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MICROBIAL CONTAMINANTS & CONTAMINATION ROUTES IN FOOD INDUSTRY

Julkaisu on saatavana

VTT SYMPOSIUM 248

Microbes able to establish and develop in the processing environment and

on food products pose a potential problem in terms of food safety and
quality. Initially it is the capability to adhere to a surface that gives an
advantage towards the majority of microbes being present. Other
important capabilities are competitive growth and long survival. Although
pathogenic microbes rarely cause the spoilage of foodstuffs (due to their
slow or non-existent growth in foods at low temperatures) they and their
toxic secondary metabolites naturally form a health risk to the consumer.
To guarantee the safety of foodstuffs microbial contamination must be
detected and traced as early as possible and also at low levels. In addition,
the safety risks related to the microbial contamination must be evaluated
as accurately as possible. To establish corrective (and for the future also
preventive) actions exact knowledge on the process hygiene level of the
production plant is needed. Characteristics of important contaminants,
contamination routes and factors enhancing microbial contamination of
process line are important items covered in this Microbial contaminants
and contamination routes in food industry -seminar by international
lecturers and participants who have provided research studies and group
work material published in this seminar proceedings.

VTT SYMPOSIUM 248

MICROBIAL CONTAMINANTS &

CONTAMINATION ROUTES IN FOOD
INDUSTRY
1st Open Seminar arranged by
SAFOODNET Food Safety and Hygiene
Networking within New Member States
and Associated Candidate Countries

VTT SYMPOSIUM 248

Keywords:
microbial contaminants, contamination routes,
pathogens, food industry, process hygiene,
quality, molecular biological methods,
packaging, antimicrobial effects

MICROBIAL CONTAMINANTS &

CONTAMINATION ROUTES IN FOOD INDUSTRY
1ST OPEN SEMINAR ARRANGED BY
SAFOODNET FOOD SAFETY AND HYGIENE NETWORKING
WITHIN NEW MEMBER STATES AND ASSOCIATED CANDIDATE
COUNTRIES; FP6-022808-2006
ESPOO, FINLAND, JANUARY 2223, 2007

EDITED BY
GUN WIRTANEN & SATU SALO
VTT TECHNICAL RESEARCH CENTRE OF FINLAND

ISBN 978-951-38-6319-7 (soft back ed.)

ISSN 0357-9387 (soft back ed.)
ISBN 978-951-38-6320-3 (URL: http://www.vtt.fi/publications/index.jsp)
ISSN 1455-0873 (URL: http://www.vtt.fi/publications/index.jsp)
Copyright VTT Technical Research Centre of Finland 2007

JULKAISIJA UTGIVARE PUBLISHER

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VTT Technical Research Centre of Finland
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phone internat. +358 20 722 111, fax +358 20 722 7071

Cover picture Antti Huovinen

Edita Prima Oy, Helsinki 2007

PREFACE
Food Safety and Hygiene Networking within New EU Member States and
Associated Candidate Countries (SAFOODNET FP6-022808) is a specific
support action EU-project building-up a sustainable network in food safety. It
aims at knowledge sharing to prevent risks related to microbial hazards, to find
future RTD needs and apply for RTD funding in food processing and packaging
safety. The action focuses towards Czech Republic, Denmark, Estonia, Finland,
Hungary, Latvia, Slovenia and Turkey in the pilot actions, seminars, and
workshops on process hygiene and end product safety. Interested researchers and
SME representatives from other new EU countries and ACCs are encouraged to
participate in the activities. The objectives of SAFOODNET are to:
1) disseminate knowledge from national and international food safety projects in
open seminars, workshops, practical exercises, RTD activities and pilot actions
resulting in new research projects for food industry especially SMEs;
2) establish an expert group (EG) in which authorities, scientists, industrial
representatives build-up or strengthen existing networks and identify specific
needs for future RTD activities in food safety and 3) bridge networks within the
new EU, fostering scientific co-operation and knowledge transfer in food safety.
The first seminar arranged by SAFOODNET to a targeted audience of industrial,
research and authority people is titled Microbial contaminants and
contamination routes in food industry and it is dealing with characteristics of
important contaminants, contamination routes and factors enhancing microbial
contamination of process line. The forthcoming activities in SAFOODNETproject are 2 open seminars in risk assessment and management to disseminate
safety issues; 2 workshops incl. practical exercises in microbial risk assessment
and management to disseminate methodologies and methods; networking
through project web-site (http://safoodnet.vtt.fi); establishment of an EG to
suggest and evaluate pilot actions for future needs in RTD activities;
performance of pilot actions in food safety suggested by the EG in food industry
especially in SMEs as a base for future RTD activities and building-up a new
channel for scientific co-operation within the new EU.

CONTENTS
PREFACE ....................................................................................................... 3
LECTURES .................................................................................................. 9
BIOFILM FORMATION IN FOOD PROCESSES................................................... 10
MICROBIAL ECOLOGY IN MANUFACTURING PAPER-BASED
PACKAGING MATERIALS FOR USE IN FOOD INDUSTRY .................................. 16
THE RELEVANCE OF PSYCHROTROPHIC SPOILAGE MICROBES
IN FOOD HYGIENE ........................................................................................ 20
IMPACT OF COMPOSITION AND METABOLITES OF INTESTINAL
MICROBIOTA ON FOODBORNE INFECTIONS ................................................... 24
CLASSIFICATION AND DIFFERENTIATION OF SALMONELLA SP.
USING DNA BASED TECHNIQUES ................................................................. 28
ENTERIC PATHOGENS PREVALENCE IN FOOD PRODUCTS AND
MECHANISMS OF SUPPRESSION BY PROBIOTIC LACTIC ACID BACTERIA ........ 31
EFFECT OF MAINTENANCE ROUTINES IN FOOD PROCESSING
ON PRODUCTION HYGIENE ........................................................................... 36
HYGIENICALLY INTEGRATED SYSTEMS.......................................................... 39
METHODS FOR EVALUATION OF PROCESS HYGIENE ..................................... 42
MOLECULAR TECHNIQUES AND MICROSCOPY IN BACTERIAL
DETECTION AND TYPING............................................................................... 46
LEGAL ASPECTS OF FOOD SAFETY ............................................................... 51
AIRBORNE BIOCONTAMINATION IN CLEANROOMS AND CONTROLLED
ENVIRONMENTS ........................................................................................... 56
ANTIMICROBIAL EFFECTS...................................................................... 65
EMERGENCE OF ANTIMICROBIAL RESISTANCE IN CAMPYLOBACTER JEJUNI
ISOLATED FROM BROILER CHICKENS IN ESTONIA .......................................... 66

INHIBITION OF PSEUDOMONAS FLUORESCENS BY ALGINATE

FILMS INCORPORATING LACTOPEROXIDASE SYSTEM .................................... 67
ANTIMICROBIAL EFFECTS OF CHLORINATED AND OZONATED
WATER FOR REDUCING MICROBIOLOGICAL CONTAMINATION
DURING BROILER PROCESSING .................................................................... 69
DETECTION OF LISTERIA MONOCYTOGENES IN GROUND TURKEY
BY IMMUNOMAGNETIC SEPARATION AND DETERMINATION OF THE
ANTIBIOTIC SUSCEPTIBILITY ......................................................................... 71
ANTAGONISTIC ACTIVITY OF LACTOBACILLUS PLANTARUM STRAINS
AGAINST ENTEROPATHOGENS ...................................................................... 72
THE IMPROVEMENT OF CHEESE QUALITY WITH HUMAN
LACTOBACILLUS PLANTARUM ADDITIVE......................................................... 74
ANTIBIOTIC SUSCEPTIBILITY PATTERN OF SALMONELLA SPP.,
LISTERIA MONOCYTOGENES AND CLOSTRIDIUM PERFRINGENS
ISOLATES FROM TURKEY MEAT ................................................................... 76
CONJUGATIONAL TRANSFER OF ANTIBIOTIC RESISTANCES IN
SALMONELLA TYPHIMURIUM ......................................................................... 78
HYGIENE & QUALITY................................................................................ 79
DIFFERENTIATION OF STAPHYLOCOCCUS EPIDERMIDIS ISOLATES FROM
RAW MATERIAL AND FOODSTUFFS AND FOOD CONTACT SURFACES
BASED ON THE ABILITY TO FORM BIOFILM .................................................... 80
MICROBIOLOGICAL QUALITY OF TURKISH CHEESES MANUFACTURED
FROM PASTEURIZED MILK PRODUCED IN COMPANIES HAVING
THE HACCP SYSTEM................................................................................... 82
INVESTIGATION OF MOULDS IN SALTED YOGHURTS
(CONCENTRATED YOGHURT)........................................................................ 83
EFFECT OF PLUM SAUCE, POMEGRANATE SAUCE AND LEMON
JUICE ON MICROBIOLOGICAL QUALITY PARAMETERS OF
TRADITIONAL RAW MEAT-BALL (I KFTE).................................................. 85

MOLECULAR BIOLOGICAL METHODS................................................... 87

PFGE GENOTYPING AND ANTIMICROBIAL SUSCEPTIBILITY
OF CAMPYLOBACTER IN RETAIL POULTRY MEAT IN ESTONIA ........................ 88
IDENTIFICATION OF ENTEROCOCCUS SPP. BACTERIA ISOLATED FROM
FOOD AND OTHER ENVIRONMENTAL SAMPLES BY PCR METHOD ................. 90
RESISTOPATHOCHIP TOOL FOR RAPID MOLECULAR
CHARACTERIZATION OF SALMONELLA STRAINS ............................................ 92
DETECTION OF BACTERIAL TOXINS IN FOODSTUFF ....................................... 94
INCIDENCE OF LISTERIA MONOCYTOGENES IN RETAIL
CHICKEN MEAT AND ESTABLISHING RELATIONSHIP WITH
SOME BACTERIA BY LOGISTIC REGRESSION ................................................. 95
PATHOGENES .......................................................................................... 97
HYGIENE SURVEY IN ESTONIAN DAIRIES AND PATHOGEN
ANALYSIS OF AUTHENTIC SAMPLES .............................................................. 98
INCIDENCE OF LISTERIA MONOCYTOGENES IN DIFFERENT
KIND OF MEAT PRODUCTS ON LATVIAN RETAIL MARKET ............................... 99
THE PREVALENCE OF CAMPYLOBACTER SPP. AT THE
FARM AND RETAIL LEVEL............................................................................ 100
DETECTION AND IDENTIFICATION OF CAMPYLOBACTER
POULTRY CARCASSES............................................................................... 101
SURVIVAL OF ESCHERICHIA COLI, STAPHYLOCOCCUS AUREUS, BACILLUS
CEREUS AND LISTERIA INNOCUA DURING REFRIGERATED STORAGE OF
ALGAM JUICE A TRADITIONAL FERMENTED TURKISH BEVERAGE ........... 102
ISOLATION AND IDENTIFICATION OF LISTERIA SPECIES AND LISTERIA
MONOCYTOGENES FROM CHICKEN AND CHARACTERIZATION OF
AUTOLYSIS OF LISTERIA MONOCYTOGENES ISOLATES ................................ 104
PROCESSING & PACKAGING ............................................................... 105
EFFECTS OF PACKAGING TREATMENT, STORAGE CONDITION AND LENGTH
OF STORAGE PERIOD ON EXTERNAL AND INTERNAL TABLE EGG QUALITY..... 106

THE EFFECT OF VACUUM AND MODIFIED ATMOSPHERE PACKAGING

ON GROWTH OF LISTERIA MONOCYTOGENES IN RAINBOW TROUT
(ONCORYNCHUS MYKISS) FILLETS .............................................................. 107
THE EFFECTS OF DIFFERENT NITRATE LEVELS AND STARTER CULTURE ON
THE REDUCTION OF E. COLI O157:H7 DURING SUCUK PRODUCTION .......... 108
EFFECT OF PROCESSING METHODS ON TOTAL CATECHIN CONTENTS
OF TEA (CAMELLIA SINENSIS) ..................................................................... 109
FOOD SAFETY PROVIDING SYSTEM FOR AUDITING OF PRIVATE
LABEL PRODUCER IN 20042006............................................................... 110
FOOD HYGIENE ACTIVITIES AT THE INSTITUTE OF PUBLIC
HEALTH IN OSTRAVA .................................................................................. 112
SHELF LIFE OF SEA BASS DURING ICE STORAGE ........................................ 113
GROUP WORKS...................................................................................... 115
BACTERIAL FOODBORNE PATHOGENS OF CONCERN .................................. 116
FOOD PROCESS HYGIENE, EFFECTIVE CLEANING AND SAFETY
IN THE FOOD INDUSTRY .............................................................................. 129
SAFE PROCESSING AND PACKAGING OF FOODS ......................................... 145
UTILIZATION OF MOLECULAR TECHNIQUES FOR THE IDENTIFICATION OF
SALMONELLA FROM FOODSTUFFS .............................................................. 151
ANTIMICROBIAL EFFECTS IN FOOD AND PREVENTION OF
CONTAMINATION IN FOOD INDUSTRY .......................................................... 159
APPENDIX 1: PARTICIPANT LIST
APPENDIX 2: SEMINAR PROGRAMME

LECTURES

BIOFILM FORMATION IN FOOD PROCESSES

Gun Wirtanen & Satu Salo
VTT, Espoo, Finland

Microbes inhabiting contact and environmental sites in food processing are

mostly harmful, because microbial communities that form in critical places
contaminate the surfaces and consequently the products made in that particular
process. Some microbes naturally have a higher tendency to produce biofilm than
others, but biofilm can generally be produced by any microbe under suitable
conditions i.e. on moist surfaces with some nutrients. This is the case especially if
the process equipment is not hygienically integrated in the process line or the
cleaning and disinfection procedures are not properly designed to remove the
organic soil from the process surfaces. Equipment that causes problems in food
processing and packaging include slicing and cutting equipment, filling and
packing machines, conveyors, plate heat exchangers and tanks with piping.
Listeria monocytogenes has been found on equipment and process surfaces,
which are difficult to clean. Such equipment can thus cause microbial
contamination in food processing. Therefore the design of the equipment and
process line in the food processing and packaging industry is important for
preventing formation of the biofilm and so improving process and production
hygiene. Furthermore, pathogens in biofilms have been found to be more
resistant to many types of disinfectants.
Harmful microbes may enter the manufacturing process and reach the endproduct in several ways, e.g. through raw materials, air in the manufacturing
area, chemicals employed, process surfaces, or factory personnel. It is also
important to remember that about 8596% of a biofilm consists of water, which
means that only 25% of the total biofilm volume is detectable on dry surfaces. A
biofilm consists of microbial cell clusters with a network of internal channels or
voids in the extracellular polysaccharide and glycoprotein matrix, which allows
nutrients and oxygen to be transported from the bulk liquid to the cells. Once a
biofilm has been formed, it can be a source of contamination for foods passing
through the same processing line. At present the most efficient means for

10

limiting the growth of microbes are good production hygiene, the sensible
running of the process line, and the well-designed use of cleaning and
decontamination processes. Common foodborne pathogens such as Bacillus
cereus, Staphylococcus aureus, L. monocytogenes, Mycobacterium paratuberculosis, Clostridium perfringens, Escherichia coli O157:H7, Salmonella
Typhimurium, Campylobacter jejuni, and Yersinia enterocolitica have readily
been found to produce biofilms on surfaces. This presentation deals with biofilm
formation of common foodborne pathogens.
Salmonella is a non-spore-forming rod-shaped, mostly motile Gram-negative
bacterium belonging to the family Enterobacteriaceae, in which approximately
2,200 serotypes are recognised. Foods commonly associated with the disease
include raw meats, poultry, eggs, milk and dairy products. Milk-borne
salmonellosis is common in parts of the world where milk is neither boiled nor
pasteurised. It occurs, but much less frequently, in developed countries where
the main products implicated are pasteurised milk, powdered milk and certain
cheeses. Several groups have reported that Salmonella has formed biofilm on
various types of surfaces used in the food processing industry. These studies
have shown that Salmonella spp. can form biofilms on food contact surfaces and
that the cells in biofilms are much more resistant to sanitizers compared with
planktonic cells.
E. coli is a Gram negative, rod-shaped bacterium. Because many microbes from
faeces are pathogenic in animals and humans, the presence of the intestinal
bacterium E. coli in water and foods indicates a potential hygiene hazard. Most
strains of E. coli are harmless. However, a few strains with well-characterised
traits are known to be associated with pathogenicity. Those causing the greatest
concern in water and foods are the five major groups of intestinal pathogens i.e.
the enterohaemorrhagic, the enterotoxigenic, the enteroinvasive, the enteropathogenic and the enteroaggregative E. coli. E. coli has been isolated from a
large number of foods and drinks e.g. dairy products, fresh soft cheeses,
Camembert and Brie cheeses, vegetables, meat, fermented meat sausage, poultry
and fish products, water and apple cider. These agents can cause outbreaks of
diarrhoea. However, food-poisoning outbreaks caused by dairy products
contaminated with E. coli have been rare. E. coli O157:H7 has been isolated from
raw milk and bulk tank milk samples in the range of 0 to 10%. E. coli O157:H7 is
often transmitted to humans via unpasteurised milk. E. coli can also survive for

11

extended periods of time in several types of acidic foods e.g. cheese and yogurt.
Acid-adapted E. coli O157:H7 has shown enhanced survival and prevalence in
biofilms on stainless steel surfaces.
Campylobacter spp. are microaerophilic, very small, curved and thin Gram
negative rods. Ingesting milk with as few as 500800 cells can cause illness.
Since the infective dose is quite low and the food may in many cases contain
only a few cells, liquid enrichment methods are normally required before plating
on a selective medium in order to detect contamination with C. jejuni or C. coli.
Successful detection of these organisms requires incubation at 42 C under
microaerophilic conditions. Laboratory tests have shown that in optimum
circumstances campylobacter is able to form a biofilm on stainless steel and
glass in 2 days. The number of viable C. jejuni determined by using a direct
viable count was greater than by using culturing techniques, which indicates that
C. jejuni cells can form a viable but nonculturable state within the biofilm. Both
determination methods showed that biofilms enhance the survival of C. jejuni
during a 7-day period at 12 C and 23 C. Food residues or moisture improve the
chances of survival by campylobacter on surfaces. It also survived very well on
wooden surfaces because the pores in the wood protect the cells from oxygen.
L. monocytogenes is a facultatively anaerobic Gram-positive, non-spore-forming
short rod that is widely distributed in nature. It is a non-host specific pathogen.
Listeriosis may occur sporadically or epidemically. The organism has been
isolated from raw milk, mastitic milk and pasteurised milk. Foodstuffs
associated with listeriosis outbreaks also include cold-smoked and gravad
rainbow trout products, sliced cold cuts, soft cheese, butter, ice-cream and
coleslaw. Examples of epidemic sources are: coleslaw in Canada 1981,
unpasteurised milk in the USA 1983, Mexican-style soft cheese in USA 1985, a
pork product in France 1992, chocolate milk in the USA 1994, soft cheese in
Switzerland 1995, rainbow trout in Sweden 1997, corn in Italy 1997, hot dogs in
the USA 199899 and butter in Finland 1999. Of the 13 different
L. monocytogenes serotypes, only three (1/2a, 1/2b and 4b) have been
predominantly implicated in human diseases. It has been reported that healthy
people can be carriers of L. monocytogenes. L. monocytogenes is able to grow in
many environments: it can grow at low oxygen tensions, in high salt
concentrations and over a wide range of pH and temperatures. The bacterium
can survive for a limited time in up to 25% salt at 4 C. Hygiene monitoring in

12

the food processing industry is important, because L. monocytogenes can

colonise and form biofilms in food processing environments and on surfaces.
L. monocytogenes has been found to form biofilms on common food contact
surfaces e.g. plastic, polypropylene, rubber, stainless steel and glass. Sources of
Listeria in food plants are conveyor belts, cutters, slicers, coolers, freezers,
brining and packaging machines and drains.
S. aureus is an opportunistic pathogen, which is a Gram-positive, aerobic, nonspore-forming catalase positive rod. Nasal and skin carriages are frequent
vehicles in the transportation of S. aureus. Its occurrence in sour milk products
such as yoghurt is worthy of investigation, as it is present in relatively high
numbers in raw milk. Staphylococci have been shown to grow rapidly during the
initial fermentation. Similar behaviour by S. aureus has previously been reported in
both yoghurt and cheese. S. aureus was isolated in a total of 7% of food contact
sites and 8% of environmental sites from 10 SMEs that produce high risk foods
in the United Kingdom. The source of S. aureus almost always originated from
food handlers or from utensils previously contaminated by humans.
Contamination of food can occur as a result of poor hygienic practices in any
part of the food chain. Resistance to oxidative disinfectants has mainly been
associated with biofilm formation. For a 4-log kill of S. aureus in biofilms on
stainless steel, polystyrene and glass in a nutrient flow 50 and 600 times higher
concentrations of benzalkonium chloride and hypochlorite were needed than for
suspended cells, respectively.
B. cereus is a Gram-positive, aerobic, spore-forming rod, normally present in
soil, dust, and water. B. cereus cells are large and motile. The organism produces
a number of toxins with distinct diarrhoeal and emetic syndromes. B. cereus
occurs extensively in the environment but despite the fact that it is a common
contaminant in raw milk, food-poisoning outbreaks caused by dairy products
contaminated with B. cereus have been rare. In a dairy product survey B. cereus
was found in 52% of ice-creams, 35% of soft ice creams, 29% of milk powders,
17% of fermented milks and 2% of pasteurised milks and fruit-flavoured milks.
Indicators of a prolonged contamination problem caused by mesophilic B. cereus
strains early in the production chain have also been shown in dairy plants.
Additional contamination of milk by the B. cereus biofilm has been shown to
occur in the filling machine. Different Bacillus spp., and among them B. cereus,
have been found on liquid packaging boards and blanks and could thus be an

13

additional source of biofilms containing Bacillus spp. Furthermore, spores of

B. cereus are reported to possess a pronounced ability to adhere to the surface of
stainless steel, which is commonly used in food processing. Spores of B. cereus
have been shown to adhere, germinate and multiply on the stainless steel
surfaces of a tube heat exchanger. The attachment of B. cereus in process lines
may act as a continual source of post-pasteurisation contamination.
C. perfringens is a spore-forming, Gram-positive, anaerobic, non-motile rod
which forms large, regular, round and slightly opaque and shiny colonies on the
surface of agar. There are 5 types of C. perfringens: A, B, C, D and E, which
produce different types of toxins. Spores of some strains are resistant to heat
treatment at 100 C for more than l hour. Cases of C. perfringens food poisoning
from new food sources, because it is so adaptable and prolific, have helped to
show how our perceptions and understanding of safe food change with new
knowledge. C. perfringens can be found in the normal flora of the intestinal
tracts of both animals and humans, as well as in soil, clothing, and skin. It has
been found in virtually all environments tested, including water, milk and dust.
In view of its widespread presence in moist soil, its presence in air and dust in
kitchens, catering and food processing environments is not surprising. Food
products besides dairy products commonly contaminated with C. perfringens are
poultry, fish and vegetables.
The genus Mycobacterium contains approximately 50 species, which are divided
into rapid growers, slow growers and the human leprosy bacillus. Mycobacteria
are weakly Gram-positive, non-motile, slender, non-spore-forming, rod-shaped,
aerobic and free-living in soil and water. They are widely distributed in nature
and have been isolated e.g. from natural and piped waters, unpasteurised milk,
butter and vegetables. They do not produce appreciable amounts of toxin
substances and do not cause food poisoning. Infections in humans and animals
may be caused by most of the slowly growing mycobacteria e.g. M. avium and
M. xenopi. The only rapidly growing pathogenic species are M. chelonae and
M. fortuitum. A pilot plant pasteuriser was used to examine the heat resistance of
M. avium subsp. paratuberculosis (M. paratuberculosis) during HTST
pasteurisation using raw milk samples under various time and temperature
conditions. Results indicated that low numbers of M. paratuberculosis may also
survive extreme HTST treatments. The ability of mycobacteria to colonise
experimental drinking water distribution systems has been investigated, and it

14

was found to be present in biofilm within an hour of introduction for over 9

weeks. It was constantly present in outlet water samples and in biofilm samples.
References to this extended abstract can be found in the book chapter:
Wirtanen, G. & Salo, S. 2005. Biofilm risks. In: Handbook of hygiene control in
the food industry. Lelieveld, H., Mostert, T. & Holah, J. (eds.). Cambridge:
Woodhead Publishing Ltd. Pp. 4668. ISBN 1-85573-957-7.

15

MICROBIAL ECOLOGY IN MANUFACTURING

PAPER-BASED PACKAGING MATERIALS
FOR USE IN FOOD INDUSTRY
Laura Raaska
VTT, Espoo, Finland

Packaging serves as a major defence against external hazards. However

undesirable interactions between packaging material and food can give rise to
potential problems e.g. migration of packaging components or penetration of
micro-organisms through packages. The importance of hygiene and microbial
management in the industry manufacturing packaging material has increased
considerably as a result of the changes in legislation, tighter international
competition and increasing customer requirements. The contact time between
package with food has extended due to longer selling periods and extended
transport time from production plant to market. Furthermore the package is not
any more only used as a cover of food product during transportation and storing
but often also as a tray in which food can be warmed up and served as well.
Simultaneously today consumer prefers minimally processed food with no
preservatives, low fat, sugar and salt content and a long time of use making in a
way food more susceptible for microbial contamination. Important microbial
growth enhancing changes such as increased use of recycled fibre as a raw
material and decreased consumption of water (closing of water circulation
system) has taken place as well in manufacturing of paper and board that also
stress the importance of hygiene and efficient microbial management. Due to
these important changes manufacturers of food packaging materials as important
raw material suppliers for food industry have become increasingly conscious of
customer demands relating to concerns of food safety. Today, most food and
packaging material plants have a hygiene and safety management system built
on the HACCP concept and good manufacturing practice systems.
The date a legal requirement exists for chemical quality of raw materials and
food packages, but microbiological requirements are usually missing. The
legislation on food contact materials does not give any specifications for the

16

microbiological quality of the materials. The Policy Statement of Council of

Europe (CoE) concerning paper and board materials and articles intended to
come into contact with food states that materials intended to come in contact
with food should be of suitable microbiological quality taking into account the
intended end use and that for materials intended to come in contact with aqueous
and/or fatty foodstuffs particular attention should be paid to pathogens.
The risk connected to packaging material is the potential transfer of food
spoilage or pathogenic organisms to the packed food. Most packaging materials
have proven to be completely impervious to micro-organisms. About 1% of the
micro-organisms found in the board has been estimated to transfer. This
indicates that transfer is possible but at low rate. Moreover, the microbial load of
fresh food products is incomparably large relative to the amounts of microorganisms that could permeate through package that this phenomenon can
usually be neglected However even a low number of bacteria in the packaging
material could be of concern for e.g. aseptic foods, if bacteria migrate across the
package. The routes of contamination from the packaging material to food
include the surface, cutting dust or direct contact with the raw edge of the
paperboard.
Production of fibre-based food contact materials include several phases in which
microbiological problems can occur. However it must be stressed that the threat
of microbial contamination from packaging materials is more theoretical while
food itself is always the most prominent source of microbial contamination.
There are no published cases were micro-organisms originating from packaging
material have migrated into the food, multiplied there and caused illness of a
human. However there are cases when micro-organisms originating from
packaging materials have spoiled the food product. Paper and board machines
offer micro-organisms a favourable environment to grow and multiply, and
hence their numbers may be quite high without causing significant problems for
runnability or end product safety. Therefore, it is more important to determine
the nature of the micro-organisms (e.g. pathogen, slime producer, toxin former,
producer of off-odours) than their exact numbers.

17

Microbiological problems in the paper industry can be grouped as follows:

1. Micro-organisms causing spoilage of raw materials, since many microorganisms break down cellulose fibres, starch, casein and rosin sizing e.g.
Pantoea agglomerans and Bacillus subtilis spoil starch.
2. Micro-organisms causing problems in the process: producers of slime and
deposits e.g. Burkholderia cepacia, Deinococcus spp. and Bacillus spp.
3. Micro-organisms threatening process safety: micro-organisms harmful to
human health in raw materials, process environment and end products e.g.
Bacillus cereus, Klebsiella pneumoniae and Staphylococcus aureus.
4. Micro-organisms reducing the quality of end product: micro-organisms
harmful to human health and their metabolic products (toxins), microorganisms that can cause colour defects in products and micro-organisms
harmful to the hygiene of the end product e.g. Bacillus cereus,
Staphylococcus aureus, coliforms and moulds.
5. Micro-organisms that can cause smell or taste defects, such as the volatile,
foul-smelling compounds produced during microbial metabolism e.g.
Clostridium spp., Desulfovibrio spp. and actinobacteria.
In paper machine environment the micro-organisms are to be found colonising
surfaces in organised biofilm. The microbial ecology and parameters that affect
the well-being of the microbial community in industrial environments is poorly
known. Microbial ecology of industrial environments is challenging to be
studied because great variation in the cell density and composition of microbial
population exist over space and time as natural and environmental factors affect
microbial growth and as a consequence the qualitative and quantitative
composition of the microbial community. However to be able to control the
microbial growth efficiently with very targeted actions we need knowledge on
the species, their characteristics and changes in the microbial community over
time and space. One approach is to perform microbiological surveys were
samples are taken from the raw materials, process environment and the end
product, micro-organisms detected and identified by using both conventional
and modern methods based on molecular biology.
Based on research results obtained hitherto the numbers of potential pathogens
are very low, most of the potential pathogens have been shown to be not virulent
18

and the migration of micro-organisms from packaging materials into food is very
unlikely. However competence in microbiology is required to perform reliable
risk assessment and to focus the risk assessment measures efficiently and costeffectively. Furthermore microbiological risk assessment and risk management
activities at industry level require significant amount of microbiological analysis
performed by using sufficiently sensitive and rapid detection methods. While
traditional cultivation methods are far too slow compared to the turnover of
fibre-based products and in many cases not sensitive enough to detect pathogens
that may cause illness at very low concentration there has been a clear need for
novel rapid and sensitive detection as well identification methods.

19

THE RELEVANCE OF PSYCHROTROPHIC SPOILAGE

MICROBES IN FOOD HYGIENE
Elina Vihavainen
University of Helsinki, Helsinki, Finland

Microbial metabolism of organic mater is a naturally occurring biological

process. When this activity leads to unappealing and unacceptable foodstuff, this
process is considered microbial spoilage. Since many foods provide ideal
conditions for the growth of micro-organisms, spoilage is a constant concern in
the modern food processing industry causing severe economic losses to the
manufacturers and retailers. Microbial spoilage is also a concern to the worlds
food supply contributing to a vast amount food that is wasted. This presentation
will focus on bacteria that are to grow and cause spoilage in refrigerated foods,
with particular reference to lactic acid bacteria. The relevance of psychrotrophic
spoilage LAB in food hygiene will be addressed in examples of contamination
studies carried out in collaboration with the Finnish food operators.
Microbial food spoilage is a complex process. Bacterial growth to high numbers
is considered a prerequisite for the development of sensory defects in many
different types of products. The sensory signs of bacterial spoilage are many and
varied; they may be visual (slime, swelling, discoloration), or be apparent by
smell or taste (off-odours and off-flavours). These signs are typically a result of
the accumulating by- and end-products formed during metabolic activity of
bacteria. Many studies have shown that from the great variety of bacteria
contaminants present in the initial population of a cold-stored product, only a
small fraction are able to grow and predominate the so called spoilage
population. The bacteria responsible for quality deteriorations are often called
specific spoilage organisms (SSO). Many factors together, such as product
type (physical and chemical characteristics, preservatives) and the way it is
processed, packaged and stored select the bacteria that are able to survive,
compete and grow to high levels, and hence cause spoilage.

20

In large-scale manufacturing of meat and meat products, the objective is often to

achieve the longest possible shelf-life to meet the demands of trade and logistics.
The traditional way to manage microbial spoilage of meat products relies on
good hygiene, appropriate cold-storage and advanced packaging techniques such
as packaging under vacuum or modified atmospheres. Due to these current
packaging techniques, psychrotrophic lactic acid bacteria (LAB) usually
predominate the microbial populations in cold-stored, packaged meats whereas
the growth of aerobic, gram-negative organisms (e.g. Pseudomonas spp. and
Shewanella putrefaciens) is restricted. In case of temperature abuse,
Enterobacteriaceae may also reach high levels and contribute to rapid spoilage.
Psychrotrophic LAB are a versatile group of organisms. Depending on the
nutrients available and the various processing steps (heat treatment,
preservatives) conducted, the growth of specific spoilage LAB can be very rapid
and they may cause severe quality deteriorations. The genera associated with
quality deteriorations in meat and meat products include Carnobacterium,
Lactobacillus and Leuconostoc. LAB spoilage of meats is typically manifested
as sour off-odours and off-flavours attributed to lactic acid, acetic acid or
volatile fatty acids. This fermenting type of spoilage is often accompanied by
gas accumulation and bulging of the package, or formation of visible slime or
green discoloration on the meat surface. The growth of spoilage LAB in meat
products is difficult to control due to their resistance to environmental conditions
that would inhibit most other bacteria such as low pH, refrigeration temperatures
and packaging in the absence of oxygen. In general, LAB are also considered
relatively resistant to nitrite which is used by meat industry to control bacterial
growth in various processed meat products.
Food may become contaminated with spoilage LAB at all stages of processing
(harvesting, processing, storage). Since many of the psychrotrophic spoilage
bacteria are wide-spread in nature, contaminated raw material or ingredients are
often thought to be major sources of spoilage bacteria. Besides being brought
into production area along with the raw materials, spoilage bacteria can be
recovered in the processing plant air or they may contaminate the product via
workers. Spoilage bacteria may also build up in high numbers in processing
equipments, and processing environment may even select certain types of SSO
to form an in-house population. In many cases, control of SSO is dependent on
maintaining good plant sanitation and high-quality ingredients. To avoid

21

contamination, it is also advisable to handle fermented meat products separately

from cooked meats.
Solving food hygienic problems caused by spoilage bacteria has proven
challenging. Psychrotrophic bacteria are not equal in their spoilage potential;
therefore contamination analysis requires conventional sampling and culturing
techniques together with molecular typing methods. A careful sampling plan is
also essential to detect the spoilage bacteria among the background microbial
populations and to trace their routes through the production chain. A set of
molecular typing or characterization methods and an identification database is
often needed, since for example LAB or Pseudomonas spp. cannot be reliably
identified by conventional phenotypic techniques. To study spoilage LAB
diversities, we have established an identification database at Department of Food
and Environmental hygiene, University of Helsinki, Finland. In this library,
ribotyping patterns (= 16S and 23S rRNA gene HindIII restriction fragment
length polymorphism patterns) are used as the operational taxonomic units in the
numerical analysis and currently it comprises ribopatterns of all relevant foodassociated LAB including over 200 type and reference strains. To identify and
characterize LAB isolates, the similarities between new strains (ribopatterns)
and the type strains can be visualized in the form of dendrogram. Once the
contaminants or the SSO have been identified, control of food spoilage requires
understanding of their ecology, physiological properties and metabolism. This
knowledge is fundamental to determine and implement improvements to prevent
the contamination and hinder the survival or growth of the SSO in the product.
Bacterial spoilage continues to be an important factor limiting the shelf-life of
fresh meat and meat products. Many meat operators consider a spoilage incident
potentially as damaging to the product brand as a foodborne outbreak. Since
foodborne pathogens are rarely responsible for spoilage, and most of the
psychrotrophic spoilage bacteria are rarely associated with foodborne outbreaks,
microbiological safety and microbial spoilage are, however, often considered
two separate issues. In general, there are no management tools systematically
planned out to control spoilage bacterial contamination during meat processing.
In many cases, management of spoilage bacterial contamination is based on
good manufacturing process and hygienic practices. However, experience and
research have shown that good hygienic practices alone cannot guarantee
complete avoidance contamination with certain spoilage bacteria, such as LAB.

22

More information about psychrotrophic spoilage bacteria thriving in food

ecosystems and their contamination routes are necessary to determine adequate
management decisions with regard to controlling this type of spoilage. However,
pre-existing microbial control strategies, such as hygiene prerequisites and risk
assessment approaches, which have been designed to control food safety could
also be applied in managing microbial food spoilage.

23

IMPACT OF COMPOSITION AND METABOLITES

OF INTESTINAL MICROBIOTA ON
FOODBORNE INFECTIONS
Marika Mikelsaar
Department of Microbiology, University of Tartu, Estonia

Several scientists of the early 20th century (L. Pasteur, I. Metchnikoff, T.

Escherich) concluded that variety of diseases could be attributed to the
disruption of the indigenous microflora, allowing pathogenic microorganisms to
multiply in host and cause disease. Further, with development of antimicrobial
therapy it was clearly shown that the disruption of normal microbial ecology of
gut increased the prevalence of enteric infections and there were emerging
protracted cases of gastroenteritis. Very complicated and variable pathogenesis
of enteric, mainly food borne infections has still dimmed the relationship
between host normal microbiota and the hosts susceptibility to infection. The
composition of intestinal microflora e.g. microbiota (IMF) of healthy individual
comprises around 2000 species, their communities express both common and
individual features in a host. The lecture deals with the definitions of normal
indigenous intestinal microbiota, its differences in particular niches of the
intestine and the genetically determined stability of host microflora. The functions of
IMF, particularly the role of microbial metabolism in health and disease has been
largely studied in vitro, in experimental models, volunteer and clinical trials. The
main impact comprises the influences on GI mucosal morphology, digestion,
peristaltics, host metabolism, immunity and improvement of colonisation resistance.
New methods and tools have provided investigators with rich databases on
composition of IMF collected by the culture based methods, molecular tools (FISH,
PCR-DGGE, PFGE, RT-PCR, etc. mRNA expression), by morphological
evaluation (Gram staining, electronmicroscopy, immuno-morphological detection
by specific antibodies). For studies of IMF metabolism several methods have been
applied: the culture based methods for detection of organic acids, lipo-and
proteolytic activity but also the estimation of the metabolic endproducts by gaschromatography, high pressure liquid chromatography, Maldi-TOFF spectrometry,
SDS-PAGE. HostIMF interactions could be evaluated by morphological

24

assessment of physiological/pathological lesions in organs, assessment of cellular

defence (Macrophages, PMN leukocytes, T and B lymphocytes in tissues, searching
for serum antibodies, interleukines, MF, PMN, Toll like receptor, TLR activation.
Mechanisms of action of IMF against pathogens can be divided as follows:

Indigenous IMF specific competitive exclusion of infectious agents (antiadhesion factors, competition for nutrients, antimicrobial metabolites,
bacteriocins, suppression of toxin production, altogether sustaining the
balance of composition of micro-flora). Still, a few is known about the
stability of metabolism exceptional in some diseases.

IMF and host-specific immune response towards foreign antigens of

microbial origin by enhancing the activity of macrophages, increasing the
amount of natural killer and NKT -cells and the level of interferon-,
stimulating the production of IgA, IgG and IgM antibodies.

In host epithelial cells the induction of some non-specific protective factors

as cytokines, mucus, short chain fatty acids (SCFA), epithelial tight junctions
and cells protecting factors like zonulins and defensins against pathogens.

The normal state of host microbial ecology (relations between IMF and host
characteristics) in different body locations is described as eubiosis. In eubiosis
(good life) the particular MF of different body regions is expressing several well
balanced functional characteristics that are controlling the morphology, several
mucosal functions, the natural and adapted immune mechanisms, altogether
resulting in general welfare of healthy persons. In opposite, the imbalance of
microflora, resulting in impaired microbial ecology and disturbance or loss of
general welfare, was in Europe called dysbiosis (formerly dysbacteriosis by
Nissle in Germany). In Anglo-American literature the imbalance in the normal
relationships between various groups of resident and transient microorganisms
are characterized by clinical syndromes or disease entities (malnutrition,
antibiotic associated diarrhea, inflammatory bowel disease etc.). The IMF
associated infectious pathologies can be induced either by:

multiplication of facultatively pathogenic host microbes or

infections by enteric pathogens where host derived altered function of

normal microflora or

deprivation of some important components of normal microflora.

25

The deprivation of colonisation resistance (CR) by IMF is the real basis for
increased susceptibility to different infectous agents, incl food-borne pathogens.
Metschnikov (1907) has proposed that re-establishing the disturbed composition
or metabolism of micro-biota might prevent disease and restore health. A
century later the concept of microbial interference therapy (MIT) has well
survived: maintenance of health or restoration of diseased functions of host by
introducing living microbes. During centuries people have consumed different
fermented milkproducts. It is now known that several bioactive peptides against
high blood pressure, blood coagulation, depression are available in fermented by
lactobacilli milk. Probiotics are one category of functional food. Probiotics
(meaning for life) can be defined as live bacteria of the normal human
microbiota that beneficially affect the host health by improving the microbial
balance. The main aims are: a) to enrich the composition of human microbiota
with non-harmful biologically active bacteria; b) maintain sound intestinal health
by fighting against infectious microbes; c) restore the imbalance of microflora
after infections and antibiotic therapy. Antagonistic activity of probiotic bacteria
against intestinal pathogens helps to prevent intestinal infections. Correction of
imbalance of microflora by probiotics reduces the number of infections with
facultative pathogens accidentally colonising host after antibiotic therapy or due
to viral infection. Probiotic bacteria are applied in food and pharmaceutical
industry. In food industry they provide additional healthy value to different
milk-based products. Pharmaceutical biotherapeutic preparations (food
additives) of probiotic bacteria can be consumed alone or in combination with
prebiotics (fructo-oligosaccharides). The latter non-absorbable carbohydrates
reach the colon and provide a good substrate for probiotics. These combinations
are called synbiotics. Probiotics can be used also with different medicines or
other substances if they did not distrub their influence yet provide some additive
value for usual treatment schemes. Probiotics as adjunct to antimicrobial therapy
have been applied in many infectious diseases, including travellersdiarrhea,
antibiotic-associated colitis, Clostridium difficile related infections, rotavirus
enteritis, urinary tract infections, vaginosis and many other diseases of infectious
origin.
In different clinical trials the efficacy of probiotic therapy varies largely.
Reviewing 46 double blind, randomized, prospective clinical studies with
probiotics the successful clinical outcome was found only in 56% of trials. In the

26

lecture the putative reasons of some failures are discussed which are seemingly
mainly associated with different pathogenesis of enteric, incl. food-borne
infections. Different enteric pathogens can cause gastroenteritis, diarrhea or
intoxication either by adhering or secreting the enterotoxines and disturbing the
absorptive function (viruses to intestinal villi of small intestine, ETEC, EPEC,
Salmonella), or causing the denudation of mucosal villi by cytotoxins (EIEC,
campylobacters, shigellas, salmonellas), intruding into cells, causing
inflammation and generalisation of infection (yersinias, Salmonella typhi,
EHEC) or causing haemolytic toxemia or neurological symptoms (Shigella
dysenteriae, EHEC). Considering the variability of the main attack to host it is
understandable that interference therapy by probiotics has its chance for effect
only in some types of GI infections. Some possibilities for reduction of the
intestinal infections by probiotic lactic acid bacteria are described. Problems to
be solved include the selection of probiotics with specific antimicrobial
properties, for particular target bacteria/infections and for action in defined
ecological niches. This has to stimulate the investigators for more profound
understanding of microbial ecology, pathogenesis of infections and immune
answer.
The elaboration of the antimicrobial and antioxidative probiotic Lactobacillus
fermentum ME-3 (DSM 14241) ME-3 has been performed under the regulations
of WHO/FAO (2002), following the strain identification and molecular typing,
assessment of the functional characteristics and safety by laboratory tests,
experimental animals and volunteers, conducting several clinical trials in
volunteers and diseased persons (urinary tract infection, allergy, brain stroke),
development of the necessary technology for industrial incorporation into
products and their control and at last, participating in market campaigns and
surveillance of consumers interests. The health claims of ME-3 contain
reduction of risk for salmonellosis, shigellosis and E. coli derived infections
from one hand and the enhanced anti-oxidative function of intestinal mucosa and
blood sera of host, comprising antiatherogenicity, on the other hand.

27

CLASSIFICATION AND DIFFERENTIATION OF

SALMONELLA SP. USING DNA BASED TECHNIQUES
Ivan Rychlik
Veterinary Research Institute, Brno, Czech Republic

An increased demand for differentiation of pathogenic microorganisms circulating

in the environment naturally results into an increased demand on new typing
methods. Although the traditional methods like biochemical characterisation,
serotyping or phage typing still remain valuable tools in differentiation of
bacterial pathogens, these are frequently supplemented by the methods based on
the analysis of genetic material. This is true for Salmonella sp., as well as many
other pathogenic bacteria. Salmonella can be subtyped either by isolation and
characterisation of plasmid DNA or chromosomal DNA.
Plasmids of Salmonella vary in size from 2 to more than 200 kb. The best
described group of plasmids are the virulence plasmids (50100 kb in size)
present in serovars Enteritidis, Typhimurium, Dublin, Cholerae-suis,
Gallinarum, Pullorum and Abortus-ovis. They all encode spvRABCD genes
involved in intra-macrophage survival of Salmonella. Another group of high
molecular weight plasmids are plasmids responsible for antibiotic resistance.
Since most of these plasmids are conjugative, besides storage of genetic
information, they contribute to the spread of genes in bacterial populations. The
low molecular weight plasmids are the last group of plasmids found in
Salmonella. Some of them have been shown to increase resistance to phage
infection due to the presence of restriction modification systems. Despite limited
knowledge on their function, their presence or absence is frequently used for
strain differentiation in epidemiological studies. Purified plasmids can be
resolved directly by agarose gel electrophoresis. Alternatively, prior the
electrophoresis, to increase the discriminatory power, plasmid DNA can be
digested by restriction endonucleases. Limiting factor for plasmid profile
analysis in Salmonella is the fact that the low molecular weight plasmids are
found only in about 10% of Salmonella field strains. When chromosomal DNA
is used as a target for bacterial strain differentiation, simple restriction

28

endonuclease digestion combined with conventional electrophoresis cannot be

applied because of the size of chromosomal DNA. However there are several
way how to overcome this complication.
The first possibility is to separate digested DNA by conventional
electrophoresis, blot the DNA fragments onto a nylon membrane and hybridise
the membrane with a suitable probe. To enable subsequent visualisation, the
probe must be labeled in some way. Although currently the non radioactive ways
of labeling are preferred, for explanation and understanding rather obsolete way
of radioactive labeling is more appropriate. The probe must contain a sequence
which appears in a reasonable number of copies in a bacterial chromosome.
Suitable probe can be therefore represented by genes coding for rRNA which
appears in 7 copies in Salmonella Typhimurium chromosome, or IS200
sequence. If the probe contains the rRNA sequence, the protocol is usually
described as ribotyping.
Since the hybridisation is rather time consuming and laborious procedure,
alternatives were sought. Current gold standard of DNA typing in Salmonella is
the macrorestriction analysis. During this procedure, the chromosomal DNA is
carefully purified from bacteria embedded in agarose plugs and digested by
restriction endonucleases which cut the DNA at limited number of positions.
This is usually achieved with restriction endonucleases recognizing 8 bp long
sequence like NotI or SpeI. Sometimes even some 6 bp long sequences appear
very rarely in bacterial genomes and restriction endonucleases recognizing such
sequences can be used for macrorestriction analysis as well. Typical example for
this is Salmonella chromosomal DNA and XbaI restriction endonuclease. Long
DNA fragments generated by the rare cutters are then separated by pulsed
field gel electrophoresis, which enables separation of DNA fragments hundreds
of thousand bp in size.
Spectrum of DNA fragments obtained either by ribotyping or after the
macrorestriction analysis is sometimes difficult to compare between different
laboratories. This is why, despite a widespread and dominant use of these two
protocols in bacterial DNA based typing, additional alternatives are being
sought. Three alternatives are worth at least brief mentioning.

29

The first one is based on sequencing of multiple house keeping genes and
comparison of obtained DNA sequences. Advantage of this approach is ultimate
information which can be shared by any laboratory. This approach has been used
for example for the description of recent evolution of Salmonella sp.
Another alternative is based on PCR detection of mobile DNA sequences. These
can be different plasmids, insertion sequences or integrated prophages. Indeed,
when we compared complete chromosomal sequences of 2 strains of Salmonella
Typhimurium, we observed that with a single exception, the remaining
differences observed by the macrorestriction analysis can be explained by the
acquisition or loss of prophages. When we performed multiple PCRs specific for
prophage sequences, we observed nearly the same discriminatory power as for
the macrorestriction analysis while the results were expressed by a digital code
easily comparable by any laboratory.
The last technique which can be used for strain characterization is the
microarray genomotyping. Great advantage of this method is that the whole
genome of a bacterial strain under investigation is analysed in one time. During
this procedure, labeled DNA from a strain under investigation is let to hybridise
with a DNA chip containing a whole genome of Salmonella. Disadvantage at the
moment is a rather high cost of the analysis and also the fact, that only the genes
present on the DNA chip but absent in the genome of investigated strain can be
detected. If the strain under investigation posses additional genes which are not
present on the chip, these will remain unrecognised.
In a summary, the most widespread method used in DNA typing of Salmonella
as well as many other pathogenic bacteria is the macrorestriction analysis.
Occasionally, plasmid profile analysis and/or ribotyping are being used in some
laboratories. Besides these, several other protocols of interest are tested but have
not reached that wide application so far.

30

ENTERIC PATHOGENS PREVALENCE IN FOOD

PRODUCTS AND MECHANISMS OF SUPPRESSION
BY PROBIOTIC LACTIC ACID BACTERIA
Marika Mikelsaar
Department of Microbiology, University of Tartu, Estonia

Foodborne disease, e.g. infection (FBD) is a major cause of illness and death
worldwide. FBD is caused by pathogenic microorganisms if consuming
contaminated food or drink. FBD outbreak means infection where two or more
different persons get infected after consuming contaminated food. Outbreaks
reported as food-borne, involve a single vehicle of infection and should be
identified by the epidemiological or microbiologic investigations. However,
most frequently they appear in form of sporadic cases that are not registered at
all (under-diagnostics). The FBD can emerge as noninfectious food poisoning
(hard metals), infections with live bacteria or intoxication with preformed toxins
(botulotoxin) or some toxic metabolites (putrescine, tyramine) produced by
contaminating bacteria.
Surveillance: The greatest challenge to protect population from food-borne
infection is to spread information and knowledge about the sources and routes of
transmission of pathogens into food products. The exclusion and control of these
well-estimated risk factors helps to develop safe food all over the world and to
reduce the socio-economic burden of diseases. WHO suggests applying the
effective control of food-borne disease by using the evaluated information
about food-borne hazards and the incidence of food-borne disease. Really, there
are available the estimates of the contribution of specific pathogens to the
overall extent of food-borne infection also at the national level in Estonia,
Sweden and Finland.
Socioeconomic impact: FBD figure mainly gastroenteritis, rarely food
intoxication. The typical cost of gastroenteritis is measured by disability adjusted
life years (DALY). In the Dutch population for instance the costs are 343 million
EUR, 77 EUR per case in 1999. The recent developments are associated with

31

EU programs highlighting all stages of the food chain to assure food safety. To
understand this relationship from farm to fork the epidemiological and clinical
studies are welcome, taking into account the national differences.
Incidence of FBD: In European Northern countries and Baltic States the main
food-borne pathogenic bacteria belong to the microbial family
Enterobacteriaceae, including Salmonella sp., Campylobacterium sp., Shigella
sp. and Yersinia enterocolitica. Altogether these bacteria are classified as gramnegative enteric bacteria. These bacteria are the emerging or remerging
pathogens worldwide whose significance is on the rise. The application of
molecular tools for their detection has received great attention. Of importance is
also the life threatening Gram-positive food-borne pathogen of Listeria sp.
However, the incidence of the listeria-derived infections is fortunately quite rare
in Northern countries and Estonia.
The pathogenesis of food-borne infection starts from consuming contaminated
food where the content of pathogenic bacteria is crucial for development of
disease. The Salmonella sp. needs very high population level of bacteria in food
(more than 105 microbial cells/ml) to achieve the proper infectious dose >107
bacteria by consumption of 100200 ml of the food. In contrast, the infectious
dose of Shigella species is quite low, figuring only 1000 microbial cells. The
infectious dose of Campylobacterium, Yersinia and Listeria largely depend on
the virulence factors of particular food contaminant pathogens and the food
environment, also the contact time and conditions for germinating bacteria. In
food industry the conditions are usually well described for safe food production.
Foods and risk of FBD: There are a few studies where the risk of different
foodborne diseases from different kinds of foods is evaluated (Adak et al.,
2005). In England and Wales, at 19962000 the most frequent etiologic agents
of FBD were campylobacteria, followed by twice lower incidence of
Clostridium perfringens and Yersinia spp. infections, the Salmonella sp. together
with different E. coli infections (ETEC, EPEC, EIEC, EagEC) figuring the 4th
and 5th in the list. However, it should be mentioned that in nearly 50% of cases
the infectious agent was left unknown though the microbiological diagnostics is
very highly evaluated in UK. The similar data are provided also in textbooks.
The Listeria monocytogenes driven infections were quite rare (221 cases), yet
the mortality rate was 30%! In this paper the foods were classified into broad
32

food groups, such as poultry, and more specific food types, e.g. chicken, and the
percentage of outbreaks due to each type of food for each pathogen was
calculated. This offers a good overview about disease impact according to food:
out of 1,724,315 studied cases for the 5y period there were only 4% of cases
where the food handlers were the source of infections; in the cases left, most
illness was attributed to eating poultry (30%), complex foods (27%) and red
meat (17%). These studies show mainly the national/ethnic habits in food
consumption. Therefore it is not astonishing that chicken consumption
accounted for more disease, deaths and healthcare usage than any other food
type in UK. Only 5% of patients were infected by eating plant-based foods e.g.
vegetables, fruit and rice and it seems that the consumption of milk products is
not very high as only 6% of FBD were associated with dairy products. In
Estonia, quite specific food was incriminated to cause outbreaks: the combined
food as puddings, creams, sweets, potato salads.
Reviewing the literature of the recent food outbreaks worldwide, there were
found very similar data to the common textbooks, concerning the association of
specific pathogens with particular food. Campylobacter sp. caused FBD is found
after consuming poultry, in US annually >1 million people are infected,
developing a long duration diarrhea; poultry has been the main source also in
Denmark (2004); in Sweden the campylobacter gastroenteritis is clearly linked
with water supply and livestock (2004). For Salmonella enteritidis the main
contaminated foods listed are hens eggs (Denmark, 2004), complex food, e.g.
school-lunch dessert (Japan, 2001), eggs-salad (US, 2003) but also as a quite
unusual foodstuff the imported peanuts from Australia, causing the spread of
FBD in Canada, England and Scotland (2001). The problem of antibiotic
resistance of salmonellas and failure of treatment, development of carriage is the
serious problem bound to FBD. Quite unexpectedly the FBD was caused by
Halva from Sesame seeds produced in Turkey and afterwards the contaminated
Halva food product had been imported to Germany, Sweden, Norway and
Australia. Though Listeria sp. infections do emerge rarely but the threat for
pregnant women, neonates and elderly persons with immunodeficiency can
become a growing problem. In Japan, a nationwide survey of human Listeria
monocytogenes infection revealed 83 cases of listeriosis per year with an
incidence of 0.65 cases per million of the population (2002). Shigella sp.
infections are usually associated with food handlers: in US it was described a
multi-state outbreak in 2000 by commercially prepared food, a dip with cheese

33

which was hand-broken to be added to dip. The education of large populations

could help to avoid FBD caused by handlers in manufactures with decreased
hygiene standards.
The dangerous food-borne agent E. coli O157:H7 caused infections are not very
frequent in Nordic and Baltic countries, showing only some single cases that are
mainly associated with consumption of red meat and milk. E. coli O157:H7
(EHEC) has been linked to salami in Canada (1999), to milk in Denmark (2004),
found among staff of a large hospital in Sweden (2004). Very similar results on
water contamination by EHEC in farms wells were assessed both in US (2003)
and Scotland (2006). The waterborne outbreaks clearly need more attention, as
in local food production the water is very closely associated with food chain. In
the sparse population of Northern Scotland, Sweden, Norway and Finland, the
groundwater is used without disinfection and the bacteria could survive for long
time. Campylobacter jejuni and C. coli caused three waterborne outbreaks in
Finland (2003). The viruses (Norovirus, Norwalk-like virus) are causing massive
water related outbreaks of gastroenteritis in different Nordic countries, the
viruses can also be transmitted to foods.
Improvement of food safety by LAB probiotics: Probiotics are live bacteria of
human origin having beneficial influence on hosts health. Viable lactic acid
bacteria (LAB) are largely used for designing functional food, e.g. probiotics. In
community there is a growing need for functional food (FF) to get support
against infections, atherosclerosis associated diseases like cardiovascular heart
disease, brain stroke but also against metabolic syndrome, obesity and cancer
(Ljungh and Wadstrm, 2006) Thus, consumers interest, granting the success in
market, is a good starting point for development of probiotics. However, only
some 10 to 15 y lasting evidence based research can result in internationally
accepted product. Next, the innovative thinking of stakeholders and enterprises
is of utmost importance to manage with financial pressure and nationwide strict
health claim regulations. Recently the consensus for health claims on FF
products was elaborated by FUFOSE and PASSCLAIM (DOI10.1007/s00394005-1104-3) allowing the use of the health claims of either enhanced function or
disease risk reduction.
The National culture collection of lactobacilli at Dept. Microbiology of
University of Tartu provides excellent basis for search of LAB strains of human
34

origin with high antimicrobial activity. However, the development of a probiotic

has to pass the long route from scientific ideas, nation-wide evidence based
research, patenting process, transfer of technology to dairy and cheese
enterprises, before to be delivered into the hands of consumer.
A meanwhile step in probiotic development affording safety of food products is
described. The food product serving as carrier of probiotic strain (to be delivered
to host organism) can get some additive value from the functional properties of
the probiotic candidate strain, in order to strengthen the food safety.
Mechanisms of action of probiotics against pathogens in raw material and food
products comprise specific competitive exclusion of infectious agents: a)
competition for nutrients, b) production of antimicrobial metabolites,
bacteriocins, c) suppression of toxin production by contaminating pathogens,
altogether sustaining the balance of composition of specific micro-flora
developed in food-product.
Together with Estonian Technology Agency we have elaborated several
products which safety is granted due to the high antagonistic activity of
incorporated LAB probiotic candidate strains. The metabolites, the organoleptic
indices and laboratory, experimental animal studies for safety should be
performed to get acceptance from local Veterinary and Food Board and
Consumer protection Board.

35

EFFECT OF MAINTENANCE ROUTINES IN FOOD

PROCESSING ON PRODUCTION HYGIENE
Kaarina Aarnisalo
VTT, Espoo, Finland

Contamination of foods by food handlers has been identified as one of the most
important cause of foodborne outbreaks. The regulation on food hygiene (EU)
No 852/2004, Annex II states, that food-handlers must be supervised and
instructed in food hygiene matters commensurate with their work activity.
Contamination from personnel can be direct or indirect. Direct contamination
can happen by contact between the body and the food product. In indirect
contamination, people act as vectors and transfer contamination from one area or
surface to another .Maintenance personnel do not actually touch the raw
materials or food products, but they presumably touch a multitude of surfaces in
contact with unpacked products. Maintenance personnel dismantle machinery
for cleaning procedures and reassemble it after cleaning in addition to
maintaining the operation of machinery during production.
L. monocytogenes is a pathogenic bacterium that survives in low temperatures
and is problematic for food industry. It attaches to and grows on different
surfaces even at low temperatures, tolerates anaerobic conditions and a wide pH
range and may persist in food processing equipment. L. monocytogenes can be
transferred from processing surfaces to foods. In the presentation, results from
two projects Hygienic equipment in food industry and Food industry, safety
and hygiene management, co-ordinated by VTT, are presented.
A study was made on the hygienic working practices of maintenance personnel
in Finnish food industry. The aim of this work was to study the level of hygiene
knowledge and the hygienic practices of maintenance personnel in the food
industry in order to identify aspects that could have a negative effect on
equipment hygiene and that should be improved. A mail survey was sent in
spring 2002 to maintenance personnel, food-handlers, quality managers and
cleaning personnel in 106 Finnish food companies. Answers were obtained from

36

23.6% of the plants. Most of the maintenance personnel (n=59) work in the
production area continuously (61%) or at least 5 times (23.7%) during a work
shift. Of the maintenance personnel, 42.4% reported having touched surfaces
with contact in food often or always and while working in the production area
55.9% of them usually wore gloves. However, only 13.6% washed their hands
after smoking and 23.7% before starting to work without gloves. 33.9% of
maintenance personnel found they had not received enough information about
hygiene commensurate with their work. Studies about food hygiene were not
included in their basic education (89.9%). Most (63.6%, n=55) of the
maintenance personnel knew where they could find the hygiene rules. One third
(32.2%) of the respondents answered that they washed their tools once a day or
always after work. The others answered that they washed their tools more
seldom and 32.2% only once a year or never. Almost all (89.8%) of the
maintenance personnel had personal tools which they themselves cleaned. For
tools that are in common use, no persons were in charge of the cleaning in
71.9% of all the cases reported. The quality managers were asked if they had
ever received consumer complaints, which could be linked directly to the work
of maintenance personnel. Such complaints were rarely or seldom reported by
62.5% of them (n=16).
Based on the results, hygienic working practices of maintenance personnel
should be improved and more hygiene education is needed. Clear hygiene rules
targeted to maintenance personnel should be available for all maintenance
personnel. Maintenance personnel are a potential source of contamination of
food products due to the nature of their work. They move a lot between the
production area and their workshop and they have to touch a lot production
surfaces. The proper use of protective clothing, washing of hands and tools as
well as avoiding foreign bodies left on the production lines should be targeted
when the hygienic working practices are developed for maintenance personnel.
In addition to the questionnaires, the working practices of maintenance
personnel were studied at four food companies and microbial samples of the
tools, protective clothing, hands and working environment were analysed for
total aerobic bacteria, Enterobacteriaceae and L. monocytogenes. The amount of
aerobic bacteria on maintenance personnels clothes, work environment and
tools, was, on average high (514 cfu/cm2). Enterobacteriaceae were not found
in samples except high numbers on one of the gloves (100 cfu/cm2).
L. monocytogenes was found from a tool, a screwdriver, which was in common
37

use by maintenance personnel which emphasizes need for regular cleaning and
disinfecting of the tools. Listeria spp. was found from another tool and
protective clothing of personnel as well indicting, that maintenance personnel
have possibility to transfer Listeria in the food plant.
Another study was made on survival and growth of L. monocytogenes in
lubricants used in the food industry. Lubricants in food-processing are needed in
maintenance of the equipment to e.g. decrease friction and wear and to increase
the efficiency of systems. The survival and growth of three L. monocytogenes
strains in 10 lubricants (synthetic and mineral-oil based) used in the food
industry, and rapeseed oil, was investigated at room temperature (20 C) and
refrigerated (5 C). Additionally, the transfer of L. monocytogenes from
lubricants to stainless steel surfaces and vice versa was investigated. Though the
amount of L. monocytogenes in most lubricants, both pure and soiled, decreased
significantly (p < 0.05) during the 14-d test period, lubricants may act as sources
of contamination on the basis of the results obtained on the survival of
L. monocytogenes. In general, temperature had significant effect (p < 0.05) on
listericidal effect of lubricants contrary to soiling (p > 0.05), however the effect
of both factors was dependent on lubricant (p < 0.05). The results clearly showed
that L. monocytogenes survived in synthetic conveyer belt lubricant diluted in
water. In addition, L. monocytogenes was transferred significantly (p < 0.05)
from stainless steel surfaces into conveyer-belt lubricants and into mineral-oil
based hydraulic oil and was transferred from lubricants to stainless steel surfaces
as well. The results emphasize need for careful use of lubricants during
maintenance work and need for regular change of lubricants and cleaning and
disinfecting of surfaces between the change. Further as conclusion of the studies
made, improvements in hygienic working practices of maintenance personnel are
required with respect to minimizing the occurrence of L. monocytogenes, as well.
References to this extended abstract can be found in the articles:
Aarnisalo, K., Tallavaara, K., Wirtanen, G., Maijala, R. & Raaska, L. 2006. The
hygienic working practices of maintenance personnel and equipment hygiene in
the Finnish food industry. Food Control, 17, 10011011 & Aarnisalo, K.,
Raaska, L. & Wirtanen, G. 2007. Survival and growth of Listeria monocytogenes
in lubricants used in the food industry. Food Control, 18, 10191025.

38

HYGIENICALLY INTEGRATED SYSTEMS

Lotte Dock Steenstrup
BioCentrum-DTU, Kgs Lyngby, Denmark

The demands that are placed on product and process within the food production
industry are interconnected. Therefore, it is not appropriate to use a mainly
sequential approach to designing a production facility, where you first address
the primary function of the product, i.e. product quality, and then consider other
issues like safety, hygienic design, cleaning, flexibility, and traceability. There is
a need for integration of these issues, in order to make the best decisions and to
balance the different needs.
Frequently during the sequence of designing, fabricating, installing, contracting,
making design changes, or maintaining a production assembly, a line, or a
facility, poor decisions are made because the sequential approach to problemsolving is used. In that case, we may unintentionally create hazards in the
process line, like leaving a valve on a branch closed, thus creating a dead end, or
just placing equipment incorrectly, making cleaning very difficult.
Another important issue in obtaining an optimally running line is to make sure
that it is operated in a systematic way. One way to ensure high performance is to
carry out implementation of HACCP and GMP, which deals primarily with
hygiene, cleaning and critical control point monitoring. Furthermore, high
performance is ensured by employing change management, by establishing and
maintaining documentation with regard to installation, automation, operation,
maintenance and cleaning and by proving the operation and performance of the
equipment before routine use.
In order to deal with these issues a guideline was developed by a subgroup of the
European Hygienic Engineering & Design Group (EHEDG). Part of the scope of
the guideline is to a) describe integration of entities, up to and including the
manufacture and supply of goods, in order to produce safe food or related
products cost effectively; and b) describe integration topics that can affect

39

hygienic design, for example installation, operation, automation, cleaning and

maintenance, especially those that are common or a frequent cause of failure.
The guideline has the task of linking and supporting currently available EHEDG
guidelines, laws and standards (such as EN1672-2). Entitled Integration of
Hygienic and Aseptic Systems, the document is in review stage and will be
published early this year.
The guideline established that the hygienic system being developed must
conform to all specified requirements (the specified requirements may originate
from, for example, legislation, users, product quality or safety). The integrated
approach also includes determining specifications for issues such as product
flow, control strategy, automation, maintenance, change management, and
training of personnel. Furthermore, hygiene risk assessment is a necessity (for
installed manufacturing systems, HACCP). A failure-mode-and-effect analysis
(FMEA), which is a structured equipment-based safety tool based on risk
assessment of the consequences of failure of any entities within a process, may
also be carried out.
The integration process comprises of a set of actions that are done in order to
successfully complete a hygienic integration of entities. These actions are for
example to define stakeholder requirements, analyze and specify design, install
entity, qualify installation, validate and qualify operation etc.
For each integration action entities must have at least a prospective validation
identifying probable failure modes. Hygienic integration should be carried out
on a modular basis, with entities that have already satisfied the hygiene
requirement for integration. Instructions must cover: installation, operation,
cleaning, sterilization (if applicable) and maintenance. Concurrency with design
and validation activities other than those concerned with hygiene is a
prerequisite. For an unassigned module or assembly, the provisionally intended
process or processes and product(s) must be defined in a prospective list.
Going through for example the action to define stakeholder requirements, the list
of requirements should produce a conceptual design of the entity or entities
under examination. Every time such a step is completed, the flow diagram takes
the user through a confirmation step, making sure there is compliance between

40

intake information and the outcome from this analysis. For example, if the user
in the conceptual design forgot to take some legislation issues into consideration,
the user should be able to notice this before going on to specify the design. The
flow chart also prompts a record of data produced during the decision process
and also a record of the decision itself.
When the verification of the hygienic performance is done (this is the last action
point in the integration step), the entity has successfully been integrated. It can
then be implemented in the specific process it was assigned for, or handed over
to the user. If the entity has not been assigned to a particular product or process,
then it can just be added to the library of hygienically verified entities.
The intention of the guideline recommendations is to avoid hazards that might
otherwise be produced, which can be of microbiological, chemical or particulate
nature in the consumable product. Hereby, the risks of recalls, lawsuits, and
damage to reputation are also minimized. By supporting the integrated approach
the guideline is also intended to reduce other non-safety problems, for example,
environmental impact, costs, and excessive use of resources like water,
chemicals or energy. The overall outcome of a successful implementation of this
approach is optimal cost efficiency of both the construction phase and the
finished design.

41

METHODS FOR EVALUATION OF PROCESS HYGIENE

Satu Salo, Erna Storgrds & Gun Wirtanen
VTT, Espoo, Finland

The control of quality is essential in order to provide wholesome and safe food
products to consumers. The intention of food companies is to minimize the risk
of epidemics and spoiled food stuffs caused by pathogens and other harmful
microbes. The requirements for better hygiene has raised via development of
food industry which includes prolonged self life, centred production and long
transportation, automated cleaning systems, reduced cleaning time, demands for
environment safe cleaning agents.
Hygiene can be defined with different aspects such as the amount of dirt,
protein, polysaccharide and organic residuals, biofilm, dead and/or live microbes
generally or specific pathogens and other harmful microbes. Also, hygiene can
be detected from different sources; from final product, raw materials, process
equipment, environment surfaces, air and personnel. This variation means that
sample to be diagnosed can be in gas, liquid or solid form or the sample can be a
surface of equipment. Sampling from surfaces is very challenging; either the
measuring has to be done direct from surface quickly and without harming the
surface or the dirt has to be detached from the surface as properly as possible
without harming surface or microbes to be detected.
Hygiene detection of the food processing equipment and surfaces is one
important part of the quality control. By sampling the surfaces it can be possible
to prevent bad quality, it is a big help in tracing the contamination sources and in
optimizing cleaning systems. For choosing a suitable method for detection it is
important to know what kind of information is needed. Also it is important to
figure out the wideness of sampling (the amount of samples and the frequency of
sampling) when choosing the method. Quantification of micro-organisms from
surfaces is difficult because of strong microbial adherence and because the cells
grow in layers, forming biofilms. By collecting data it is possible to notice the
changes in trendlines. Also the setting of limit values is depending on the sample

42

and the method used. The threshold of detection of adhering micro-organisms

can vary according to the enumeration technique employed, and some
techniques underestimate the number of micro-organisms on a surface.
The problems associated with the repeatability and reproducibility of studying
biofilm microbes are well known. The methods currently most often used in
surface hygiene control are the cultivation method based on detached samples
(swab, sponge, biofilm reagents, ultrasonics) and contact plates or slides, the
ATP bioluminescence method (measurements directly from the surface or from
swabbed sample), protein residues, impedance based method and microscopying
methods such as epifluorescence microscopying.
Various researches have been made to test and develop alternative methods for
sampling from surfaces at VTT within several projects. Often used test matrix
has been microbiological soil dried or grown onto surface of stainless steel plate.
The reference method has been the conventional cultivation using swabbed
sample.

SWABS AND SPONGES

The sample for hygiene controlling is generally detached from surface with
sterile swab or sponge. In the swabbing method the process area of interest is
swept with cotton, dacron, ryon or alginate tipped stick to collect possible microorganisms. The collected micro-organisms are then released into an appropriate
solution for subsequent cultivation. The advantage of the method is that it
provides good access in confined process areas. The method is also easily
adaptable to many detection techniques.
Comparison of three types of swabs was done by testing cotton-, dacron-, and
alginate-tipped swabs. The results of cultivation and epifluorescence
microscopying after swabbing showed that there were not big differences
between different types of swabs. Thus microscopying shows that cotton tipped
swab removes slightly more biofilm than the others. The special property of
calcium alginate swab is that it is degrading easily. That is advantage when the
cells have to be released into an appropriate solution for cultivation but a
disadvantage during swabbing.

43

DETERGENT BASED SOLUTIONS FOR DETACHING BIOFILM

Various substances are known to improve the detachment of biofilms. The mild
alcohol-, surfactant- and enzyme-based detergent blends has been tested and
these detergent based solutions can be used to improve the detachment of
biofilms either with swabs moistened in detergent solution or by spraying
detergent solution onto sampling surface before swabbing with dry swab.
Contact plates can be used for flat surfaces; in this case the assumption is made
that the micro-organisms stick to the agar surface and start to multiply. The
method is easy to use and labour saving because it is not necessary to transfer
the microbes from the swab to the cultivation medium. However, this method is
also based on detachment of surface-bound micro-organisms, which is the
limiting factor in the swab method.
Ultrasonication has proved to be an effective technique for the removal of
biofilms from surfaces. In ultrasonication high-frequency vibration leads to
strong formation of very small bubbles that hit the surface at high speed, which
in turn causes detachment of surface-bound micro-organisms and biofilms. The
technique has mainly been applied for detaching biofilm bacteria from test slides
in bath sonicators. The method cannot be applied as such for routine control of
process hygiene.
It has been reported that cells counted by direct microscopy consistently give
results one log unit higher than the cultivation method. In our experiments
epifluorescence microscopy clearly revealed that even vigorous swabbing only
detached a small part of the actual biofilm and cells within it. This means that
techniques based on swabbing only provide limited information about the true
surface hygiene.
The advantage of measuring ATP directly from the surface is that the assessment
can be undertaken while the micro-organisms remain surface-bound. The ATP
method is especially suitable for hygiene monitoring, as it detects both living
micro-organisms and other organic residues in the sample. However, a severe
deficiency of the method is its rather high detection limit for bacteria that may
be further increased in the case of cells stressed by cleaning or disinfection. Thus

44

the method is rather unreliable in situations in which even very low numbers of
residual micro-organisms after cleaning are to be monitored.
An obstacle to the use of both the contact plate technique and ATP
measurements directly from surfaces is that both methods require a flat, even
surface of a certain dimension. Unfortunately, critical points in the hygienic
aspect are often poorly accessible, typically located in confined spaces in the
process.

45

MOLECULAR TECHNIQUES AND MICROSCOPY IN

BACTERIAL DETECTION AND TYPING
Johanna Maukonen
VTT, Espoo, Finland

Microorganisms in the food industry are mostly harmless but some may be
harmful to the process and/or safety of the product. Therefore, the control of
harmful microorganisms is essential. Industrial processes that deal with any
biological material provide nutrients and conditions for microorganisms to grow
either as plankton cells in the circulating process waters or as sessile in the
shelter of biofilm on surfaces. Due to the spatial and temporal heterogeneity of
industrial samples and technical problems related to sampling in the industrial
environment, obtaining a representative sample from certain foods and foodrelated industry is a demanding task. In addition, microbes are often tightly
attached to the surfaces and the process equipment may contain parts that are
difficult to access.
Fluorescence microscopy is widely used in microbial ecology studies. There are
several advantages in the use of fluorescence microscopy. It is fast and rather
easy to use, it allows the visualization of spatial distribution of cells in the
sample, and with suitable combination of fluorescent stains, the differentiation
between viable and dead cells is possible. However, direct identification of
microbes is not possible with conventional fluorescent stains. Distinguishing
cells on the basis of morphology is therefore important, because fluorochromes
are not specific for bacterial species or genera. Numerous fluorescent stains are
used for detection of both biofilm and plankton cell samples to study the
viability and/or the total number of microorganisms. The most commonly used
stains for the detection of total number of bacteria are acridine orange (AO) and
4,6-diamidino-2-phenylindole (DAPI). There are several stains that target either
viable bacteria or non-viable bacteria. The most commonly used viabilitystaining system for industrial samples is LIVE/DEAD BacLight Viability Kit
(Molecular Probes, USA).

46

A molecular technique used for detection of pathogens must be capable of

detecting low numbers of target bacteria in samples which may contain
considerable background of interfering microorganisms and several matrixderived compounds that may hamper the detection. In microbial community
analysis the method should allow detection of different groups or species present
in the ecosystem with similar efficacy to avoid biases in evaluation of species
distribution and complexity of the microbiota. Biases may already be introduced
by sample handling, as well as during the extraction of nucleic acids from
microbes in the sample. Sensitivity of the molecular method can be improved by
enrichment culture, but this also precludes attempts to quantify the number of
target organisms in the sample. Molecular techniques can be utilized in the
detection and identification of microbes in two ways: a) identification is
performed directly from sample material, or b) identification is based on
combined culture and molecular detection. A sample matrix studied plays an
important role when the decision between the two choices is made. If the matrix
is known to contain factors that can inhibit e.g. PCR reaction and which are
difficult to remove, it is often best to use the combination of a culture technique
and a suitable molecular technique. There are two major techniques applied in
the molecular detection and identification of bacteria, PCR and hybridization.
When molecular tools were first introduced for detection and identification of
microbes, hybridization methods were widely applied. The rapid evolvement of
PCR techniques has led to the present situation where hybridization is mainly used
in combination with PCR. However, a technique called in situ hybridization, in
which bacteria are detected in their natural microhabitat, has proved useful in
applications where enumeration of the target organisms is warranted. Recent
development of DNA microarrays allows, due to the automatisation of the
procedure, simultaneous identification of huge number of specific sequences by
hybridization.
Microbial ecology studies are often based on ribosomal RNA (rRNA) or rDNA
sequences. 16S rDNA sequences can be used to infer phylogenetic relationships
and to identify unknown microbes by database comparisons. It has also been
proposed that rRNA content is appropriate for assessing changes in
metabolically active bacterial populations, since rRNA content depends on
bacterial activity. Besides rDNA gene, other target genes can be used for
molecular detection of selected microbial groups/species from food and industrial
samples. Genes associated with virulence factors, such as toxin producing

47

listeriolysin O (hlyA) gene in L. monocytogenes, are commonly used for detection

of food-borne pathogens. In addition, genes coding for physiological properties e.g.
cold shock protein genes that are present in psychrotrophic B. cereus -group strains
have been used as target molecules for detection.
Genetic fingerprinting techniques can be used to characterize bacterial
communities or single bacterial isolates. Genetic fingerprinting techniques of
microbial communities provide a pattern or a profile of the community diversity
based upon the physical separation of unique nucleic acid sequences.
Community analysis techniques are relatively easy and rapid to perform and they
allow simultaneous analysis of multiple samples enabling the comparison of
genetic diversities of microbial communities from different habitats, or studying
the behaviour of individual communities over time. Community analysis can be
performed with techniques such as denaturing gradient gel electrophoresis
(DGGE), temperature gradient gel electrophoresis (TGGE), and single-stranded
conformation polymorphism (SSCP). DGGE/TGGE analysis combines a direct
visualization of bacterial diversity and the opportunity to subsequently identify
community members by DNA fragment sequence analysis or hybridization with
specific probes.
Fingerprinting of bacterial isolates can be performed by a variety of techniques
including e.g. ribotyping, amplified ribosomal DNA restriction analysis
(ARDRA), pulsed-field gel electrophoresis (PFGE), randomly amplified
polymorphic DNA (RAPD), repetitive element sequence-based-PCR (rep-PCR),
and amplified fragment length polymorphism (AFLP). All these techniques aim
at differentiating bacterial isolates at the subspecies level, preferably even at the
strain-level. Prior to molecular techniques phenotypic methods such as biotyping
and serotyping were used for the bacterial strain differentiation. These
techniques are still used today, but with molecular techniques more reliable and
often less laborious fingerprinting can be achieved. Regardless of whether
phenotypic or genotypic techniques are applied, the fingerprinting is preceded by
culture and single-colony subculture steps. Thus, even though PCR and
hybridization can be used both in bacterial detection and fingerprinting, the
techniques applied differ in a profound way: while detection methods are able to
find the target organisms in a sample containing hundreds of other bacteria,
fingerprinting methods are not genus or species specific and can therefore be
applied to bacterial pure cultures only.

48

The emergence of new detection and real-time methods is linked to the need for a
better assessment of the microbiological quality of products. This objective can be
reached through an increase in detection specificity and a reduction in analysis
time. In particular, the in situ techniques should enable progress in the
understanding of the ecology of complex microbial communities in minimally
disturbed samples. The most important weakness of culture-independent methods is
that the taxonomic interpretation of data appears problematic. Although various
new detection methods are applied to detect microorganisms from the industrial
environment, the use of culture techniques will persist since the international
standard methods for detection and enumeration of pathogens are based on
cultivation. In addition, in many industrial quality control laboratories resources
for the use of new molecular methods are inadequate.
In routine food control PCR-assays may shorten the time needed to identify e.g.
L. monocytogenes, although enrichment may be necessary prior to the detection.
By using virulence-associated genes as primers or probes, the presence of
pathogenic species can be rapidly determined. However, dead bacterial cells
may constitute a problem in basic-PCR-detection in hygiene control. For
example, heat-treated samples may contain dead or damaged cells with no
relevance to the product safety, however, the dead bacteria may still create
positive signals due to the stability of the DNA molecules. In some
circumstances (when the RNAse activity of the bacterial population is not
destroyed in the sample prior to analysis) RT-PCR can be applicable in assessing
the viable and active populations in samples. DNA-based detection methods,
especially PCR, may gradually replace traditional methods for assaying
microorganisms in food. When applicable (e.g. when no enrichment step is
required) real-time PCR, which enables the quantification of target sequences, can
prove highly useful for the rapid analysis of food pathogens. However, PCRdetection of pathogens, in food samples is still time-consuming, particularly in the
case of large-scale testing. High throughput methods, such as dot blot
hybridization using microarrays, have promising future potential for the routine
diagnostic and quality control procedures in industrial settings.
One of the challenges for microbial ecology is to gain more information below the
bacterial community, genera, and even species level. Subspecies level identification
is especially important when a source of contamination is traced in an industrial
environment. DNA fingerprinting techniques provide effective molecular tools to

49

identify and type microorganisms to subspecies level. While typing of the microbial
isolates is performed, e.g. to trace a contamination source, the importance of
including sufficient numbers of isolates from each sample site should be
remembered. Once efficiently integrated, the typing techniques provide precise
information on the heterogeneity of target bacterial population at a given time/space
combination. However, fingerprinting methods are laborious and time consuming
since isolation and cultivation of a large number of bacterial isolates cannot be
avoided. Another limitation is that the unculturable strains present in natural
ecosystems cannot be reached with typing methods.
In conclusion, bacterial detection, identification, and typing from industrial samples
remains a laborious task, mainly due to the fact that frequently large numbers of
samples need to be analyzed. Development of automated techniques that allow high
throughput analysis of large numbers of samples will greatly facilitate the studies
on industrial microbial ecology in the future.
References to the methods described in this abstract can be found in the review
article of Maukonen, J., Mtt, J., Wirtanen, G., Raaska, L., Mattila-Sandholm,
T. & Saarela, M. 2003. Methodologies for the characterization of microbes in
industrial environments: a review. Journal of Industrial Microbiology and
Biotechnology, 30, 327356.

50

LEGAL ASPECTS OF FOOD SAFETY

Tiina Saron
Estonian Dairy Association, Tallinn, Estonia

The aim of this paper is to share the Estonian experience in implementation and
enforcement of the veterinary and food safety acquis. In addition, a broad
overview about the EU food safety legislation is given. The paper consists of
three parts. The first part describes the existing EU food safety legislation and
food control system. The second part describes the creation of food safety
legislation and supervision system in Estonia. The third part concentrates on
analysing the level of compliance with the EU production and processing
requirements in Estonian dairy and meat industry. The amount of investments
needed to achieve the fulfilment of the requirements is also evaluated. In
addition, the impact of food safety measures on trade is looked at.
In recent years food safety and food quality have been the issues increasingly
dominating public and political debate. A major reason for this is the tremendous
development that the entire food sector has gone through both in terms of
technology and production. At the same time the debate has also questioned
whether the consumers demands and expectations are being met. Food safety
and quality are of the greatest importance for the consumers and their sensitivity
in that respect is very high. Food safety, nutrition and health considerations,
consumer information and environmental policies lead to regular review and
amendment of Community legislation with respect to food products, the related
production methods and enforcement mechanisms.
The EU food law aims at ensuring a high level of protection of human life and
health, taking into account the protection of animal health and welfare, plant
health and the environment. This integrated farm to fork approach is now
considered a general principle for EU food safety policy. Food law, both at
national and EU level, establishes the rights of consumers to safe food and to
accurate and honest information. The EU food law aims at harmonising existing
national requirements in order to ensure the free movement of food and feed in

51

the EU. The food law recognises the EUs commitment to its international
obligations and will be developed and adapted taking international standards into
consideration, except where this might undermine the high level of consumer
protection pursued by the EU.
There is no doubt that the safety of foodstuffs has the absolutely highest priority
for every government but food safety issues are also crucial in trade.
International trade in high-value food products has expanded enormously over
the last decades, fueled by changing consumer tastes and advances in
production, transport, and other supply-chain technologies. The cost of
complying with food safety standards has been a major source of concern in the
international development and among the countries in transition.
Achieving a standard of food safety is necessary for the development of internal
and external markets. The respective Legal framework and appropriate
supervising system must be created. Harmonisation of the acquis
communautaire and particularly meeting the technical requirements demand
substantial investments, but as a result the opening of EU internal market is
possible. Many aspects of standards compliance do not require large investments
or sophisticated technical or administrative capacities. The most significant
challenge is often building broad awareness about the need for proper food
safety measures and facilitating the broad adoption of good agricultural and
manufacturing practices. A coherent regulatory framework and a system to
assess compliance and conformity are also needed.
The EU food law aims at ensuring a high level of protection of human life and
health, taking into account the protection of animal health and welfare, plant
health and the environment. This integrated farm to fork approach is now
considered a general principle for EU food safety policy. Food law, both at
national and EU level, establishes the rights of consumers to safe food and to
accurate and honest information. The EU food law aims at harmonising existing
national requirements in order to ensure the free movement of food and feed in
the EU. The food law recognises the EUs commitment to its international
obligations and will be developed and adapted taking international standards into
consideration, except where this might undermine the high level of consumer
protection applied by the EU.

52

Food safety, nutrition and health considerations, consumer information and

environmental policies lead to regular review and amendment of Community
legislation with respect to food products, related production methods and
enforcement mechanisms.
The regulatory framework in which EU food industry businesses operate is a key
determinant of their competitiveness, growth and employment performance.
With regard to food trade, formally the development of trade measures has been
towards adjustment and transparency, but in practise the agricultural trade has
remained a subject to trade restrictions of different kind. Although the main
purpose of practising non-tariff measures is the protection of humans, animals,
plants and environment, these are often used to restrict trade. Non-tariff barriers
are a result of differences in food legislation from one country to another.
Examples include diverging labelling requirements, different residue limits or
lacking recognition of test results.
The EU food safety policy includes the whole of the animal and human food
chain. It provides extensive legislation and outlines the responsiblity of
producers and suppliers in helping to ensure a safe quality of the food supply.
The EU regulations are amongst the most stringent in the world.
In the scope of the association agreement with the European Union Estonia took
an obligation to accept all policies and objectives of the EU without any
reservations. That obligation had the main impact on the preparations of
legislation and control systems. The accession procedure with the EU has
highlighted the fulfilling of the technical requirements set for processing of
agricultural products in Estonian food industry. A special feature of Estonia is
that all processing facilities are EU fit without a transition period for adapting
the EU food safety requirements. Only Cyprus is in the same position. Generally
speaking Estonia fulfilled the main goal to join the EU without any transitional
period.
Meeting the technical requirements demands substantial investments and as a
result the opening of the EU internal market is possible. It is important to
achieve optimal level of investments and it is not rational to invest in unutilized
processing capacities. Estonia was unfortunately not able to reach this goal, as in
some sectors investments were not made in time; in others over-investing

53

occurred. On one hand, food safety is the main priority of the government and of
the food processing enterprises as well. All food operators have to take care of
that and make necessary investments. On the other hand, the role of the
government is to realize institutional reform and apply the legal framework in
the field of food safety. Obligation of compliance with the food safety
requirements directly influences the number of operating enterprises. As a matter
of fact it is not easy to specify the appropriate period and measures for
implementing the requirements in the transition economies.
Today our lifestyles are different from those of the past. There is an increasing
number of single-person households, one-parent families and working women
that have changed the food preparation and consumption habits. A positive
outcome of this has been the development in food technology, processing and
packaging techniques to help ensure the safety and wholesomeness of the food
supply. The quality and safety of food depends on the efforts of everyone
involved in the comlpex chain of agriculture production, processing, transport,
food production and consumption. As the EU and World Health Organisation
(WHO) formulated it food safety is a shared responsibility from farm to fork.
Maintaining the quality and safety of food throughout the food chain requires
both operating procedures to ensure the wholesomeness of food and monitoring
procedures to ensure operations are carried out as intended.

FURTHER READING
1. The Impact of Food Safety and Agricultural Health Standards on
Developing Country Exports, Summary of Report No 31302, Poverty
Reduction and Economic Management Trade Unit, and Agriculture and
Rural Development Department, World Bank, 2005. 13 pages.
2. http://europa.eu/scadplus/prinversion/en/s84000.html
3. http://europa.eu/scadplus/prinversion/en/lvb/f84006.html
4. http://europa.eu/scadplus/printversion/en/lvb/f84003.html
5. http://europa.eu/scadplus/printversion/en/lvb/f84001.html
54

6. http://europa.eu/scadplus/printversion/en/lvb/f84002.html
7. http://europa.eu/scadplus/printversion/en/lvb/f84004.html
8. White Paper on Food Safety, Commission of the European Communities,
Brussels, 2002.
9. Regulation (EC) No 178/2002 of the European Parliament and of the
Council of 28 January 2002 laying down the general principles and
requirements of food law, establishing the European Food Safety
Authority and laying down procedures of food safety, OJ L.
10. www.agri.vet.ee
11. Saron, T. The Impact of Technical Measures to Estonian meat and dairy
industry, MBA thesis, 2002, Tartu.

55

AIRBORNE BIOCONTAMINATION IN CLEANROOMS

AND CONTROLLED ENVIRONMENTS
Bengt Ljungqvist & Berit Reinmller
Building Services Engineering, KTH, Stockholm, Sweden
In cleanrooms the main source of biocontamination is people. The concentration
of airborne biocontamination depends upon the number of people present, their
level of activity, and the clothing system used. In controlled areas the supply air
may be of the same cleanliness as in the cleanroom but processes and people are
not controlled in the same rigid way. Microbiological methods used for
monitoring air in controlled areas e.g., during operational or dynamic state
should be able to measure both higher and lower concentrations of airborne
microorganisms. There are several methods of measuring the airborne
contamination and many published reports show that the results as a number of
colony forming units per cubic meter (CFU/m3) depend upon the equipment
used. The difference in results between microbiological air samplers often
depend upon physical parameters of the samplers. These parameters, together
with d50-value (cutoff size) are discussed. The d50-value is the aerodynamic
particle diameter where 50% of the particles are collected in the sampler and
50% are not collected. In order to evaluate the collection efficiency of impaction
samplers, a simplified mathematical model will be presented and examples given.

INTRODUCTION
In cleanrooms the main source of biocontamination is people. The concentration
of airborne biocontamination depends upon the number of people present, their
level of activity, and the clothing system used. Usually, the processes carried out
in cleanrooms and clean zones are well controlled and do not contribute to the
airborne biocontamination in the areas. The microbiological monitoring methods are
challenged by the task to measure low concentrations of airborne biocontamination.
In controlled areas the supply air may be of the same cleanliness as in the critical
areas but processes and people are not controlled in the same rigid way.
Microbiological methods used for monitoring air in controlled areas e.g., during

56

operational or dynamic state should be able to measure both higher and lower
concentrations of airborne biocontamination
Monitoring of airborne viable particles can be considered as a specific form of
aerosol measurement. The term aerosol means an assembly of liquid or solid
particles in a gaseous medium (e.g., air) stabile enough to enable observation
and measurement. Generally, the size of aerosol particles is in the range 0.001
100 micron. The measuring devices collect particles from the air and give the
collected viable microorganisms a possibility to multiply and be detected as
Colony Forming Units (CFUs).
Particle size, shape and density determine the behavior of particles in air. A
commonly used term in aerosol science and technology is the aerodynamic
particle diameter, which is the diameter of a unit-density sphere (1 g/cm3) having
the same settling velocity as the irregularly shaped particle being studied. The
particle diameter is in the literature also called equivalent diameter. Reference to
the aerodynamic diameter is useful for describing settling and inertial behavior.
Large particles e.g., skin flakes, might have an inertial behavior similar to that of
a particle with smaller equivalent diameter. The motion of a particle is of
concern for impaction devices (e.g., slit-to-agar samplers, sieve-samplers,
centrifugal samplers) and for settling plates.

SAMPLING EFFICIENCY
PHYSICAL EFFICIENCY
The physical sampling efficiency of an aerosol sampler is influenced by inlet or
extraction efficiency and by separation efficiency.

Inlet or extraction efficiency is a function of the inlet design of the

sampler and its ability to collect particles from the air in a representative
way and transport the particles to the impaction nozzle or the filter.
Separation efficiency is the ability of the sampling device to separate
and collect particles of different sizes from the air stream by impaction
onto the collection medium or into the filter medium.

The physical sampling efficiency is the same whether the particles consist of
single microorganisms, carry microorganisms, or are nonviable (inanimate). The
57

physical sampling efficiency is based on physical characteristics of the sampling

device such as airflow, orifice shape, and orifice size. The d50 (cutoff size)
describes the aerodynamic equivalent particle diameter removed by 50% from
the air stream and impacted. The d50 can be calculated as follows:

d 50 =
where

Dh
Stk50

U
C

9 D h Stk50
U C

(1)

= Viscosity of air (Pa s)

= Hydralic diameter of the air inlet nozzle (m)
= Stokes number that gives 50% collection efficiency
(non-dimensional)
= Particle density (kg/m3)
= Impact velocity (m/s)
= Cunningham correction factor used for particles smaller
than 1 m (non-dimensional).

Impactor collection data is usually given in terms of an aerodynamic d50 ( = 1

g/cm3 = 1000 kg/m3) and the results of impactor measurements expressed in
terms of aerodynamic diameter. The Cunningham correction factor could for
particle sizes discussed here mostly is chosen to 1. For smaller particles need
more accurate estimations.
It could be mentioned that such a correction for particles with diameters of 1 m
and 0.5 m a reduction will occur with 8% and 14% respectively. The Stk50
number is often chosen to 0.24 to 0.25 for inlet nozzles. Most impaction
sampling devices have sharp cutoff characteristics, meaning that almost all
particles larger than that of d50 are collected. However, it is not yet common for
manufacturers of microbiological samplers to present the d50-value of their
equipment. Equation (1) can be simplified by using constant factors for air
viscosity, particle density, and the Cunningham correction factor. The
expression for d50 expressed in m will approximately become

d 50 =
where

Dh
U

40 Dh
U
=
=

(2)

Hydraulic diameter of the inlet nozzle (mm)

Impaction velocity (m/s).
58

For a round opening the hydraulic diameter Dh is the hole diameter. For a
rectangular long slit (length much larger than the width) the hydraulic diameter
will approximately be twice the slit width.
EXAMPLE 1
Calculate the d50-value for an impaction sampler (Sieve Sampler) with a
sampling air volume flow of 100 liters per minute and a lid with 200 holes of a
diameter of 1 mm. The ratio between the airflow and the total area of the holes
gives the impaction velocity to 10.6 m/s. With the aid of Equation (2) the
d50-value will be estimated to 1.94 m.

d 50 =

40 1
1.94
10.6

EXAMPLE 2
Calculate the d50-value for an impaction sampler (Slit Sampler) with a sampling
air volume flow of 50 liters per minute and a rectangular inlet slit 1 mm wide
and 25 mm long. The ratio between the airflow and the total area of the slit gives
the impaction velocity to 33.3 m/s. With the aid of Equation (2) the d50-value will
be estimated to 1.55 m.

d 50 =

40 2
1.55
33.3

EXAMPLE 3
Calculate the d50-value for an impaction sampler (Sieve Sampler) with a
sampling air volume flow of 100 liters per minute and a lid with 12 holes of a
diameter of 10 mm. The ratio between the airflow and the total area of the holes
gives the impaction velocity to 1.8 m/s. With the aid of Equation (2) the d50-value
will be estimated to 14.9 m.

d 50 =

40 10
14.9
1 .8
59

Information of the d50-value is an important factor when selecting the

appropriate equipment for cleanrooms, clean zones and controlled environments.
However, the user should also be aware that in a controlled environment with
cleanroom-dressed operators as main contamination source, the aerodynamic
equivalent particle size of viable particles usually is smaller than e.g., in
operation rooms. A study by Ljungqvist and Reinmller (Cleanroom Clothing
Systems, People as a Contamination Source. Bethesda, MD, PDA. River
Growth, IL, DHI Publishing LLC, 2004) of the generation of viable particles
from cleanroom dressed operators reported the viable particle size distribution
according to results the Andersen 6-stage Sampler (cascade sampler). The
results shown as percentage of airborne CFU separated by the Andersen 6-stage
Sampler are illustrated in Figure 1. The figure shows that approximately one
third of the viable aerobic particles recovered by the Andersen 6-stage Sampler
have an aerodynamic diameter below 2.1 m.

Percent %

60

40

20

0
0.65 - 2.1

2.1 - 4.7

Particle size

>4.7

Figure 1. Aerodynamic particle size distribution in percent of airborne aerobic

CFU, measured with an Andersen 6-stage Sampler (cascade sampler), during
evaluation studies of operators dressed in new modern cleanroom clothing
systems (Ljungqvist and Reinmller, 2004).

60

BIOLOGICAL EFFICIENCY
The biological sampling efficiency, mostly below the physical sampling
efficiency, is the ability to maintain the viability of the microorganisms during
separation and collection in combination with the ability of the collection
medium to support growth.
Guidance on the evaluation of biological efficiency is presented in the ISO
14698-1 (ISO 14698-1. 2003. Cleanroom and associated controlled environments
Biocontamination control Part 1: General Principles and methods. Geneva:
International Organization for Standardization) in the informative annex B. The
method described is based on a method by Clark, Lach and Lidwell (The
performance of the Biotest RCS centrifugal sampler. J. Hosp. Infect., 1981, 2,
181186) and can not be carried out in common or general microbiological
laboratory. The test should preferably be performed in an independent test
laboratory specialized for this kind of tests. The results of the tests are expected
to be provided by the manufacturer of the air sampler.
The method makes use of airborne particles of different sizes containing spores
of Bacillus subtilis var. niger NCTC 10073 which survives the sampling
conditions. To obtain the concentration of spores in the test chamber a
membrane filter method is used. The concentration obtained from the tested
sampler is compared to the concentration from the membrane filter method at
five particle sizes in the range between 0.8 and 15 m. For each test at least 10
experiments should be carried out. The efficiency of the tested sampler is
calculated using the following equation:
Efficiency of sampler (%) =

test sampler count

100
total count ( from membrane sampler )

(3)

Measuring the biological efficiency with microorganisms typically found in the

cleanroom is suggested as a better method by Whyte (Collection efficiency of
microbial methods used to monitor cleanrooms. Eur. J. Parent. Pharmaceut. Sci.,
2005, 10:2, 4350). Whyte also points out the importance of testing the air
sampler including the tube extensions if tube extinctions are used.

61

AIR SAMPLING
There are three main methods for collecting particles that are used for
microbiological air monitoring: Impaction, filtration and sedimentation.
Impaction and filtration are considered active sampling techniques and require
collection of a known volume of air. Sedimentation is the passive collection of
particles by fall out or settling into an open Petri dish with suitable medium.
The purpose of active air sampling procedure is to collect particles at a
representative location, separate the particles from the sampled air, and trap the
particles onto a suitable medium without affecting the viability of the
microorganisms, and without altering the air flow pattern in the sampling region.
The selection of the most appropriate sampling device for a particular
application depends upon the following factors:

Physical characteristics of the sampling equipment

The type of viable particle to be sampled (single spores or cells that are
carried by non-viable particles)
The equivalent size of particles to be collected
The sensitivity of the viable particles to the sampling procedure
The expected concentration of CFUs in the environments
The ability to detect low concentrations of CFUs in a reliable way
The time and duration of the sampling
The sampling location.

ISO 14698-1 considers air samplers that collect viable particles by direct
impact of particles on nutrient media and filtration samplers that collect particles
on special filters suitable for active sampling in clean zones with low
biocontamination.
The impaction velocity should be high enough to separate particles down to
approximately 1 micron and low enough to avoid mechanical damage to the
cells. For cleanroom applications, 1 cubic meter should be sampled in a
reasonable time without drying the collection medium.

62

SUMMARY
To interpret the results from viable air sampling, the user should understand the
dynamics of sampling and collection of particles on the collection medium.
Results of 0 CFU per cubic meter in manned cleanrooms could indicate that the
sampling process, sampling location or the collection media, incubation
temperature and time have not been optimized.
It is important to be aware of the limitations of each sampling method. Results
from one sampling method must not be compared with results obtained by
another method without careful investigation. To improve the evaluation of
controlled environments based on achieved results, the air sampler used has to
be specified. An air sampler should be selected based upon a thorough
evaluation of the characteristics of the sampler, the sampling conditions and
sampling requirements.

63

64

PARTICIPANT ABSTRACTS

ANTIMICROBIAL EFFECTS

65

EMERGENCE OF ANTIMICROBIAL RESISTANCE IN

CAMPYLOBACTER JEJUNI ISOLATED FROM
BROILER CHICKENS IN ESTONIA
Mati Roasto, Kadrin Juhkam, Terje Tamme, Ari Hrman, Liidia Hkkinen,
Mari Reinik, Avo Karus & Marja-Liisa Hnninen
Estonian University of Life Sciences, Tartu, Estonia

The development of antimicrobial resistance in Campylobacter jejuni and C. coli

is a matter of increasing concern. Since campylobacteriosis is transmitted to
humans particularly via food of animal origin, the presence of antimicrobial
resistant Campylobacter isolates in broiler chickens has important public health
impacts. The aim of this study was to determine resistance patterns of
Campylobacter spp. isolated from Estonian broiler chickens. The strains were
isolated from faecal samples collected at a large Estonian chicken
slaughterhouse, and from meat samples collected at the retail level in Estonia. A
total of 131 C. jejuni isolates were collected during 13 month period, and tested
by Minimal Inhibitory Concentration (MIC) based broth microdilution method
(VetMICTM). Resistance to one or more antimicrobials was detected in 104
isolates (79.4%). A high proportion of the isolates were found to be resistant to
enrofloxacin (96/131, 73.3%) and nalidixic acid (99/131, 75.6%). Multidrug
resistance (to three or more unrelated antimicrobials) was detected in 36 (27.5%)
isolates and all of those were resistant to enrofloxacin. Our results showed that
multidrug resistance was significantly associated with enrofloxacin resistance
(p < 0.01) and the use of enrofloxacin may select multiresistant strains.

66

INHIBITION OF PSEUDOMONAS FLUORESCENS BY

ALGINATE FILMS INCORPORATING
LACTOPEROXIDASE SYSTEM
Fatih Y.G. Yener, Figen Korel & Ahmet Yemeniciolu
Izmir Institute of Technology, Urla, Izmir, Turkey

Growth of microorganisms on food surfaces is a major cause of food spoilage.

Especially, microbial contamination of ready-to-eat products is of serious
concern to consumer health. Since microbial growth in foods occurs mainly at
surface, use of antimicrobial sprays and dips could inhibit growth for a while.
Edible films having antimicrobial substances could be a possibility to provide
microbial stability for foods if antimicrobial substances in edible films are
slowly released to food surface and remain at high concentrations at surface for
longer time. Currently, chemicals such as organic acids, inorganic acids, or
alcohols, are incorporated into edible films. Health concerns of consumers
related to chemical agents caused an increasing demand to use of natural
biopreservatives such as antimicrobial enzymes and bacteriocins in antimicrobial
packaging. Lactoperoxidase system has been found to exhibit antimicrobial
properties and its use has been suggested as a preservative in foods.
Lactoperoxide system, with lactoperoxidase, thiocyanate and H2O2, is an
inhibitory system naturally found in milk. This system inhibits many of the
pathogenic and spoilage microorganisms including Pseudomonas fluorescens,
Listeria monocytogenes and Escherichia coli. The objective of this research was
to assess antimicrobial activity of alginate films incorporated with
lactoperoxidase system against P. fluorescens at different concentrations of H2O2
and thiocyanate. Alginate films were prepared by using 2% (w/v) alginic acid
solution and addition of 0.020.03 mg lactoperoxidase per g of alginic acid
solution. Film solution was spread onto glass Petri dishes and dried at room
temperature for 3 d. After drying, 0.3 M CaCl2 was pipetted into plates to crosslink the dried films. Discs (1.3 cm), which were cut aseptically from dried films,
were used in antimicrobial activity tests. Sterile nutrient broth (3 ml) was
inoculated with P. luorescens culture (0.5 ml), grown overnight at 26 C.
Thiocyanate (1 ml, 4 mM) and H2O2 (0.1 ml, 0.2, 0.4, 0.8 mM) were also added

67

to the medium and the test tubes were incubated at 26 C/24 h. To observe the effect
of antimicrobial system at different conditions, varying thiocyanate (14 mM) or
H2O2 (0.20.8 mM) concentrations were applied in different sets of experiments.
P. fluorescens counts were monitored at 0, 6 and 24 h using Nutrient agar and
plates were incubated at 26 C/48 h. The results of the experiments showed that
at constant H2O2 concentrations an increase in thiocyanate concentration did not
correlated well with growth inhibitory effect. In contrast, at constant thiocyanate
concentration, increase of H2O2 concentration increased the inhibitory effect on
P. fluorescens. In the first 6 h of the incubation, at constant thiocyanate
concentrations levels the P. fluorescens inhibition changed between 0.5 and 1.7
log reduction. After 24 h of incubation, counts were increased by 3.05.0 log
compared to 0 h. At extended periods of incubation of reaction mixtures,
inhibitory effect of lactoperoxidase was generally ended due to H2O2 and
reactive thiocyanate products exhaustion. It is likely that at the studied
concentrations, H2O2 component of lactoperoxidase system is a critical limiting
factor for the obtained antimicrobial effect. These results revealed that
lactoperoxidase system could have a potential to be incorporated into alginate
films for several food applications. However, further studies are needed to
increase antimicrobial activity for longer time.

68

ANTIMICROBIAL EFFECTS OF CHLORINATED AND

OZONATED WATER FOR REDUCING
MICROBIOLOGICAL CONTAMINATION DURING
BROILER PROCESSING
C. Dokuzlu & Metin Guldas
Karacabey Vocational School, Uludag University, Bursa, Turkey

In this study, effectiveness of ozonated (1.5 mg/l) and chlorinated (30 mg/l)
washing water as antimicrobial agent for killing some harmful microorganisms
on broiler carcass, equipment surfaces and personnel hands at pre-determined
control points were investigated. On broiler carcass the total aerobic mesophilic
bacteria (TAB), Escherichia coli, Staphylococcus and Micrococcus spp. and
Salmonella spp. were analyzed at fourteen different control points. On
equipment surfaces the occurrence of the same bacteria except for Salmonella
spp. were analysed at forty-two different control points. The hands of fifteen
persons from three different production sections were also examined.
Furthermore, TAB, E. coli and yeast/mould as air contamination source were
enumerated in the rooms for hanging living broilers, for evisceration and for
portioning and packaging. All microbiological analyses were performed using
four replicates. In the broiler carcass treated with ozone and chlorine; the TAB
counts were reduced by 81% and 61%, E. coli by 98% and 56% and
Staphylococcus and Micrococcus spp. by 85% and 60%, respectively. No viable
cells of Salmonella were recovered in the samples treated with ozone and
chlorine after washing. The following microbial counts were determined on the
equipment surfaces in evisceration, chilling and portioning/ packaging during
processing: TAB 414482 colony forming units (cfu)/cm2, Staphylococcus and
Micrococcus spp. 2888 cfu/cm2 and E. coli 5134 cfu/cm2. The microbial loads
on personnel hands from evisceration, portioning and grading/packaging
sections were determined 175-306 cfu/cm2 for TAB, <10 cfu/cm2 for E. coli and
1580 cfu/cm2 for Staphylococcus and Micrococcus spp. TAB, E. coli and
yeast/mould as of air contamination indicator were determined 800, 588 and <10
cfu/plate on alive chicken hanged, while the same microorganisms were
determined 223, 11 and 19 cfu/plate in the broiler eviscerated, respectively.
69

TAB, E. coli and yeast/mould in packaging and portioning were also determined
as 30, <10 and <10 cfu/plate. As a conclusion the raw material was determined
as the primary contamination source based on the results obtained from the
control points examined and the hands of the personnel hands as well as the
equipment surfaces were determined as secondary contamination sources. Our
study demonstrated that ozonated water was very effective in reducing the
populations of TAB, E. coli, Staphylococcus and Micrococcus spp. and
Salmonella on chicken. It was also a good measure in preventing crosscontamination of process environments.

70

DETECTION OF LISTERIA MONOCYTOGENES

IN GROUND TURKEY BY IMMUNOMAGNETIC
SEPARATION AND DETERMINATION OF THE
ANTIBIOTIC SUSCEPTIBILITY
Naim Deniz Ayaz & rfan Erol
Faculty of Veterinary Medicine, Ankara University, Ankara, Turkey

The objective of this study was to determine the incidence of Listeria

monocytogenes in packaged ground turkey from different producers sold in
Ankara and to detect the antibiotic susceptibility in isolates obtained.
Immunomagnetic separation (IMS) was used for the isolation of L. onocytogenes
from ground turkey samples and disc diffusion method was used to determine
the antibiotic susceptibility of the isolates. In the present study, a total of 180
ground turkey samples were analysed during 1 year period (15 samples each
month) and in 32 (17.7%) of the samples tested L. monocytogenes were detected.
For the incidence of L. monocytogenes seasonal differences was not significant.
Twenty seven (87.37%) of the 32 isolates were resistant to at least one antibiotic,
21 (65.62%) of them were resistant to more than one antibiotic. It was
determined that 22 (68.75%) isolates were resistant to ampicillin and 26
(81.25%) to penicillin G. In addition to these, 13 isolates were intermediately
resistant to erythromycin and 6 isolates to streptomycin. In conclusion, the
incidence of L. monocytogenes in raw ground turkey was found to be high and
most of the isolates were resistant to widely used antibiotics. Based on the
results, the ground turkey should be produced under suitable hygienic and
technological conditions for the prevention of public health. Also, the use of
antimicrobials as prophylactic or growth promoter agent should be controlled
systematically by the governmental authorities.

71

ANTAGONISTIC ACTIVITY OF LACTOBACILLUS

PLANTARUM STRAINS AGAINST
ENTEROPATHOGENS
Pirje Htt, Epp Songisepp* & Marika Mikelsaar
Department of Microbiology, Tartu University, Tartu, Estonia
*Bio-Competence Centre of Healthy Dairy Products, Tartu, Estonia

The foodborne illnesses are the most widespread health problems. The most
common bacteria that cause foodborne infections are Campylobacter,
Salmonella and Listeria spp. There are studies showing inhibition of these
enteric pathogens by human origin indigenous lactobacilli and probiotic
Lactobacillus strains. The aim was to investigate the antagonistic activity of
eight Lactobacillus plantarum strains and one commercial probiotic strain
against different enteropathogens. The antagonistic activity of 8 human
L. plantarum strains and one commercial probiotic strain (L. plantarum 299v)
was assessed against Listeria monocytogenes ATCC 13932, L. monocytogenes
ATCC 51774, Salmonella enterica subsp. enterica ATCC 13076, and 3
reference strains of Campylobacter jejuni. The antagonistic activity of
lactobacilli against selected bacteria was assessed using a streak line method.
The media used were: modified MRS medium (MRS medium without
triammonium-citrate and sodium-acetate; pH 7.2) for testing Listeria and
Salmonella, Columbia Agar Base supplemented with 7% horse blood and 1%
Vitox for C. jejuni. Lactobacilli were seeded on agar plates and incubated under
microaerobic environment at 37 C for 48 h; the growth was inactivated with
chloroform gas for 2 h. The following target bacteria L. monocytogenes and
S. enterica grown in peptone water for 18 h (turbidity 109 cfu/ml), C. jejuni
suspension in Brucella broth adjusted to McFarland density of 34. The target
bacteria were seeded in duplicate perpendicular to the streak line of lactobacilli
on the respective media. The plates cultivated with L. monocytogenes and S.
enterica were incubated in aerobic environment at 37 C for 18 h. C. jejuni was
grown in micro-aerobic environment at 37 C for 48 h. The antagonistic activity
of lactobacilli against pathogens was measured in millimetres the width of the
inhibition zone extending from the culture line of lactobacilli. L. plantarum 299v

72

and 4/8 putative probiotic L. plantarum strains expressed strong inhibition (mean
24.9 versus 19.2 mm; p<0.001) against Listeria and Salmonella strains. The
antagonistic activity of all tested lactobacilli against C. jejuni was low (mean 3.5
to 14.0 mm). The inhibition activity of the four stronger putative probiotic L.
plantarum stains was similar to the commercial L. plantarum 299v ones. The
inhibition of lactobacilli is strain specific. Due to the antagonistic ability against
enteric pathogens the four tested L. plantarum strains could be applied as
promising probiotic candidates to avoid food-borne illnesses.

73

THE IMPROVEMENT OF CHEESE QUALITY WITH

HUMAN LACTOBACILLUS PLANTARUM ADDITIVE
Epp Songisepp, Merle Rtsep, Janne ksti*, **, Kersti Zilmer*, **,
Pirje Htt* & Marika Mikelsaar*
Bio-Competence Centre of Healthy Dairy Products, Tartu, Estonia
*Department of Microbiology, University of Tartu, Estonia
**Department of Biochemistry, University of Tartu, Estonia

Secondary flora putatively contaminating the cheese milk plays an important

role in the cheese quality. However, contaminating bacteria, entered during
manufacture or from milk can cause pasty texture and off-flavors in cheese and
could be detrimental to human health. Aim of the present study was to find the
best antagonistic adjunct starter from human Lactobacillus plantarum strains for
the semi-hard Edam-type Estonian cheese in order to develop a cheese with
evaluated bioquality. Two L. plantarum strains were selected due to their
relatively good conjugated linoleic acid (CLA) production from linoleic acid in
different media, strong antagonistic activity in vitro against various
enteropathogens (Listeria monocytogenes, Salmonella enteritidis, Shigella
sonnei) and cheese non-starter microflora. Three batches of Estonian cheese (1.2
tons each) were prepared: a regular cheese (control) and two experimental
probiotic cheeses containing human Lactobacillus sp additive. The suspension of
lactobacilli (inoculation rate 107 CFU/g of cheese) was added together with the
cheese starter into the pasteurized cow milk before rennet coagulation. Cheese
microflora was seeded in decimal dilutions on different culture media. The
antimicrobial activity of prepared cheeses against various enteropathogens was
measured. The CLA content of cheese was determined by the modified method
of Ahotupa et al. (1998). The survival of both added L. plantarum strains in
cheese was excellent (9 x 108 and 2 x 108 CFU/g cheese). Both prototype cheeses
were able to suppress non-starter microflora other than lactobacilli. The
antimicrobial activity of prepared prototype cheese with strain no 1 remained
almost at the same level in comparison with the data of the pure culture in vitro.
The inhibition of both Yersinia enterocolitica and L. monocytogenes remained
high even at the storage temperature of the cheese at 4 oC. The experimental

74

cheese with strain no 2 revealed lower antimicrobial activity than the

incorporated strain in vitro, though significantly higher than the control cheese
without probiotic L. plantarum additive. The CLA content in experimental
cheeses ranged from 1.5 to 3.0 mg per 1 g of cheese. The prototype cheese no 1
contained two times more CLA than the cheese with the strain no 2 and the
control cheese. As a conclusion it can be stated that it is possible to control
cheese secondary flora by selecting antagonistic L. plantarum strains of human
origin. Additionally, it may support potential protection against enteric
pathogens. Besides the increased the bioquality of cheese, the higher CLA
content produced by the L. plantarum additive could also contribute to the
antimicrobial potential of cheese.

75

ANTIBIOTIC SUSCEPTIBILITY PATTERN OF

SALMONELLA SPP., LISTERIA MONOCYTOGENES
AND CLOSTRIDIUM PERFRINGENS ISOLATES
FROM TURKEY MEAT
Fatma Seda Ormanci
Ankara University Faculty of Veterinary Medicine, Ankara, Turkey

The objective of this study was to determine the antibiotic susceptibility profile
of the Salmonella spp., Listeria monocytogenes and Clostridium perfringens
isolated from brisket, leg and minced turkey meat samples obtained from 3
different producers marketed in Ankara, from June 2005 to January 2006. In this
study 55 Salmonella spp., 24 L. monocytogenes and 78 C. perfringens isolates
from 180 turkey meat samples were used for antibiotic susceptibility by disc
diffusion technique (NCCLS-National Committee for Clinical Laboratory
Standards). As a result Salmonella spp. isolates were most often found to be
resistant to tetracycline (50.9%), nalidixic acid (41.8%), streptomycin and
ampicillin (40.0%) and gentamicin (5.45%). Intermediate sensitivity was found
in 48 isolates to kanamycin. Multiple antibiotic resistance were observed as
followed: 5 isolates were resistant to 2 antibiotics, 17 isolates to 3 antibiotics and
6 isolates to 4 antibiotics. Twenty isolates of L. monocytogenes showed
resistance against penicillin (83.3%) and 18 isolates to ampicillin (75%). Nine
isolates were found intermediate sensitive to erythromycin (37.5%) and 8 isolates
to streptomycin (33.3%). All of the L. monocytogenes isolates were found
susceptible to chloramphenicol, gentamicin, tetracycline and vancomycin. In
addition to that 17 isolates were multiple resistant to 2 antibiotics. C. perfringens
isolates were most often found to be resistant to trimethoprim (100%) and
gentamicin (85.8%). Relatively lesser resistance was observed against
tetracycline (10.2%) and ciprofloxacin (6.4%). Three isolates were intermediate
sensitive to gentamicin (3.8%) and 24 isolates to tetracycline (30.7%). Multiple
antibiotic resistance were observed as followed: 58 isolates were resistant to 2
antibiotics and 11 isolates to 3 antibiotics. In conclusion, public health risk of
antimicrobial resistance in bacteria as a result of the use of antibiotics in animals
for prophylactic and growth promoter agent. Antibiotic resistance associated with
76

animal origin food has been a global concern. Therefore the Turkish regulation
which is in compliance with the EU prohibition regulation for the use of
antibiotics in animals for prophylactic and growth promoter agent should apply
in national area.

77

CONJUGATIONAL TRANSFER OF ANTIBIOTIC

RESISTANCES IN SALMONELLA TYPHIMURIUM
Helena Hradecka & Ivan Rychlik
Veterinary Research Institute, Brno, Czech Republic
Salmonella enterica serovar Typhimurium is an important pathogen with a
numerous incidences of strains resistant to more than one antimicrobial agent.
The genes responsible for multiresistance are mostly located on chromosome as
a part of Salmonella Genomic Island 1 (SGI1) and code for typical pentadrugresistant phenotype ACSSuT (ampicillin, chloramphenicol, streptomycin,
sulphonamides and tetracycline). Alternatively, antibiotic resistance genes can
be located on high molecular weight plasmids, so-called R-plasmids. The aim of
this study was to analyse mobility of antibiotic resistances in a collection of 24
multidrug-resistant S. Typhimurium strains with a special interest on resistance
markers transferable by conjugation. Plasmid-free recipient S. Typhimurium F98
strain and resulting transconjugants were analyzed by pulsed-field gel
electrophoresis (PFGE). R-plasmids that were responsible for the transfer of
resistance phenotype were determined by comparing plasmid profiles of donor
strains with the transconjugants. The conjugational transfer of antibiotic
resistance markers was observed in 12 strains. Transfer of complete phenotype
was predominant and transfer frequencies were around the value 10-3. In 4
strains (115/0, B71, F8025 and F8475) a transfer of class 1 integrons was
recorded. By DNA sequencing, the same type of integron was identified in 2
strains (115/0 and B71). This integron contained aadA1 and sul1 gene cassettes
and was localized on a plasmid of 190 kb in size. The antibiotic resistance in
F8025 strain was encoded by dfrA1, aadA1 and sul1 gene cassettes that were
localized in integron on a large plasmid of about 230 kb in size. In the last strain
F8475, a plasmid of about 210 kb was able to conjugate and carried the integron
with dfrA12, orfF and aadA2 gene cassettes. In 2 strains (9046 and 9134) an
incomplete transfer of antibiotic resistant phenotype was recorded. This
happened in approx. 10% of transconjugants and tetA and blaTEM genes coding
for tetracycline and ampicillin resistance were the least stable during the
conjugational transfer. By PFGE, we found that the loss of tetracycline
resistance and both tetracycline and ampicillin resistance correlated with the loss
approx. 10 kb and 25 kb of plasmid DNA, respectively.
78

PARTICIPANT ABSTRACTS

HYGIENE & QUALITY

79

DIFFERENTIATION OF STAPHYLOCOCCUS
EPIDERMIDIS ISOLATES FROM RAW MATERIAL AND
FOODSTUFFS AND FOOD CONTACT SURFACES
BASED ON THE ABILITY TO FORM BIOFILM
Sylva Karpkov
Veterinary Research Institute, Brno, Czech Republic

The aim of this study was to differentiate isolates of Staphylococcus epidermidis

from raw material and foodstuffs and food contact surfaces by using phenotypic
and genotypic methods detecting the ability of the strains to form biofilm. From
2000 to 2006, 197 S. epidermidis isolates were obtained from swabs of contact
surfaces on the equipment of food processing plants, raw materials and
foodstuffs produced from meat and milk. Capability of this microorganism to
form a biofilm was investigated by phenotype and multiplex PCR method.
Resistance of selected isolates to antimicrobial agents was investigated using the
micro dilution method. Staphylococcus epidermidis isolates were incubated in
brain heart infusion at 35 C for 72 h in glass tubes. Then the tubes were rinsed
with peptone water and suspect biofilm was dried turning the tubes upside down
and coloured by 0.1% safranin solution. The intensity of red colour biofilm
structure was rated visually. Total genomic DNA was obtained from a bacterial
culture, in a brain heart infusion for 24 h. The bacterial suspension was
centrifuged and the pellet washed in 500 l of sterile distilled water. The
suspension was then incubated at 80 C / 20 min and finally centrifuged at 1400
rpm for 1 min. The primers for the detection of ica genes (amplicon 546 bp),
primers UNB applied as internal control (370 bp) and primers SE705 specific for
S. epidermidis (124 bp) were used for multiplex PCR method. The amplification
mode was 96 C for 2 min, (96 C for 10 s, 55 C for 10 s, 72 C / 40 s) for 45
cycles with an extension of 72 C for 2 min. Amplification products were
separated by electrophoresis in 2% ethidium bromide-stained agarose gel. A
total of 197 S. epidermidis isolates were obtained from raw food materials, milk
and meat products and from swabs of equipment used in meat and milk
processing plants. The production of biofilm was detected in 12 isolates (6%).
Most of them originated from raw milk (4) and milk products (4). The ica
80

operon was detected in 9 isolates and the phenotypical ability to produce biofilm
was determined in 10 isolates. Different results between the phenotypical and
genotypical method were detected in 5 isolates). Resistance to antimicrobial
agents was observed in 41 isolates of S. epidermidis. The most common
resistance was to penicillin. Multiresistant isolates (resistance to 3 and more
agents) were detected in 5 cases; one of them was resistant to 8 antimicrobial
agents (penicillin, oxacillin, ampicillin-sulbactam, tetracycline, trimethoprimsulfamethoxazole, erythromycin, clindamycin, and gentamicin). We found
virulent strains of S. epidermidis with the ability to form biofilm in many food
processing environments. It is possible for the microorganisms released from
biofilm to carry resistance genes and could represent potential source of food
contamination. This may lead to food spoilage or to the transmission of diseases.

81

MICROBIOLOGICAL QUALITY OF TURKISH CHEESES

MANUFACTURED FROM PASTEURIZED MILK
PRODUCED IN COMPANIES HAVING THE
HACCP SYSTEM
Esra Agel
Tbitak Marmara Research Center, Food Institute, Kocaeli, Turkey

In Turkey approximately 45 million tons (40%) of raw milk are used for cheese
production. The production rate of white cheese is 13%, kasar cheese is 3%, and
other cheese types are 3% of milk production. The classical white cheese and
kakaval cheese are the first groups among the other cheese types on export.
Milk used in traditional cheese production is not pasteurised in Turkey;
however, pasteurized milk is used for industrial scale production. On that point,
starter culture is not used in white and kakaval cheese producing generally.
There are problems in dairy sector with regard to processing and marketing.
Producing plants having different capacities of production from the small family
plants to modern plants which has European standards and HACCP system, are
showing activity together. Most of cheese production is realised in small scale
dairies and approximately 10% of cheese is manufactured in modern plants. This
research was undertaken to carry out microbiological and chemical analyses in
the 316 white and kakaval cheese samples produced by 16 different large scale
companies having HACCP system in Marmara Region. All cheese samples are
produced with pasteurised milk. In these samples, the microbial counts were
found as follows: coliform bacteria, 4.3 x 102 cfu/g; yeast, 8.2 x 104 cfu/g; E. coli
3.9 x 102 cfu/g. Salmonella and mould were not detected in any of the samples.
Mean moisture, salt content, acidity and pH of white cheese samples were 58.7,
7.4, 1.37 and 4.63; and of kakaval cheese samples 43.5, 4.12, 3.05, 5.30. When
the results were analysed according to the Turkish Food Codex Alimentarius
limits for coliform bacteria, E. coli and yeasts, 46%, 58% and 22% of samples of
the 166 white cheese samples and 4%, 9%, 45% of the 150 kakaval cheese
samples examined were out of standards, respectively.

82

INVESTIGATION OF MOULDS IN SALTED YOGHURTS

(CONCENTRATED YOGHURT)
Il Var, Emine Aksan*, Nuray Sahan & Dilek Say
University of Cukurova, Faculty of Agriculture, Balcal-Adana, Turkey
*University of Mustafa Kemal, Faculty of Agriculture,
Alahan, Antakya-Hatay, Turkey

Salted yoghurt is a traditional milk product produced in Hatay region in Turkey.

The salted yoghurt has unique properties e.g. saltiness and sour aroma. It can be
stored for a year without spoilage and consumed in almost every meal among
other yoghurts. Besides this it contains high dry matter, fat, protein and minerals,
because it is a concentrated product. Furthermore, it is a highly nutritious
product. But, salted yoghurt provides appropriate conditions, e.g. water activity
and acidity, for the moulds to grow. Therefore contamination with mould plays
an important role in spoilage of the salted yoghurt. In this study, the aim was to
determine the type and amount of mould contaminating the production of salted
yoghurt produced of cows and goats milk. We also looked at the effects of
covering it with and without butter and at storage in different temperatures on
the type and amount of mould in the salted yoghurt. Firstly the milk was heated
(100 C, 1 min), cooled, inoculated with yoghurt culture and incubated. After
fermentation, yoghurt was divided into two batches. The first batch was strained,
the other batch was unstrained. The unstrained and strained yoghurts were
transferred into boiling pans and cooked for 110 and 50 min, respectively by
stirring. The salt was added to both batches 10 min before the end of cooking.
The salted yogurt was let to cool down at room temperature and was placed into
plastic (rigidpolycitrine) packages. One part of each batch was covered with
and without butter and stored at different temperatures (room and refrigerated
temperature). After that, the type and amount of mould in the samples were
counted according to Onions et al. (1981) and Samson and Reenen-Hoekstra
(1988). The initial amount of mould in the samples ranged between 1.44 0.16
log cfu/g and 0.46 0.10 log cfu/g. Based on the analysis in the 6th month the
amount of moulds in samples covered with butter and stored in refrigerator
temperature ranged between 2.60 0.12 cfu/g and 4.46 0.11 log cfu/g and in

83

samples covered without butter varied from 1.82 0.05 log cfu/g to 2.74 0.49
log cfu/g. The amount of moulds in to samples covered with butter and stored in
room temperature ranged, when analysed at the 6th month, between 3.26 0.24
cfu/g and 4.32 0.24 log cfu/g and in samples covered without butter in the 8th
week varied from 4.88 0.66 log cfu/g to 5.70 0.20 log cfu/g. The statistical
analysis indicates that straining and type of milk did not statistically affect the
results of mould contamination in the salted yoghurt stored in refrigerator and
room temperature. In addition the amount of mould in the samples stored in
refrigerator temperature on the 6th month were not affected significantly if
covered with or without butter, but there was statistical difference in the mould
amount between samples covered with butter and without butter. Besides that
the storage temperature did not make any statistical difference on the amount of
mould in the salted yoghurt covered with butter, but according to samples stored
at different temperatures there was a statistical difference on the amount of
moulds in samples covered without butter. In this study, Aspergillus sp. and
Penicillum sp., were commonly identified. This study showed that if salted
yoghurt is stored in the refrigerated temperature it need not be covered with
butter. But, if the salted yoghurt is stored in the room temperature, it must be
covered with butter, because otherwise, moulds in the salted yoghurt will cause
losses.

84

EFFECT OF PLUM SAUCE, POMEGRANATE SAUCE

AND LEMON JUICE ON MICROBIOLOGICAL QUALITY
PARAMETERS OF TRADITIONAL RAW MEAT-BALL
(I KFTE)
Il Var & Blent Kabak
School of Agriculture, University of ukurova, Adana, Turkey

Raw meat-ball (i kfte) is consumed in a wide region of Turkey, largely in

eastern and south-eastern Anatolia. There are no official regulations on raw
meat-ball production and therefore the ingredients, quality and quantity of
ingredients differ depending on the consumers preferences. It is traditionally
produced with a mixture of lean veal and bulgur (wheat) in a ratio of 2:1 or
1:1, dried red pepper (mostly hot pepper), salt, onion, black pepper, parsley, and
tomato and/or red pepper sauce. It is prepared as raw with no cooking process at
all. Lately, according to EU regulations, some food products in Turkey are under
scrutiny due to the high microbiological risk they carry. Hence, the traditional
raw meat-ball is one of these food products to be investigated closely. Firstly in
this research, the antibacterial effects of home-made dried plum sauce,
pomegranate sauce and lemon juice on Salmonella Enteritidis, Salmonella
Typhimurium, Escherichia coli, Staphylococcus aureus and Bacillus spp. were
determined in vitro conditions by agar-diffusion method. We found that plum
sauce, pomegranate sauce and lemon juice showed high antibacterial activity
against the tested bacteria. But, a significant reduction in their antibacterial
activity was found when they are used in diluted forms. The antibacterial activity
depends on the concentration of antibacterial agent and bacterial species;
amongst all the bacteria E. coli happens to be the most resistant against all
antibacterial agents tested in this research. In the second phase of our research,
we looked at the effect of plum sauce, pomegranate sauce and lemon juice on
some microbiological quality parameters of raw meat-balls. Salmonella spp. and
coagulase (+) S. aureus were not found in any of raw meat-ball samples.
Furthermore, there was no significant reduction in the population of coliforms
and Escherichia coli. The number of total aerobic mesophilic bacteria in raw
meat-ball samples prepared with plum sauce and lemon juice decreased
85

approximately 1 log cycle when compared to the control sample. It was also
found that pomegranate sauce had little antibacterial effect in raw meat-ball.

86

PARTICIPANT ABSTRACTS

MOLECULAR BIOLOGICAL METHODS

87

PFGE GENOTYPING AND ANTIMICROBIAL

SUSCEPTIBILITY OF CAMPYLOBACTER IN RETAIL
POULTRY MEAT IN ESTONIA
Kristi Praakle*, Mati Roasto, Hannu Korkeala* & Marja-Liisa Hnninen
Estonian University of Life Sciences, Tartu, Estonia
*University of Helsinki, Helsinki, Finland

In the study, the Campylobacter isolates from retail poultry meat in Estonia were
sero- and genotyped, and the antimicrobial susceptibility was determined.
Campylobacter were detected using the NCFA method. The DNA was digested
with SmaI and KpnI (New England Biolabs, Beverly, Mass.) (20 U per sample),
and the restriction fragments were separated with ramped pulses of 1 to 30 s and
1 to 25 s for 19 h, respectively. All Campylobacter isolates were tested by disc
diffusion method against ampicillin (25 g), ciprofloxacin (5 g), erythromycin
(15 g), gentamicin (10 g), nalidixic acid (30 g), and tetracycline (10 g)
(Oxoid), and by the E-test (AB Biodisk, Solna, Sweden) against ampicillin,
ciprofloxacin, erythromycin, and tetracycline. A total of 54 C. jejuni isolates
were serotyped using commercial Campylobacter antisera according to the
manufacturer instructions (Denka Seiken, Tokyo, Japan). Before the serotyping
test the isolates were cultured on Brucella blood agar (Oxoid) plates at 37 C for
48 h in microaerobic conditions. We studied 48 chicken (36 Estonian, 12
imported) and 22 turkey (imported) Campylobacter isolates from 580 raw
broiler chicken (396 Estonian, 184 imported) and 30 turkey (imported) meat
samples. Of the isolates, 64 were C. jejuni, 4 C. coli, and 2 Campylobacter spp.
Penner serotyping of 54 C. jejuni isolates revealed 11 different serotypes, and
22% of the isolates were nontypeable by the commercial antisera. The most
common serotypes O:1,44; O:21, and O:55 accounted for 28%, 13%, and 13%
of the isolates, respectively. Differences in serotype distribution were seen for
chicken and turkey isolates. Genotypic characterization of all Campylobacter
isolates (n = 70) was performed by pulsed-field gel electrophoresis (PFGE).
SmaI and KpnI yielded 29 and 34 PFGE types, respectively, revealing high
diversity among isolates. The serotype distribution did not show an association
with the origin of the sample, but the majority of the isolates sharing a similar

88

PFGE genotype originated from one country. High level of resistance to

ciprofloxacin (66%), nalidixic acid (66%), tetracycline (44%), ampicillin (34%),
and erythromycin (14%) were detected among the 70 Campylobacter isolates.
The simultaneous resistance to two or three antimicrobial agents occurred in
60% of the isolates. The Campylobacter isolates from turkey meat had higher
resistance to ampicillin, ciprofloxacin, nalidixic acid, and tetracycline than those
from chicken meat. None of the chicken isolates were resistant to gentamicin,
and no turkey isolates to erythromycin or gentamicin.

89

IDENTIFICATION OF ENTEROCOCCUS SPP.

BACTERIA ISOLATED FROM FOOD AND OTHER
ENVIRONMENTAL SAMPLES BY PCR METHOD
Veronika Turkov, M. Dukov, . Cupkov,
Renata Karpkov* & Marketa Pospilov*
University of Veterinary and Pharmaceutical Sciences Brno,
Brno, Czech Republic
*National Institute of Public Health Prague, Brno, Czech Republic

The aim of this study, which was supported by the project of Ministry of
Education, Youth and Sports of the Czech Republic No. MSM6215712402
Veterinary aspects of food safety and quality and project NAZV 1B53018, was
the application of the species-specific PCR method based on the detection of
sodA genes encoding enzyme mangan-dependent superoxiddismutas for the
rapid identification of bacteria Enterococcus faecalis and E. faecium. It was
studied 599 samples isolated from cattle farms in south and north Moravia,
human, milk foods as milk, butter and yoghurt. It was identified 247 samples of
Enterococcus spp. by the help of phenotypic characteristics as production of
pyrolidonyl arylamidase (PYRAtest, PlivaLachema, a.s., Brno, CZ) and growth
and appearance of colonies on blood agar (Blood Agar Base No.2, HiMedia; and
sheep blood, Bioveta, a.s., Ivanovice na Han, CZ), S-B agar and Bile Aesculin
agar (Oxoid, Basingstoke, Hampshire, England). To isolate bacterial DNA a 24h culture from the blood agar incubated aerobically at 37 C was used. Bacterial
DNA was isolated by 20% Chelex 100 (Bio-Rad Laboratories, Inc., Hercules,
CA, USA). The supernatant was used as template DNA. The species
identification of bacteria E. faecalis and E. faecium was also performed as
multiplex-PCR when apart from the species-specific sodA genes detection, the
internal control (16S rRNA segment) was also included. A product was detected
by the gel electrophoresis in 2% agarose (Serva Electrophoresis GmbH,
Heidelberg, Germany) with the following visualisation at UV on transilluminator
after the staining by ethidium bromide. Among 247 isolated strains the presence
of the species-specific sodA genes was detected 102 strains E. faecalis, 22
E. faecium, 51 strains were identified as other kinds Enterococcus spp. and 72

90

strains were no specified as Enterococcus spp. The identification of

Enterococccus spp. using conventional methods and biochemical tests based on
their phenotypic characteristics is complicated and time-consuming therefore
was designed as multiplex-PCR for the detection of genus-specific sodA genes
and internal control (16S rRNA). Present of internal control standardizes results
of method and makes it possible to reduce number mistaken reading. Simple and
rapid isolation bacterial DNA by 20% Chelex 100 is adequate for finish reaction.
The above stated PCR method based on detection sodA genes can be acceptable
for the rapid species identification of bacteria E. faecalis and E. faecium isolated
from samples of environmental, cattle, human and milk foods.

91

RESISTOPATHOCHIP TOOL FOR RAPID

MOLECULAR CHARACTERIZATION OF
SALMONELLA STRAINS
Tom Majtn, L. Majtnov*, J. Timko & V. Majtn
Slovak Academy of Sciences, Bratislava, Slovakia
*Slovak Medical University, Bratislava, Slovakia

The goal of our study is the development of DNA microarray-based tool for
rapid molecular characterization and typing of multidrug resistant and virulent
Salmonella isolates. DNA microarray contains short oligonucleotide probes (33
35 mers) designed from conservative domains according to thorough sequence
alignments. The ResistoPathoChip for Salmonella spp. includes probes for
detection of pathogenicity markers localized on the chromosomal (including
Salmonella Pathogenicity Islands PAIs) and plasmid DNA, phage-associated
genes, fimbriae, integrons and various antimicrobial resistance genetic markers.
The chip also contains Salmonella species- and serotype-specific probes and a
range of external controls for chip analysis. The HPLC-purified oligonucleotide
probes were spotted on the surface of Superepoxy slides. The preparation of
target DNA, adjustment of suitable hybridization and post-hybridization
conditions were properly optimized. The ResistoPathoChip was tested on the 24
samples of multidrug resistant clinical Salmonella isolates covering the most
prevalent serotypes in Slovakia (Typhimurium, Enteritidis and Kentucky). The
results of DNA microarray approach were validated only for antimicrobial
resistance markers by PCR as development of multidrug resistant bacteria
especially via horizontal gene transfer (HGT) causes some infections untreatable
now and antimicrobial resistance represents a major health concern. The
majority of PCR results were consistent with the microarray data and also
phenotypic susceptibility testing. Moreover, we made a substantial effort in
localization of monitored genetic markers either they are fixed on chromosome
or localized on transferable elements such as plasmids or integrons. Purified
plasmids without any traces of contaminating chromosomal DNA were
fluorescently labeled and hybridized to the oligochip. We found that genetic
determinant of resistance against cefotaxime (CTX) and ceftriaxone (CRO) as

92

extended-spectrum beta-lactams blaSHV is localized on conjugative plasmid,

which was transfered into E. coli K-12 3110 recipient strain. Microarray analysis
of selected transcojugant reveals transfer of another two resistance genes against
aminoglycosides (aph) and amphenicols (catA) along with blaSHV. The
ResistoPathoChip described here provides comprehensive information about
Salmonella genotype, especially focused on pathogenic and antimicrobial
resistance potential. Such data are necessary in epidemiological studies as well
as for food safety assessment studies with respect to dissemination of
antimicrobial resistance and prevalence of multidrug resistant pathogens.

93

DETECTION OF BACTERIAL TOXINS IN FOODSTUFF

Marketa Pospilov & Renata Karpkov
National Institute of Public Health, Brno, Czech Republic

A wide spectrum of foods is contaminated by low numbers of Staphylococcus

aureus. In case of incorrect storage of foods these bacteria can multiply and
produce enterotoxins. Staphylococcal enterotoxins are heat stable proteins,
resistant to proteolytic enzymes and may cause alimentary intoxications.
Nowadays 18 types of staphylococcal enterotoxins have been described but only
five classical toxins A, B, C, D and E can be detected routinely. Methods using
for the detection of enterotoxins in food are reverse passive latex agglutination
(RPLA) and ELISA. The sensitivity of these methods is 1 ng/g of food without
the use of complicated extraction procedures. ELFA using the automated
detection system VIDAS can detect less than 1 ng/g of food. In this study 321
ready-to-eat foods (meat and milk products, delicatessen and confectionary
products) from retail market were examined for the presence of S. aureus using
the EN ISO 6888-1 (1999) guideline. 72 samples (22%) were contaminated by
S. aureus; the contamination level did not exceed 102 colony forming units
(CFU)/g or CFU/ml. All the S. aureus isolates in our study were screened for the
ability of toxin AE production (using the RPLA test and VIDAS SET 2) and
also for the specific genes seasej encoding the production of enterotoxins
(using multiplex PCR method). Out of 72 isolates 42 (58%) harboured one of
these specific genes. The most frequently found genes were seg and sei in
37 (88%), sec in 11 (26%), seh in 5 (12%), sea in 4 (10%), seb in 3 (7%) and sed
in 2 (5%) isolates. Genes see and sej were not detected. Full agreement was
observed between the detection of enterotoxin genes sea to sed and production
of corresponding proteins in vitro. As a conclusion we recommend the use of the
VIDAS SET 2 method and the PCR method to obtain more objective results in
the food analysis. Strains of S. aureus isolated from foods are the vehicles of
new types of enterotoxin genes more frequently. Some of them have been
described as etiological agents of alimentary intoxications.

94

INCIDENCE OF LISTERIA MONOCYTOGENES IN

RETAIL CHICKEN MEAT AND ESTABLISHING
RELATIONSHIP WITH SOME BACTERIA
BY LOGISTIC REGRESSION
Ziya Gkalp Ceylan, Alper Krsad Demirkaya & Glah anaki
Faculty of Veterinary Sciences, Atatrk University, Erzurum, Turkey

The objective of this study was to determine the contamination of retail poultry
meat in Erzurum (Turkey) by Listeria monocytogenes and to evaluate its
relationship with indicator bacteria using logistic regression. The incidence of
L. monocytogenes was 32.76%, found in 38 of 116 samples. The application of
logistic regression showed that Enterobacteriaceae, Pseudomonas spp., and
B. thermosphacta populations have positive and yeast and mould have negative
relationship with L. monocytogenes presence. The results of this study
demonstrate that there is a serious risk in raw poultry meat for consumer health
in Erzurum, because of the high incidence of L. monocytogenes in the samples of
the present study. Hygienic conditions described in HACCP program should still
be enforced in order to minimize L. monocytogenes in poultry meat during the
processing.

95

96

PARTICIPANT ABSTRACTS

PATHOGENES

97

HYGIENE SURVEY IN ESTONIAN DAIRIES AND

PATHOGEN ANALYSIS OF AUTHENTIC SAMPLES
Helen Ehavald
Tallinn University of Technology, Tallinn, Estonia
(now: AS Coca-Cola HBC Eesti, Tallinn, Estonia)

Hygiene survey was performed in three Estonian dairies. Samples were collected
from process environment, equipment, packaging material. Liquid and solid
samples were taken from raw material, product from the process, final product,
wastewater and water. Samples for detecting Bacillus cereus, Listeria
monocytogenes, mycobacterial count, total bacterial count, Enterobacteriaceae
spp., coliforms, -glucuronidase positive bacteria, yeast and mould were
collected from all dairies and analyzed. Gauze tests, Rida Count and Hygicult
tests were used for detecting bacteria from surfaces. Traditional cultivation
techniques and DryCult test were used for liquid samples. Results were
compared with each other. Equipment samples were both food and non-contact,
results showed that food contact samples were less contaminated than noncontact, therefore dairies pay more attention to cleaning process of food contact
equipment. Two L. monocytogenes positive samples were found from two
dairies, both were from raw milk samples, one L. seeligeri/ivanovii was found
from food-contact equipment sample and 13 mycobacteria were found from all
dairies, two of them were identified with DNA sequence method. One was either
Mycobacterium porcinum or Mycobacterium fortuitum (both risk group 2), it
was found from wastewater. Other was Mycobacterium phlei (risk group 1) and it
was found from final product. One final product was very contaminated with
aerobic bacteria and two raw material samples were contaminated according to
Finnish law. Amounts of clean, contaminated and very contaminated
samples for each dairy were similar. As contamination from non-contact places
(environmental and equipment) may be carried to food contact places and from
there to the process line, dairies should pay more attention to cleaning processes of
these places. Carriers can be for example employees hands. Biofilms are
problematic in the food industry, because they are protective barrier for
microorganisms against sanitizers, and due to it cleaning can be more complicated.

98

INCIDENCE OF LISTERIA MONOCYTOGENES IN

DIFFERENT KIND OF MEAT PRODUCTS ON
LATVIAN RETAIL MARKET
Margarita Terentjeva & A. Brzi
Latvian University of Agriculture, Jelgava, Latvia

The aim of study was to determine incidence and number of Listeria

monocytogenes in a variety of meat products on the Latvian retail market. A
total of 209 samples of sliced, vacuum packed or modified atmosphere packed
(MAP) meat products e.g. cold smoked beef, cold smoked pork, fermented meat,
hot smoked ham, fermented cold smoked and boiled sausage, pt and poultry
were purchased from retail outlets in Latvian supermarkets. Samples were
obtained from 9 meat plants located in Estonia, Latvia and Lithuania. All
speciments were investigated 5 d after or before expiry date according to ISO
11290-1:1996 and ISO 11290-2:1996 with minor modifications (Johansson,
1998) during December 2004 April 2005. L. monocytogenes were isolated
from cold smoked, vacuum packed and sliced beef at range 56% (30/54) and
pork 80% (4/5) samples originated from one Latvian meat producer. One culture
of L.monocytogenes from pt 4% (1/24) , fermented meat (basturma) 20% (1/5)
and cold smoked, vacuum packed and sliced pork sample from another Latvian
meat processing plant 11% (1/9) were isolated. None of hot smoked ham,
fermented cold smoked or boiled sausage and poultry meat samples from
Estonia, Latvia and Lithuania were found positive. Larger proportion of cold
smoked beef, pork samples (32) contains L. monocytogenes at low level (<100
cfu/g), in six samples of cold smoked pork, beef and fermented meat samples
number of bacteria was between 100 cfu/g and 1000 cfu/g. The number of
L. monocytogenes did not exceeded 1000 cfu/g in retail meat samples. As
expected, cold smoked, vacuum packed meat products should be recognized as
high risk products, however prevalence of L. monocytogenes at low level (<100
cfu/g) at the time of consumption seems to be of low risk to the consumers.

99

THE PREVALENCE OF CAMPYLOBACTER SPP. AT

THE FARM AND RETAIL LEVEL
Kadrin Juhkam & Mati Roasto
Estonian University of Life Sciences, Tartu, Estonia

The aim of the study was to investigate the Campylobacter spp. contamination at
the farm and retail level. A total of 102 faecal samples of poultry origin were
collected in 2004. A total of 610 raw poultry meat samples were purchased from
retail outlets in Estonia. Campylobacter spp. was detected using a modification
of the NMKL Method no 119, 2nd ed. 1990, which includes the enrichment in
Preston or in Bolton broth. Enrichment media was plated out on selective
mCCDA (modified Charcoal Cefoperazone Deoxycholate agar, Oxoid). Typical
colonies were streaked on Brucella agar, and verified by the Gram-reaction,
motility-test, oxidase and catalase test, and hippurate hydrolysis. Fifty-two
samples were collected from the farm A and of those 29 (55.8%) were positive.
All samples of farm B origin were negative. Farm A samples were collected at
the beginning of October and samples from farm B were collected at the
beginning of November in order to investigate the presence of seasonal variation.
There was a seasonal variation in flock colonization with Campylobacter spp.
The overall prevalence of Campylobacter spp. in raw poultry products was 12%.
Of the isolates, 77% were identified as C. jejuni. In conclusion, raw poultry
products of Estonian origin are contaminated by Campylobacter species. The
problem appears to be more severe in small-scale operations. The Campylobacter
contamination observed in present study may indicate that the prevalence of
human campylobacteriosis in Estonia is greater than the 124 cases reported by
the Estonian Health Protection Inspectorate. An Estonian Campylobacter control
programme is currently under development, and additional studies of different
foods, at farms and processing plants are needed.

100

DETECTION AND IDENTIFICATION OF

CAMPYLOBACTER POULTRY CARCASSES
Elbieta Makiw & Lucjan Szponar
National Food and Nutrition Institute, Warszawa, Poland

The aim of the study was the consolidation of the contamination levels of poultry
carcasses, procured from the marketplace, with Campylobacter bacteria. In 2005
in the Microbiology Laboratory at National Food and Nutrition Institute prepared
the monitoring plan to determine the levels of the Campylobacter bacteria in
poultry carcasses. The monitoring tests were carried out in selected provinces by
the laboratories of Provincial Sanitary and Epidemiological Stations, pursuant to
the procedure described in the standard PN ISO 10 272:2002 Food and feed
microbiology. Horizontal method for detection of thermotolerant Campylobacter
spp.. The monitoring tests covered 240 poultry carcasses in 2005. The number
of contaminated carcasses ranged from 0% to 100%. The results of monitoring
tests in 2005 with 75.4% of all tested samples contaminated with Campylobacter
spp., are very close to the results obtained in the previous year. The monitoring
tests performed in 2005 allowed to estimate the scale of contamination of poultry
carcasses with Campylobacter spp. in Poland. Unfortunately the obtained result
is much higher than the analogical data collected in most EU countries (2.2
62.2%). Numerous epidemiological data from the Western Europe and the United
States confirm the gravity of the foodborne infections caused by thermotolerant
Campylobacter spp. Unfortunately still the source of contamination remains food
of animal origin, primarily poultry carcasses. Therefore, it is legitimate to
continue monitoring of the contamination rate of poultry carcasses with
Campylobacter bacteria. However, it is not necessary to eliminate the
contaminated poultry carcasses from the market. Special attention should be paid
to educating the society about the ways of Campylobacter infection spreading
e.g. consumption of raw or undercooked meat and/or improper hygienic habits.

101

SURVIVAL OF ESCHERICHIA COLI,

STAPHYLOCOCCUS AUREUS, BACILLUS CEREUS
AND LISTERIA INNOCUA DURING REFRIGERATED
STORAGE OF ALGAM JUICE A TRADITIONAL
FERMENTED TURKISH BEVERAGE
Aye Handan Diner Baysal & ebnem Harsa
zmir Institute of Technology, Faculty of Engineering, Urla, zmir, Turkey

algam juice (turnip juice) is a popular beverage of southern Turkey, originating

from Adana and consumed largely all over the country. Although its Turkish
name algam suyu (or shortened, algam) does literally mean turnip juice, it
is, in fact, a product of lactic fermentation. It has a turbid, dark red or purple
colour and a very strong soar taste. In the production of algam juice pounded
wheat or bulgur flour (setik), yeast, sour dough, salt, black/purple carrot (Daucus
carota), aromatic turnip (Brassica rapa var. rapa) and water are used. There is
no information on the antimicrobial effect of algam juice. The objective of this
study was to investigate survival and growth of Escherichia coli, Staphylococcus
aureus, Bacillus cereus and Listeria innocua during refrigerated storage of
algam juice. algam juice samples were also examined for their pH and
number of lactic acid bacteria (LAB) during refrigerated storage. In this study
E. coli ATCC 8739, S. aureus (turnip juice isolate), B. cereus IAM 6A5 and
L. innocua NRRL B-33314 were used as test strains. Each test strain of E. coli
(6.4 log10 CFU/ml), S. aureus (8.2 log10 CFU/ml), B. cereus (5 log10 CFU/ml)
and L. innocua (7.8 log10 CFU/ml) was inoculated into algam juice obtained
from market and their duplicates were kept at refrigeration temperature ( 4C).
A volume of 1 ml was separately taken from bottles inoculated with the test strain
at 4 h intervals and a volume of 0.1 ml of appropriate dilutions was spread plated
in duplicate on pre-dried surfaces of Violet Red Bile agar for E. coli, Baird Parker
agar for S. aureus, Tryptone Soya agar for B. cereus and L. innocua. Counting
was done after incubation at 32 C for 2448 h. In order to count LAB a volume
of 1 ml of sample was diluted and inoculum from appropriate dilutions was pour
plated with double layer in duplicate on MRS agar. LAB were counted after

102

incubation at 30 C for 2448 h. Results showed that bacteria population

inoculated into algam juice decreased rapidly and many of the bacteria were
inhibited completely after 24 h. However, the population of L. innocua after 24
and 48 h was around 2.6 log10 CFU/g and 2.7 log10 CFU/g, respectively. Survival
could be due to adaptation mechanisms used to overcome the stresses (low pH,
salinity and lactic acid). Because L. innocua may still be viable well after 48 h at
refrigeration temperature, there is a food safety risk involved. Additional studies
about the relationships between the stresses and the changes in Listeria
characteristics and acid adapted cells/pathogens are also needed.

103

ISOLATION AND IDENTIFICATION OF LISTERIA

SPECIES AND LISTERIA MONOCYTOGENES FROM
CHICKEN AND CHARACTERIZATION OF AUTOLYSIS
OF LISTERIA MONOCYTOGENES ISOLATES
Recep ibik & Figen Cetinkaya
Faculty of Veterinary Medicine, Uludag University, Bursa, Turkey

Listeria monocytogenes is an ubiquitous environmental microorganism

responsible for human listeriosis. According to the reports of the World Health
Organization, the primary mechanism of transmission of L. monocytogenes to
human is through contaminated foodstuffs. Several outbreaks associated with the
consumption of soft cheeses, milk, fish products, meat and poultry products have
been reported. In the present study, Listeria spp. and L. monocytogenes were
isolated from raw chicken legs sold in Bursa province between 2001 and 2002 and
then the isolates were isolated by biochemical test. By direct and enrichment
techniques we have isolated 25 L. monocytogenes, 12 L. innocua, 10 L. welchimeri,
1 L. seeligeri and 1 L. grayi strain. Most of the isolates demonstrated serotype 4
profiles by serotyping. The second part of the studies was contributed to the
characterization of autolysis of L. monocytogenes strains isolated from clinical
and food specimens under starvation conditions. Late exponential phase
harvested bacterial cells were transferred in potassium phosphate buffer (100
mM, pH 7) and incubated at 37 C to induce lysis. At the end of the 48 h
incubation period a heterogeneous autolysis profile was observed among the
tested strains. All the clinical strains exhibited high autolysis i.e. above 75%.
Meanwhile, the extent of food isolates was not as high as the clinical ones but
demonstrated a great variability ranging between 22 and 88%. An increase in
the amount of intracellular markers upon lysis was also measured in the higher
autolytic clinical strain UVF172, compared to low autolytic food strain
UVF114. The results obtained from the study provided evidence that autolysis in
L. monocytogenes is strain dependent and revealed that tested clinical isolates
had a higher level of autolysis than food isolates.

104

PARTICIPANT ABSTRACTS

PROCESSING & PACKAGING

105

EFFECTS OF PACKAGING TREATMENT, STORAGE

CONDITION AND LENGTH OF STORAGE PERIOD ON
EXTERNAL AND INTERNAL TABLE EGG QUALITY
Metin Petek, Fazl Alpay & Serdal Dkmen
Faculty of Veterinary Medicine, University of Uludag, Bursa, Turkey

Packaging is an important stage in providing quality eggs to buyers. It protects

the eggs from microorganisms and natural predators; prevents loss of moisture;
protects from temperatures that cause deterioration and possible crushing during
handling storage and transportation. There are many different types of egg
packages, which vary both in design and packaging material used. Cupboard
cases are commonly used by the Turkish egg producers and covering these cases
by a plastic material before marketing became mandatory after the last avian flu
epidemics. But there are no clear findings about the egg quality and shelf life
during the marketing process. This study was conducted to determine if
packaging system consisted of cardboard with plastic coverings would improve
the shelf life of eggs. The second objective was to determine the interactive
effect of different storage conditions (room or refrigeration temperature) and
length of storage period (5, 10, 15 or 20 d) with packaging treatment (with or
without plastic covering) on internal and external table hen egg quality. Freshly
laid eggs (n = 480) were collected, randomized, weighed, pointed out
numerically and divided into four groups of 120 eggs to test the length of egg
storage treatments for 5, 10, 15 or 20 d. Eggs in each egg storage treatment
group were randomized and allocated into two batches (with or without plastic
covering material) of 60 eggs to test the effect of plastic covering material. Then,
eggs were stored at room or refrigeration temperature conditions. Significant
differences were observed for the internal and some external egg quality traits
related with the length of storage period and storage conditions. All internal and
external egg quality were not significantly influenced due to main effects of
packaging treatment, except for yolk index. The internal egg quality traits
became worst by the prolonged storage particularly when the eggs were stored at
room temperature.

106

THE EFFECT OF VACUUM AND MODIFIED

ATMOSPHERE PACKAGING ON GROWTH OF
LISTERIA MONOCYTOGENES IN RAINBOW TROUT
(ONCORYNCHUS MYKISS) FILLETS
Mehtap Ylmaz, Ziya Gkalp Ceylan*, Mahmut Kocaman, Hatice Ylmaz &
Mkerrem Kaya
Faculty of Agriculture, Atatrk University, Erzurum, Turkey
*Faculty of Veterinary Sciences, Atatrk University, Erzurum, Turkey

Rainbow trout (Oncorynchus mykiss) fillets stored at 4 1 oC were analyzed with

respect to microbiological (Listeria monocytogenes, total aerobic mesophilic
count, phychrotrophic bacteria and Enterobacteriaceae) and chemical (pH, Total
Volatile Basses Nitrogen) under air (control), vacuum and gas mixture (50% CO2
: 50% N2, 80%O2 : 20% CO2 and 2.5%O2 : 7.5% N2 : 90% CO2). The minimum L.
monocytogenes counts were determined form the samples with gas packaged, and
no significant differences were found between them. It was determined that the
effect of different atmosphere conditions and storage time on total aerobic
bacterial count, phychrotrophic bacterial count and Enterobacteriaceae count
were significantly different (p<0.01). The maximum Total Volatile Basses
Nitrogen value was determined form the control group. The effects of
atmosphere condition on pH values were significant (P<0.05).

107

THE EFFECTS OF DIFFERENT NITRATE LEVELS AND

STARTER CULTURE ON THE REDUCTION OF E. COLI
O157:H7 DURING SUCUK PRODUCTION
Gzin Apaydn, Ziya Gkalp Ceylan* & Mkerrem Kaya
Faculty of Agriculture, Atatrk University, Erzurum, Turkey
*Faculty of Veterinary Sciences, Atatrk University, Erzurum, Turkey

The objective of this study was to determine the effects of nitrate levels (0, 200
and 400 ppm) and the starter culture (Staphylococcus carnosus and Pediococcus
pentosaceus) on the reduction of Escherichia coli O157:H7 (initial level of 106
CFU/g) in sucuk (Turkish dry fermented sausage). Enterobacteriacea, E. coli
O157:H7, lactic acid bacteria, Staphylococcus/Micrococcus counts, moisture,
pH, residual nitrite, nitrite/nitrate, salt analysis were also recorded.
Microbiological analysis showed that, the number of E. coli O157:H7 decreased
during the ripening period at a rate of 1.6 log unit in 14th day and a total of 3.3
log units in 21st day. The number of E. coli O157:H7 was below the detection
limit in all samples at 28th day. But the bacteria were recovered with IMS
method in all samples except control. In addition, Verotoxin was found to be
positive using PCR. There was no statistically significant difference in the
number of E. coli O157:H7 between the samples with or without starter culture
and nitrate. The effect of starter culture on the pH decrease was very significant;
the effect of the nitrate levels x ripening-storage period interaction was
significant on E. coli O157:H7 counts.

108

EFFECT OF PROCESSING METHODS ON TOTAL

CATECHIN CONTENTS OF TEA
(CAMELLIA SINENSIS)
Saziye Ilgaz & Ali Kabaogli
Atatrk Research Institute of Tea and Horticultural Products, Rize, Turkey
Young leaves of Camellia sinensis tea plant are very important sources of catechins
belong to the flavan-3-ol class of flavonoids. Tea catechins have been found to
have a number of antioxidant activities, including scavenging of such reactive
oxygen species as superoxide, hydroxyl and peroxyl radicals, inhibition of lipid
peroxidation, inhibition of 2'-deoxyguanosine oxidation in DNA to 8-hydroxy-2'deoxyguanosine and inhibition of the oxidation of low-density lipoproteins. Also it
has been declared that tea catechins had an antibacterial activity against
Helicobacter pylori and antifungal activity against Penicillium chrysogenum and
Saccharomyces cerevisiae. Due to the antioxidative and antibacterial effects of tea
catechins, it is essential to protect the great amount of tea leaf catechins while they
are processing. In this study different kinds of tea processing methods were tried for
determining the best tea manufacturing method in terms of protecting tea leaf
catechins. Two main kinds of green tea manufacturing methods were tried in this
essay; one of them was steaming and the other of them was pan firing. Besides those
green tea manufacturing methods, two black tea manufacturing methods were tried
as well. One of them was CTC method, the other was CAYKUR method. By using
those production methods four tea samples were obtained; two of them were green
and two of them were black tea samples. All of those green and black tea samples
were analysed by using high performance liquid chromatograpy (HPLC). Not
only total amount of catechins but also individual amounts of (+)-catechin, (-)epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin, and (-)-epigallocatechin
gallate were determined using HPLC. As a result of those analyses it was clearly
observed that both of produced green tea samples have got high amounts of
catechins. Steamed green tea has got the highest amount of catechines with its
13.47% Total Catechin Contents while CAYKUR black tea sample has got the
lowest amounts of catechins with its 3.23% Total Catechin Contents. As a result
of this study we can say that for obtaining high amount of catechins in dry tea
leaves it must be chosen green tea especially steamed green tea.

109

FOOD SAFETY PROVIDING SYSTEM FOR AUDITING

OF PRIVATE LABEL PRODUCER IN 20042006
Ali Tercan & Nee Asli Kisikkaya
Tbitak-Marmara Research Center Food Institute, Kocaeli, Turkey

The aim of the auditing is to develop basic facilities and to evaluate food safety
system of the private label producers for supermarket chains in Turkey. For this
purpose, food safety providing system auditing has been performed in 272 food
producers in the meat, milk, seasoning and tea sectors. Audits have been made in
three phases in different producers and cities in Turkey. First phase is on
documentation control systems, pest and cleaning files. The system files have
more questions about HACCP preliminary necessity conditions. In the auditor
controls the files where recorded and found. The second phase is field audit e.g.
personal application, engineering, design and cleaning. The auditor looks
especially at personal applications and food process surfaces in direct contact
with the products produced. The last phase is on hygiene control of personnel
and food contact surfaces. The auditor uses swabs for sampling from hands and
surfaces. The analyses are performed in the Food Institute laboratories. The
micro-organisms examined are Staphylococcus aureus and fecal coliforms on
hands of the personnel and total aerobic mesophilic bacteria and fecal coliforms
on food contact surfaces. All these parameters are evaluated by one or more
auditors to finalize the report. According to this evaluation, the report is graded
as 1000901 Excellent, 900750 Satisfactory, 750650 Acceptable and
<650 Insufficient. According to the result of 2005 in which the personal
hygiene results were included, 23% were found to be dirty whereas rest of them
met the good practices. As a result, 228 of the 272 producers got at least the
grade sufficient.
After, third part audits performed by auditors for supermarket chains in Turkey,
the topic which got most attention was microbiological contamination and
contamination routes. The microbiological results have been evaluated looking
at crucial parameters. Most of the audits have been performed mostly in the
Marmara Region in Turkey. Cold chain factors were taken into consideration

110

through samples, which have been taken from the different private label
producers, promptly sent to our institute. Furthermore, we separate samples into
different groups e.g. dairy, meat and meat products, seasoning and tea groups.
The analyses have been performed according to product groups e.g. for dairy
products coliforms, E. coli, yeast, mould, S. aureus and Salmonella. For spices
the total aerobic mesophilic bacteria, E. coli, mould and yeast, E. coli O157:H7,
Salmonella, Bacillus cereus and aflatoxin analyses are carried out. For meat and
meat products E. coli, S. aureus, mould and yeast, Salmonella, C. perfringens,
E. coli O157:H7 and Listeria monocytogenes and for tea total aerobic mesophilic
bacteria, coliforms, E. coli, yeast and Salmonella are analysed In evaluating
these parameters limits given in the Turkish Food Codex Alimentarius have been
used. Methods published by FDA, Bacteriological Analytical Manual, AOAC
International and ISO were applied during the course of study.

111

FOOD HYGIENE ACTIVITIES AT THE INSTITUTE

OF PUBLIC HEALTH IN OSTRAVA
Jaroslav imek
Institute of Public Health in Ostrava, Ostrava, Czech Republic

Institute of Public Health in Ostrava is a health care institute established by the

Ministry of Health CR. Institute of Public Health provides services in the field of
health promotion and protection. The main activities of Institute comprise
determination and measurement of various components of living and working
environment, examination of general consumption articles and food characteristics
and wide range of investigations in biological and clinical material. The
measurements of various components of living and working environment,
particularly chemical, microbiological and biological analyses and physical
factors measurement are carried out with professional evaluation at the Centre of
Hygienic Laboratories. All services are carried out in line with effective legal
rules and they are accredited according to the standard CSN EN ISO/IEC 17025.
Department of Hygienic Microbiology is a part of the Centre of Hygienic
Laboratories and it performs analyses of water, foods, drugs and process
hygiene. Basic analyse and presence of pathogens (Salmonella, Listeria, incl. E.
coli O157) in food are carried out.

112

SHELF LIFE OF SEA BASS DURING ICE STORAGE

Ivana Filipovi, K. Bojani, L. Kozainski, M. Hadiomsanovi,
N. Zdolec & . Cvrtila
Veterinary Faculty, University of Zagreb, Zagreb, Croatia

In this study sensory and microbiological parameters of sea bass meat on ice
were monitored during a 12-day storage period to obtain information about
quality and hygiene of stored fish meat. Twenty five sea bass of approximately
300 g were caught for this purpose. The sea bass had been farmed in a Adriatic
Sea fish farm. The fish were immediately ice stored in a mobile fridge at the
temperature of about 3 C. On the storage day 2, 4, 6, 9 and 12, five fishes were
subjected to sensor and bacteriological analysis. The sensor quality evaluation
was made using the Quality Index Method test (QIM) for sea bass (Icelandic
Fisheries Laboratories). The QIM includes sensor indicators of fish freshness
and their assessment using the numerical score system. Fresh fish has the
minimum (0), while the maximum number of points (20) indicates spoilt fish. In
the bacteriological analysis the fish samples were analysed for total viable count
(TVC), psychrotrophic count and number of Pseudomonas spp. In the
microbiological analysis 25 g was diluted in 225 ml of peptone saline and
homogenised in the stomacher. After serial dilution, appropriate dilution samples
(1 or 0.1 ml) were poured or spread on agar plates. TVC was determined on
Plate Count Agar (PCA) at 30 C for 72 h, psychrotrophic bacteria on PCA at
6.5 C for 10 d, and Pseudomonas spp. on Pseudomonas agar F at 25 C for 2 d.
After the completion of sensor analysis the results showed that all fish samples
taken on the second day of storage were rated first class (QIM 02 points).
Changes in the shape and clarity of eye, colour and odour of gills and
appearance of gill mucus had occurred already at day 4 of storage (mean value
of QIM 3.5 points). However, during the entire 12 d of storage, the fish
showed no substantial sensor changes indicative of spoilage, and on the day 12
they were scored 10-14 points. The TVC at 30 C did not increase considerably
during the storage of sea bass on ice (from 5.5 log10 cfu/g at day 2 to 6.2 log10
cfu/g at day 12). The psychrotrophic bacterial count increased from 4.3 log10
cfu/g (day 2) to 7.3 log10 cfu/g (day 12). At the same time, an increase in the

113

number of Pseudomonas was recorded i.e. from 3.9 log10 cfu/g (day 2) to 5.5
log10 cfu/g (day) 12 in fish meat. As conclusion the increased deviation in
characteristic appearance and odour of fresh sea bass was simultaneous with the
increase of recorded psychrotrophic bacteria and Pseudomonas spp. The
obtained results are in conformity with the literature data according to which the
principal spoilage organisms of the Mediterranean Sea fish belong to bacteria of
the genus Pseudomonas.

114

GROUP WORKS BASED ON THE LECTURES,

PARTICIPANT ABSTRACTS AND LITERATURE

115

BACTERIAL FOODBORNE PATHOGENS

OF CONCERN
Mati Roasto1, Berit Reinmller2, Raivo Vokk3, Aye Handan Diner Baysal4,
Julijana Polanc5, Tiina Veskus3, Kadrin Juhkam1,
Margarita Terentjeva6 & Elbieta Makiw7
1
Estonian University of Life Sciences, Tartu, Estonia
2
KTH, Stockholm, Sweden
3
Tallinn University of Technology, Tallinn, Estonia
4
zmir Institute of Technology, zmir, Turkey
5
Fructal, Ajdovscina, Slovenia
6
Latvian University of Agriculture, Jelgava, Latvia
7
National Food and Nutrition Institute, Warszawa, Poland

INTRODUCTION
A high level of protection of public health is one of the fundamental objectives
of food law as laid down in regulations (EC) No 178/2002 and 852/2004.
Throughout the European Union (EU) consumers are requiring the food industry
to provide them with an increasing range of safe, nutritious and healthy foods of
high sensory quality and increased shelf life. To meet the demand for healthier
food of high sensory quality, the use of additives and preservatives is being
reduced or eliminated and minimal processing techniques introduced. To
increase food safety and quality considerable amount of time, effort and money
has been spent to food safety control and management (ISO 22000:2005)
systems including better packaging methods and improved new pathogen
detection methods. Nevertheless there is still little sign within official statistics
of significant reductions in the incidence of foodborne illnesses within EU
countries. Todd (1997) reported that in the beginning of this decade 73 to 100%
of all European outbreaks with known aetiology were caused by bacteria.
Particular priority areas are species such as Listeria monocytogenes,
Campylobacter jejuni, Salmonella, and Escherichia coli O157:H7. Biofilm
formation and other problems in production environment have been in focus
lately. Wirtanen et al. (2003) reported that pathogens such as Listeria

116

monocytogenes, Salmonella Typhimurium, and Yersinia enterocolitica can

readily produce biofilms, causing severe disinfection and cleaning problems on
surfaces in the food industry. Yeasts belonging to Saccharomyces, Candida, and
Rhodotorula have been related with contamination of production environment as
well as bacteria like Legionella pneumophila, Pseudomonas spp., Gallionella
spp., and fungi of the Aspergillus, Mucor, and Penicillium strains (Wirtanen et
al., 2003). The aims of the present report are to concisely discuss the most
important bacterial pathogen contaminants with respect to associated foods,
contamination sources and routes. Results of certain studies in some project
related countries and specific aspects of the new European Food Law and the
future needs in scientific and industrial research are emphasized.

BACTERIAL FOODBORNE PATHOGENS

LISTERIA MONOCYTOGENES
L. monocytogenes is a Gram-positive and motile bacterium that is commonly
present in the environment and occurs in almost all food raw materials from time
to time. According to current knowledge the genus Listeria contains six clearly
distinguishable species (McLauchlin, 2006). The most commonly occurring
species in food are L. monocytogenes and L. innocua, however L. monocytogenes
is the only important human pathogen of the genus (Catteau, 1995). Some
studies suggest that 110% of humans may be intestinal carriers of
L. monocytogenes. It has been found in at least 37 mammalian species, both
domestic and feral, as well as at least 17 species of birds and possibly some
species of fish and shellfish. Healthy birds may asymptomatically shed
L. monocytogenes in faecal material (Skovgaard and Morgen, 1988). However,
the poultry meat is contaminated during slaughtering and processing (Rrvik et
al., 2003). It can be isolated from water, soil, silage, and other environmental
sources. L. monocytogenes is quite hardy and resists the deleterious effects of
freezing, drying, and heat remarkably well for a bacterium that does not form
spores. (Johansson, 1999; EuropeAid, 2004). L. monocytogenes is transmitted
via three main routes: contact with animals, cross-infection of newborn babies in
hospital and foodborne infection. The latter two sources result in the majority of
cases of listeriosis in humans. Listeriosis is an uncommon but the serious
foodborne disease that can be life-threatening to the elderly, people with weakened
immune system and pregnant women (Lyytikinen et al., 2000; Frye et al., 2002).

117

Associated foods: L. monocytogenes has been associated with food sources such
as raw milk, supposedly pasteurised fluid milk, cheeses (particularly soft-ripened
varieties), ice cream, raw vegetables, fermented raw-meat sausages, raw and
cooked poultry, raw meats (all types), and raw and smoked fish (Farber and
Peterkin, 1991). Its ability to grow at temperatures as low as 3 C permits
multiplication in refrigerated foods (Roasto, 2004). It can survive or even grow
at pH values as low as 4.4 and at salt concentrations of up to 14%.
In the study of Praakle et al. (2007) a total of 240 raw broiler legs (120 of
Estonian and 120 of foreign origin) from 12 retail stores in two biggest cities
(Tallinn and Tartu) of Estonia were investigated from January to December
2002. Of the raw broiler legs, 70% were positive for L. monocytogenes. The
prevalence of L. monocytogenes in broiler legs of Estonian origin (88%) was
significantly higher than in broiler legs of foreign origin (53%) (P < 0.001).
Praakle et al. concluded the high prevalence of L. monocytogenes showing
various PFGE types in the broiler legs could be caused by cross-contamination
at retail level.
Ready-to-eat meat products with a long shelf life are associated with risk of
transmission of L. monocytogenes (Farber and Peterkin, 1991). Prevalence of
L. monocytogenes in cold smoked, sliced, vacuum packaged pork products
during 15-month period from 2003 until 2004 was studied by Brzi et al.
Samples originated from 8 Latvian and 7 Lithuanian manufacturers. The
prevalences of L. monocytogenes in cold-smoked pork varied from 0 to 67% in
Latvian products and 10 to 73% in Lithuanian products (Brzi et al., 2007). In
order to identify the main risk factors associated with L. monocytogenes
contamination, all production steps were studied separately in each meat
processing plant. Brzi et al. suggested that brining by injection was a
significant (P < 0.05) factor in contamination. Moreover, long cold-smoking
times (12 h) had a significant (P < 0.014) predictive value for a sample to test
positive for L. monocytogenes. The cold-smoking temperatures between 24 and
30 C can provide an inhibitory effect on presence of L. monocytogenes. Low
number of L. monocytogenes at the end of shell-life (< 100 cfu/g) can be
explained by use of starter cultures during processing, which have an antilisterial
effect, and affect multiplying of L. monocytogenes in pork products.

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It is recognised that presence of L. monocytogenes in almost all raw foods

cannot be completely eliminated, but through the application of effective
hygienic measures, it is possible to reduce its incidence and levels in food
products. In order to ensure the safety of food products, growing, harvesting
handling, storage, processing and food supply systems must be managed by food
handlers in such a way as to reliably control the growth of Listeria
monocytogenes and to prevent from multiplying to potentially harmful levels,
>100/g (Commission Regulation, 2005).
CAMPYLOBACTER JEJUNI
Campylobacter jejuni is a Gram-negative slender, curved, non-sporing motile
rod. It is a microaerophilic organism, which means it has a requirement for
reduced levels of oxygen. It is relatively fragile, and sensitive to environmental
stresses (e.g., 21% oxygen, drying, heating, disinfectants, and acidic conditions).
Because of its microaerophilic characteristics the organism requires 3 to 5%
oxygen and 2 to 10% carbon dioxide for optimal growth conditions. This
bacterium is now recognized as an important enteric pathogen. Before 1972,
when methods were developed for its isolation from faeces, it was believed to be
primarily an animal pathogen causing abortion and enteritis in sheep and cattle.
Surveys have shown that Campylobacter spp. is the most common registered
bacterial causes of human intestinal infections in many developed countries
(Hnninen et al., 2003). Campylobacter jejuni subsp. jejuni and C. coli are the
main cause of Campylobacter enteritis in human (Nachamkin and Blaser, 2000).
C. jejuni is responsible for 8090% of campylobacteriosis. It causes more
disease than Shigella spp. and Salmonella spp. combined (Nachamkin and
Blaser, 2000). Although C. jejuni is not carried by healthy individuals in the US
or Europe, it is often isolated from healthy cattle, chickens, birds and even flies.
It is sometimes present in non-chlorinated water sources such as streams and
ponds. In industrialized countries, including Western Europe, US, Canada,
Australia and New Zealand, the rate of human Campylobacter infections has
been increasing steadily. In 2004 a total of 183 961 human cases of
campylobacteriosis were reported from 25 Member States of European Union.
The Community incidence was 47.6 cases per 100 000 population. (EFSA,
2006.) An estimated 2.5 million cases of Campylobacter infection occur each
year in the United States, and 80% of these cases have been found to be the
result of foodborne transmission (Bhaduri and Cottrell, 2004).

119

Associated foods and environment: Campylobacter spp. is widespread in nature,

not only in wildlife but also among food animals such as cattle, sheep, swine,
and avian species as commensally organisms (Friedman et al., 2000). The avian
species are the most common host for Campylobacter, probably because of their
higher body temperature (Skirrow, 1977). Monitoring studies indicate that most
chicken flocks are colonised with C. jejuni. Intestinal colonisation usually leads
to contamination of the final product, which cannot be prevented in the
processing plant.
Studies carried out in slaughterhouses have shown that the main source of the
spread of C. jejuni on poultry carcasses is their intestinal contents (Stern and
Robach, 2003). Campylobacter spp. colonization in commercial poultry flocks is
widespread in many countries. Studies in Europe indicate flock prevalences
ranking from 18 to over 90%, with northern countries showing a lower
proportion of positive flocks (Barrios et al., 2006). A recent monitoring study in
Poland showed that 75.4% of chicken carcasses is contaminated with
Campylobacter species. It is well established that poultry products are a vehicle
for foodborne campylobacteriosis and they are suspected to be an important
source of infection (Roasto et al., 2005).
Other foods (mainly of animal origin) must be considered as potential sources of
infection. Campylobacter have also been isolated from such food items as raw
milk, pork, beef, lamb, and seafood (Duffy et al., 2001). The presence of
Campylobacter spp. in raw materials and products of animal origin may
represent a source of infections, however, a real health hazard exists only when
meat consumed is raw or undercooked (Domingues et al., 2002). The other
major hazard may be a result of improper hygienic habits and disregard of Good
Manufacturing Practice (GMP) principles. This is related to the transfer of
bacteria from raw meat to other foodstuffs (cross-contamination).
Effective quality-control programme in Estonian large-scale poultry processing
plant accounted for the lower contamination levels of fresh chicken meat
compare to contamination level with the same type of products of small-scale
plant (Roasto et al., 2005). Altogether, 279 samples of Estonian raw chicken
meat (breasts, carcasses, legs, minced meat, thighs and wings) were analysed
during 2000 and 2002 (Roasto et al., 2005). Of these, 90 were collected directly
from the end of the slaughter line of a small-scale poultry meat plant and 189

120

from traditional market halls of Tartu town. All chicken meat samples from
market halls were sold fresh and unpacked. Of the raw chicken products of
Estonian origin, 15.8% were positive for Campylobacter. The prevalence of
Campylobacter in the products (breasts, carcasses, thighs and wings) of the
small-scale poultry meat plant (35.6%) was significantly higher than in those
originated from the large-scale company (6.3%) (P<0.001). In order to reduce
the incidence of campylobacteriosis in humans a number of preventive measures
are needed throughout the way from farm to table.
SALMONELLA
Salmonella spp. are facultatively anaerobic, Gram-negative, straight, non-sporeforming small rods, which are usually motile with peritrichous flagella. Salmonella
is a genus within the family Enterobacteriaceae in which approximately 2200
serotypes are recognised. Some of these strains are specifically adapted to hosts
and largely restricted to them, e.g. S. Typhi in man and S. Dublin in cattle. The
growth range for salmonellae is 547 C at pH 4.09.0, with optimum growth at
3537 C and pH 6.57.5. Salmonellae are not particularly salt-tolerant, although
growth can occur in the presence of 4% sodium chloride. The lower limit of
water activity (aw) permitting growth is 0.93 (Mead, 1993).
Associated foods: A wide variety of foods have been implicated in outbreaks of
illness caused by many different serotypes of Salmonella: raw meats, poultry,
eggs, milk and dairy products, fish, shrimp, frog legs, yeast, coconut, sauces and
salad dressing, cake mixes, cream-filled desserts and toppings, dried gelatine,
peanut butter, cocoa, and chocolate. Various Salmonella serotypes have long
been isolated from the outside of eggshells. The present situation with
S. Enteritidis is complicated by the presence of the organism inside the egg, in
the yolk. This and other information strongly suggest vertical transmission, i.e.,
deposition of the organism in the yolk by an infected layer hen prior to shell
deposition. Foods other than eggs have also caused outbreaks of S. Enteritidis
disease. Salmonella still is the most frequently recorded pathogen in the
production chain of food of animal origin. At present the predominant serotypes
are S. Enteritidis and S. Typhimurium. This is true especially considering the
most important meats from pig and poultry. In areas such as Scandinavia
measures against this pathogen have been traditionally more thoroughly
endeavoured, finally resulting in a lower prevalence of Salmonella in these
121

countries compared to Continental Europe (EuropeAid, 2004). Whatever the

Salmonella serotype, effective controls for minimising/eliminating the hazard of
Salmonella from foods involve control of the following steps: raw materials,
personal and environmental hygiene, process conditions, post-process
contamination, retail and catering practices, consumer handling.
ESCHERICHIA COLI O157:H7
E. coli is a facultatively anaerobic, non-spore forming, Gram-negative rod within
the family Enterobacteriaceae. They form part of the natural gastro-intestinal
microflora of man and warm-blooded animals. Because many microbes from
faeces are pathogenic in animals and humans, the presence of the intestinal
bacterium E. coli in water and foods indicates a potential hygiene hazard.
Normally E. coli serves a useful function in the body by suppressing the growth
of harmful bacterial species and by synthesizing appreciable amounts of
vitamins. Although most E. coli are harmless commensal organisms, there are
many pathogenic strains capable cause a variety of illness in humans. There are
six recognized groups of pathogenic E. coli (EPEC, ETEC, EIEC, EaggEC,
EHEC, NTEC). Each group has different virulence traits and mechanisms of
pathogenity (Duffy, 2006). Currently, there are four recognized classes of
enterovirulent E. coli (collectively referred to as the EEC group) that cause
gastroenteritis in humans. Among these is the enterohemorrhagic (EHEC) strain
designated E. coli O157:H7. E. coli serotype O157:H7 is a rare variety of E. coli
that produces large quantities of one or more related, potent toxins that cause
severe damage to the lining of the intestine. These toxins (verotoxin (VT), shigalike toxin) are closely related or identical to the toxin produced by Shigella
dysenteriae.
Associated Foods: Undercooked or raw hamburger (ground beef) has been
implicated in many of the documented outbreaks, however E. coli O157:H7
outbreaks have implicated alfalfa sprouts, unpasteurized fruit juices, dry-cured
salami, lettuce, game meat, and cheese curds. Raw milk was the vehicle in a
school outbreak in Canada.

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LEGISLATION
Commission Regulation (EC) No 2073/2005 of 15 November 2005 on
microbiological criteria for foodstuffs constitutes that foodstuffs should not
contain micro-organisms or their toxins or metabolites in quantities that present
an unacceptable risk for human health. Regulation (EC) No 178/2002 lays down
general food safety requirements, according to which food must not be placed on
the market if it is unsafe. The use of microbiological criteria should form an
integral part of the implementation of HACCP (hazard analysis and critical
control points) based procedures and other hygiene control measures. According
to Article 4 of Regulation (EC) No 852/2004, food business operators are to
comply with microbiological criteria. This should include testing against the
values set for the criteria through the taking of samples, the conduct of analyses
and the implementation of corrective actions, in accordance with food law and
the instructions given by the competent authority. Article 5 of Regulation (EC)
No 2073/2005 is laying down specific rules for testing and sampling, according
to which the ISO standard 18593 shall be used as a reference method. Food
business operators manufacturing ready-to-eat foods, which may pose a L.
monocytogenes risk for public health, shall sample the processing areas and
equipment for L. monocytogenes as part of their sampling scheme. Food safety
and process hygiene criteria are given in chapter 1 and 2 of the Commission
Regulation (EC) No 2073/2005 for microbiological criteria for foodstuff.

FUTURE NEEDS
There is research need for more sensitive, reliable, and cost-effective tools,
particularly sampling methodologies, for analysing food and environmental
samples (e.g., high priority commodities include produce, eggs, and seafood) for
microbial pathogens, where frequency and extent of contamination are expected
to be low, for identification and evaluation of relevant characteristics of different
forms of product packing and handling on the safety of a variety of foods.
Developing modelling techniques to assess microbial behaviour in various
foods, human exposure and dose-response relations to certain foodborne
pathogens (e.g., enumerative detection methods for pathogens), potential risk of
those pathogens causing human illness, and the setting of safety performance
standards to regulate microbial content of food, determining the population
trends with respect to food safety knowledge, attitudes, and practices, especially
123

behaviours that may be significant risk factors for foodborne illness (e.g., food
consumption, in-home food preparation and handling) are the other aspects
needed research. The microbiological safety of food has been advanced
substantially by the introduction and implementation of HACCP. HACCP
provides a systematic conceptual framework for identifying hazards and
focusing efforts on the proper functioning of key food production, processing,
and marketing steps. HACCP cannot be expected to control unknown hazards,
such as emerging foodborne pathogens. There is a need to re-examine how food
is produced, processed, marketed, and prepared to identify conditions that
contribute to emergence. For example, organic acids are used extensively
throughout the food industry to control spoilage and pathogenic microorganisms
(Baysal and Unluturk, in press).
The changing epidemiology of foodborne disease calls for improved surveillance
including rapid sub-typing methods, cluster identification, and collaborative
epidemiological investigation (including case-control studies). Also examined
was the need for better integrated, coordinated, and standardized animal disease
surveillance and health monitoring programs. The new problems of foodborne
disease require new control and prevention strategies to ensure that food in both
domestic and international trade is safe. Topics included a need for
multidisciplinary teams that can provide just in time research; for basic
research to explain factors associated with food production and processing that
contribute to new foodborne microbial threats; for prompt evaluation and
implementation of innovative preservation methods (e.g., food irradiation) to
meet consumer demand for fresh foods; for the use of emerging molecular
methods (e.g., DNA hybridisation and polymerase chain reaction) to examine
emerging foodborne disease organisms; and for models to predict the probability
of a particular microbial event (e.g., growth and death), which may be useful in
the design of HACCP programs and in defining processes, formulations, and
storage conditions to yield foods with acceptable shelf life and safety
characteristics.

CONCLUSION
It is a long way from the new borne food animal to the consumers table. On one
hand, there exist several stages independent from each other, where the persons
involved do not always contact. On the other hand, there are a lot of
124

circumstances and hazards, which may or may not constitute a risk to humans.
As a consequence, measures should be taken especially, where the prevalence of
pathogens has been high, i.e. hygiene in the primary production, immunisation,
logistic slaughter or measures in cleaning and disinfection the site. The
horizontal and vertical transfer of pathogens must become under tighter control:
the routes of the agent via transport to the abattoir are not at all safe. There is not
point, where Salmonella or other pathogens would be safely prohibited to invade
the human food chain. It is obvious, that the inspection service by the authorities
cannot afford the total of surveillance in every production process. The hygienic
status of intermediate products and end products is particularly dependent on the
circumstances of previous stages of production. In consequence hygiene is an
issue of day-to-day practice and checks must be carried out frequently. As a
consequence the authorities have to rely more in the responsibility of the plant.
So the role of the authorities is presently in reconsideration in order to focus the
available resources on the essentials of surveillance. This is true also with
respect to future additional tasks of surveillance in husbandry, which possibly
demands more personnel in the future. It should be emphasized that the producer
is responsible for the product and should do everything to guarantee it.

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23. Praakle, K., Roasto, M., Korkeala, H. & Hnninen, M.-L. 2007. PFGE
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128

FOOD PROCESS HYGIENE, EFFECTIVE CLEANING

AND SAFETY IN THE FOOD INDUSTRY
Helen Ehavald1, Ajda alej2, Hulya alikan3, Emine Aksan4,
Sylva Karpikova5, Blent Kabak6, Martina Weiss7,
Esra Agel8 & Glah Karahan9
1
As Coca-Cola HBC Eesti, Tallinn, Estonia
2
Tina d.o.o., Komenda, Slovenia
3
Ulker Gida San. Ve Tic. As, Istanbul Turkey
4
University of Mustafa Kemal, Alahan, Antakya-Hatay, Turkey
5
Veterinary Research Institute, Brno, Czech Republic
6
School of Agriculture, University of Cukurova, Adana, Turkey
7
Zito Group, Ljubljana, Slovenia
8
Tbitak Marmara Research Center, Food Institute, Gebze, Kocaeli, Turkey
9
Abalioglu Feed Soybean and Textile Company, Izmir, Turkey

INTRODUCTION
Understanding the term food safety along the food chain has various aspects.
One can start with technology integrating regulation and continuing with
nutrition and finishing with consumer. In the food safety program we should be
able to identify all hazards, analyze them, evaluate the likelihood of their
occurrence and identify measures for their control. A hazard is a biological,
chemical, physical agent or condition with the potential to cause an adverse
health effect or spoilage. Today one can manage food safety based on HACCP
through good agriculture, good manufacturing practice, good hygiene practice,
good transport practice, good storage practice, good catering practice and good
laboratory practice. The objective of this group work is to review the different
approaches to assure good hygiene and consequently the safety of the food.

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FOOD PROCESS HYGIENE

CROSS-CONTAMINATION
In the early days, sources of contamination included insects, rodents, sticks,
stones, straw, wood, sand and dirt. People have learned to avoid these
contamination sources; they can be eliminated by maintaining physical
cleanliness in and around the food plant. Looking beyond these sources of gross
contamination, today one is concerned with sources of microbiological
contamination. Microorganisms may come from people working in the plant or
handling the food, from raw products, from the food materials or ingredients or
from lack of proper cleaning of the food plant equipment or the food plant. A
third problem area is potential chemical contamination. This source of
contamination can be controlled by proper usage of pesticides. That is, using the
right pesticides on the right time, and in the right amount. This same principle
applies to chemicals used in the manufacturing of the food, that is, the proper
use and application of food additives and/or chemicals used in the cleaning and
sanitizing of the food plant.
Preventing cross-contamination is one step to eliminate food borne illness.
Cross-contamination of food is a common factor in the cause of food borne
illness. Foods can become contaminated by microorganisms (bacteria and
viruses) from many different sources during the food preparation and storage
process. What is cross-contamination? Cross-contamination is the contamination
of a food product from another source. There are three main ways crosscontamination can occur: from food to food, from people to food or from
equipment to food.
Food can become contaminated by bacteria from other foods. This type of crosscontamination is especially hazardous if raw foods come into contact with
cooked foods. Here are some examples of food-to-food cross-contamination:
o

In a refrigerator, meat drippings from raw meat stored on a top shelf

might drip onto cooked vegetables placed on lower shelf.

Raw chicken placed on a grill touching a steak that is being cooked.

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People can also be a source of cross-contamination to foods. Some examples are:

o
o

Handling foods after visiting the toilet without properly hand washing.
Touching raw meats and then preparing vegetables without washing
hands between tasks.

Using an apron to wipe your hands between handling different foods, or

wiping a counter with a towel and then using it to dry your hands.

Contamination can also be passed from kitchen equipment and utensils to food.
This type of contamination occurs because the equipment or utensils were not
properly cleaned and sanitized between each use. Some examples are:
o

Using unclean equipment, e.g. slicers, can openers, utensils to prepare

food.

Using a cutting board and the same knife, when cutting different types
of foods, such as cutting raw chicken followed by salad preparation.

Storing a cooked product, such as a sauce, in an unsanitized container

that previously stored raw meat.

Cross-contamination is the transfer of biological or chemical contaminants to

food products from raw foods, food handlers, or the food handling environment.
The type of cross-contamination most frequently implicated in food borne illness
occurs when pathogenic bacteria or viruses are transferred to ready-to-eat foods.
Follow these steps to prevent cross-contamination and reduce hazards to food:
o

Wash your hands thoroughly between handling different foods or after

using the toilet.

Wash and sanitize all equipment and utensils that come in contact with
food.

Avoid touching your face, skin, and hair, or wiping your hands on
cleaning cloths.

Store foods properly by separating washed or prepared foods from

unwashed or raw foods.

Try preparing each type of food at different times, and then clean and
sanitize food contact surfaces between each task.

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Many cross-contaminations can be avoided through better employee

practices, separation of raw and ready-to-eat foods, and plant design to
prevent cross-contamination.
AIRBORNE MICROFLORA

Bacteria, fungi, algae, protozoa, and viruses float in air currents. The microbial
flora of air is not constant and is highly variable or transient. The numbers of
microorganisms in the air range from 10 to 10,000 per cubic meter.
Microorganisms do not multiply in the air; their reproduction is uncommon in
air because of lack of moisture and nutrients. The presence of micro-organisms
in air is almost entirely accidental. Indoor air is generally dry and contains no
available microbial nutrients; microorganisms cannot grow in air but can survive
in association with dust particles. Dust particles in air come from clothing,
bedding and similar textiles human and animal skin scales and hair dried soil and
similar contaminating sources. Dust particles are dry and highly charged on the
outside but may be moist in the centre. The different types of micro-organisms
that are normally detected in air include bacteria, fungal spores and some yeast:
o

Bacterial endospores and exospores of e.g. Bacillus, Clostridium,

Streptomyces.

Vegetative cells of many bacterial species mainly gram positive bacteria

which are more resistant to drying. Bacteria from human and animal
skin, hair, respiratory aerosols etc. mainly Gram positive bacteria.

Fungal spores of many species.

Airborne microorganisms in food processing plants are extremely hazardous

because of the economic and health problems they may cause. Because of the
rich nutrients and moisture, microorganisms can reproduce at an incredible rate
in the foods. Beside this atmospheric micro flora, sneezing, talking, laughing,
falling hair, using soiled laboratory coats, as well as shedding from hands and
arms contributed to the microbial contamination of air in food processing plants.
Immediate washing of floors containing materials that may support microbial
growth has been observed to be critical in reducing the airborne counts in food
processing plants. The raw materials in processing plants contribute a major part
of the microbial air contamination. From these raw materials, microorganisms
may be deposited into the air during handling and processing of the raw materials.
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Virtually all micro-organisms can be transmitted from air to food surface due to
their bioaerosols form. The pathogens are extremely sensitive to the environment
and cannot be outside of the host for any length of time. Only a few pathogens
may be transmitted by air. Some airborne pathogenic bacteria, such as
Staphylococcus aureus, spore form the antrax-causing bacterium Bacillus
anthraci are well known to exist. But, no data are available to contaminated food
by air-borne pathogens. But there is a potential risk for foods produced in
livestock farms. Contamination from airborne yeasts, moulds and other
microorganisms can result in shortened shelf life of food. A few researches
about air micro flora in different food production are as follow:
o

Donnelly (1977) researched airborne microbial contamination in a

winery bottling room. He isolated Acetobacter, Lactobacillus, Bacillus,
Saccharomyces, Rhodotorula, and Serratia species from the air.

Strm and Blomquist (1986) monitored concentrations of airborne

fungal particles in the sorting departments of two fruit warehouses
during the handling of moldy citrus fruits. They found that
concentrations of airborne moulds varied between 105 and 108 colonyforming units per cubic metre (CFU m3). They counted that the highest
mean value recorded 7.7 x 108 spores m3 and showed that 8595% of
the micro-organisms were <3.5m. They indicated that the dominant
flora consisted of Penicillium italicum and Penicillium venucosum.

Ellerbroek (1997) examined bacterial loads in the air at different locations

of a poultry slaughtering and processing plant and found that relatively
high mean counts of airborne Enterobacteriaceae (log 3.2 cfu/m3) were
recovered in the bird reception area and at the entry to the evisceration
room (log 2.6 cfu/m3), while low mean counts of Enterobacteriaceae
were recovered in the cutting and deboning area (log 1.0 cfu/m3). He
found that the microbes near evisceration were predominantly
Staphylococcus spp., Moraxella spp., Micrococcus spp., Acinetobacter
spp., and Klebsiella spp. In the reception area mainly Micrococcus spp.,
Staphylococcus spp., Corynebacterium spp., yeasts and Flavobacterium
spp. were found and in the chilling rooms Corynebacterium spp.,
Acinetobacter spp., Moraxella spp., and yeasts.

Salustiano et al. (2003), researched the microbiological air quality at

processing areas in a dairy plant. For all processing areas, they

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determined that the numbers of mesophilic aerobic bacteria, yeast and

moulds were higher than 90 CFUm-3, which is the maximum value
recommended by American Public Health Association. They stated that
the results showed a difference (p<0.05) for the Staphylococcus aureus
numbers (from <1.0 to 4.3 CFUm-3) at processing areas.
o

Northcutt et al. (2004) researched total aerobic bacteria, moulds/yeasts,

coliforms and Pseudomonas in the air in three shell egg processing
operations. They found that highest counts for total aerobic bacteria (5.9
log cfu/ml air), moulds/yeasts (4.0 log cfu/ml air) and coliforms (2.5 log
cfu/ml air) were found in the hen house. Highest counts for
Pseudomonas were found in the hen house (3.2 log cfu/ml air) and
behind the egg washer (3.5 log cfu/ml air). Lowest counts for total
aerobic bacteria (2.5 log cfu/ml air) and moulds/yeast (2.7 log cfu/ml
air) were found in the post-processing cooler.

Soldatou et al. (2006) isolated primarily Gram-positive, catalase

negative cocci, micrococci and bacilli from the air of cheese factories.
They also stated that airborne contaminants exhibit acidifying and
proteolytic activities and may thus contribute to cheese ripening and
flavour. However, they indicated that room air quality may be the cause
of unsatisfactory shelf life.

Contamination of microorganisms from air may be reduced practically by good

ventilation, use of face masks, controlled temperature and RH and establishing
pressure differentials between rooms. In current, different technical methods are
used for prevention of air-borne microorganisms:
o

Air filtration a typical HEPA (High Efficiency Particulate Air) filter

will filter micron sized particles at about 95% efficiency. In theory,
HEPA filters should be highly effective against bacteria and fairly
effective against viruses.

Ultraviolet irradiation Microorganisms are uniquely vulnerable to the

effects of light at wavelengths at or near 253.7 nm due to the resonance
of this wavelength with molecular structures. Ultraviolet light possesses
the right amount of energy to break organic molecular bonds and
damages the microorganisms. The outdoor die-off rate, which is due to
the ultraviolet component in sunlight, varies from one pathogen to

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another, but can be anywhere from a few seconds to a few minutes for a
9099% killing of viruses or contagious bacteria. Spores, and some
environmental bacteria, tend to be resistant and can survive much longer
exposures.
o

Outdoor air purging Airborne microorganisms can be removed by

purging with outside air, which is naturally sterilized.

Electrostatic precipitation is commonly used to remove particles

including microorganisms from airstreams.

Negative air ionization has the potential to reduce the concentration of

airborne microorganisms. The effect results from the ionization of
bioaerosols and dust particles that may carry microorganisms.

In addition, photocatalytic oxidation, air ozonation, carbon adsorption,

passive solar exposure, ultrasonic atomization, microwave atomization,
pulsed light have been researched for control of airborne microorganisms.
SURFACE HYGIENE AND BIOFILMS

A biofilm is a complex aggregation of microorganisms marked by the excretion

of a protective and adhesive polysaccharide and glycoprotein matrix. Biofilms
may contain spoilage and pathogenic microorganisms. All this microorganisms
within biofilm are protected from sanitizers increasing the likelihood of survival
and subsequent contamination of food. Biofilms also play an important role in
the spreading of resistant microbes. Planktonic bacteria are sensitive to the
antimicrobial and disinfectants, but biofilm bacteria are more resistive. Within
the high dense bacterial population, efficient horizontal transfer of resistance and
virulence genes takes place. This increases the risk of reduced shelf and disease
transmission. The ability to form biofilm is therefore considered to be an
important virulence factor. Microbes inhabiting contact and environmental sites
in food processing are mostly harmful, because these microbial communities are
formed in critical process places.
Some microbes naturally have a higher tendency to produce biofilm than others,
but biofilm can generally be produced by any microbe suitable conditions. Once
a biofilm has been formed, it can be a source of contamination for foods passing
through the same processing line. The attachment of bacteria with subsequent

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development of biofilms in food processing environments is a potential source of

contamination of finished product. This is the case especially if the process
equipment is not hygienically integrated in the process line or the cleaning and
disinfection procedures are not properly designed to remove the organic soil
from the process surfaces. The surfaces of equipment used for food handling,
storage, or processing are recognized as major sources of microbial
contamination. Areas that are more prone to biofilm development include dead
ends, joints, valves, and gaskets. In such places bacteria can remain on
equipment surfaces even with acceptable cleaning systems in place. The
microbes in thin biofilms can be removed from the surface only by the
combination of the physical and mechanical cleaning methods. These
microorganisms can not be removed only by spraying or washing methods and
the decontamination can not be obtained. This point causes the cross
contamination and recontamination.
In the food industry, it is very hard to find the best food grade cleaning chemical
or disinfectants. Is has to be taken into consideration if the concentration of the
chemical is not strong enough the effectiveness of it will be passive and the
insufficient cleaning of the surface will not display an antimicrobiological effect.
Besides, the equivalent chemical of the food grade chemicals are considerable
cheap and do not has a legal necessity. For that reason manufacturers prefers
these chemicals.
The investment cost of the chemical and physical cleaning applications is an
additional cost for the manufacturers. Beside, the tolls used in mechanical
cleaning (brush, cloth ext.) are as important as the contact time with the surface
of the chemical and it increases the effect of the cleaning. This informations is
determined detailed in the chemical usage instructions. The need of ambience
ventilation for the heavy cleaning chemicals or evaporation attributed acid and
base applications prove us that mechanical cleaning is the easiest and the safest
solution for every condition. At the food manufacture production lines, even it is
installed with the new technology designed machines, the line tendency to dirty
it self and the food hidden to the dead points can be removed only by the
mechanical cleaning.

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TRAINING OF PERSONNEL IN HYGIENE AND FOOD SAFETY

An important way to prevent food contamination is to maintain a high standard
of personal hygiene and cleanliness. Even healthy people carry food poisoning
bacteria on their bodies. By touching parts of the body, such as nose, mouth, hair
and including clothes, you can spread bacteria from hands to the food. It is
essential that personnel are well informed and educated about hygiene rules and
recommendations in the food industry. Training of employees and managers
needs to be performed on a continuous basis.
A major reason for improved personal hygiene is the dreaded of foodborne
illness outbreak. When this occurs, the reputation and life of a company can be
severely harmed. The management must ensure that their hygiene procedures are
adequate to safeguard the product quality and hinder or at least minimise
outbreaks to few isolated cases of low severity. The HACCP system helps in
prioritising process steps focusing on elimination or at least reduction of hazards.
Most infectious intestinal diseases are spread through contaminated hands via
the fecal-oral transmission route. It has been estimated that between 25 and 40
percent of all foodborne illnesses are caused by poor personal hygiene. This
includes outbreaks traced to food processing and service establishments as well
as in the home environment. Specific outbreak-causing organisms have varying
degrees of personal hygiene involvement as contributing causes. Therefore, the
food industry personal hygiene program is a prerequisite in most food processing
environments. If food handling step is deemed to be a critical control point
(CCP), then hand contact should be avoided/eliminated or hand washing should
be monitored, documented and verified (MDV). In addition to monitoring the
employees hand washing behaviour through use of documentation forms filled
out by managers, there are a number of newly introduced hand wash monitoring
systems, which provides automatic documentation satisfying the MDV
requirement. It must be remembered that the personal hygiene must not be
neglected, even though the proportion of foodborne illness caused by products
produced in the food processing is low. With current large-scale production and
distribution channels for food products, a single flawed practice in a processing
plant or lapse of hygiene by a single individual can cause national or
international product recalls. Sometimes bare hand contact with food under quite
exaggerated circumstances has been described to occur. Even low level of hand

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contact e.g. handling sliced pineapple, tomato or ice cubes has been shown to
initiate outbreaks. Also extremely hazardous activities have been documented
e.g. plunging hands into milk containers to change malfunctioning spigot,
repeated and repeated tasting food with fingers, using bare hands to remove meat
from bones or mixing salad by hand. Such hand contact should not be tolerated
and alternatives should be sought e.g. gloves, food handling utensils, tongs or
bakery papers.

EFFECTIVE CLEANING IN THE FOOD INDUSTRY

MECHANICAL CLEANING
Mechanical cleaning processes are good alternatives to traditional solvent-based
cleaning operations. They reduce waste production and eliminate potential safety
problems with the handling and usage of toxic, ozone-depleting, and oftenflammable solvents. These cleaning processes are many and varied. Cleaning of
almost any piece of equipment, surface, or component is possible if it is study
enough to withstand the friction and force produced by the mechanical work of
cleaning operations such as sanding, grinding, polishing, brushing, and scraping.
These methods might not be appropriate for precision or delicate parts and the
stripping rate varies with the type of paint, coating condition and coating
thickness. The following benefits have been seen:
o

Significant reduction in the amount of hazardous waste generated

compared to chemical stripping.

Reduces hazardous waste disposal costs.

Provides solvent cost savings.

Reduces the number of hours required for paint stripping in comparison

to chemical stripping.

Reduces worker exposure to solvent, hazardous waste and hazardous air

emissions.
DRY AND WET CLEANING

In food industry the efficient cleaning is very important part of the whole
production. The whole point of cleaning is to remove all dirt (residues of food,
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biofilms of microorganisms). Because of the different types of food industries,

there are different types of cleaning e.g. dry cleaning, wet cleaning and cleaning
with enhanced mechanical physical forces. The dry cleaning is used in bakery
industry and wet cleaning is in production of beverage and in all kind of industry
where we are dealing with moisture and grease. Mechanical physical cleaning
is used mostly to prepare the working desk or other working tools before using
the wet cleaning. The chemical cleaning is mostly used is places where all other
ways of cleanings are not suitable.
The dry cleaning is mostly used in bakery industry because of the specific
characteristics and types of ingredients. The flour is present in the air all the time
and because of that the water can cause a lot of damage especially in cauldron.
On the other hand with water you can easily flush away all the dirt. In bakery
industry the compromise between dry and wet cleaning and the whole
production most is done. On daily and weekly bases the dry cleaning is used and
the wet cleaning is used on monthly bases. In the process of dry cleaning the
broom, grater and the special types of vacuum cleaners are the most important
help. But the same broom or grater most not be used for working desk and for
the floor for instance. In that purpose the brooms are marked with labels in
different colours (green colour for working desks, red colour for toilettes, yellow
colour for floor, etc.).
The wet cleaning on daily bases is useable only for some parts of the machines
which have become in contact with wet or greasy ingredients. Those cleanings if
possible most be performed in special places marked only for that. In production
of fresh cakes-patisserie the dry and wet cleaning are used. There is very
important to separate facilities where the sponge cakes are baked and where the
crmes are prepared. In first part of the production only the dry cleaning is used
and in second part of the production only the wet cleaning in used. In good
hygiene practice those two ways of cleaning most never mixed. In baking tin the
water can do a lot of damage, on other hand only the dry cleaning is useless in
production of crmes. Only the thorough hygiene practice in those facilities is
sufficient. Regardless of the type of the cleaning, cleaning is necessary at home
as in production facilities at work.

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CIP CLEANING
Cleaning-in-place (CIP) is a method for cleaning equipment quickly and
efficiently without needing to dismantle the equipment. The cleaning solution
are taken from a storage tank, circulated in the process tanks and pipelines, and
returned to the storage tank (www.worldpumps.com). Typically a CIP system
would include a solution reservoir, a supply pump, and a return circuit in the
case of a tank or other similar vessel, a spray device and some type of return
pump may also be included. Any closed vessel (tanker, tank, vat, chamber,
hopper, or bin) can be CIP cleaned using one of the following spray devices:
disc, stick, ball, ring or tanker spray. Also, any closed circuit, including piping,
pumps, valves, and plate heat exchangers can be cleaned in place. The success in
cleaning any vessel or pipeline circuit in place depends on five considerations:
time, temperature, concentration and mechanics.
Considering the disinfection technology you have to distinguish between
appropriate procedures of closed piping systems (CIP cleaning in place) and
containers with large quantities of circulated solutions and good turbulence,
good microbial suspension, simple temperature application between cold (4 to
10 C) to heated (4060 C) and generally good pre-rinsing. In closed cleaning
systems pre-rinsing with cold water is carried out to remove loose soil, and the
CIP treatment in normally performed using a hot cleaning solution, but a cold
solution can also be used in handling fat-free product. In dairy CIP cleaning the
warm alkaline cleaning solutions, normally of 11,5% sodium hydroxide
(NaOH), is heated to 7580 C and the cleaning time is 1520 min. Cleaning step
before disinfection in CIP program must be effective enough to affect biofilm.
The separate disinfection, carried out after the cleaning operation, deals with less
soiling, but is an additional step. If the extent of soiling is not too excessive one
would therefore normally carry out a combined cleaning and disinfection agents.
In this context, the choice of cleaning and disinfecting agents must depend
among other parameters on the type and intensity of soiling and contamination
considering also the material quality of the process equipment. The CIP
disinfection technology can be generally applied with low concentration of
active ingredients. Disinfectants used in the food processing industry include
oxidizing agents, hypochlorite, hydrogen peroxide, ozone and peracetic acid,

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denaturizing agents, alcohol-based products, non-oxidizing and surface tension

diminishing agents, and enzyme based compounds.
DISINFECTANTS
Disinfectants are applied as the final step in the cleaning program and
disinfection should be performed again if time between completion of
disinfectant program and startup exceeds four hours. Different microorganisms
vary considerably in their sensitivity or resistance to disinfectants. In order to be
effective, a disinfectant must:
o

provide a broad spectrum of proven activity

demonstrate a reduction in viable organisms for which it comes in

contact

work in a broad temperature range and in a variety of pH conditions

work in the presence of organic material (such as urine, feces, or blood).

Before deciding to use a disinfectant, consider whether a more appropriate

method is available. The main use of chemical disinfection is for heat-labile
equipment where single use is not cost effective. Some of these items (e.g.
bronchoscopes) require high-level disinfection. A limited number of disinfectants
(e.g. glutaraldehyde 2%, 6% hydrogen peroxide, 0.20.3% peracetic acid) can be
used for this purpose. If a sporicidal action is required, immersion in 2%
glutaraldehyde for at least 3 hours is required.
Chemical disinfectants must be made up freshly to the correct concentrations
according to the manufactures instructions and discarded after the correct
period of time or number of uses. They should be stored in clean bottles with
plastic stoppers. When the bottle is empty it should be thoroughly cleaned before
re-filling. Partially empty bottles should not be topped up since this will
encourage contamination with and multiplication of disinfectant resistant organisms.
The object must be thoroughly rinsed with sterile water after disinfection. If
sterile water is not available, freshly boiled water can be used. After rinsing,
items must be kept dry and well protected from being recontaminated.

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FURTHER READING
1. Aarnisalo, K., Tallavaara, K., Wirtanen, G., Maijala, R. & Raaska, L. 2005.
The hygienic working practices of maintenance personnel and equipment
hygiene in the Finnish food industry. Food Control, 17, 10011011.
2. Adhkari, A., Sen, M.M., Gupta-Bhattacharya, S. & Chanda, S. 2004.
Volumetric Assessment of Airborne Fungi in Two Sections of a Rural
indoor Dairy Cattle Shed. Environm. Int., 29, 10711078.
3. Angelilo, I.F., Viggiaini, N.M.A., Rizzo, L. & Bianco, A. 2000. Food
handlers and foodborne diseases: knowledge, attitudes, and reported
behaviour in Italy. J. Food Prot., 63, 381385.
4. Atanassova, V. & Ring, C. 1999. Prevalence of Campylobacter spp. in
poultry and poultry meat in Germany. Int. J. Food Microbiol., 51, 187190.
5. Brabban, A.D., Nelsen, D.A., Kutter, E., Edrington, T.S. & Callaway, T.R.
2004. Approaches to controlling Escherichia coli O157:H7, a foodborne
pathogen and an emerging environmental hazard. Environm. Rev., 6, 208229.
6. Donnelly, D.M. 1977. Airborne Microbial Contamination in a Winery
Bottling Room. Am. J. Enol. Vitic., 28, 176181.
7. Doyle, M.P., Zhao, T., Meng, J. & Zhao, S. 1997. Escherichia coli
O157:H7. In: Doyle, M.P., Beuchat, L.R. & Montville, T.J. (Eds.) Food
Microbiology Fundamentals and Frontiers. ASM Press, Washington D.C.
Pp. 171191.
8. Ellerbroek, L. 1997. Airborne microflora in poultry slaughtering
establishments. Food Microbiology, 14(6), 527531.
9. Jablonski, L.M. & Bohach, G.A. 1997. Staphylococcus aureus. In: Doyle,
M.P., Beuchat, L.R. & Montville, T.J. (Eds.) Food Microbiology
Fundamentals and Frontiers. ASM Press, Washington D.C. Pp. 353375.

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10. Kapperud, G., Gustavsen, S., Hellesnes, I., Hansen, A.H., Lassen, J., Hirn, J.,
Jahkola, M., Montenegro, M.A. & Helmuth, R. 1990. Outbreak of
Salmonella typhimurium infection traced to contaminated chocolate and
caused by a strain lacking the 60-megadalton virulence plasmid. J. Clin.
Microbiol., 28, 25972601.
11. Kerouanton, A., Hennekinne, J.A., Letertre, C., Petit, L., Chesneau, O.,
Brisabois, A. & De Buyser, M.L. 2007. Characterization of Stahyloccocus
aureus strains associated with food poisining outbreaks in France. Int. J.
Food Microbiol., in press.
12. Nachamkin, I. 1997. Camphylobacter jejuni. In: Doyle, M.P., Beuchat, L.R.
& Montville, T.J. (Eds.) Food Microbiology Fundamentals and Frontiers.
ASM Press, Washington D.C. Pp. 159170.
13. Northcutt, J.K., Jones, D.R., Ingram, K.D., Hnton, A. & Musgrove, M.T.
2004. Airborne Microorganisms in Commercial Shell Egg Processing
Facilities. Int. Poultry Sci., 3, 195200.
14. Orth, R. 1998. The inportance of disinfection for the hygiene in the dairy
and beverage production. Int. Biodeterior. Biodegrad., 41, 201208.
15. Rangel, J.M., Sparling, P.H., Crowe, C., Griffin, P.M. & Swerdlow, D.L.
2005. Epidemiology of Escherichia coli O157:H7 outbreaks, United States,
19822002. Emerg. Infect. Dis., 11, 603609.
16. Rezende, C.L.E., Mallinson, E.T., Gupte, A. & Joseph, S.W. 2001.
Salmonella spp. Are affected by different levels of water activity in closed
microcosms. J. Ind. Microbiol. Biotechnol., 26, 222225.
17. Rocourt, J. & Cossart, P. 1997. Listeria monocytogenes. In: Doyle, M.P.,
Beuchat, L.R. & Montville, T.J. (Eds.) Food Microbiology Fundamentals
and Frontiers. ASM Press, Washington D.C. Pp. 337352.
18. Salo, S. 2006. Evaluating hygiene and cleaning efficiency of food process
surfaces based on experimental data and modelling. BioCentrum-DTU.

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19. Salustano, V.C., Andrade, N.J., Brando, S.C.C., Azeredo, R.M.C. & Lma,
S.A.K. 2003. Microbiological air quality of processing areas in a dairy plant
as evaluated by the sedimentation technique and a one-stage air sampler.
Braz. J. Microbiol. 2003, 34, 255259.
20. Soldatou, H., Psoni, L., Tzanetakis, N. & Litopoulou-Tzanetaki, E. 2006.
Populations, types and biochemical activities of aerobic bacteria and lactic
acid bacteria from the air of cheese factories. Int. J. Dairy Technol., 59,
20002008.
21. Strm, G. & Blomquist, G. 1986. Airborne Spores from Mouldy Citrus Fruit
A Potential Occupational Health Hazard. Ann. Occup. Hyg., 30, 455460.
22. Wirtanen, G., Salo, S., Helander, I.M. & Sandholm-Mattila, T. 2000.
Microbiological methods for testing disinfectants efficiency on
Pseudomonas biofilm. Colloids and Surfaces B: Biointerfaces, 20, 3750.
http://www.engr.psu.edu/ae/iec/abe/topics.asp
http://www.rcw.raifoundation.org/biotechnology/MScBioinformatics/microbilogy/
lecture-notes/lecture-29.pdf
http://ohioline.osu.edu/b604/index.html
http://www.answers.com/topic/pathogen-transmission
http://www.worldpumps.com
http://www.cfsan.fda.gov
http://www.foodquality.news.com
http://www.foodsafetycentre.com
http://www.eurosurveillance.org
http://www.melrosechem.com/english/publicat/dairy%20industries/cip.pdf

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SAFE PROCESSING AND PACKAGING OF FOODS

Ziya Gkalp Ceylan1, Saziye Ilgaz2, Nee Asli Kisikkaya3, Iva Lovin Kukman4,
Barbara Pellis5, Metin Petek6, Jaroslav imek7 & Jana Ramus8
1
Faculty of Veterinary Sciences, Atatrk University, Erzurum, Turkey
2
Atatrk Research Institute of Tea and Horticultural Products, Rize, Turkey
3
Tbitak Marmara Research Center, Food Institute, Gebze, Kocaeli, Turkey
4
Mercator-Emba d.d., Ljubljana, Slovenia
5
Pivovarna Union d.d., Ljubljana, Slovenia
6
University of Uludag, Bursa, Turkey
7
Institute of Public Health in Ostrava, Ostrava, Czech Republic
8
CCIS, Ljubljana, Slovenia

Pure, safe, wholesome food is the minimum expectation of todays consumer.

The food industry respect different approaches to assure safety food and to gain
consumer trusts with implementation of different standards (ISO 9001-2000,
ISO 22000, HACCP, IFS, BRC, etc.). The EU legislation put into force a several
requirements to assure safety food for protection consumers health and to help
the producers. Food processors must have enough knowledge of safe processing
methods and a range of management skills to assure that this knowledge is
continuously applied. Properly controlled production, packaging, storage and
distribution practices minimize the risk of product spoilage and health risk. In
order to provide the required assurance of product safety and stability each food
industry must develop procedures, criteria and control system appropriate to
their situation. Therefore, the risk of food poisoning is minimized only by
designing and implementing appropriate preventive measures. This means
understanding its causes, determining the required preventive and remedial
measures and managing food handling so that these measures are always used.
The basis for a positive assurance of product safety and stability in each food
processing company is a Hazard Analysis Critical Control Point (HACCP)
concept. Before any HACCP principles are taken into consideration the processing
plant have to be aware of good manufacturing and good hygienic practices to
facilitate in controlled production conditions and in good sanitary manners.

145

The scope of food processors should be minimal deterioration of raw ingredients,

which means to use the minimal thermal degradation of food and mechanical
forces during processing and minimum usage of preservation chemicals with
choosing suitable packaging materials and methods. Today consumers prefer
minimally processed food with no preservatives and a long shelf life. It is very
important to know some aspects of microbiological safety in food preservation
technologies as pathogens as well as spoilage organisms should always be
assumed to be present in every stage of processing and packaging.

RAW MATERIALS
Any unprocessed (raw) ingredients may be expected to contain pathogens or
spoilage microbes, e.g. milk, vegetables, meat, dried herbs, etc. For certain
ingredients, e.g. which will receive no kill step during processing,
microbiological monitoring of becoming incoming batches may be carried out
more regularly than those undertaken thermal processing to conform that they
conform to the specification for that ingredient. So it is very important to obtain
high quality raw materials and establish quick methods to determine raw
material quality. It depends on the further processing but in many cases
microbiological parameters should not only be according to regulation
guidelines but many times below them. It is to emphasize that ingredients must
be segregated from finished products. Ingredients must be stored so that
microbiological growth is either prevented or minimized. Chilling or freezing is
a stage which inhibits growth of microbes. It must be strictly controlled as
germination and outgrowth of surviving spores should be prevented. For
example, for chill ingredients, storage should be at no more than 5 C and
preferably less than 3 C and the storage time needs to be limited.
When we discuss about HACCP programmes which determine safe food
processing from farm to fork, we may not forget about on farm hygiene
programmes, when the animals becoming a food are reared. The new legislation
(Regulation EC 852/2004) has not declared the principles of HACCP approach
into the primary production, but the producers have to establish very closed rules
to those that HACCP are, especially from the part of hygienic preprogrammes.
Disease challenge by viruses, bacteria and fungi presents a mayor threat to
profitable production for instance in poultry production. Biosecurity, in other
words reducing the number of infectious organisms in the environment, is the

146

most effective form of protection. It means that on farm HACCP system, we

should establish CCPs which determine control points at which pathogen
reduction can take place as a part of a biosecurity programme:
o
o
o
o
o
o
o

site security: transport sanitation, wheel dips and foot dips,

personnel hygiene: protecting clothing, hand hygiene and showering in
and out,
water system: sanitize the drinking water,
aerial: fogging the house to control airborne pathogens,
litter: clean litter can be sprayed to reduce infection,
rodent control: integrated pest management programme and
terminal disinfection programme of broiler house.

The great challenge for control of hazardous bacteria e.g. thermotolerant

campylobacter in poultry industry is in water monitoring and proper use of
sanitizers for decontamination of biofilm, which has been identified as a possible
source for transmission of campylobacter. Also other means of controlling this
bacterium have been developed and should be implemented into the broiler
house in the future. The food processes records must be kept to show that the
biosecurity programme is in place and implemented correctly and continuously.
Records should be kept of products used, critical limits, cleaning schedules and
any corrective action provide documentation for control and for monitoring.
Such means show that the control of animals as raw materials entering the
slaughtering process is taken care of.

PROCESSING
Since so many processing methods may be applied to foods it would be
impractical to detail each in this abstract. The objective during manufacture is to
maintain the safety of a food by preventing microbial contamination during
packaging. All equipment must be of suitable design and construction and
thoroughly cleaned and sanitized before use, which means rigorous application
of cleaning schedules as part of GMP. Some food processing requires a manual
process. To minimize contamination good personnel hygienic practices must be
enforced, but environmental contamination must not be forgotten and these
operations should be take place in a high care area.

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Each process used must have its appropriate controls applied during
manufacture. It means that through risk assessment potential hazards are
identified and suitable preventive measures undertaken and any critical control
point (CCP) taken into control. For example, drying of salami will need the
temperature and time of drying to be specified, and the allowed tolerances for
that process stage. Pasteurization of liquids will be regulated in the usual way by
specified temperature being achieved, correct flow-rate insuring that the liquid is
held for the specific time and a flow rate diversion valve fitted to reject underprocessed liquid. Pasteurization (cooking) of meat, preserves, etc. requires
temperature monitoring of the slowest heating portion of the product batch to
insure that the pasteurization temperature is reached. Both cooking and cooling
of products must be monitored for temperature and time. Whatever control is
applied, instruments used for measurement must be accurately calibrated.
Process parameters must be recorded and verified.
Todays customers have become more and more aware of quality in foods and
have an increased expectation of consistency in all quality parameters. With the
large scale of modern food industry and nationwide distribution of products, a
small number of viable organisms have a potential to contaminate large volumes
of products, resulting in severe financial losses for the producers affected.
Fruit juices were until recently considered to only be susceptible to spoilage by
yeasts, moulds and lactic bacteria. Spoilage by these organisms was prevented
by the acidic pH of fruit juices and heat treatment applied during the hot-fillhold process. Despite these control measures an increasing number of spoilage
causes of fruit juices and fruit juices products due to contamination by
thermophilic acidophilic bacteria have been reported.
The thermophilic and acidophilic characteristics of Alicyclobacillus spp. allow
resistence to current pasteurization process and the ability to produce off flavors
unacceptable in juices. The first Alicyclobacillus spp. was isolated in 1982 from
spoiled apple juice and was implied as the causative agent for spoilage. It is
currently one of the microorganisms of concern in the fruit juice industry. The
most likely source of contamination of fruit juices is from fruit contaminated by
soil during harvest or introduced into the manufacturing process by unwashed or
poorly washed raw fruit. Water and sugar can also be a source of contamination.
Till now acidophilic spore formers were isolated from various environmental

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sources including garden soil, forest soil, apple, pear and orange juices. The
major off-flavors associated with the spoilage caused by Alicyclobacillus spp. is
guaiacol which is generally accepted as the predominant metabolite associated
with the smoky taints in fruit juice. Spores of Alicyclobacillus survive
pasteurization conditions of 95 C for 2 min and grow within a pH range of 2.5
to 6.0. During the heat treatment of foods pathogens and most non-spore
forming microorganisms are killed but a heat process sufficient to destroy all the
microbial spores will have a harmful effect on the organoleptic quality of the
product. Procedures that requires attention is the optimization of the membrane
filtration which can assist in recovering low numbers of Alicyclobacillus spp.
spores and many fruit juice manufactures are currently utilizing this method. The
quality of membrane filters differ greatly among different manufactures and this
should be carefully assessed for their ability to retain Alicyclobacillus spores.

PACKAGING
Packaging materials are of many types, but they serve as a major defence against
external hazards. However undesirable interactions between packaging material
and food can give rise to potential problems e.g. migration of packaging
components or penetration of micro-organism through packages. The importance
of hygiene and suitable packaging material and method is very important.
One of the safest methods of packaging is aseptically filling. In aseptic
packaging, a product is sterilized, cooled and transported under sterile conditions
to a container which has also been sterilized. The product is filled and sealed
under sterile conditions into the container. The end product is hermetically
sealed for prolonged periods of time at ambient conditions. Depending upon the
material, packaging sterilization may be accomplished by heating, use of
chemical or radiation. The sterility of filling can be maintained by air filters,
positive pressure, vaporized H2O2, nitrogen and heat.
The other very used method for liquid product is hot filling. The product is
pasteurized, kept hot in holding tank, filled hot into the container or other kind
of packaging and sealed/closed. Hot filling of products must be monitored for
temperature.

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FINAL PRODUCT
It is important to emphasize that once processed and packed food must be
avoided to any cross-contamination which might lead to spoilage or health risk.
Finished products must be undertaken to some monitoring to verify the
appropriate processing and to determine their quality and complying with
regulation. It is important that methods should be quick and reliable.

CONCLUSIONS
Despite all activities for food protections the industry meets many problems
connected with lack of knowledge in complex food matrix as well as in the
cooperation with stakeholders. Based on our discussion we found that there are
some very interesting research topics for industry e.g. Alicyclobacillus detections
in soft drinks, better detection of Campylobacter in drinking water in broiler
houses and improving microbiological quality of water, biofilms in processing,
comparison detections methods for catechins in tea, which can be used to inhibit
spoilage bacteria and pathogens, improving safety and shelf-life of egg products
in different packaging materials and spore formers e.g. Bacillus cereus in
complex matrix such as liquid chocolate syrup.
The industry needs better communication with research and scientific
institutions to get better knowledge on e.g. unknown or less known
microorganisms, package materials and improved risk assessment. Furthermore,
the industry needs also better cooperation with food chain stakeholders
especially in the primary production to improve trust and reduce costs in
controlling materials entering the process plants. The big challenge in the
controlling area are quick tests results using reliable method of checking the
hygiene and therefore money and knowledge should be put into development of
quick detection methods e.g. biochemical methods.

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UTILIZATION OF MOLECULAR TECHNIQUES

FOR THE IDENTIFICATION OF SALMONELLA
FROM FOODSTUFFS
Recep ibik1, Tom Majtn2, Marcela Malcov3, Marketa Pospilov4,
Merle Rtsep5, Valerija irovnik6, Veronika Turkov7 & Il Var8
1
Faculty of Veterinary Medicine, Uludag University, Bursa, Turkey
2
Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia
3
Veterinary Research Institute, Brno, Czech Republic
4
National Institute of Public Health, Brno, Czech Republic
5
Bio-competence Centre of Healthy Dairy Products, Tartu, Estonia
6
Ljubljanske mlekarne, Ljublijana, Slovenia
7
University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic
8
School of Agriculture, University of ukurova, Adana, Turkey

INTRODUCTION
Detection and identification of pathogens on live food animals and plants as well
as in processed food and water is essential for ensuring the safety of food
products for human consumption. Foodborne illnesses are defined as diseases,
usually either infectious or toxic in nature, resulting from the consumption of
food contaminated with pathogenic bacteria, viruses, parasites or their toxins.
Such contamination usually arises from improper handling, preparation or food
storage. Good hygiene practices before, during, and after food preparation can
reduce the chances of contracting an illness. The action of monitoring food to
ensure that it will not cause foodborne illness is known as food safety. Recently,
reductions in the incidence of some foodborne infections in the USA and
European countries have occurred, in part caused by implementation of Hazard
Analysis Critical Control Point (HACCP) program aimed at improving food
safety through the improved science-based regulation of food production and
quality control. HACCP programs are designed to identify and prevent
biological (i.e. pathogenic bacteria), chemical (i.e. toxins) or physical (i.e.
metals) risks posed by contaminated food.

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Most common bacterial foodborne pathogens are Salmonella spp., Listeria

monocytogenes, Campylobacter spp. and Escherichia coli O157:H7. From
further foodborne bacterial threats we should mention Bacillus cereus, Coxiella
burnetii, Shigella spp., Streptococcus spp., Vibrio spp. and Yersinia enterocolitica.
Salmonella is a rod-shaped, Gram-negative enterobacteria that causes typhoid
fever, paratyphoid fever and foodborne illness. They are motile and produce
hydrogen sulfide. At least 2400 different serovars of Salmonella exist in the
nature among these nearly 20 types are responsible from human salmonellosis.
They are spread in intestines of different animals including mammals, birds,
snakes, turtles and lizards. Hens, chickens, gooses, ducks, pheasants, pigeons,
cattle and swine are often infected with salmonella. People becomes infected
directly with eggs, meat (poultry, pork, beef), milk and dairy products made
from raw milk or from milk that was thermal treated with low temperatures
(below pasteurization temperature), ice cream, chocolate, waters, vegetables,
fruits, sprout seeds, mussel, shrimp and mollusc. Salmonella was named after
Daniel Elmer Salmon, an American veterinary pathologist who, together with
Theobald Smith first discovered the bacterium in 1885. The scientific
classification of Salmonella was:
Kingdom:
Phylum:
Class:
Order:
Family:
Genus:

Bacteria
Proteobacteria
Gamma Proteobacteria
Enterobacteriales
Enterobacteriaceae
Salmonella.

Every year, approximately 40,000 cases of salmonellosis are reported in the

United States. Because many milder cases are not diagnosed or reported, the
actual number of infections may be thirty or more times greater. Salmonellosis is
more common in the summer than winter. Children are the most likely to get
salmonellosis. Young children, the elderly, and the immunocompromised are the
most likely to have severe infections. It is estimated that approximately 600
persons die each year with acute salmonellosis.
According to EC regulation for safety foods (Commission regulation (EC) No.
2073/2005 of 15. November 2005 on microbiological criteria for foodstuffs)

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Salmonella must be absent in 25 g except by minced meat and meat preparations

made from poultry meat intended to be eaten cooked, minced meat and meat
preparations made from other species than poultry intended to be eaten cooked,
mechanically separated meat. In this case Salmonella must be absence in 10 g.

BIOFILM FORMATION BY SALMONELLA

Salmonella Enterica, a cause of bacterial gastroenteritis, has reached world wide
distribution. As other inhabitants of human and animal gastrointestinal tract have
evolved special mechanism for persisting outside of host, surviving the
environment and disseminate along the food chain. This benefit consists in the
ability to form specialized surfaces structures with adhesives abilities which
leads to the formation of highly organized bacterial community, a biofilm. A
biofilm is a community of microbes embedded in an organic polymer matrix,
adhering to a surface. Its formation is an organized multi-step process which
leads to the formation of multi-cellular structure and is a main virulence
determinant in many bacterial infections. In general, the specific physiology of
biofilms especially slow growth and the barrier function of the extracellular
biofilm matrix determine resistance to antibacterial compounds.
Structures involving in multi-cellular behavior and subsequent biofilm formation
are composed of protein or of polysaccharides. The most important are
adhesives structures; thin aggregative fimbriae (or curlifimbriae), voluminous
exopolymer cellulose and newly described capsular polysaccharide. Those
structures contribute to the three dimensional structure of biofilm in cooperation
with many regulators and biofilm associated proteins. Curli fimbriae and
cellulose are the most important component of extra-cellular matrix with
protective function. Increasing resistance to antibiotics is an important event in
bacterial species especially in bacteria causing infectious diseases in humans and
animals. Genes of antibiotic resistance may influence changes in the cell surface
properties, such as hydrophobicity and charge, which are relevant to the initial
stages of biofilm formation and may contribute to the multi-cellularity behavior.

ISOLATION AND IDENTIFICATION OF SALMONELLA

Salmonella is usually isolated on Mac Conkey agar, XLD agar or DCA agar.
Because they cause intestinal infections and are greatly outnumbered by the
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bacteria normally found in the healthy bowel, primary isolation requires the use
of a selective medium, so use of a relatively non-selective medium such as
CLED agar is not often practiced. Numbers of Salmonella may be so low in
clinical samples that stools are routinely also subjected to enrichment culture
where a small volume of stool is incubated in a selective broth medium, such as
selenite broth or Rappaprt Vassiliadis soya peptone broth overnight. These
media are inhibitory to the growth of the microbes normally found in the healthy
human bowel, while allowing salmonellae to become enriched in numbers.
Salmonella may then be recovered by inoculating the enrichment broth on one or
more of the primary selective media. On blood agar, they form moist colonies
about 2 to 3 mm in diameter. They usually do not ferment lactose.
Traditional methods, though reliable and efficient, require several days to weeks
before results are obtained. Furthermore, phenotypic properties by which the
bacteria are identified may not always be expressed; and when expressed, they
may be difficult to interpret and classify. Another disadvantage of traditional
methods is that cells which are viable, but otherwise non-culturable cannot be
detected, e.g. some stressed Campylobacter spp. These methods therefore do not
meet requirements of making rapid assessments of microbiological safety
Classical methods of isolation and identification of Salmonella in foodstuffs
samples are chosen in accordance with ISO 6579 standard and can require up to
7 and at least 4 d. Method includes primary and secondary selective enrichment
of foodstuffs, isolation and identification of isolates with morphological,
biochemical and serological tests. Types of Salmonella tests available:
o
o
o

Conventional culture: Reference method v. optimum method for sample

type/purpose
Rapid Methods: IMS, ELISA, PCR, Conductance Impedance not fully
optimised for faeces and environmental samples in all labs
Serology: Eg. ELISA for SE, STM, Mix ELISA.

Meanwhile since the conventional methods are time consuming and results can
be obtained in 4 to 7, studies are conducted on the development of new and
quicker methods. PCR-based methods offer theoretically many advantages over
traditional microbiological methods but they should be evaluated prior
implementation in routine testing. Appropriate procedure of PCR method is

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faster than standard microbiological methods, it has the same relative accuracy,
relative sensitivity and relative specificity and means good basis for further
inter-laboratory validation.

SEROTYPING AND PHAGE TYPING

Historically, the classification of Salmonella enterica into strains has been based
on methods such as biotyping, serotyping, antibiotic resistance profiles phage
typing, and plasmid typing. Salmonella serotypes are defined on the basis of
somatic (O) antigens which define the serogroup and flagellar (H) factor antigens,
both are present in the cell wall of Salmonella. More than 2,400 serovars of
Salmonella exist and the prevalence of the different serovars changes over time
but S. Enteritidis and S. Typhimurium are the most frequently reported serovars.
The combination of three PCRs enables to completely serotype organisms
belonging to the Salmonella species. Two multiplex PCRs distinguish the most
common first- and second-phase antigens. A third multiplex PCR identify the
most common serogroups (O:B; O:C1; O:C2; O:D and O:E). This method was
found to be very helpful in the laboratory as an alternative method for typing
strains causing outbreaks, and it can be used to supplement conventional
serotyping, since it is also applicable to motionless and rough strains.
Phage typing of salmonella has the longest tradition. The first system was
established in 1943. The basis of Anderson phage typing system is a collection
of Salmonella phages which were propagated on particular hosts of the species
S. Typhimurium. The Anderson typing phages used worldwide today are
aliquots of phage stocks prepared decades ago. In 1987, it was described a phage
typing scheme for S. Enteritidis. The most common phage types of S. Enteritidis
varies between countries; while phage type (PT) 4 is reported to be dominant in
most countries in Western Europe, PT 8 is common in North America and also a
few European countries.

IDENTIFICATION OF SALMONELLA BY MOLECULAR TECHNIQUES

Alternative rapid methods have been developed to overcome problems such as
time- and labor- intensiveness of the traditional approach. Based on varying
analysis principles and targets, these methods can be divided into a number of
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categories, i.e. modified and automated conventional methods, bioluminescence,

cell counting using flow cytometry and immunological methods. The most
promising are those methods based on direct analysis of nucleic acid from
foodborne pathogens. However, their common disadvantage is inability to
distinguish between vital and dead cells. Several methodologies with different
underlying techniques are based on nucleic acids (mainly DNA) detection:
o

Nucleic acid hybridization Hybridization techniques are based on the

formation of double stranded complexes between complementary
molecules of either DNA or RNA. DNA or RNA probes in a single
stranded form are used for the analysis. The specificity of a
hybridization assay is completely controlled by the nucleotide sequence
of the detection probe. The first step in these genetic methods is the
isolation of nucleic acids, usually followed by labeling (mainly
fluorescent). Following hybridization step allows the labeled sample
DNA to hybridize to probe DNA. Washing and analysis follow.
Microarray technology is the most sophisticated form of nucleic acids
hybridization principle.
Amplification methods Because of the greater sensitivity, DNAbased methods which include an amplification step have become
increasingly popular. The most popular method of amplification is the
polymerase chain reaction (PCR) technique. In this method, numerous
thermal cycles are applied and special thermostable DNA synthesizing
enzymes are used, allowing exponential growth of target DNA
molecules in each cycle. Starting from a single target DNA or RNA
sequence, more than one billion product sequences can routinely be
synthesized within few hours. This quantity of DNA can be either
visualized on special gel or used in other downstream procedures. Many
PCR protocols for the detection of foodborne bacteria and viruses have
been described
Molecular subtyping methods can identify different strains within a
species, generating data useful for taxonomic or epidemiologic
purposes. Nucleic acid-based methods have the advantage over
phenotypic identification methods of not being influenced by
environmental conditions of the cells, because the nucleotide sequence
of the DNA does not change during growth. In the restriction fragment
length polymorphism RFLP technique, DNA is cleaved by restriction

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enzymes and the resulting fragments are separated by gel

electrophoresis. In Pulsed-field gel electrophoresis (PFGE) restriction
enzymes digest the complete genome and large DNA molecules are
resolved by continuous reorientation of the electric field during gel
electrophoresis. This technique is considered to have superior
reproducibility and resolving power when compared to previous and
some other similar techniques.
The microarray technology currently dominates gene expression profiling
studies, but begins to play an increasingly important role in genomic studies,
toxicology research or drug discovery. Additionally, this technology provides
valuable attributes for the needs of multiple organisms identification. The
potential of determining several thousands of unique and precisely defined
genetic features (such as distinctive features of individual species or organisms)
in a single analysis within relatively short time period offers an excellent
solution for the needs of simultaneous detection of several pathogens with their
varying forms. This potential has not escaped the interest of several research
groups and several works have been published on this topic.
Two main types of DNA arrays can be applied in detection of multiple
organisms: genomic microarrays and oligochips. Genomic microarrays comprise
whole genome, genomic fragments from a cDNA library or ORFs from a strain
of microorganism. Genomic microarrays can also be adapted to include genomic
DNA from several related strains of a microorganism, referred to as a mixed
genomic microarray. Genomic microarrays are useful for pathogen identification
based on fingerprinting. Oligonucleotide arrays generally involve
oligonucleotides between 18 and 70 nucleotides in length. They are often used
for pathogen detection and genomic analysis because these arrays can consist of
multiple sequence variants of a target gene allowing for more sensitive
detection. Therefore, DNA microarrays can be adapted to combine effective
strategies for detecting and identifying pathogenic isolates from food samples,
while providing information for the characterization and potential determination
of the pathogenicity of the isolate.
The most severe challenge of the detection of pathogens in food systems using
microarray analysis is the sample preparation step. Particularly, DNA or RNA
extraction from pathogens present in complex food samples as well as detection

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of foodborne pathogens present in low levels could be extremely challenging.

DNA microarray detection of amplified PCR products was 32 times more
sensitive than traditional end-point detection: agarose gel electrophoresis. Thus
oligochip combined with PCR amplification could be a sensitive method for
detecting foodborne pathogens. For the purpose of food safety surveillance,
researchers were able to detect 55 CFU/ml of enterohemorrhagic E. coli isolated
from chicken rinse.
Despite significant improvements in speed, sensitivity, complexity of obtained
information advantage over traditional methods, one of the aspects of microarray
analysis with optical detection is its relatively intense procedural complexity.
However, this complexity can be optimized. Several works addressing the use of
biosensors based on non-optical detection principle have been published. The
greatest advantage of biosensor is the omission of labeling step in sample
preparation, significantly reducing cost and labeling-associated problems.
Furthermore such biochips can be used as simple portable micro-devices for
convenient real-time pathogen detection.

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ANTIMICROBIAL EFFECTS IN FOOD AND

PREVENTION OF CONTAMINATION IN
FOOD INDUSTRY
Pirje Htt1, Epp Songisepp2, Metin Guldas3, Terje Tamme4,
Fatma Seda Ormanci5, Naim Deniz Ayaz5, Figen Korel6, Kaarina Aarnisalo7,
Helena Hradeck8 & Marika Mikelsaar1
1
Department of Microbiology, Tartu University, Tartu, Estonia
2
Bio-Competence Centre of Healthy Dairy Products, Tartu, Estonia
3
Dept. of Food Technology, Uludag University, Bursa, Turkey
4
Department of Food Science and Hygiene, Institute of Veterinary Medicine and
Animal Sciences, Estonian University of Life Sciences, Tartu, Estonia
5
Faculty of Veterinary Medicine, Ankara University, Ankara, Turkey
6
zmir Institute of Technology, Urla, zmir, Turkey
7
VTT, Espoo, Finland
8
Veterinary Research Institute, Brno, Czech Republic

INTRODUCTION
The most commonly recognized food-borne infections are caused by
Campylobacter jejuni and Salmonella spp. In the past 25 years, Listeria
monocytogenes has become increasingly important as a food-associated
pathogen. Though, an annual incidence of human listeriosis is between two and
ten reported cases per million in EU countries. Because of its high case fatality
rate, listeriosis ranks among the most frequent causes of death due to food-borne
illness. L. monocytogenes and C. jejuni has been shown to adapt to different
environmental stress factors, including disinfectants. Salmonella enteritidis, C.
jejuni and L. monocytogenes may also form a biolfim which helps to survive in the
environment. For that reason, the food industrial hygiene has become important to
avoid contamination of raw food and food products with these pathogens.
The aims of the present report are to describe and discuss different antimicrobial
methods that prevent the contamination and transmission of food-borne
pathogens. The antimicrobial methods could be divided such as physical
activities (agents) and chemical agents (e.g. sanitizers, antibiotics).

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PHYSICAL ACTIVITIES (AGENTS)

CLEANING OF EQUIPMENT
Since cleaning and sanitizing may be the most important aspect of a sanitation
program, sufficient time should be given to outline proper procedures, and
parameters. Detailed procedures must be developed for all food-product contact
surfaces (equipment, utensils, etc.) as well as for non-product surfaces such as: nonproduct portions of equipment, overhead structures, shield, walls, ceilings, lighting
devices, refrigeration units, heating, ventilation and air conditioning (HVAC)
systems, and anything else which could impact food safety.
Equipment cleaning methods can be categorized with regard to cleaning method as
follows: a) Mechanical cleaning often referred to as cleaning-in-place (CIP) requires
no disassembly or partial disassembly; b) Cleaning-out-of-place (COP) can be
partially disassembled and cleaned in specialized COP pressure tanks & c) Manual
cleaning requires total disassembly for cleaning and inspection.
THERMAL SANITIZATION
Thermal sanitization involves the use of hot water or steam for a specified
temperature and contact time. On the other hand, the effectiveness of an
antimicrobial agent depends on its chemical and physical state, treatment conditions
(such as water temperature).
Hot-water sanitizing, through immersion (small parts, knives, etc.), spray
(dishwashers), or circulating systems, is commonly used. The time required is
determined by the temperature of the water. Typical industrial requirements for use
of hot water in dishwashing and utensil sanitizing applications specify: immersion
for at least 30 sec. at 77 C for manual operations; a final rinse temperature of 74 C
in single tank, single temperature machines and 82 C for other machines.
The primary advantages of hot-water sanitization are: relatively inexpensive,
easy to apply and readily available, generally effective over a broad range of
microorganisms, relatively non-corrosive, and penetrates into cracks and
crevices. Hot-water sanitization is a slow process which requires come-up and
cool-down time; can have high energy costs; and has certain safety concerns for
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employees. The process also has the disadvantages of forming or contributing to

film formations, and shortening the life of certain equipment or parts thereof
(gaskets, etc.).
FILTRATION
The guarantee of food quality, and particularly food safety, places an onerous
responsibility on all food processors. EU Regulations (EN 2900029004) define
this responsibility not only in terms of the quality of the end produce, but also
the precautions which must be taken in the preparation and processing of foods
to minimize the risk of contamination.
For all food products, and especially for those which are heat sensitive (e.g. milk
or egg-based) and subject to minimal heat treatment, all aspects of the process
must be rigorously controlled to prevent microbial contamination. This includes
not only the processing equipment, but also the processing environment
including the air, which may reduce product shelf life, and in certain
circumstances, pose a serious hazard to food safety.
While air filtration is now standard practice in food processing environments,
significant increases in aerial microbial counts may occur intermittently due to
failure to adhere to Good Manufacturing Practices and/or failure of the air
filtration system due to design, inadequate maintenance, malfunction, etc.
Frequent and effective monitoring of the air sterility, in the process environment
is therefore essential to alert processors of the potential risks, and the possible
need for corrective action. However, the lack of reliable quantitative air
sampling techniques, coupled to the uncertainty about the behaviour or control
of micro-organisms within air filtration systems, present serious obstacles to
effective control of processing environments.
FREEZING IN FOOD PRESERVATION
According to EU Directive 89/108, the temperature of quick-frozen foods must
be stable and maintained at -18 C or colder at all points. The temperature of
frozen foods must be -12 C or colder, and this temperature must be maintained.
Directive 89/108 requires that after quick-freezing the product temperature must
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be -18 C or colder after thermal stabilization. This is useful wording, as it

means that the freezing process can be stopped before the core temperature is
-18C. It is sometimes recommended that for practical purposes the freezing
process can be stopped when the core temperature is -10 C or colder. For most
products, this would mean that the average temperature is the same or colder
than the stipulated storage temperature.
The freezing process can be considered ended when all points in the food are
-18 C or colder. The length of this stabilization period depends on the type of
food (thermal conductivity), how it is packaged, and how the temperature
distribution in the food is at the beginning of stabilization. In some countries,
there has been legislation requiring that a core temperature of -18 C must be
reached in less than 1 hour for small pieces (steaks), 2 to 6 hours for medium
sized pieces, e.g. poultry and roasts, and 24 hours for large packs, e.g. cartons
with boned meat. Frozen foods must be stored at -12 C or colder, quick-frozen
foods at -18 C or colder. Some frozen foods, e.g. beef, broilers, butter, have a
fairly long storage life even at -12 C, while foods such as lean fish require
storage temperatures around -28 C in order to reduce the quality loss and have a
long storage life.
The EU Directive 92/1 requires that storage facilities must have installed a
temperature recording device. The refrigerating capacity must be sufficient to
obtain a maximum difference between evaporator temperature and room
temperature of 7 C. The QFF Directive requires that the temperature of quickfrozen foods must be maintained at -18 C or colder at all points in the product,
with possibly brief upward fluctuations of no more than 3 C during transport. It
is often recommended, e.g. given in the cooking instructions on the label, that if
frozen foods must be thawed before cooking, this should be done in a refrigerator.
PASTEURIZATION
Pasteurization is achieved by a treatment involving (i) a high temperature for a
short time (at least 72 C for 15 s); (ii) a low temperature for a long time (at least
63 C for 30 min); or (iii) any other combination of time and temperature
conditions to obtain an equivalent effect, such that the products show, where
applicable, a negative reaction to an alkaline phosphatase test immediately after
such treatment.
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Ultrahigh temperature (UHT) treatment is achieved by a treatment (i) involving

a continuous flow of heat at a high temperature for a short time (not less than
135 C in combination with a suitable holding time) such that there are no viable
micro-organisms or spores capable of growing in the treated product when kept
in an aseptic closed container at ambient temperature; and (ii) sufficient to
ensure that the products remain microbiologically stable after incubating for 15
days at 30 C in closed containers, or for 7 days at 55 C in closed containers, or
after any other method demonstrating that the appropriate heat treatment has
been applied.
PRESSURE
Food preservation using high pressure is a promising technique in food industry as it
offers numerous opportunities for developing new foods with extended shelf-life,
high nutritional value and excellent organoleptic characteristics. High pressure is an
alternative to thermal processing. The resistance of microorganisms to pressure
varies considerably depending on the pressure range applied, temperature and
treatment duration, and type of microorganism. Generally, gram-positive bacteria
are more resistant to pressure than Gram-negative bacteria, moulds and yeasts; the
most resistant are bacterial spores. The nature of the food is also important, as it may
contain substances which protect the microorganism from high pressure. Despite the
introduction of food standards obligatory in EU countries, epidemiologists believe
that 75% of food-borne diseases are caused by bacteria.
High pressure is an alternative to thermal processing. The resistance of
microorganisms to pressure varies considerably depending on the pressure range
applied, temperature and treatment duration, and type of microorganism. The first
high pressure processed food products appeared in Japan in the early 1990s. In
Europe, high pressure processing (HPP) of foods was rather at the stage of research
or pilot production in the last decade. EU legislation included HPP foods in the
novel food category. EC Novel Food regulation (EC 258/97) has introduced a
statutory pre-market approval system for novel foods across the whole of the
European Union. Recently, rapid progress of HPP toward commercial exploitation
has been achieved, but still the process requires close collaboration between
researchers, food and equipment manufacturers, as well as proper financial support.

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SONICATION
Ultrasound is energy generated by sound waves of 20,000 or more vibrations per
second. Presently, most developments of ultrasonic (sonication) for food
applications are nonmicrobial in nature. High frequencies in the range of 0.1 to
20 MHz, pulsed operation and low power levels (100 mW) are used for nondestructive testing. Industrial applications include texture, viscosity and
concentration measurements of many solid or fluid foods; composition
determination of eggs, meats, fruits and vegetables, dairy and other products;
thickness, flow level and temperature measurements for monitoring and control
of several processes; and non-destructive inspection of egg shells and food
packages. Researchers also listed direct process improvements such as cleaning
surfaces, enhancement of dewatering, drying and filtration, inactivation of
microorganisms and enzymes, disruption of cells, degassing of liquids,
acceleration of heat transfer and extraction processes and enhancement of any
process dependent upon diffusion. It is evident that ultrasound technology has a
wide range of current and future applications in the food industry.
The bactericidal effect of ultrasound is generally attributed to intracellular
cavitation. It is proposed that micro-mechanical shocks are created by making
and breaking microscopic bubbles induced by fluctuating pressures under the
ultrasonication process. These shocks disrupt cellular structural and functional
components up to the point of cell lysis.
PULSED ELECTRIC FIELD
Pulsed electric field (PEF) processing is a non-thermal method of food
preservation that uses short bursts of electricity for microbial inactivation and
causes minimal or no detrimental effect on food quality. PEF can be used for
processing liquid and semi-liquid food products. PEF processing offers high
quality fresh-like liquid foods with excellent flavour, nutritional value, and
shelf-life. Since it preserves foods without using heat, foods treated this way
retain their fresh aroma, taste, and appearance. PEF processing involves treating
foods placed between electrodes by high voltage pulses in the order of 2080 kV
(usually for a couple of microseconds). The applied high voltage results in an
electric field that causes microbial inactivation.

164

ELECTROPORATION
This method is used to transform a wide variety of microorganisms and requires
a brief exposure to a high-voltage electric field to introduce genetic material into
a microorganism. Electroporation is the most popular technique for introducing
genetic material in microorganisms because of its simplicity, efficacy and
versatility. Electroporation is the phenomenon in which a cell exposed to high
voltage electric field pulses temporarily destabilizes the lipid bilayer and
proteins of cell membranes. The plasma membranes of cells become permeable
to small molecules after being exposed to an electric field, and permeation then
causes swelling and eventual rupture of the cell membrane. The main effect of
an electric field on a microorganism cell is to increase membrane permeability
due to membrane compression and poration.
OHMIC HEATING
Ohmic heating is an advanced thermal processing method wherein the food material,
which serves as an electrical resistor, is heated by passing electricity through it.
Electrical energy is dissipated into heat, which results in rapid and uniform heating.
Ohmic heating is also called electrical resistance heating, Joule heating, or electroheating, and may be used for a variety of applications in the food industry. Like
thermal processing, ohmic heating inactivates microorganisms by heat. Additional
non-thermal electroporation type effects have been reported at low-frequency (50
60 Hz), when electrical charges can build up and form pores across microbial cells
however, it is not necessary to claim such effects since heating is the main
mechanism. The shelf life of ohmically processed foods is comparable to that of
canned and sterile, aseptically processed products. In various countries, including
Italy, Greece, France, ohmic heating has been used to produce a low-acid particulate
product in a can, as well as pasteurized liquid egg.

CHEMICAL AGENTS
SANITIZERS
The selection of a sanitizer depends on the type of equipment to be sanitized, the
hardness of the water, the application equipment available, the effectiveness of
the sanitizer under site conditions, and cost. Sanitizing compounds which
165

contain phenols impart strong undesirable odors and flavors to foods and should
not be used. Thorough cleaning is essential before using a sanitizer. Sanitizers
are less effective when food particles or dirt are present on equipment surfaces.
Typical disinfecting agents used in the food industry include quaternary
ammonium compounds (QACs), chlorine, alcohols, hydrogen peroxide,
peracetic acid and iodofors.
Legislation concerning disinfectants include Directive 98/8/EC of the European
Parliament and of the Council on the placing on the market of biocidal products.
Member States had to transpose the rules before 14 May 2000 into national law.
The Biocidal Product Directive aims to harmonise the European market for
biocidal products and their active substances and to protect humans, animals and
the environment (http://ec.europa.eu/environment/biocides/index.htm).The new
REACH Regulation (EC) No 1907/2006 of the European Parliament and of the
Council and Directive 2006/121/EC of the European Parliament and of the
Council were published in the Official Journal on 30 December 2006. The aims
of the proposed new Regulation are to improve the protection of human health
and the environment while maintaining the competitiveness and enhancing the
innovative capability of the EU chemicals industry. REACH will also give
greater responsibility to industry to manage the risks from chemicals and to
provide safety information on the substances. REACH will enter into force on 1
June 2007 (http://ec.europa.eu/enterprise/reach/index_en.htm).
Many commonly used disinfectants have been shown to be effective against
L. monocytogenes in suspension. Other factors affecting on efficacy of
disinfectants against L. monocytogenes include e.g. the concentration and effect
time as well as temperature and pH of the use solution. L. monocytogenes strains
can vary in resistance against disinfectants. L. monocytogenes has been shown to
adapt to different environmental stress factors, including disinfectants. The
adaptation may occur especially when the disinfectant is present in the
environment in sub lethal amounts. Differences in the susceptibility and adaptive
response of strains to disinfectants have been observed, and it has been
suggested that such differences could influence the survival of the strains in the
food processing plants. Generally, the recommendations of use-concentration of
disinfectants should be followed. However, these may not always be sufficient
against L. monocytogenes.

166

Future investigations include further studies on mechanisms of adaptation and

differences between strains. Besides disinfectants, other technologies for
reducing use of water and chemicals in disinfection in food plants should be
investigated.
ANTIBIOTICS
Antibiotics are chemical substances, either produced naturally by microorganisms or manufactured synthetically, that are lethal to other bacteria.
Antibiotics are used for the treatment of bacterial infections in both humans and
animals. The emergence of antibiotic resistance as a serious problem in human
medicine has prompted concerns about the public health implications of
antibiotic use in agriculture. Antimicrobial resistance has emerged among
Campylobacter mainly as a consequence of the use of antimicrobial agents,
especially fluoroquinolones, macrolides, and tetracyclines in food animal
production. In the recent study a high number of Campylobacter isolates from
Estonian poultry had increased antimicrobial resistance to ciprofloxacin,
tetracycline. The use of the antibiotics as growth promoting agents is be
forbidden in EU countries since January 2006 (Regulation (EC) No 1831/2003
of the European Parliament and of the Council).

FUTURE NEEDS
New technologies are needed for surveillance of food-borne disease and food
monitoring. These include typing pathogens, different in vitro, animal and
clinical testing. New research and development are required in food industry
such as application of antimicrobial surface materials and green technologies
(enzymes).

FURTHER READING
1. Aarestrup, F.M. & Engberg, J. 2001. Antimicrobial resistance of
thermophilic Campylobacter. Vet. Res., 32, 311321.
2. Aarnisalo, K., Salo, S., Miettinen, H., Suihko, M.-L., Wirtanen, G., Autio,
T., Lundn, J., Korkeala, H. & Sjberg, A.-M. 2000. Bactericidal

167

efficiencies of commercial disinfectants against Listeria monocytogenes on

surfaces. J. Food Safety, 20, 237250.
3. Aarnisalo, K., Lundn, J., Korkeala, H. & Wirtanen, G. 2007. Susceptibility
and adaptive responses of Listeria monocytogenes strains to disinfectants
and chlorinated alkaline cleaners in cold temperatures. LWT, 40, 10411048.
4. Aase, B., Sundheim, G., Langsrud, S. & Rrvik, L. 2000. Occurrence of and
possible mechanism for resistance to a quaternary ammonium compound in
Listeria monocytogenes. Int. J. Food Microbiol., 62(1/2), 5763.
5. Alter, T. & Scherer, K. 2006. Stress response of Campylobacter spp. and its
role in food processing. J. Vet. Med. B Infect. Dis. Vet. Publ. Health., 53,
351357.
6. Anon. 1998. Guide to Minimize Microbial Food Safety Hazards for Fresh Fruits
and Vegetables. U.S. Department of Health and Human Services. Food and
Drug Administration, Center for Food Safety and Applied Nutrition (CFSAN),
http://www.foodsafety.gov/~dms/prodguid.html#ii.
7. Anon. 2000. Kinetics of Microbial Inactivation for Alternative Food Processing
Technologies Ultrasound. U. S. Food and Drug Administration Center for Food
Safety and Applied Nutrition, http://www.cfsan.fda.gov/~comm/ift-us.html.
8. Anon. 2003. Codex Alimentarius Commission. Report of the Thirty-Fifth
Session of the Codex Committee on Food Hygiene. Alinorm 03/13A. Session of
the Codex Committee on Food Hygiene.
9. Anon. 2004. Hygiene of foodstuffs. Regulation (EC) No. 852/2004.
10. Anon. 2005. Legal responsibilities of food businesses and food handlers 2005
legislation (EC Directive 852/2004 Hygiene of foodstuffs).
11. Anon. 2006b. Guide to Minimize Microbial Food Safety Hazards of Fresh-cut
Fruits and Vegetables, http://www.cfsan.fda.gov/~dms/prodgui2.html.
12. Best, M., Kennedy, M.E. & Coates, F. 1990. Efficacy of a variety of
disinfectants against Listeria spp. Appl. Environ. Microbiol., 56(2), 377380.
13. Earnshaw, A.M. & Lawrence, L.M. 1998. Sensitivity to commercial
disinfectants, and the occurrence of plasmids within various Listeria
monocytogenes genotypes isolated from poultry products and the poultry
processing environment. J. Appl. Microbiol., 84, 642648.

168

14. El-Kest, S.E. & Marth, E.H. 1988. Temperature, pH and strain of pathogen
as factors affecting inactivation of Listeria monocytogenes by chlorine. J.
Food Prot., 51, 622625.
15. Endtz, H.P., Ruijs, G.J., van Klingeren, B., Jansen, W.H., van der Reyden,
T. & Mouton, R.P. 1991. Quinolone resistance in Campylobacter isolated
from man and poultry following the introduction of fluoroquinolones in
veterinary medicine. J. Antimicrob. Chemother., 27, 199208.
16. Engberg, J., Aarestrup, F.M., Taylor, D.E., Gerner-Smidt, P. & Nachamkin,
I. 2001. Quinolone and macrolide resistance in Campylobacter jejuni and C.
coli: resistance mechanisms and trends in human isolates. Emerg. Infect.
Dis., 7, 2434.
17. Jacobs-Reitsma, W.F. 1997. Aspects of epidemiology of Campylobacter in
poultry. Vet. Quart., 19, 113117.
18. Jemmi, T. & Stephan, R. 2006. Listeria monocytogenes: food-borne
pathogen and hygiene indicator. Rev. Sci. Tech., 25, 571580.
19. Heir, E., Lindstedt, B.-A., Rtterud, O.-J., Vardund, T., Kapperud, G. &
Nesbakken, T. 2004. Molecular epidemiology and disinfectant susceptibility
of Listeria monocytogenes from meat processing plants and human
infections. Int. J. Food Microbiol., 96, 8596.
20. Hoover, D.G. 1997. Minimally processed fruits and vegetables: Reducing
microbial load by nonthermal physical treatments. Food Technol., 51, 6669, 71.
21. http://ec.europa.eu/environment/biocides/index.htm
22. http://ec.europa.eu/enterprise/reach/index_en.htm
23. Komorowski, E.D. 2006. New dairy hygiene legislation. Society of Dairy
Technology, http://www.blackwell-synergy.com/doi/pdf/10.1111/j.14710307.2006.00245.x, 5 p.
24. Krysinski, E.P., Brown, L.J. & Marchisello, T.J. 1992. Effect of cleaners and
sanitizers on Listeria monocytogenes attached to product contact surfaces.
J. Food Prot., 55, 246251.
25. Lundn, J. Autio, T., Markkula, A., Hellstrm, S. & Korkeala, H. 2003.
Adaptive and cross-adaptive responses of persistent and non-persistent
Listeria monocytogenes strains to disinfectants. Int. J. Food Microbiol., 82,
265272.

169

26. Mafu, A., Roy, D., Goulet, J. & Magny, P. 1990. Attachment of Listeria
monocytogenes to stainless steel, glass, polyprophylene, and rubber surfaces
after short contact times. J. Food Prot., 53, 742746.
27. OCallaghan, D., Murphy, P., Lynch, D. & Coleman, S. 1999. Improved Food
Safety through Sterility of Air in Food Processing and Packaging. The Dairy
Products Research Centre Moorepark, http://www.teagasc.ie/research/reports/
dairyproduction/3989/eopr-3989.htm.
28. Piddock, L.J., Ricci, V., Stanley, K. & Jones, K. 2004. Activity of antibiotics
used in human medicine for Campylobacter jejuni isolated from farm
animals and their environment in Lancashire, UK. J. Antimicrob.
Chemother., 6, 303306.
29. Praakle-Amin, K., Roasto, M., Korkeala, H. & Hnninen, M.-L. 2007. PFGE
genotyping and antimicrobial susceptibility of Campylobacter in retail
poultry meat in Estonia. Int. J. Food Microbiol., in press.
30. Ramaswamy, R., Jin, T., Balasubramaniam, V.M. & Zang, H. 2003. Pulsed
Electric, Ohio State University, http://ohioline.osu.edu/fse-fact/pdf/0002.pdf.
31. Ramaswamy, R., Balasubramaniam, V.M.
http://ohioline.osu.edu/fse-fact/pdf/0004.pdf.

&

Sastry,

S.K.

2003.

32. Schmidt, R.H. 2003. Basic Elements of Equipment Cleaning and Sanitizing in
Food Processing and Handling Operations, FS14. Series of the Food Science
and Human Nutrition Department, Institute of Food and Agricultural Sciences,
University of Florida, http://edis.ifas.ufl.edu/FS077.
33. Smith, K.E., Bender, J.B. & Osterholm, M.T. 2000. Antimicrobial resistance
in animals and relevance to human infections. In: Nachamkin, I. & Blaser,
M.J. (Eds.) Campylobacter. 2nd ed. American Society for Microbiology,
Washington, D.C. Pp. 483495.
34. Tuncan, E.U. 1993. Effect of cold temperature on germicidal efficacy of
quaternary ammonium compound, iodofor and chlorine on Listeria. J. Food
Prot., 56, 10291033.
35. Wirtanen, G., Storgards, E. & Mattila-Sandholm, T. 2003. Biofilms. In:
Caballero, B., Trugo, L. & Finglas, P. (Eds.) Encyclopedia of Food Science
and Nutrition. Academic Press, London. Pp. 484489. ISBN: 0-12-227055-X.

170

APPENDIX 1: PARTICIPANT LIST

1. Esra Agel, Tbitak Marmara Research Center, Food Institute, Gebze,
Kocaeli, Turkey
2. Emine Aksan, University of Mustafa Kemal, Alahan, Antakya-Hatay, Turkey
3. Naim Deniz Ayaz, Faculty of Veterinary Medicine, Ankara University,
Ankara, Turkey
4. Hulya alikan, Ulker Gida San. Ve Tic. A, Istanbul, Turkey
5. Ziya Gkalp Ceylan, Faculty of Veterinary Sciences, Atatrk University,
Erzurum, Turkey
6. Recep ibik, Faculty of Veterinary Medicine, Uludag University, Bursa, Turkey
7. Aye Handan Diner Baysal, zmir Institute of Technology, Urla, zmir, Turkey
8. Helen Ehavald, AS Coca-Cola HBC Eesti, Tallinn, Estonia
9. Ivana Filipovi, University of Zagreb, Zagreb, Croatia
10. Metin Guldas, Karacabey Vocational School, Uludag University, Bursa, Turkey
11. Helena Hradecka, Veterinary Research Institute, Brno, Czech Republic
12. Pirje Htt, University of Tartu, Tartu, Estonia
13. Saziye Ilgaz, Atatrk Research Institute of Tea and Horticultural Products,
Rize, Turkey
14. Kadrin Juhkam, Estonian University of Life Sciences, Tartu, Estonia
15. Blent Kabak, School of Agriculture, University of ukurova, Adana, Turkey
16. Sylva Karpkov, Veterinary Research Institute, Brno, Czech Republic
17. Nee Asli Kisikkaya, Tbitak Marmara Research Center, Food Institute,
Gebze, Kocaeli, Turkey
18. Figen Korel, zmir Institute of Technology, Urla, zmir, Turkey
19. Iva Lovin Kukman, Mercator-Emba d.d., Ljubljana, Slovenia
20. Elbieta Makiw, National Food and Nutrition Institute, Warszawa, Poland
21. Tom Majtn, Institute of Molecular Biology, Slovak Academy of
Sciences, Bratislava, Slovakia
22. Marcela Malcov, Veterinary Research Institute, Brno, Czech Republic
23. Vida Mari, Perutnina Ptuj, Ptuj, Slovenia
24. Fatma Seda Ormanci, Faculty of Veterinary Medicine, Ankara University,
Ankara, Turkey

1/1

25. Glah Pekdemir, Abalolu Feed Soybean and Textile Company, zmir, Turkey
26. Barbara Pellis, Pivovarna Union d.d., Ljubljana, Slovenia
27. Metin Petek, University of Uludag, Bursa, Turkey
28. Julijana Polanc, Fructal, Ajdovina, Slovenia
29. Marketa Pospilov, National Institute of Public Health, Brno, Czech Republic
30. Mati Roasto, Estonian University of Life Sciences, Tartu, Estonia
31. Merle Rtsep, Bio-competence Centre of Healthy Dairy Products, Tartu, Estonia
32. Ajda alej, Tina d.o.o., Komenda, Slovenia
33. Jaroslav imek, Institute of Public Health in Ostrava, Ostrava, Czech Republic
34. Valerija irovnik, Ljubljanske mlekarne, Ljublijana, Slovenia
35. Epp Songisepp, Bio-competence Centre of Healthy Dairy Products, Tartu, Estonia
36. Terje Tamme, Estonian University of Life Sciences, Tartu, Estonia
37. Margarita Terentjeva, Latvian University of Agriculture, Jelgava, Latvia
38. Veronika Turkov, University of Veterinary and Pharmaceutical Sciences
Brno, Brno, Czech Republic
39. Il Var, School of Agriculture, University of ukurova, Adana, Turkey
40. Martina Weiss, Zito Group, Ljubljana, Slovenia
41. Kaarina Aarnisalo, VTT, Espoo, Finland
42. Marika Mikelsaar, University of Tartu, Tartu, Estonia
43. Johanna Maukonen, VTT, Espoo, Finland
44. Laura Raaska, VTT, Espoo, Finland
45. Jana Ramus, CCIS, Ljubljana, Slovenia
46. Berit Reinmller, KTH, Stockholm, Sweden
47. Ivan Rychlik, VRI, Czech Republic
48. Satu Salo, VTT, Espoo, Finland
49. Tiina Saron, Estonian Dairy Association, Viimsi, Estonia
50. Lotte Dock Steenstrup, BioCentrum-DTU, Lyngby, Denmark
51. Tiina Veskus, Tallinn University of Technology, Tallinn, Estonia
52. Elina Vihavainen, University of Helsinki, Helsinki, Finland
53. Raivo Vokk, Tallinn University of Technology, Tallinn, Estonia
54. Gun Wirtanen, VTT, Espoo, Finland

1/2

APPENDIX 2: SEMINAR PROGRAMME

Monday, January 22, 2007
9.009.15

Coffee

9.159.30

Welcome

9.3010.00

Biofilm formation in food processes;

Gun Wirtanen, VTT, Finland

10.0010.30

Microbial ecology in manufacturing paper-based

packaging materials for use in food industry;
Laura Raaska, VTT, Finland

10.3011.15

The relevance of psychrotropic spoilage microbes in food

hygiene;
Elina Vihavainen, University of Helsinki, Finland

11.1512.15

Lunch

12.1513.00

Enteric pathogens prevalence in food products and

mechanisms of suppression by probiotic lactobacilli;
Marika Mikelsaar, University of Tartu, Estonia

13.0013.45

Classification and differentiation of Salmonella sp. using

various DNA based techniques;
Ivan Rychlik, VRI, Czech Republic

13.4514.15

Coffee and tea

14.1515.00

Composition of intestinal microflora in health and disease

and its association with food-borne diseases;
Marika Mikelsaar, University of Tartu, Estonia

15.0017.30

Group works incl at least 30 min for preparation of

presentations

16.0017.30

Coffee and tea

17.3018.30

Group work presentations

19.0021.00

Dinner

2/1

Tuesday, January 23, 2007

9.009.30

Effect of maintenance routines in food processing on

production hygiene especially on Listeria contamination
routes; Kaarina Aarnisalo, VTT, Finland

9.3010.00

Hygienically integrated systems in food processing to

avoid microbiological contamination;
Lotte Dock Steenstrup, BioCentrum-DTU, Denmark

10.0010.30

Coffee

10.3011.00

Methods for evaluation of process hygiene;

Satu Salo, VTT, Finland

11.0012.00

Molecular techniques and microscopy in bacterial

detection and typing;
Johanna Maukonen, VTT, Finland

12.0013.00

Lunch

13.0014.00

Food safety legislation and food control systems

according to the new EU food law;
Tiina Saron, Estonian Dairy Association, Estonia

14.0014.30

Coffee

14.3015.30

Air microbiology;
Berit Reinmller, KTH, Sweden

15.3015.50

Concluding remarks

2/2

Series title, number and report

code of publication

VTT Symposium 248

VTTSYMP248
Author(s)

Wirtanen, Gun & Salo, Satu (eds.)

Title

MICROBIAL CONTAMINANTS & CONTAMINATION ROUTES

IN FOOD INDUSTRY
Abstract

Microbes able to establish and develop in the processing environment and on food
products pose a potential problem in terms of food safety and quality. Initially it is the
capability to adhere to a surface that gives an advantage towards the majority of microbes
being present. Other important capabilities are competitive growth and long survival.
Although pathogenic microbes rarely cause the spoilage of foodstuffs (due to their slow
or non-existent growth in foods at low temperatures) they and their toxic secondary
metabolites naturally form a health risk to the consumer. To guarantee the safety of
foodstuffs microbial contamination must be detected and traced as early as possible and
also at low levels. In addition, the safety risks related to the microbial contamination must
be evaluated as accurately as possible. To establish corrective (and for the future also
preventive) actions exact knowledge on the process hygiene level of the production plant
is needed. Characteristics of important contaminants, contamination routes and factors
enhancing microbial contamination of process line are important items covered in this
Microbial contaminants and contamination routes in food industry -seminar by international
lecturers and participants who have provided research studies and group work material
published in this seminar proceedings.

ISBN

978-951-38-6319-7 (soft back ed.)

978-951-38-6320-3 (URL: http://www.vtt.fi/publications/index.jsp)
Series title and ISSN

Project number

VTT Symposium
0357-9387 (soft back ed.)
1455-0873 (URL: http://www.vtt.fi/publications/index.jsp)

8673

Date

Language

Pages

May 2007

English

170 p. + app. 4 p.

Name of project

Commissioned by

SAFOODNET

SAFOODNET; FP6-022808-2006

Keywords

Publisher

microbial contaminants, contamination routes,

pathogens, food industry, process hygiene,
quality, molecular biological methods,
packaging, antimicrobial effects

VTT Technical Research Centre of Finland

P.O. Box 1000, FI-02044 VTT, Finland
Phone internat. +358 20 722 4404
Fax +358 20 722 4374

ESPOO 2007

Publikationen distribueras av

This publication is available from

VTT
PL 1000
02044 VTT
Puh. 020 722 4404
Faksi 020 722 4374

VTT
PB 1000
02044 VTT
Tel. 020 722 4404
Fax 020 722 4374

VTT
P.O. Box 1000
FI-02044 VTT, Finland
Phone internat. + 358 20 722 4404
Fax + 358 20 722 4374

ISBN 978-951-38-6319-7 (soft back ed.)

ISSN 0357-9387 (soft back ed.)

ISBN 978-951-38-6320-3 (URL: http://www.vtt.fi/publications/index.jsp)

ISSN 1455-0873 (URL: http://www.vtt.fi/publications/index.jsp)

MICROBIAL CONTAMINANTS & CONTAMINATION ROUTES IN FOOD INDUSTRY

Julkaisu on saatavana

VTT SYMPOSIUM 248

Microbes able to establish and develop in the processing environment and

on food products pose a potential problem in terms of food safety and
quality. Initially it is the capability to adhere to a surface that gives an
advantage towards the majority of microbes being present. Other
important capabilities are competitive growth and long survival. Although
pathogenic microbes rarely cause the spoilage of foodstuffs (due to their
slow or non-existent growth in foods at low temperatures) they and their
toxic secondary metabolites naturally form a health risk to the consumer.
To guarantee the safety of foodstuffs microbial contamination must be
detected and traced as early as possible and also at low levels. In addition,
the safety risks related to the microbial contamination must be evaluated
as accurately as possible. To establish corrective (and for the future also
preventive) actions exact knowledge on the process hygiene level of the
production plant is needed. Characteristics of important contaminants,
contamination routes and factors enhancing microbial contamination of
process line are important items covered in this Microbial contaminants
and contamination routes in food industry -seminar by international
lecturers and participants who have provided research studies and group
work material published in this seminar proceedings.

VTT SYMPOSIUM 248

MICROBIAL CONTAMINANTS &

CONTAMINATION ROUTES IN FOOD
INDUSTRY
1st Open Seminar arranged by
SAFOODNET Food Safety and Hygiene
Networking within New Member States
and Associated Candidate Countries