately 5-10%. Gels areeasily applied through high and low pres-sure systems or from specific portableelectric pumped units and physically ad-here to the surface (Holah, 2003).

Fogging disinfection implies dispersal offinely disposed droplets of a disinfectantwithin a room. The intention of foggingis to ensure that all regions and equip-ment in the room receive an adequate ap-plication of the disinfectant. Fogging sys-tems are costly, but could be cost efficientand also result in improved hygiene ifused appropriately (Bore and Langsrud,2005). Only few studies have investigatedthe use of fogging. Bagge-Ravn et al,(2003) compared the efficacy of peraceticacid-based fogging with hypochloritebased foam in a salmon smokehouse. Theresults indicated that the procedure basedon fogging gave similar or better reduc-tion in micro-organism than the foam-based method.

Russell (1999) found that fogging is suc-cessful for the disinfection of horizontal,upward facing surfaces but is ineffectivefor disinfecting vertical surfaces, under-sides and dismantled equipment. Theseneed spraying. It was also pointed outthat fogging must be regarded as an ad-ditional safeguard only and must not re-place traditional cleaning and disinfectionroutines. Unwanted processing materialleft on surfaces prevents the fog reaching

the micro-organisms and can deactivatecertain chemicals.

Holah (2003) has in his review given atstatus of the foams, mist, gels and fog-ging systems and compared them.Readers are referred to read more inHolah’s review (Holah, 2003).

Disinfectants

Chlorine, a strong oxidant, and chlorinereleasing compounds have been widelyused as disinfectants in the food industry(Holah, 2003) for example for washingfresh produce and sodium hypochloritehas been used in several CIP systems(Birks, 2003).

Chlorine has been shown to have the abi-lity to depolymerise the polymeric matrixof biofilm so that it can also detach bac-terial cells. Carpentier (unpublished re-sults) found that chlorinated alkaline de-tergents were the most effective inremoval of Pseudomonas fluorescens bio-film cells from stainless steel. Acidic andneutral detergents detached no more cellsthan water.

Chlorine compounds can be corrosive toequipment and hazardous to health, andshould always be handled with care andin the appropriate concentrations (Holah,2003). Chlorine and chlorine-derivedcompounds are now considered undesi-rable due to the health concerns basedon toxicity and ecological concerns andare gradually being withdrawn from use.

Ozonated cold water/ozone. Ozone is apowerful antimicrobial substance due toits potential oxidizing capacity and it iswidely used as a disinfectant in a varietyof applications. Ozone (O3) is formed by


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addition of an oxygen atom to moleculardiatomic oxygen. The triatomic oxygenis highly unstable and rapidly degradesby releasing the third oxygen atom. Thetri-oxygen molecule of ozone is the se-cond most powerful oxidizer after fluo-rine. There are many advantages ofozone in the food industry such as foodsurface hygiene, sanitation of food plantequipment, treatment of food plantwaste and reuse of waste water (Guzel-Seydim et al, 2004). Guzel-Seydim et al,(2000) found that on ozonated waterpretreatment was better than pre-treat-ment with hot water. However the diffe-rence was slight. The investigations havebeen done in steady-state systems withthe only movement coming from thebubbles of ozone and the correspondingbubbles of air for the hot water cleaning.Ozone treatment was cold (10 °C) andhot water was at 40 °C.

Ozone can be applied both as a gas andin ozonated water. When used in com-bination with water it provides a strongsterilising agent several times more po-werful than chlorine, suitable for equip-ment and production area washdown,thus eliminating the need for hot wateror hazardous chemicals (Birks, 2003).Some recent studies have examined newmethods of applying ozone by foggingozonated water and charging it electros-tatically to increase the effectiveness ofapplicationon vertical surfaces (Birks,2003).

Pascual et al, (2007) have in a reviewdescribed the use of ozone as a disinfec-tant agent, as well as it uses to clean anddisinfect surfaces and equipment, andthe environmental impact of cleaningand disinfection using ozone. A table is

presented which summarize findings onefficacy of ozone on various surfaces andmicro-organisms. Moderate doses ofozone (between 0.5 ppm and 3.5 ppm)both in gas form and as ozonated waterwere sufficient to achieve significant mi-crobial reductions. When ozone is ap-plied as a gas, the necessary exposuretimes 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 orderto increase the capability of organicmatter removal, a high temperature anda high concentration of dissolved ozonein ozonated water was most effective.

Guzel-Seydim et al, (2004) reviewed theuse of ozone in the food industry andconcluded that there are sound advan-tages of ozone applications in food in-dustry. Ozone does not leave any residuedue to its quick decomposition. Howeverrestrictions should be applied to humanexposure to ozone. In humans, ozoneprimarily affects the respiratory tract.

Dosti et al, (2005) found that the killingeffect for microorganisms on surfaceswas better with ozone than heat and ch-lorine. Microorganisms in biofilm havethe same “resistance” to ozone and ch-lorine.

Sanitising agents based on chlorine arenow considered undesirable due to he-alth concerns. The interest in ozone asan alternative to chlorine and other che-mical disinfectants in cleaning and disin-fection operations is based on its highbiocide efficacy, wide antimicrobial spec-trum, absence of by-products that aredetrimental to health, and the ability tobe generated it on demand, in situ, wit-


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hout a storage requirement. It also hasthe significant advantage of being envi-ronmental friendly that reduces the com-pany’s environmental cost (Pascual et al,2007).

Electrolysed strong acid water (ESAW) &Electrolysed weak acid water (EWAW)also known as “activated water”. ESAWand EWAW have a high oxidation reduc-tion potential. These solutions can beprepared using salt and tap water, andlose their oxidative and acidic propertieswhen exposed to the environment. Theprocess of generation of electrolyzedwater is simple: electrolyzed water is ge-nerated by the electrolysis of a 0.1%concentration of NaCl solution in deio-nized water (Mahmoud, 2007). ESAWhas a pH value between 2.3 and 2.7while EWAW has a pH value between5.0 and 6.0. Mahmoud (2007) has re-viewed electrolysed water and its use forfood decontamination.

Urata et al, (2003) investigate the disin-fection of endoscope and found thatESAW and EWAW were effective aftermechanical cleaning of upper gastro-in-testinal endoscopes, and could, there-fore, be used in the endoscopy unit.Electrolysed water is more effective forkilling than ozonated water (for micro-organisms found on medical equip-ment). However it also has a corrosive ef-fect on the steel.

Electrolyzed oxidizing water is createdwhen electric current flowing throughtwo electrodes – immersed in a weaksalt solution and separated by a mem-brane – produces an alkaline and anacidic solution. Research results haveshowed that with 7.5-10 min the elec-

trolyzed oxidizing water was as effectiveas conventional treatments in removingorganic matter from the milk pipes.Other claims have been more obscure orless transparent (Napper, 2007).

Additional considerations

To compare sensitivity of different bacterialstrains to disinfectants, it is very importantto standardize the assays used with respectto biomass. Also, the system must be cali-brated to allow a measurable reduction, i.e.some bacterial cells must remain after theactivity. DTU Aqua has developed such met-hodology for planktonic bacteria, attachedbacteria and biofilm bacteria (Kastbjerg andGram 2009). The set-up uses, deliberately,very low concentrations of disinfectants toachieve measurable reductions that can bestatistically compared. We hypothesisedthat the cause of persistence could be theappearance of persister cells within a L. mo-nocytogenes population. This has been stu-died in collaboration with KU-Life and wehave not found indications of such persistersub-populations using FRIM to measure in-tracellular pH of surface attached L. mo-nocytogenes (Kastbjerg et al, 2009a).

Since processing equipment is the mostcommon source of L. monocytogenes asproduct contamination, the influence ofdisinfectants on phenotypic behaviour isimportant. Especially, PathogenCombathas in collaboration with Professor HanneIngmer, KU-Life determined if sub-lethalconcentrations of disinfectants has any ef-fect on the virulence of L. monocyto-genes. Our data, currently being preparedfor publication, indicate that indeed somedisinfectants may either upregulate ordown regulate the central virluence regu-lator, prfA (Kastbjerg et al, 2009b).


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Comparison of persistent and non-persistent strains (e.g Listeria mo-nocytogenes) with respect to theirsensitivity to disinfections (Incimaxx)

A collection of persistent and non-persis-tent Listeria monocytogenes strains hasbeen tested for sensitivity to a commonacidic disinfectant (Incimaxx). DTU Aquahas not been able to demonstrate a sys-tematic difference in tolerance betweenthese two groups. A sub-group of thesehas been tested against one other disin-fectants (Triquart). As with Incimaxx, nosystematic strain difference was detected.This has been published in Kastbjerg andGram (2009).

The effect of simple foodpreservation parameters (e.g. NaCl)on disinfectants

The effect of simple food preservation pa-rameters has been studied. DTU Aqua hasdemonstrated that when grown with 3-5%NaCl, most L. monocytogenes strains auto-aggregate and stick to plastic surfaces. InPathogenCombat, we have for the firsttime demonstrated that the adhesion tostainless steel is also enhanced by presenceof moderate levels of NaCl and 1/2-1 loghigher cell counts are seen on steel surfacesif L. monocytogenes has been grown withmoderate levels of salt than if grown inmedia with 1/2-1% NaCl. We have foundthat NaCl protects planktonic cells of L. mo-nocytogenes against the acidic disinfectant,Incimaxx. This protective effect is, however,not seen for surface spotted bacteria.Measurment of intracellular pH (collabora-tion with WP1) has confirmed that NaClprotects that planktonic cells against thestress encountered when exposed to the di-

sinfectant. These results were presented atIAFP summer 2008 and are being publishedin Kastbjerg and Gram (2009). Using mea-surement of intracellular pH by FRIM(Kastbjerg et al, 2009a), it has also been cle-arly demonstrated that bacteria grown withmoderate NaCl levels are more tolerant todisinfectants as measured by a higher intra-cellular pH.

ConclusionWithin the last few years several newapproaches for cleaning agents and cle-aning methods in the food industry havebeen described. Many of these are phy-sical/mechanical methods rather thanchemical agents. One of the most pro-mising procedures seems to be ultra-sound, which can be used in criticalplaces in closed equipment and forpieces of open equipment. Ozonatedwater has potential as a disinfectant,lacking some of the toxicity/environ-mental issues associated with somemore conventional biocides.

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Problemática de limpieza ydesinfección y ventajas deluso del ozono Las operaciones de limpieza y desinfecciónson imprescindibles para la industria agro-alimentaria ya que aseguran el manteni-miento de los niveles de higiene exigibles ynecesarios para producir con unos nivelesde calidad adecuados. Sin embargo, estasoperaciones llevan asociado un elevado im-pacto medio ambiental en lo que se refierea consumos de agua, productos químicosy energía, así como en la generación deaguas residuales y residuos sólidos. En lamayor parte de los casos estos impactosson los más relevantes que se producen enlas instalaciones agroalimentarias.

Habitualmente, los procedimientos delimpieza se diseñan atendiendo única-mente a su eficacia higiénica, olvidandoel impacto global que dichas actuacionestienen sobre el medio ambiente. No es ex-traño encontrar en las industrias agroali-mentarias un consumo desmesurado deagua y productos químicos, elevada pro-ducción de residuos y residuos de en-vases, alto consumo de energía térmica yeléctrica, etc. Otro aspecto medio am-biental que debería considerarse es la ge-

neración de subproductos peligrosos dela desinfección.

En lo que respecta al consumo de agua, lasinstalaciones de procesado de alimentosconsumen diariamente grandes cantidadesde agua (por ejemplo, una industria lácteapuede consumir entre 1 y 5 m3 por Tm deleche procesada, una cervecera puede con-sumir entre 4 y 8 Hl por Hl de cerveza ela-borado). Normalmente, las operaciones delimpieza son las que mayor consumo deagua conllevan en la industria alimentaria.La limpieza de depósitos y redes de tube-rías son las actividades que mayor consumode agua implican. Normalmente este tipode elementos se limpian mediante sistemasCIP (clean in place).

En cuanto a las aguas residuales generadas,se debe tener en cuenta que en la industriaalimentaria, casi toda el agua utilizada enlas distintas actividades se convierte en unefluente de agua residual (por ejemplo, enel sector lácteo se generan 3-5 m3 por Tmde leche procesada, en el sector cervecero2,5-7,2 hl/hl de cerveza elaborada). En elcaso del sector lácteo, no se precisa aguapara la formulación del producto final, perose utiliza una gran cantidad de agua y segenera gran cantidad de aguas residuales

Aplicación de nuevas técnicas ecoeficientes de limpieza y desinfección ensistemas CIP, basadas en el uso del ozono.Proyecto OZONECIPV. Martínez


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en las actividades de limpieza y desinfec-ción de conducciones, depósitos, filtros, etc.Habitualmente esta agua residual contieneN, P, COT y cloruros como principales ele-mentos contaminantes.

El proyecto OZONECIP que se describe enel siguiente apartado experimenta contécnicas de limpieza y desinfección “máslimpias” que conjuguen la variable higié-nica y la variable ambiental manteniendola viabilidad económica de estos procesos.De esta forma se asegurarán los rendi-mientos higiénicos y además se alcanzaráun menor impacto medio ambiental. Esteplanteamiento coincide con la filosofía delas Mejores Técnicas Disponibles (MTDs).

Es conveniente recordar que el cloro, es undesinfectante con importantes ventajas quelo han hecho prácticamente universal en lasinstalaciones agroalimentarias. Sin em-bargo, hay que tener en cuenta algunasdesventajas relacionadas con la generaciónde derivados del cloro, especialmente triha-lometanos, cloraminas o clorofenoles, quehan sido relacionados con enfermedadescomo el cáncer y vinculados a otros efectosadversos, y por ello se buscan alternativas,siendo el ozono una de las más promete-doras con la ventaja adicional de su mejorresultado medio ambiental. A continuaciónse destacan las principales característicasfuncionales por las cuales el ozono resultade gran interés para las operaciones de lim-pieza y desinfección:

• El ozono tiene un mayor oxidante que elresto de desinfectantes químicos, con loque se ahorra agua y tiempo. La acciónantimicrobiana de O3 está basada en sualto potencial de oxidación (2,07 V),mucho mayor que los de otros com-

puestos químicos 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 mi-croorganismos mediante la oxidación delas paredes celulares de los mismos, quecon su ataque quedan desintegradas(lisis). Este es un mecanismo muy dife-rente al del cloro, el cual se difunde através de la pared celular haciendo lapared susceptible del ataque enzimático.

• Ahorro de consumo de agua: el ozonono genera residuos, por lo que no esnecesario el uso de aguas de aclarado.El agua ozonizada que ha sido usada enla desinfección puede ser reutilizadanuevamente en las etapas de limpiezaprevias, directamente o después de unanueva ozonización.

Proyecto OZONECIP

Introducción

El proyecto OZONECIP (LIFE 05 ENV/E/000251) es un proyecto cofinanciado porla Comisión Europea a través del programaLife-Environment cuyo objetivo fue contri-buir a la reducción del impacto ambientalde las operaciones de limpieza y desinfec-ción a través de una técnica innovadora,más eficiente en términos medio ambien-tales, consistente en el empleo de ozonocomo agente desinfectante alternativo a losagentes químicos de desinfección tradicio-nales en el ámbito de la limpieza y desin-fección de equipos cerrados mediante sis-temas CIP (Clean in Place).

El proyecto se circunscribe a los sectoreslácteo, vinícola y cervecero como usua-rios intensivos de los sistemas tipo CIP ysuficientemente representativos para

otros tipos de subsectores interesados:zumos, bebidas refrescantes, etc. El pro-yecto tuvo una duración de 36 meses,iniciándose el 1 de diciembre de 2005.Para su realización, además de AINIAcentro tecnológico como coordinadorde proyecto, han participado otros cen-tros europeos: Bionord en Alemania y laUniversidad de Gdansk en Polonia, asícomo socios industriales: BodegasDomecq en España, la cervecera Inbev yla láctea MG Lang en Alemania.

Durante el primer año se realizaron unaserie de revisiones del estado del arte delas tecnologías implicadas: CIP y ozoniza-ción y lograr su adecuada integración. Porotro lado se revisaron otros aspectos notecnológicos que pudieran afectar la via-bilidad del sistema propuesto.

Paralelamente, durante el primer año serealizaron una serie de trabajos de campoen industrias colaboradoras para obtenerdatos reales del impacto ambiental pro-

vocado actualmente por las operacionesde limpieza y desinfección de depósitos yequipos cerrados.

Como ejemplo de resultados obtenidosen el sector lácteo, destacar que el con-sumo de agua está principalmente ligadoa las operaciones de limpieza y desinfec-ción representando de forma general al-rededor del 80%. Por otro lado, elarrastre de producto en las aguas de la-vado supone un incremento considerablede la carga orgánica del vertido.

La siguiente tabla muestra los resultadosobtenidos por AINIA en diferentes em-presas lácteas en los procesos convencio-nales de limpieza y desinfección tipo CIPde depósitos de almacenamiento, fer-mentadores y pasteurizadores.

Con la información obtenida a través delas actividades del primer año, el se-gundo año se diseñó y construyó unprototipo piloto que permite simularprocesos de lavado CIP convencionales

Aplicación de nuevas técnicas eco eficientes de limpieza y desinfección en sistemas CIP...

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Tabla 1. Muestreo de aguas residuales en sector lácteo.

pH Conductividad DQO Nitrógeno PO4-P(unidades) (mS/cm) (mg/L) (mg/L) (mg/L)

Leche 6,66 5,25 160,500 590 1.680 Yogur 4,15 155 184,500 370 980

Tabla 2. Aguas residuales de limpieza de equipos lácteos.

pH (unidades) Conductividad (µS/cm) DQO (mg O2/L)

1º Enjuagado con agua 8,1-11,61 430-1.700 28-1.465Lavado alcalino 12,8-13,11 13,280-39.200 196-568

2º Enjuagado con agua 8,63-13,22 485-18.760 32-1.190 Lavado ácido 2,49-2,50 4,840-9.920 428-958

3º Enjuagado con agua 2,65-4,97 1,170-6.940 31-428 Último enjuague 7,00-8,00 412-1.040 30-60

de cualquier tipo y ensayar ciclos alter-nativos basados en el empleo de aguaozonizada de forma que fuera posiblemonitorizar ambas opciones y obtenerdatos comparativos en relación a su res-pectivo impacto ambiental en términosde agua consumida y características delvertido generado.

La planta piloto se divide en tres sistemas:sistema generación de ozono, sistema CIP ysistema objetivo a limpiar y desinfectar (de-pósito de acero inoxidable 316 de 500 litros).El conjunto cuenta con un PLC que gobierna

el sistema ozono y un PLC principal que go-bierna el sistema en su conjunto. La plantase opera desde un SCADA que permite lacomunicación con el PLC principal y modi-ficar las secuencias y condiciones de opera-ción de los diversos ensayos a efectuar. Lasimágenes a continuación muestran el as-pecto del CIP y del depósito objetivo.

El tercer y último año de proyecto se rea-lizaron ensayos comparativos de ciclos delimpieza habituales en la industria y ciclosalternativos basados en el empleo delozono, que se describen a continuación.


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• Water in

1 Water2 Alkali solution3 Acid solution4 Ozonated water5 Disinfectant injection6 Ozonation system: formed by injector, 3 ozonegenerators and 5 PSA units7 Target vessel

• Drain

1

6

2 3 4

5

7

Prototipo OZONECIP.

• Entrada de agua

1 Agua.2 Solución alcalina.3 Solución ácida.4 Agua ozonizada.5 Inyección de desinfectante.6 Sistema de ozonización

formado por inyector, 3 generadores de ozono y 5 unidades PSA.

7 Tanque diana.

• Drenaje

OZONECIP ensayos comparativos:

materiales y método

Con objeto de disponer de datos con-cretos del impacto ambiental de las ope-raciones de limpieza y desinfección, serealizaron ensayos en planta piloto, eje-cutando ciclos de limpieza convencio-nales y ciclos basados en el empleo deagua ozonizada con objeto de obtenerdatos comparativos de la eficiencia hi-giénica y del impacto ambiental deambas metodologías.

Como ejemplo se describen seguida-mente los ciclos empleados en los ensayosrealizados con leche:

En los ensayos se empleó un depósito deacero inoxidable de 500 litros que para lasdiversas pruebas fue ensuciado con:

• Vino en rama y con vino inoculado concepas de Brettanomyces y de bacteriasacéticas.

• Cerveza inoculada con Lactobacilllus brevi,Pectinatus cerevisiphiluy y Saccharomycescerevisiae.

• Leche inoculada con Bacillus pumilis,Listeria inocua y Saccharomyces cerevisiae.

En cada ensayo se controló la eficienciade la limpieza y desinfección medianteplacas RODAC, analizándose la carga mi-crobiana en la superficie interior del de-


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Tabla 3. Protocolos de limpieza y desinfección convencionales.

DCIP1 DCIP2tiempo conc tiempo conc

Enjuague con agua 2 2 Lavado con hidróxido sódico 5 2% 5 0,5% Enjuague con agua 2 2 Lavado con ácido nítrico 2 1,5% 2 1,5% Enjuague con agua 2 2 Desinfección con ácido peracético 5 0,5% 5 0,5% Enjuague con agua 2 2

Tabla 4. Protocolos de limpieza y desinfección alternativas con ozono.

O3CIP4 O3CIP5

tiempo conc tiempo conc Enjuague con O3 a vertido 2 1 ppm 2 1 ppm

Lavado con hidróxido sódico 5 2% 5 0,50%

Enjuague con O3 a vertido 2 1 ppm 2 1 ppm

Lavado con ácido nítrico 2 1,50% 2 1,50%

Enjuague con O3 a vertido 2 1 ppm 2 1 ppm

Enjuague con O3 en recirculación 4 1 ppm 4 1 ppm


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pósito antes y después de la limpieza y lacalidad de las últimas aguas de enjuague.Paralelamente se controló el volumen deagua consumida (por tanto el volumen devertido) y se determinó la carga orgánicadel vertido generado.

Resultados del proyecto

De los ensayos realizados de limpieza ydesinfección CIP con vino, cerveza y lechese obtuvieron resultados de eficacia dedesinfección de superficies y aguas de en-juagado y de calidad ambiental de lasaguas residuales generadas.

Seguidamente se describen como ejemplolos resultados obtenidos en las pruebasrealizadas con leche contaminadas conuna mezcla de microorganismos: Bacilluspumilis, Listeria inocua y Saccharomycescerevisiae a una concentración media por

microorganismo de 105 unidades forma-doras de colonia/100 ml.

La tabla 5 muestra los resultados de la efi-cacia de desinfección. Los resultadosdieron valores aceptables en las superfi-cies del depósito muestreadas, no identi-ficándose diferencias significativas entrelos tratamientos con ácido peracético yozono. De igual forma los resultados mi-crobiológicos del agua del último en-juague también dieron valores aceptables.

La tabla 7 muestra los resultados ambien-tales de muestras integradas de las aguasresiduales generadas en las pruebas delimpieza y desinfección. Como principalesresultados, destacar el menor consumo deagua obtenido en la pruebas con ozonodebido a que no es necesario la realiza-ción de un enjuague final, junto con la re-ducción del COD y la carga orgánica en

Tabla 5.

Superficie sucia Superficie limpia Último enjuague Superficie Superficie ufc/25 cm2 ufc/25 cm2 ufc/100 ml sucia ATP limpia ATP

Bacillus pumilisCIP1 > 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 inocuaCIP1 > 100 < 1 < 1 9.500 128 CIP2 > 100 < 1 < 1 9.200 90 O3CIP4 > 100 < 1 1 7.900 140

O3CIP5 > 100 < 1 8 13.525 85

Saccharomyces cerevisiaeCIP1 > 100 < 1 < 1 9.500 128 CIP2 > 100 8 < 1 9.200 90 O3CIP4 > 100 < 1 5 7.900 140

O3CIP5 > 100 < 1 < 1 13.525 85

términos de reducción de gramos de COD(más del 75%) cuando se usa el ozonocomo agente desinfectante.

Consideracioneseconómicas

Coste del equipamiento

Un equipo completo de ozono para laaplicación en un sistema CIP está com-puesto principalmente por un generadorde ozono, un sistema de alimentaciónde gas (aire u oxígeno concentrado), in-yector, tanque de reacción y destructorde ozono gas, equipos de medida deconcentración de ozono gas en alimen-tación, ozono disuelto y ozono am-biental, unidad de control y bomba derecirculación.

Para un sistema CIP convencional las ne-cesidades de higienización se deter-

minan generalmente mediante el ta-maño y la potencia de la bomba de cir-culación, o por la capacidad de genera-ción de ozono medido en g O3/h. Unavez definidos estos términos es cuandose determinan las especificaciones de loscomponentes del sistema. Un ejemploorientativo de coste de un sistema deozono para una unidad convencionalCIP, se específica en la siguiente tabla:

La cuantificación incluye: el generadorde ozono, inyector, destructor de ozonoresidual, medidor de concentración deozono disuelto, unidad de control y es-tructura de soporte. Considerando queel prototipo utilizado en el proyectoOZONECIP, utilizaba principalmentepara las pruebas un generador de 30 gO3/h, dicho prototipo pude utilizarsepara la higienización de equipos a nivelindustrial.


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Tabla 6. Calidad ambiental del agua del último enjuague.

pH Conductividad DQOunidades µS/cm mg O2/l

CIP1 7,71 1.068 4,1 CIP2 7,81 1.044 7,1 O3CIP4 8,06 1.057 5,5

O3CIP5 8,1 1.074 5,6

Tabla 7. Calidad ambiental de las aguas residuales generadas.

pH Conductividad DQO Volumen Carga unidades µS/cm mg O2/l l g COD

CIP1 9,81 2.860 1.425 1.401 1.996 CIP2 4,85 1.800 1.320 1.399 1.847 O3CIP4 12,03 2.900 795 551 438

O3CIP5 3,2 2.680 740 563 417


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Coste de uso

Cuando se evalúa el coste de explotacióndel uso del ozono en sistema CIP, al igualque en un CIP convencional hay que teneren cuenta los siguientes aspectos:

• Consumo de energía.

• Consumo de agua.

• Consumo de productos químicos.

• Costes de mantenimiento.

Consumo de energía

En función de la concentración de ozonoproducida por el generador el consumopuede variar entre 80 W y 1.500 W. En lasiguiente tabla se describe el consumomedio de energía en función de la pro-ducción de ozono. El rango de consumopuede variar en función del fabricante,tipo de sistema de generación de ozono,electrodos, etc.

El consumo de energía para la generaciónde ozono es relativamente baja compa-rada con la requerida para calentar las so-luciones detergentes y desinfectantes ensistemas CIP convencionales.

Consumo de agua

El consumo de agua se vio claramente re-ducido cuando se utilizó ozono y el agua

ozonizada era recirculada en circuito ce-rrado para su posterior uso. En laspruebas de limpieza y desinfección conozono OZONECIP se redujo alrededor deun 50% el consumo de agua.

Consumo de productos químicos

Cuando se utiliza agua ozonizada comoagente desinfectante no se requieren pro-ductos químicos adicionales.

Costes de mantenimiento

En los cálculos de mantenimiento espe-rados para un sistema CIP basado en eluso del ozono, hay que considerar deforma adicional los generados por el sis-tema de ozonización.

Destacar que los costes de mantenimientodel sistema de ozonización son relativa-mente bajos puesto que ninguna de las

Tabla 8. Cuantificación de diferentes equipos de ozono, conforme con Air-Tree EuropeGMBH (precios de 2006).

Flujo de agua Producción de ozono Coste de la inversión en €

2 m3/h 10 g/h 20.000

5 m3/h 20 g/h 25.000

10 m3/h 40 g/h 35.000

20 m3/h 100 g/h 40.000

Tabla 9. Consumo medio de energía enfunción de la producción de ozono.

Producción Consumo mediode ozono de energía 4 g O3/h 80-180 W

10 g O3/h 200-1.000 W

40 g O3/h Alrededor de 1.500 W

partes del mismo requiere un cambio pe-riódico. Entre los costes del sistema más sig-nificativos se incluyen los relacionados conla calibración de los equipos de medición.

Conclusiones Con los datos medio ambientales obte-nidos en el proyecto OZONECIP se con-firma la importancia del impacto gene-rado en las operaciones de limpieza ydesinfección tal como recogía cualitativa-mente el BREF y lo complementan cuan-titativamente con valores referidos a ope-raciones particulares, con los siguientescomentarios generales:

• En torno al 80% del agua total consu-mida en los sectores estudiados se con-sume en operaciones de limpieza y de-sinfección (valor aún mayor en el casode bodegas elaboradoras de vino).

• La operación manual de los sistemas CIPes una práctica aún muy extendida enla industria. La automatización y la mo-nitorización de los sistemas conduciríaa una mayor reproducibilidad de lasoperaciones y a ahorros en los actualesniveles de consumo de agua de en-juague u agentes de limpieza.

• Los primeros enjuagues y las solucionescon desinfectante son generalmentevertidas.

• Valores para diferentes operaciones delimpieza han sido obtenidos. Tales valoresconstituyen sólo una referencia del ordende magnitud de la carga contaminante,los rangos son muy amplios dado que laoperación manual causa una gran varia-bilidad en función de la toma o ausencia

de buenas prácticas y de la propia periciade los operadores. Así, se observan picosde pH (pH=12 y pH=3) y de conducti-vidad. La carga orgánica es muy variable.

• La segregación de las primeras aguas deenjuague extremadamente cargadas esun factor clave para mejorar significativa-mente la calidad del vertido. Se debe ex-tremar la precaución en no sobredosificardetergentes ni desinfectantes puesto quesi se sobredosifica se requieren ingentescantidades de agua para eliminar es-pumas y asegurarse de que no quedanrestos de productos químicos en losequipos. Además puede conferir toxi-cidad a las aguas residuales.

• En los tres productos utilizados en laspruebas (vino, leche y cerveza) se haconseguido una reducción del 50% enel consumo de agua cuando se aplicabaagua ozonizada como agente desinfec-tante.

• La reducción de la carga orgánica entérminos de gramos de COD fue de un50% cuando se usó agua ozonizadacomo agente desinfectante.

• En todas las pruebas se obtuvieron va-lores de eficacia de desinfección de lassuperficies del depósito higienizadocon agua ozonizada similares a los ob-tenidos con el ácido peracético. En loque concierne a los resultados micro-biológicos de las aguas de enjuague,cabe destacar que algunas muestrascon tratamiento con ozono presen-taron valores más elevados que los ob-tenidos con el ácido peracético. Estopuede ser debido a que el ozono con


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el paso del tiempo no alcanzó unaconcentración residual aceptable en elmedio al transformarse en oxígeno.

• La revisión tecnológica realizada entorno a la integración de las tecnolo-gías del ozono en los sistemas CIPmuestra que sería relativamente sen-cillo de implantar en sistemas CIP yaexistentes, adoptando ciertas medidasadicionales. Así, cabe revisar la com-patibilidad de los materiales que en-trarían en contacto con el ozono, engeneral las instalaciones son de aceroinoxidable por lo que sólo habría querevisar componentes en válvulas,codos, sondas, etc. Por otro lado, seproduce un pase de ozono desde lafase acuosa a la fase gaseosa en losequipos, tales como depósitos, por loque podría ser conveniente, segúnequipos y productos, el forzar la salidadel aire rico en ozono empujándolocon un gas inerte: nitrógeno o conaire y destruir el ozono residual con ul-travioletas. Por último, no debe olvi-darse que el ozono es un gas tóxico ydebe prevenirse la exposición de lostrabajadores al mismo. El actual valorVLA-ED (valor de exposición diaria)ante el ozono es 0,1 ppm. El “InstitutoNacional de Seguridad e Higiene en el

Trabajo” además indica los siguientesvalores en función de la actividad:

• Así, los sistemas deben incorporar sen-sores de nivel de ozono en ambiente quedetengan el sistema en caso de detectarfugas y actúen en la renovación rápida delaire ambiente del área afectada.

En definitiva, como consecuencia del pro-yecto OZONECIP puede apuntarse que laincorporación de las tecnologías delozono en nuevos protocolos de lavado ydesinfección en sistemas CIP nuevos yexistentes es factible, adoptando ciertasmedidas ya apuntadas anteriormente, ob-teniendo una eficacia en la higiene y de-sinfección igual o superior que el sistematradicional basado en el uso exclusivo deagentes químicos pero con un potencialgran ahorro en la cantidad de agua nece-saria para efectuar la operación y una me-jora en la calidad del vertido de aguas re-siduales generado.

ppm mg/m3

Trabajo pesado 0,05 0,1 Trabajo moderado 0,08 0,16 Trabajo ligero 0,1 0,2 Trabajo pesado, 0,2 0,4 moderadoligero (≤ 2 horas)

TLV para concentración de ozono en ambientes detrabajo en España.

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