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DAIRY MICROBIOLOGY
A Practical Approach
DAIRY MICROBIOLOGY
A Practical Approach
Editor
Photis Papademas
Department of Agricultural Sciences
Biotechnology and Food Science
Cyprus University of Technology
Limassol, Cyprus
p,
A SCIENCE PUBLISHERS BOOK
CRC Press
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Dedication
The book is dedicated to

the late Dr. R.K. Robinson for leading the way
and to my son Nicholas for making it worthwhile.
Preface
Dairy products have been consumed in numerous forms over

centuries by a vast majority of people (including the At-Risk Groups)
from infancy. Besides nutrition, dairy products are responsible for
pathogens and beneficial bacteria in humans; hence it is of paramount
importance to have up-to-date knowledge on dairy microbiology.
The objective of this book is to provide a scientific background
to dairy microbiology by re-examining the basic concepts of general
food microbiology and the microbiology of raw milk while offering
a practical approach to the following aspects: well-known and
newfound pathogens that are of major concern to the dairy industry,
e.g., Cronobacter sakazakii and its importance to infant formula
milk or Mycobacterium avium subspecies paratuberculosis (MAP)
that might be connected to chronic human diseases (Crohn’s); the
role of dairy starter cultures in manufacturing fermented dairy
products; in developing novel functional dairy products through the
incorporation of probiotic strains; insights in the field of molecular
methods for microbial identification; controlling dairy pathogens
owing to the compulsory application of food safety management
systems (FSMS) to the dairy industry.
I hope that the book will provide dairy professionals and students
alike, the latest information on this vast topic. Finally, I would like to
sincerely thank the contributors both for their academic excellence
and their “hands-on” experience that they openly offered for the
successful completion of this project.
Photis Papademas
Department of Agricultural Sciences
Biotechnology and Food Science
Cyprus University of Technology
Limassol, Cyprus
Contents
Dedication v
Preface vii
1. Basic Concepts of Food Microbiology 1
Peter Raspor, Sonja Smole Smožina and Mateja Ambrožič
2. The Microbiology of Raw Milk 22
Apostolos S. Angelidis
3. Dairy Pathogens: Characteristics and Impact 69
Photis Papademas and Maria Aspri
4. Dairy Starter Cultures 114
Thomas Bintsis and Antonis Athanasoulas
5. Application of Probiotics in the Dairy Industry: 155
The Long Way from Traditional to Novel Functional Foods
Adele Costabile and Simone Maccaferri
6. Application of Molecular Methods for Microbial 177
Identification in Dairy Products
Paul A. Lawson and Dimitris Tsaltas
7. Application of Food Safety Management Systems (FSMS) 217
in the Dairy Industry
Thomas Bintsis
Index 237
Color Plate Section 239
CHAPTER 1
Basic Concepts of Food

Microbiology
Peter Raspor,1,* Sonja Smole Smožina2 and
Mateja Ambrožič2
INTRODUCTION
Food has never been safer than it is today, but at the same time it has also
never been at greater risk. Concerns over food safety and quality as well as
its production, processing and preservation have enhanced the importance
of food microbiology as it encompasses the study of microorganisms which
can have beneficial as well as harmful effects on the quality and safety of
food. Food microbiology has begun to implement all necessary precautions
to improve quality and safety of food products. The milk industry was one
of the first to use food microbiology to select relevant starter cultures, to
eliminate spoilage and reduce zoonoses, which were transmitted along
food supply chain.
It is important to stress that milk production and processing was on the
front lines of implementation of technical and regulatory novelties like novel
process design, innovative equipment with most advanced pasteurization
and sterilisation concepts, but also with variety of products that had more
1
Vice Dean for Research and International Cooperation, Faculty of Health Sciences, University
of Primorska, Head of the Institute for Food, Nutrition and Health, Polje 42 SI – 6310, Izola,
Slovenia.
Email: [email protected]
2
Chair of Microbiology, Biotechnology and Food Safety, Department of Food Science and
Technology, Biotechnical Faculty, Jamnikarjeva 101, 1000 Ljubljana, Slovenia.
Emails: [email protected]; [email protected]
* Corresponding author
2 Dairy Microbiology: A Practical Approach
sophisticated functional properties like cheese with bacteriocins, cottage

cheese with natural extracts, probiotics enhanced with prebiotics, and
phytosterols enriched milk products, etc.
Current Perspective Through Selected Past Events

Milk and milk products are indispensable components of our diet and are
valuable sources of essential and nonessential nutrients. Milk is as ancient
as mankind itself, because the natural role of milk is to nourish and provide
immunological protection to the mammalian baby. Milk and dairy products
have been recognised as a significant part of human diet as early as 8000–
6000 BCE (Kervina 2006). In that period of history, ancient man learned to
domesticate species of animals and to use by-products such as milk. The
practice of preserving milk may be also as old as the domesticated animals
and the use of fire, because milk is recognised as a highly perishable food
item. Honey and milk are synonymous with welfare and there is even a
dairy product mentioned in the Old Testament of the Holy Bible, which
was translated by Luther as butter (Kervina 2006).
Although the collection and use of animal milk for human consumption
can be traced back to the earliest societies of our history, the concept of
milks sold in markets developed during the industrial revolution in the
19th century due to rapid growth of urban areas and improved and faster
distribution chains and transportation facilities like railway and steamers
(Eckles et al. 1936). These fast transport and cooling techniques prevented
the undesirable changes in products and was the reason for the market
expansion. Historically market milks have been defined as fluid milk
products sold for direct human consumption. Until the end of 19th century,
market milk was primary raw milk, collected from the farm, distributed
fresh to the consumer and consumed fresh (Boor and Murphy 2002).
Milk and dairy products are important components of a healthy diet,
but if consumed raw or unpasteurized, they can become a health hazard
due to possible contamination with pathogenic bacteria. These bacteria can
originate from clinically healthy animals from which milk is derived or from
environmental contamination occurring during collection and storage of
milk. In the first half of the 19th century scientists like William Dewees and
Gail Borden (Holsinger et al. 1997) began to recommend that milk should
be heated prior to consumption to increase its shelf life.
Pasteurization is a process based on the 1860s experiments of
microbiologist Louis Pasteur (Westhoff 1978). He discovered that the use
of temperatures (50–60°C), which did not alter the original characteristics
of fluids, for a few minutes could prevent or delay microbial spoilage.
Spoilage is a term used to describe the deterioration of a food’s texture,
colour, odour or flavour to the point where it is unappetising or unsuitable
Basic Concepts of Food Microbiology 3
for human consumption (Arvanitoyannis et al. 2009). Microbial spoilage

of food often involves the degradation of protein, carbohydrates and fats
by the microorganisms and their enzymes. In milk the microorganisms
that are involved in spoilage are psychrotrophic organisms that survive
pasteurization or post-pasteurization contamination (Arvanitoyannis et al.
2009). With this discovery the ability to store and distribute milk far away
from the farm has increased. Although milk adulteration was the primary
concern at that time (Accum 1820, Sinclair 1906), the association of disease-
causing organisms with raw milk was also becoming more evident. The
epidemics like cholera, tuberculosis, typhoid fever, scarlet fever, anthrax,
foot and mouth disease, and diphtheria (Straus 1913, Westhoff 1978), which
killed thousands of people in the cities in the late 19th century, were among
the human diseases that had been recognised as being transmitted through
raw milk consumption, but data for thermal destruction of pathogenic
microorganisms in milk were very limited, contrasting and confusing
(Westhoff 1978). For example, North reported in 1925 that at least 26 reports
appeared in the literature between 1883 and 1906 on the thermal death of
M. tuberculosis, which was at that time considered to be the most heat
resistant pathogen associated with milk. Reported times and temperatures
ranged from 50°C to 100°C and 1 min to 6 h (North 1925).
The first application of pasteurizing heat treatments to milk may have
been suggested and performed by Soxhlet, who pasteurized bottled milk
for infants (Holsinger et al. 1997). The first commercial pasteurizer was
made in Germany in 1882 (Sarg 1896) and pasteurization on a commercial
scale quickly became common practice in Denmark and Sweden in the
mid-1880s (Westhoff 1978). Initially, commercial pasteurization of milk was
not readily accepted by consumers, but many companies had adopted the
process in secret (Pegram 1991).
In the beginning of the 20th century, heat treatment of milk was slowly
adopted. But, the extent and duration of commercial heat treatments lacked
uniformity among processors’ operation. To address these gaps, extensive
research was conducted to establish both a standard for pasteurization
and standards for pasteurization machinery (Westhoff 1978). The entire
development required exchanging and sharing experiences and better
control. In 1903, the 1st world dairy congress was organised in Brussels
(Smith 1964). The International Dairy Federation was formed and numerous
countries became members and contributed to the developments and
legislations in dairy processes the world over.
Only after the World Wars in the 1920s and 1930s did milk consumption
get promoted by politics as a normal and healthy food. The commercial
success of the “holding” method and its acceptance as an adequate
method of pasteurization resulted in the first pasteurized milk ordinance
being published in the issue of Public Health Reports in 1924 in USA.
Pasteurization was defined as a heating process of not less than 61.1°C

for 30 min in approved equipment, because experiments had pointed out
defects in pasteurizing machinery (Westhoff 1978).
Although the holding method was the most widely used, new
equipment designs like plate heat exchangers were being applied for use
as high-temperature short-time (HTST) pasteurization methods. The first
HTST pasteurization standards were included in the 1933 U.S. Public
Health Service Milk Ordinance and Code (Westhoff 1978). Although
pasteurization standards had now been established, even as late as 1938,
milk-borne outbreaks were still responsible for 25% of all outbreaks due to
contaminated food and water (United States Public Health Service/Food
and Drugs Administration 2011).
The rickettsia responsible for Q-fever, Coxiella burnetii was first
described by Derrick (1937). Brucella species are zoonotic bacteria, which
are the principal causative agents of brucellosis in livestock and humans.
Early investigations of Q-fever had already demonstrated that this organism
was more heat resistant than M. tuberculosis and could be isolated from
pasteurized milk that was processed according to minimum standards. In
1956 the pasteurization temperature was raised to 63°C to ensure destruction
of C. burnetii, which was associated with Q-fever (Westhoff 1978).
Nowadays pasteurization is the principle method used to eliminate
C. burnetii from milk, but throughout history many legal definitions of
milk pasteurization appeared in the regulations with regards to time
temperature combinations. The Codex Committee on Food Hygiene in
2004 defined pasteurization as a microbiocidal heat treatment aimed at
reducing the number of any pathogenic microorganisms in milk and
liquid milk products, if present, to a level at which they do not constitute
a significant health hazard. Pasteurization conditions are designed
to effectively destroy the organisms Mycobacterium tuberculosis and
C. burnetii. According to validations carried out on whole milk, the minimum
pasteurization conditions are those having bactericidal effects equivalent
to heating every particle of the milk to 72°C for 15 seconds (continuous
flow pasteurization) or 63°C for 30 minutes (batch pasteurization) (Codex
Alimentarius Commission 2004).
Starter Cultures
In the middle of the 19th century, Pasteur and others finally demonstrated
that microorganisms cause food spoilage. Thousands of years before this
sensational experimental proof, man had developed actions for preventing
spoilage based on the use of experiences to identify suitable methods to
prevent this spoilage. Raw milk in its natural state is highly perishable
because it is susceptible to spoilage from naturally occurring enzymes and
contaminating microorganisms. The preservation of food by fermentation

is one of the oldest techniques used, because fermentation was simply the
unavoidable outcome that resulted when raw food materials were left in
an unpreserved state by endogenous microflora (Hutkins 2006). The oldest
food processes include the baking of yeast leavened breads, brewing of beer,
sake and wine and production of yoghurt and cheese.
Dairy starter cultures have a long history in the production of fermented
milk products dating back several thousand years. The oldest method
simply relies on the indigenous microorganisms present in the raw material.
These cultures consist primarily of several members of the lactic acid
bacteria. Lactococcus lactis as the workhorse of the dairy starter cultures or
at that time called “Bacterium lactis” was the first bacterium to be isolated
from a mixed population in pure culture and scientifically described by
Joseph Lister in 1873 (Lister 1878).
The use of dairy starter cultures dates well before any knowledge of
bacteria, and knowledge of which microbes are involved is quite recent
and it is still not known for all dairy starter cultures used. In the beginning
of 20th century the term “lactic acid bacteria” (LAB) started to be used to
refer to milk souring organisms. The monograph by Orla-Jensen in 1919
formed the basis of the present classification of LAB (Orla-Jensen 1921). The
criteria used by Orla-Jensen such as cellular morphology, mode of glucose
fermentation, temperature ranges of growth and sugar utilization patterns
are still very important for the classification of LAB. The advent of modern
taxonomic tools like molecular and biological methods has increased the
number of LAB genera from the four originally recognised (Lactobacillus,
Leuconostoc, Pediococcus and Streptococcus) by Orla-Jensen.
Starter cultures have rapidly become an integral part of food industry.
They consist of different microorganisms that are inoculated directly into
food materials to overwhelm the existing flora and bring about desired
changes in the finished products.
The essential roles of LAB starter cultures are (Tamime 2002):
• The production of lactic acid as a result of lactose fermentation.
• The production of volatile compounds that contribute toward the
flavour.
• Possessing a proteolytic or lipolytic activity that may be desirable
(maturation of cheese).
• Production of other compounds like alcohol (koumiss, kefir).
• The acidic conditions and production of bacteriocins prevents the
growth of pathogens as well as many other spoilage organisms.
LAB are the backbone of the dairy starter culture industry. The world
over there is a huge diversity of LAB and consequently also a diversity
of fermented milk products. Tradition and experiences tell us that some

fermented milk products can provide additional health benefits to the
consumer. LAB are generally considered as beneficial microorganisms,
some strains even as health promoting bacteria or probiotics to differentiate
them from the technological cultures used to manufacture fermented milks
(Fuller 1992). LAB constitute a group of gram positive bacteria united by
morphological, metabolic and physiological characteristics, which produce
lactic acid as one of the main fermentation products of carbohydrates (Von
Wright and Axelsson 2011). The main LAB that compromise dairy starters
are in the genera Lactococcus, Streptococcus, Leuconostoc, Enterococcus, and
Lactobacillus (O’Sullivan 2006). Most of the time starter cultures are not made
of a pure single strain but are often a mixture of many different strains and
species. Dairy cultures also contain microorganisms outside the general
LAB classification and these include certain bifidobacteria, brevibacteria,
propionibacteria and fungi (O’Sullivan 2006). However, some genera like
Streptococcus, Lactococcus, Enterococcus and Carnobacterium also contain
strains that are recognised as human or animal pathogens (Von Wright
and Axelsson 2011).
Challenges for Starter Cultures
On one side finding the right culture for the right application often includes
the simplification of a process or adding an attribute value to the consumer,
who is the last link in food supply chain. Optimal speed of acidification,
reaching target viscosity, developing a new flavour profile or safety of
the final dairy product are just some issues concerning added values in
production lines. Bacteriophages and their impact on starter cultures
are still a challenge for milk starter developers. On the other side novel
starter technology concept is eliminating the huge natural biodiversity of
microorganisms and consequently also products, which were offered by
traditional technologies. The future activities on the production side are
challenged with the issue of how to preserve the natural heritage of many
dairy products in this regard.
Milk-borne Pathogens
Since ancient time milk and products derived from milk have been part
of the human diet. Milk is a complex source of nutrients which includes
protein, carbohydrate, lipid, vitamins and minerals. The components that
make it nutritious for humans also provide ideal growth medium for
producing both beneficial and detrimental microorganisms.
Milk-borne infections were relatively common before the advent of

pasteurization in the late 19th century and even today, illness related to
consumption of unpasteurized and also pasteurized dairy products remains
a public health problem (Anaelom et al. 2010, European Food Safety
Authority 2012). Thus, before the adoption of routine pasteurization, milk
was an important vehicle for the transmission of a wide range of diseases.
Pasteurization and improvements in veterinary medicine have seen a
very large reduction in the incidence of milk-borne diseases. Historically
the most serious human diseases disseminated by the consumption of
contaminated raw milk are tuberculosis and brucellosis as representatives
of zoonotic diseases. Zoonoses are infections and diseases that are naturally
transmissible directly or indirectly, for example via contaminated foodstuffs,
between animals and humans (European Food Safety Authority 2012).
Tuberculosis is a serious disease of humans and animals caused by the
bacterial species of the family Mycobacteriaceae, more specifically by the
species in the Mycobacterium tuberculosis complex. Tuberculosis occurs in
humans and many animal species including animals used for production
of food like meat and raw milk for human consumption. Mycobacterium
bovis is the causative agent of tuberculosis in animals used for production
of food. The main transmission routes of M. bovis to humans are through
contaminated raw milk and raw milk products or through direct contact with
infected animals. Properly controlled heat treatment of milk inactivates M.
bovis and this treatment has had a major impact on reducing the importance
of milk as a vehicle of transmission of M. bovis, although consumption of
unpasteurized milk and milk products continues to represent a hazard
in relation to M. bovis. In the previous years, human infections due to
M. bovis and human brucellosis cases were rare in the EU. In 2009 the EU
notification rate for human tuberculosis due to M. bovis was 0.03 cases per
100,000 population and for brucellosis was 0.07 cases per 100,000 population
(European Food Safety Authority 2012).
The concepts of “produce, sell and buy local” and also “back to nature”
represent growing consumer demands for natural, unprocessed or at
least minimal processed foods. These concepts have resulted also in an
increased consumption of raw milk, even though numerous epidemiological
studies have shown that raw milk is a food safety hazard (Oliver et al.
2009). Although pasteurization eliminates pathogens and consumption
of unpasteurized dairy products is uncommon, dairy associated disease
outbreaks continue to occur. During 1993–2006 in USA there were 121 dairy
associated outbreaks. Of the 121 outbreaks, 54% involved cheese and 46%
involved fluid milk. Of the 54% outbreaks involving cheese, 42% involved
cheese made from unpasteurized milk. Of the 46% outbreaks involving
fluid milk, 82% involved unpasteurized milk (Langer et al. 2012).
Raw milk contains numerous microorganisms that originate from the

animal itself or from the environment, but at the same time raw milk is
known to contain antimicrobial compounds like lactoperoxidase, lactoferrin,
lysozyme and immunoglobulin. The role of these compounds is to confer
a degree of protection on neonates of the species from which the milk was
obtained and at the same time to protect the mammary gland itself from
infection (Griffiths 2000).
The fermentation process is the result of the presence of microorganisms
(bacteria, molds, yeast or combinations of these) and their enzymes in milk.
Microorganisms present in milk can be classified into group of pathogenic
and spoilage organisms, although some may play a dual role. The yeasts
play a role in fermentation and maturation, as well as in spoilage of dairy
products. The role of yeasts as spoilage organisms in dairy products is linked
with their physiological characteristics and requirements like nutritional
requirements, enzymatic activities, low water activities, etc. (Jakobsen
and Narvhus 1996, Hansen and Jakobsen 2004). Nevertheless, yeasts have
been used as starter cultures in specific applications in the dairy industry
like kefir, koumiss and soft cheeses. Spoilage organisms are capable of
hydrolysing milk components such as protein, fat and lactose in order
to yield compounds suitable for their growth. Such reactions can lead to
spoilage of milk, manifested as off-flavours and odours, and changes in
texture and appearance. Salmonella spp., E. coli, Campylobacter spp., Listeria
monocytogenes, Staphylococcus aureus, Streptococcus spp., Yersinia spp. and C.
Burnetii are the most frequent pathogens and several publications have been
published on the subject (Jayarao et al. 2006, LeJeune and Rajala-Schultz
2009, Oliver et al. 2009). Numerous other microorganisms such as lactic acid
bacteria, micrococci, Bacillus spp., Enterobacteriaceae, Pseudomonas spp., etc.
are also part of the initial biota of raw milk. Composition and levels found
depend on the health status of the herds and the hygiene conditions under
which the milk is collected (Chambers 2002). Levels and composition of
the initial microflora are influenced by factors like health status of animals
(e.g., contamination from the udder infections, udder diseases, faecal
contamination of the udder, inhibitory substances or veterinary drugs used
to treat diseased animals) and environmental sources of contamination (e.g.,
personnel; air borne and water borne contamination; contamination from
milking and storage equipment at the farm level; contamination during
transportation; and contamination at the dairy). Levels of initial microflora
can be maintained if appropriate hygiene programs are implemented to
control the initial contamination. Such programs include mastitis control
programs; farm management and environmental provisions including feed;
milking machine and milking procedure hygiene programs that include
disinfectants and disinfectant rotation; and on farm cooling programs.
Challenges for Milk-borne Pathogens
Milk-borne pathogens are traditionally considered as microorganisms,

which can cause illness of the consumer if milk is not properly heat treated
and maintained in the cold chain. Recently it has become obvious that
viruses can be also transmitted by the milk supply route. Current life style
is asking for natural and fresh products from all food sources and due
to that habit some consumers are seeking out raw unpasteurised milk.
Knowing the exact milk characteristics is a big challenge for food safety
issues. Secondly, the food supply chain asks for longer and longer shelf life
of food items, which represents an additional possibility for a number of
psychrothrophic organisms that can survive and multiply in refrigeration
supply chains. Unfortunately some psychrothrophic microorganisms are
pathogenic like Listeriamonocytogenes. Keeping all these issues in mind,
we have to pay attention to the milk supply chain for novel milk-borne
pathogens, but also take care not to ignore traditional ones, which are
coming back like Mycobacterium.
Microbiology as Tool for Food Safety and Quality Issues

With important changes in lifestyles, demography, environmental
conditions and shifts from the local to the global in the food trade, food
supply is growing rapidly in size and diversity. Rising incomes and mobility
have given rise to the demand to know how and where food is produced
and to be assured of its safety. The increased demand for safer food has
resulted in the development and introduction of new food safety standards
and regulations to reach a higher level of food safety (Aruoma 2006).
Food quality and safety are important drivers for management of food
processing and production systems in agribusiness as well as in the food
industry. In the last decades consumers have become very critical about food
quality and food safety due to several incidents of contaminated food like
BSE, dioxin poisoning in feed and pathogenic strains of E. coli in sprouts. In
order to build and maintain the trust of consumers in food quality and safety,
quality management systems (QMS) and food safety management systems
(FSMS) are used to control the quality and safety of foods (Raspor and
Ambrožič 2012). Quality is divided into aspects of product safety, products
quality and total quality, which embrace products safety and quality (Raspor
and Jevšnik 2008). QMS refers to all activities that organizations use to
direct, control and coordinate quality, including formulating a quality
policy, setting quality objectives, quality planning, control and assurance
and improvement (ISO 9000:2000 2000). A FSMS involves that part of
the QMS that is specially focused on food safety. Nowadays in the food
industry quality assurance systems include good manufacturing practice
(GMP), good hygiene practice (GHP), hazard analysis and critical control
points (HACCP) and including private standards as well. Quality assurance
is a term used for describing the control, evaluation and audit of a food
processing system.
Food safety defined according to Codex Alimentarius as assurance
that food will not cause harm to the consumer when it is prepared and/
or eaten according to its intended use (Codex Alimentarius Commission/
RCP 1 2003) is an international challenge requiring close cooperation
between producers, processors, retailers and consumers on the one side and
governmental and nongovernmental organisations on the other side. There
are many factors affecting food safety such as global trade, socio-economic
and technological development, urbanization and agricultural land use. In
a food safety program we should be able to identify all hazards, analyse
them, assess them, assess the likelihood of their occurrence and identify
measures to their control. Hazard is a biological, chemical or physical
agent in, or condition of, food with the potential to cause an adverse health
effect (Codex Alimentarius Commission 2003). The aim of HACCP based
systems is to ensure that food is produced safely. HACCP is a tool which
identifies, evaluates, and controls hazards throughout the food chain from
primary production stages to final consumption which are significant for
food safety (Codex Alimentarius Commission 2003). It assesses hazards
and establishes control systems that focus on prevention rather than relying
mainly on end-product testing. A HACCP plan proves that the controls are
in place and that the system is functioning effectively (Food and Agriculture
Organization 1998).
In spite of major advances in science and technology, microbial food-
borne illnesses are considered a significant public health issue (European
Food Safety Authority 2012). To provide safety, stability and quality of food
products detection of microbial contamination is therefore important. The
spectrum of food-borne infections has been changing over time, as well
established pathogens have been controlled or eliminated (Tauxe 2002) and
new ones have emerged because of the changed consumer eating habits and
patterns. In most cases food-borne diseases are associated with diarrhoea,
vomiting, other gastrointestinal and/or extra intestinal manifestations, but
secondary complications can occur. Managing the microbiological food
safety risk with the goal of reducing the burden of microbial food-borne
illnesses is still one of the important challenges today due to the fact that
factors affecting the microbiological food safety are changing dramatically.
Over time we have been witnessed to rapid and huge technological changes
in food processing and production procedures and also due to the changes
in methods of microbiological analysis.
Microbiological criteria are tools that can be used in assessing the safety
and quality of foods. They are necessary to assist in setting critical limits in
HACCP systems, verification and validation of HACCP procedures, other

hygiene control measures and in shelf life studies where storage trials
and challenge tests are needed (Institute of Food and Science Technology,
Professional Food Microbiology Group 1997). In microbiological risk
assessments, knowledge of microbial population distribution and numbers
in food forms an essential part of the information required, in conjunction
with population exposure, infective dose and pathogenicity of organisms.
When mathematical models are used to predict microbial growth, survival
or decline, it is also necessary to appreciate the number that are inevitable
and those that cause concern or signal the end of shelf life (Institute of Food
and Science Technology, Professional Food Microbiology Group 1997).
Microbiological criteria not only give guidance, but also set microbiological
criteria defining the acceptability of the processes and for setting a limit
above which a foodstuff should be considered unacceptably contaminated
with the microorganisms for which the criteria are set (EC 2005).
Due to the reasons related to sampling, detection and unequal
distribution of microorganisms in the food matrix, microbiological testing
of finished food products done alone is not adequate to guarantee the safety
of tested product. The safety of foodstuffs is mainly ensured by preventive
approaches, such as good practices and application of procedures based on
HACCP principles, but nevertheless microbiological criteria should form an
integral part of the implementation of HACC based procedures and other
hygiene measures (EC 2005).
Good practices or prerequisite programs (International Organization
of Standardization 22000:2005 2005) are an essential element of food
safety systems, but are often neglected as basics for successful food safety
management systems. Good practices are basic conditions and activities,
which are necessary to maintain a hygienic environment throughout the
food chain suitable for the production, handling and provision of safe end-
products and safe food for human consumption (International Organization
of Standardization 22000:2005 2005).
Challenges for Milk Quality and Safety
Consumers in developed countries ask for food products that are high and
consistent in quality, possible to get in broad assortments throughout the
year and also for competitive prices. All food producers and processors are
responsible for quality products and to guarantee safety of foods. Collecting
products from animals leads to transfer of microorganisms from animals
to those products. Contamination of milk may also arise at any stage of
milking and also during the later stages such as farm storage, transport and
processing. That is why milk and milk products present a unique challenge
for food safety, because numerous microorganisms including bacteria,
yeasts and moulds constitute the complex ecosystem present in milk and
fermented dairy products. Beside microbiological hazards chemical hazards
also present hazards to the public health, because toxic chemicals present in
animals’ bodies can be shed into the milk. Chemical contaminants include
industrially derived contaminants like dioxins, furans, PCBs and elemental
heavy metals; biologically derived contaminants like mycotoxins and
phytotoxins; pesticides and residues of plant agrichemicals like pesticides,
and residues of animal remedies (O’Mahoney et al. 2009).
At this point climate changes should also be mentioned because it
may have an impact on the occurrence of food safety hazards at various
stages of any food chain, not just the milk supply chain. Climate change
is widely recognized globally as the major environmental problem to be
faced. There are multiple pathways through which climate related factors
may impact food safety, such as changes in temperature, extreme weather
events and others. Climate change may also affect aspects related to food
safety systems such as agriculture, animal production and trade (Tirado
et al. 2010). The experts selected feed related issues like raw materials,
pasture, silage, storage and manufacturing feed and also animal health as
the most critical factors that affect the occurrence of food safety hazards
due to climate change (van der Spiegel et al. 2012).
An increasing number of people are consuming raw unpasteurized
milk although numerous epidemiological studies have shown clearly that
raw milk can be contaminated by a variety of detrimental microorganisms
(Oliver et al. 2009). Reasons for increased interest in raw milk consumption
is seen in enhanced nutritional qualities and consequently in health benefits.
However, pasteurization reduces spoilage and eliminates pathogens.
Spoilage microorganisms (sporoforms) in raw milk cannot be completely
eliminated and growth can take place readily. For this reason shelf life of
pasteurized milk, even when refrigerated, is limited. In many countries sale
of raw milk for direct consumption is restricted or completely prohibited
because of the potential risk to public health (LeJeune and Rajala-Schultz
2009) but a small amount of milk is still sold as raw or unpasteurized. In
conventional distribution two main heat treatments, pasteurization and
sterilisation are used for milk sold in the retail sector. The main aims of
heat treatment developed for market milks are to eliminate or reduce
the microbial population associated with pathogens, potential spoilage
organisms and degradative enzymes (Lewis and Deeth 2009). A very good
microbial quality of raw milk is also important to prevent production
losses and to achieve an optimal shelf life of dairy products. To ensure a
good microbial quality of milk, quality assurance systems of dairy farms
are being developed and bacteriological schemes are being implemented.
Many aspects of food safety and quality management are involved in the
control of the microbial contamination, especially on farms as main areas
of microbiological concerns and the maintenance of a cold chain along

milk supply chain.
As mentioned above, dairy farmers as well as processors have to
adopt practices of production and processing that satisfy the demands of
consumers. Good dairy farming practice underpins the production of milk
that satisfies the highest expectations of the food industry and consumers.
On farm practices should also ensure that milk is produced by healthy
animals under acceptable conditions for the animals and in balance with the
local environment (Food and Agriculture Organization and International
Dairy Federation 2004).
Milk Supply Chain Issues

These days, production and processing are fragmented around the world.
Food reaches consumers via supply chains that link many different
organizations, companies and other partners, which could stretch across
multiple borders. A food supply chain is a network of food related
businesses involved in creation and consumption of food products, where
food products move from farm to table (Selvan 2008). All actors within the
food supply chain are linked by material, capital and as well as information
flows, which are necessary due to the obligatory traceability system.
The global population is growing rapidly. Increasing population growth
with economic development is resulting in increased demand for high
quality food. Dairy products promote the good health and wellbeing of
people, because they are an important part of a balanced diet contributing a
majority of essential amino acids in our nutrition. The world dairy market is
constantly growing and evolving (International Dairy Federation 2010). The
amount of milk produced and consumed globally in 2009 reached a level of
more than 703 million tonnes (International Dairy Federation 2010). By the
IDF statistics in the total world milk production, cow milk represents around
84% and buffalo milk around 13%. Goat, sheep and camel milk represents
a minor portion of the total world milk production. Milk production is
a very important element of the whole dairy chain. The most important
milk production regions are Europe and south Asia, which provide more
than 50% of the global milk production. European countries alone provide
25.2% of the global milk production (International Dairy Federation 2010).
Dairy farmers’ production systems are continuously being challenged to
combine the responsibility of protecting human and animal health, animal
welfare and the environment. This is increasingly becoming an important
demand on economic husbandry. Recent studies by the United Nations
Food and Agriculture Organization (Food and Agriculture Organization)
have drawn attention to the considerable environmental footprint of the
global livestock industry. Global climate change is also challenging the dairy
industry to develop sustainable initiatives to reduce their environmental
impact. Carbon footprint has become a widely used term and concept of
global climate change. Carbon footprint by its definition is the total amount
of greenhouse gas (GHG) emissions associated with a product, along its
supply chain, and sometimes includes emissions from consumption, end of
life recovery and disposal. It is usually expressed in kilograms or tonnes of
carbon dioxide equivalent (Food and Agriculture Organization 2010). FAO
reported (Food and Agriculture Organization 2010) that the dairy sector in
2007 emitted 1,969 million tonnes CO2-eq of which 1,328 million tonnes are
attributed directly to milk. Recent studies have estimated that cradle-to-
farm gate emissions of milk globally contribute 4.0% of global greenhouse
gas emissions such as methane, nitrous dioxide and carbon dioxide. The
overall contribution of the global milk production, the processing and
transportation to global GHG emissions is estimated at 2.7%. Or put in
another way the global average of GHG emissions per kilogram of milk
and related milk products is estimated at 2.4 kg CO2 equivalent. This
estimation includes emissions associated with milk production, processing
and transportation of milk and milk products only. A significant source of
emissions in the dairy supply chain is methane, produced from the natural
digestive process of cows. Nitrous oxide and carbon dioxide are also by-
products of dairy production.
Dairy industry worldwide has engaged to lower the GHG emissions in
next key areas (Global Dairy Agenda for Action 2009):
• Emissions reduction (i.e., optimising animal feeding, optimising use
of resources and optimising manure management).
• Energy efficiency (i.e., on farm energy use in milking and refrigeration,
optimised processing, renewable energy).
• Transport efficiency (i.e., optimised milk collections, optimised
transport and distribution).
• Reduction in loss of milk and milk products (i.e., shelf life improvement,
reduce household waste).
• Resource efficiency (i.e., recycling of packaging, increase recovery of
waste, use of packaging with lowest environmental impact).
• Management (i.e., development of a global standard for measuring
monitoring and reducing GHG emissions).
Transport is highly related to almost all human activities. It stimulates
economy, improves people’s wellbeing and comfort, but at the same
time contributes to negative environmental impacts such as pollution,
depletion of ozone layers, depletion of resources, global warming, waste,
noise, vibration, barriers and congestion in populated areas (Hesse and
Rodrigue 2004, Anderson et al. 2005, Russo and Comi 2010). Food reaches
our plates nowadays via different routes and the present logistic challenge
for food suppliers. Transportation and distribution are one of the most
visible elements of our current food system. We can see trucks labelled
with commercials of food giants on our roads every day. Transport of food
and agricultural produce is a significant component of goods transport as a
whole (Gebresenbet et al. 2011). Considering the importance of maintaining
the food supply system and reducing the environmental impact of the
transport system, increasing the efficiency of food distribution appears
to be a major challenge not only in the dairy sector, but in the food sector
in general. The transportation of foodstuffs is having a considerable
environmental impact through fuel used for transportation and energy
used for refrigeration. According to EUROSTAT statistics road plays a
predominant role in European countries in both passengers and goods
transportation. 46% of total goods required for transportation are done so
by roads. Road transports consumed 26% of total final energy consumption
of the 27 member countries of European Union. Statisatics showed that
road transportations are responsible for emitting 93% of greenhouse gases
of total European transport (Statistical Office of European Communities
2009). Road transportation of agricultural products and foodstuffs is largely
a national operation, where majority of goods were transported less than
150 km, although certain foods are moved and delivered over considerable
distances by road (Statistical Office of European Communities 2011).
Since the dairy industry concentrate their primary production and
processing facilities we cannot ignore the fact that they substantially
contribute to carbon footprint. In addition to this, food transport refrigeration
is a critical link in the food chain, not only in terms of maintaining the
temperature integrity, but also its impact on energy consumption and CO2
emissions (Tassou et al. 2009). Refrigerated storage is one of the most widely
practiced methods of preserving perishable foods like dairy products. It is
important in maintaining the safety and quality of many perishable foods
and it enables that food is supplied to their destination. Refrigeration
stops or reduces the rate at which changes occur in food. These changes
can be microbiological (i.e., growth of microorganisms), physiological (i.e.,
ripening, respiration), biochemical (i.e., browning reactions, lipid oxidation
and pigment degradation) and/or physical (i.e., moisture loss). An efficient
and effective cold chain is designed to prove the best conditions for slowing
and preventing these changes for as long as it is practical (James and James
2010), but the data suggest that currently the cold-chain accounts for
approximately 1% of CO2 production in the world. The cold chain is vital
part of modern global trade as it impacts on all food commodities.
Current Microbial Issues

The analysis of foods for the presence of both pathogenic and spoilage
microorganisms is a standard practice for ensuring food quality and safety.
Modern quality management and control systems such as GMP and HACCP
systems require methods and techniques that can be used on-line and give
results in real-time. Especially in the food industry there is a need for more
rapid methods to provide adequate information on the possible presence
of pathogens in raw materials and finished products, for manufacturing
process control and for the monitoring of cleaning and hygiene practices.
Large scale production lines become even more sensitive for processing
mistakes, which could result in unsafe milk products.
Microbiological assessment of quality and safety of foods generally and
also milk traditionally relies upon the enumeration and specific detection of
pathogenic and spoilage microorganisms. Conventional culturing methods
are slow, material and labour intensive (de Boer and Beumer 1999). Modified
versions facilitate obtaining results rapidly. Over the past two decades many
improvements have been seen in both conventional and modern methods
for the detection of microorganisms in food. Non-traditional testing methods
relying on physical, chemical, immunological or molecular principles have
been introduced to supplement or replace conventional testing methods
(Deak 2009). Rapid techniques are particularly useful in modern procedures
of quality management and control systems such as HACCP to ensure the
microbiological quality and safety of foods in a preventive way that cannot
be attained by end-product testing.
In the past decades the control of the safety of foods has been mainly
carried out by product testing rather than process control. The main
problem with doing end-product testing is the high number of samples
to be eliminated before one can decide on the safety of the product batch.
Microbiological methods are needed within a HACCP approach for risk
assessment, the control of raw materials, the control of the process line
and the production line environment, and for validation and verification
of the HACCP program. Further development of on-line microbiology is
important for rapid monitoring on HACCP plans.
Challenges for Fast and Reliable Microbiological Analytics
The industry decision-makers, facing a scenario of increasingly more

demanding milk hygiene standards feel the necessity of taking reliable and
quick decisions about the milk and milk products (i.e., before the unloading
of the tank lorries at the dairy). Microbiological control of raw milk quality
at the dairy plant is therefore crucially important. This step is considered
a critical control point (CCP) in the HACCP plans. Under the European
Union (EU) law, 105 bacteria ml−1 at this point of the process are the critical
limits enforced (EC 2004).
The availability and application of culture-independent tools that
enable a detailed investigation of the microflora and microbial biodiversity
of food systems has had a major impact. However, it has become apparent
that approaches that include a culturing step can lead to inaccuracies due
to species present in low numbers being outcompeted in laboratory media
by numerically more abundant microbial species (Hugenholtz et al. 1998)
or the fact that others may simply not be amenable to cultivation in the
laboratory (Head et al. 1998). For these reasons approaches to access the
microbial composition of food have had to change dramatically. To address
this, there has been an increased focus in recent year on the use of culture
independent investigations through the direct analysis of DNA or RNA
from food without a culturing step (Quigley et al. 2011). The shift from
culture dependent assessment to culture independent analysis has led to a
revolution in food microbiology.
Advent of biotechnology has greatly altered food testing methods.
Improvements in the field of immunology, molecular biology, and
automation and computer technology continue to have a positive effect
on the development of faster, more sensitive and more convenient and
reliable methods in food microbiology. Further, development of on-line
microbiology, including ATP bioluminescence and cell counting methods,
is important for rapid monitoring of cleanliness in HACCP programs.
However, the important challenge is still sample preparation. More
research is needed on techniques for separating microorganisms from the
food matrices. The possibilities of combining different rapid methods,
including immunological and DNA based methods are occurring more and
more regularly in milk microbiology. Analytical technology is improving
constantly and the current generation of assays potentially has the capability
for near real time and online monitoring of multiple pathogens. Modern
methods are based on molecular biology techniques like PCR, RFLP, DNA
microarray assay, immunological techniques like ELISA, biophysical
and biochemical principles with the application of biosensors like
bioluminescence sensor, bio-analytical sensors utilizing enzymes, electrical
impedimentry and flow cytometry (Mandal et al. 2011).
Concerns over milk safety and quality as well as production, processing
and preservation have increased the importance of food microbiology.
Raw milk is known to comprise a diverse microbial community. The high
nutritional value of raw milk, its high water content and near neutral
pH allows the growth of many, maybe even all microorganisms. These
microorganisms include microflora of technological relevance like starter
cultures as well. On the other hand the presence of spoilage bacteria can
have significant negative effects on the quality of milk and milk products,
while one of the most difficult and fundamental issues in food safety is
the detection of pathogens that can have severe effects on human health.
Current transformation of methods to fast and reliable microbiological
analytics is a real challenge since it also needs rapid improvement of the
skills and knowledge of diary/food microbiologist to be able to serve the
system in a fast and reliable way.
Conclusions
The food/milk supply chain is emerging as the most integrated system on
the globe. Milk production, processing and consumption are connected to
human nutrition requirements and the ability of current systems to keep
quality and safety of milk and milk products during assigned shelf life
periods. Novel technologies are offering efficient primary production and
secondary processing with less and less impact on quality reduction, and
at the same time promoting safety of final product to be consumed.
Substantial development was realised by environment engineering and
process design, which was efficiently underpinned with fast and reliable
analytical methods to trace physical, chemical and microbial hazards. The
development in last fifteen years is slowly fusing together novelties on
processing, technical and regulatory level. It is offering us new solutions
in safe and quality management practices which can deliver numerous
traditional and manmade novel products with the intention to serve needs
of contemporary consumer who have less knowledge about food and less
time for learning and handling basics nutritional needs.
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CHAPTER 2
The Microbiology of Raw Milk

Apostolos S. Angelidis
INTRODUCTION
Raw milk quality is of utmost importance for the dairy industry because
the principle requirement for the production of high quality dairy products
is the production and use of high quality raw milk. This chapter aims to
cover the main aspects of the microbiology of raw milk and is confined
to the study of the milk of the most common domestic lactating animal
species, i.e., cows, goats and sheep. The chapter discusses aspects pertaining
mostly to bacteria (eubacteria) and to a lesser extent to fungi and viruses.
Other groups of (micro) organisms that might be occasionally present in
raw milk such as protozoa (e.g., Toxoplasma, Cryptosporidium), helminths or
other parasites are not covered.
Legislative Requirements for Raw Milk in the EU

Specific rules governing the production and handling of raw milk are
laid down in Regulation (EC) No. 853/2004 of the European Parliament
and of the Council. The same regulation contains legislative requirements
pertaining to the composition [(Microbiological, Somatic Cell Count (SCC)]
of raw milk. As of July 1, 2012, Regulation 853/2004 has been corrected
once and amended by 12 more Commission or Council Regulations. As is
the case for other European Regulations, the reader is advised to consult
Assistant Professor, Laboratory of Milk Hygiene and Technology, School of Veterinary

Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki
54124, Greece.
The Microbiology of Raw Milk 23
the most recent version of Regulation 853. A way to do this is by performing a

search for the Regulation in its consolidated form (i.e., including amendments
and corrections) through an official web site. For instance, the reader can
freely access the most recent consolidated form of Regulation 853 (and other
European Union Law) by accessing the Eur Lex website (http://eur-lex.europa.
eu/advanced-search-form.html?qid=1404982845826&action=update, accessed
July 10, 2014) and clicking on the “Consolidated Legislation” tab.
Annex I of Regulation 853 contains the definitions of “raw milk”, “milk
production holding” and “processed dairy products”, whereas Annex III
lists specific requirements for different categories of animal-origin foods
(e.g., different categories of meat and meat-products, fishery products,
eggs, etc.). Section IX of Annex III is devoted to raw milk, colostrum, dairy
products and colostrum-based products. According to the requirements
laid down, raw milk should come from animals in good health and not
displaying symptoms of infectious diseases that could be transmitted to
humans through milk—including diseases of the genital tract, diarrhea,
fever, or recognizable mastitis. Also the lactating animals must not have
undergone illegal treatment as defined in Directive 96/23/EC. Special
provisions are listed for two highly severe infectious zoonoses, brucellosis
and tuberculosis and the interested reader should consult the Regulation for
more detailed legislative requirements such as control plans and exceptions
with competent authority authorization. The same section outlines general
construction and cleaning requirements for milk production and storage
premises, and milking and milk-storage equipment. The hygiene of
milking is emphasized and so is the identification of animals undergoing
medical treatment, for which proper milk withdrawal periods must be met.
Emphasis is also given to milk cooling requirements. Hence, unless milk is
going to be processed within two hr after milking or a higher temperature is
deemed necessary for technological reasons for the manufacture of certain
dairy products, upon milking “milk must be cooled immediately to not more
than 8°C in the case of daily collection, or not more than 6°C if collection is
not daily”. The chill chain must be maintained throughout all steps of milk
storage and transport and the temperature of milk should not exceed 10°C
upon arrival at the destination establishment (e.g., milk processing plant).
Regulation 853 states that raw milk should not contain antibiotic
residues above the allowable limits specified in Regulation (EEC) No.
2377/90. Regulation 853 also lists the criteria for raw milk with respect to its
total mesophilic microbial content (plate count at 30°C) and SCC. Raw milk
SCC is recognized as a reliable indicator of cow udder health. In addition,
raw milk SCC is inversely related to cheese yield and quality. Hence, for
cows’ milk the SCC rolling geometric average over a two-month period,
with a minimum of two samples per month, should not exceed 400,000. For
microbiological counts, the rolling geometric average over a three-month
period (with at least one sample per month) should not exceed 100,000 cfu/mL.
The production of milk from lactating animals other than bovine (e.g., sheep,
goats or buffalos) often presents practical difficulties. For instance, in south-
western Europe, a considerable number of sheep and/or goat farms are
located in mountainous regions, distant from milk processing facilities. The
farms’ herd size is often small and therefore does not produce sufficient
revenues for the producers to invest on mechanization. Therefore frequent
pick-up and collection of milk is not cost-effective or even plausible. In
some cases access to electrical power is not for granted and automated
milking systems and/or milk cooling equipment are not easy to install
and/or operate. These limitations may have led European legislators to
(understandably) adopt a more lenient limit with regards to the microbial
content of milk from “other species”, which is currently set at 1,500,000 cfu/
mL. However, much better milk microbiological quality is required (i.e., a
maximum of 500,000 cfu/mL) for raw milk from species other than cows,
if such milk is intended to be used for the manufacture of dairy products
in a fashion that does not involve a heat treatment (sanitation) step. An
example where the “500,000 cfu/mL” criterion applies is the use of raw
sheep milk for the manufacture of cheeses that undergo a minimum ripening
time of two or more months. No SCC limits are listed in Regulation 853
for the milk of species other than bovine. This is likely the result of the fact
that the SCC in small ruminants’ milk (particularly in goats’ milk) varies
considerably, even among healthy animals and several non-infectious
factors (i.e., factors other than inflammation of the mammary gland) can
influence SCC. Milk secretion in small ruminants, particularly in goats,
is apocrine in nature, and cytoplasmic particles, often similar in size to
milk somatic cells, are normally present in their milk (Souza et al. 2012). A
great body of research worldwide has been allocated on the investigation
of (non-pathological) factors affecting milk SCC levels in sheep and goats,
milk and such factors include, amongst others, parity, stage of lactation,
or even sample handling, sample storage practices and measuring method
(Paape et al. 2001, Contreras et al. 2007, Raynal-Ljutovac et al. 2007, Souza
et al. 2012). Currently, there does not seem to be a consensus on establishing
a unique and scientifically justifiable discriminatory SCC standard (limit)
for sheep and goat milk quality because research studies on the SCC of
small ruminant milk have sometimes resulted in conflicting outcomes and
conclusions. Hence, at present, no legislative upper limits regarding small
ruminant raw milk SCC values are in place in the EU.
As mentioned previously, the microbiological and SCC (where
applicable) upper allowable limits for raw milk are expressed as geometric,
rather than arithmetic means (averages). When evaluating bulk tank SCCs
or microbial counts the use of geometric averages helps to decrease the
variation or impact of high or low counts (often the result of sampling or
measurement error) on the mean. An example illustrating the “smoothing”

effect of geometric means is illustrated using the hypothetical data presented
in Table 1.
Table 1. Arithmetic and geometric average of total mesophilic counts obtained from the
analysis of ovine bulk tank milk samples (n = 5) collected randomly over a three-month period.
Sampling no. Microbial counts (cfu/mL) Log microbial counts (log cfu/mL)
1 820,000 5.91
2 950,000 5.67
3 3,900,000 6.54
4 1,200,000 5.85
5 720,000 5.71
Arithmetic mean 1,518,000 6.084
Geometric mean 1,212,895 10 6.084
The geometric mean of a range of n values can be easily calculated.

Probably the easiest way of calculation is with the use of spreadsheet
computer packages. In Microsoft Excel, for instance, one can use the
GEOMEAN function. If the user has no access to a software package, the
geometric mean can be calculated with a simple calculator by multiplying
all the n values and then taking the 1/n root of the product. In the above
example, the “manual” calculation of the geometric mean would be as
follows: (820,000 × 950,000 × 3,900,000 × 1,200,000 × 720,000)0.2. Alternatively,
one can rely on the definition of the geometric mean (i.e., the antilog of the
mean of the log counts). Hence the user can calculate the log (to the base
10) cfu/mL value for each observation and then calculate the mean of the
log counts (here equal to 6.084). The anti-log of this value (106.084) is equal to
the geometric mean. In the hypothetical scenario provided above, it can be
seen that the use of the arithmetic mean would result in an average microbial
count that would exceed the 1,500,000 EU limit for raw ovine milk. The use
of the geometric mean, however, helps to reduce the influence of extreme
values (e.g., of the measurement taken during the 4th sampling point (i.e.,
3,900,000)) resulting in a final average value that is within the EU limit.
As the dairy industry in developed countries is moving towards the
production of dairy products with extended shelf-life, it is anticipated that
greater demands will be placed on raw milk in terms of its quality.
Pre-Harvest Food Safety—Raw Milk Production

Livestock species including ruminants may serve as reservoirs of critical
pathogenic microorganisms and several food-borne hazards (both
microbiological and chemical) can potentially originate during animal
production. Many food-borne pathogens (e.g., Salmonella spp., Campylobacter

spp., and Shiga-toxin producing Escherichia coli) can have habitats in
lactating mammals (gastrointestinal tract and hide) and can therefore
contaminate raw milk during milking. The contamination of animals is
through ingestion of food or water contaminated with fecal material. Such
pathogens further spread in the (farm) environment via animal feces and
farm practices such as the use of recycled wastewater and non-treated
manure as fertilizer.
The need for application of preventive food safety programs at the
farm level arose as a result of the risks and consequences associated with
the presence of food-borne hazards (pathogens and chemical residues)
in raw milk. Food safety is an imperative issue for consumers, food
manufacturers and government officials (Hoffmann et al. 2012) and it is now
well recognized that food safety measures and approaches must be applied
throughout the entire food chain, i.e., “from farm to table”. Preventive
measures and approaches applied during the pre-harvest phase of milk
production aiming at minimizing food-safety risks include the prerequisite
programs such as Good Agricultural Practices (GAP) and Good Hygienic
Practices (GHP). In addition, the widespread and indubitable success
of HACCP at the food industry (processing) level (van Schothorst and
Kleiss 1994) prompted its application on the farm (on-farm HACCP-type
programs). On-farm, HACCP-type programs for chemical hazards are
feasible to develop and implement. A successful example is the 10-point Milk
and Dairy Beef Residue Prevention Program that was originally developed
by the American Veterinary Medical Association and the National Milk
Producers Federation in 1993. This protocol originally included 10 “critical
control points” and was designed to minimize veterinary drug residues in
milk and meat. Since its first publication, the protocol has undergone some
revisions and the latest revised version (2014 edition) is freely available
at: http://www.nationaldairyfarm.com/sites/default/files/2014%20
Residue%20Manual_WEB.pdf. In the US, the Grade “A” Pasteurized Milk
Ordinance (Anonymous 2011) specifies the standards for production,
handling, transportation, processing, testing and sale of milk.
It has been argued that the application of on-farm, HACCP-type
programs (Cullor 1997) during the primary production of milk may not be
effective or adequate to significantly improve food safety. This is mainly due
to the lack of definitive critical control points that could eliminate or control
identified microbiological hazards (Sperber 2005). Nonetheless, quantitative
molecular methods, such as real time PCR, have been developed in the last
decade which can target and quantify specific pathogens in environmental
samples as well as in raw milk (Amagliani et al. 2012). Such methods
are considerably faster and often more sensitive than conventional
culture-dependent methods. In addition, recent applications make use
of protocols in “multiplex” format, enabling the identification of more

than one pathogen at the same time (Omiccioli et al. 2009). The significant
shortening in the required time needed for analysis offers an advantage
for highly perishable foods requiring continuous monitoring, such as raw
milk. Hence, such methods may be used for more effective prevention at
the pre-harvest level. Newer attempts to reduce pathogen contamination
in the farm environment include the use of bacteriophages, or the addition
of probiotics in the animal feed. These and additional current strategies
(e.g., vaccination) to reduce food-borne pathogens in food animals have
been reviewed by Oliver et al. (2009b) and practical food safety interventions
during dairy production are reviewed by Ruegg (2003). A prototype
application of a HACCP-type program in two parts of the production
process (i.e., milk harvest and treatment of cows) has been described by
Lievaart et al. 2005. In addition, the application of preventive, food-safety
measures and approaches during the pre-harvest phase, as well as during
milk collection, storage and transport has led to the improvement of milk
quality in terms of its microbiological count (Nada et al. 2012).
Sources of Microbial Contamination of Raw Milk

Milk is considered nature’s perfect food for mammals and many benefits to
human health have been associated with the consumption of high-quality
milk and dairy products (Kliem and Givens 2012). Freshly milked raw milk
contains active antimicrobial components (briefly discussed later in this
chapter) which, depending mainly upon raw milk storage temperature,
inhibit the growth of contaminating microorganisms for a short period
of time. However, raw milk has a high aw value (ca. 0.99), is only slightly
acidic (pH typically equal to 6.6–6.7) and is a medium rich in energy sources
(lactose, proteins and lipids) as well as a source of a plethora of preformed
building blocks for microbial metabolism and biosynthesis. Even under the
currently available most optimal conditions of routine milk collection, some
level of contamination of raw milk is practically inevitable. Hence, unless
raw milk is properly handled and stored, contaminating microorganisms
will eventually proliferate to high populations and lead to milk spoilage.
Milk is synthesized in specialized epithelial cells in the mammary gland
of mammals. In cows, the udder is divided into two halves by the median
suspensory ligament and each half further divided into two individual
quarters, resulting in a total of four anatomically “independent” mammary
glands (“quarters”). In the udder of goats and sheep there are only two
mammary glands (“halves”) (Nickerson and Akers 2001).
The parenchyma of the mammary gland is composed of glandular tissue
(alveoli, which are the milk producing structures), ducts and connective
tissue. Milk biosynthesis takes place in the alveolar epithelial cells, using
precursors supplied by the animal’s bloodstream. The (biosynthesized)

milk components are released into the lumen of the alveolus. Alveoli form
clusters (lobules) and the synthesized milk is drained via small ducts, which
eventually converge into larger intralobular ducts. The parenchymal tissue
of the mammary gland essentially consists of lobes formed by a cluster of
lobules and drained by yet larger ducts. The large duct emerging from the
lobe is called interlobar duct and milk is eventually collected (drained) into
the mammary gland cistern (Nickerson and Akers 2001). During milking,
the milk passes through the teats (i.e., through the teat cistern) and exits
the udder through the teat canal. The smooth muscles surrounding the teat
canal, aided by the presence of keratin in the canal lumen, help to maintain
the canal closed between milkings and therefore prevent bacterial invasion
from the external environment (teat skin, floor, etc.) into the teat cistern.
In healthy animals the synthesized and secreted milk is sterile. In
lactating animals affected by intra-mammary infections (IMI), i.e., mastitis,
the causative microorganisms will be present, in the milk usually in high
numbers, depending on the severity of the infection. It should be noted,
however, that occasionally in mild or less severe cases of mastitis the
enumeration (or rarely even the detection) of pathogens in milk is not
guaranteed. Occasionally, intermittent secretion can occur for some IMI
pathogens. Even in healthy lactating animals, unless milk is drawn aseptically
from the udder following a proper aseptic collection protocol, freshly milked
raw milk will contain contaminating microorganisms at low to moderate
levels. These initial microbial concentrations can vary considerably among
lactating animals or among animals of different farms, but they are typically
in the range of several hundreds to few thousands of cfu/mL (Morse et al.
1968). There are many possible sources of microbiological contamination
of raw milk. Some of the obvious sources of milk contamination at the
immediate environment of the milking parlor include the animal hide and
the teat/udder skin surfaces that are contaminated with organisms from the
bedding material or dirt from the ground, the air (dust), the milker’s hands,
the milking equipment, the animal feed and the water supply (Rendos et
al. 1975, Hogan et al. 1989, Desmasures et al. 1997b, Murphy and Boor
2000, Zdanowicz et al. 2004). A recent study evaluated the diversity of the
microbiota on cow teat skin using culture-dependent methods and direct
molecular approaches (Verdier-Metz et al. 2012). The study pointed out a
great diversity in the bacterial communities that may reside on teat skin.
In fact, the authors reported that the microbial clones corresponded to 66
identified species, mainly belonging to bacterial genera commonly found
in raw milk (Enterococcus, Pediococcus, Enterobacter, Pantoea, Aerococcus and
Staphylococcus), as well as to several unidentified species.
The teat canal, i.e., the opening through which milk is secreted, can
be also colonized by microorganisms. For instance, the microbiological
investigation of different anatomical sites of nulliparous heifers revealed

the presence of different coagulase-negative Staphylococcus spp. in samples
taken aseptically from the teat canals (White et al. 1989). Further studies have
also confirmed the presence of Staphylococcus spp. as well as the presence of
additional species of bacteria such as Streptococcus uberis and Escherichia coli
in the teat canals of lactating cows that were free of clinical mastitis (Paduch
et al. 2012). A detailed study using a culture-independent approach (PCR-
amplified 16S rRNA gene sequencing) revealed a broader diversity in the
bacterial species residing in the teat canals of lactating dairy cattle. Among
the species identified were bacteria commonly associated with soil, water
and the gastrointestinal tract of cows, as well as uncultured bacteria (Gill et
al. 2006). The teat canal is the usual port of entry for infectious pathogens
responsible for IMIs in lactating ruminants. A study in dairy cattle revealed
a positive association between teat end hyperkeratosis (typically induced by
machine milking) and teat canal microbial load of environmental pathogenic
bacteria. E. coli bacterial counts and the presence of this pathogen in teat
canals were associated with the hyperkeratosis scores of teat ends (Paduch
et al. 2012). Less obvious reservoirs of microbial contamination of raw milk
are biofilms formed in the interior of rubber or stainless steel equipment
used for milk collection, storage and transport. Biofilms are complex
microbial communities residing in a matrix or organic matter (proteins,
polysaccharides and DNA) adhered to solid surfaces (Marchand et al. 2012).
Thorough investigations have been conducted involving the
microbiological examination of multiple environmental samples from sites
within dairy farms including air samples, samples from animal sites and raw
milk. These studies have shown that the diversity and numbers of different
microbial groups in raw milks can be influenced or at least correlated with
several factors including herd management practices, sampling stage (farm
vs. dairy bulk tank) farm hygiene, season and milking practices. Most of the
bacterial and fungal species found in milk are also recovered from the stable
and milking parlor environments. However, some bacterial species were
isolated only from raw milk and their origin remains unknown (Vacheyrou
et al. 2011). Less intense hygienic milking practices seem to preserve a wider
bacterial diversity in raw milk (Verdier-Metz et al. 2009), whereas differences
in the microbiological composition can be observed between milk sampled
from farm tanks (more Gram-positive isolates) and milk sampled from
the dairy bulk tanks after being stored at low temperatures (more Gram-
negative isolates) (Fricker et al. 2011). These and other extensive studies
(Callon et al. 2007, Fricker et al. 2011, Tormo et al. 2011, Mallet et al. 2012)
have also highlighted the great microbial diversity in raw milk and have
led to the identification of bacterial species that had not been previously
associated with raw milk. Finally, these and other studies have highlighted
the necessity of combining culture-dependent and culture independent

methods for a more accurate representation of the raw milk microbiota.
Enumeration of the Raw Milk Microbial Flora

Accurate and efficient methods for quantifying the microbial load of
incoming milk are of great importance to the milk industry and milk
producers. Total viable counts are used as indicators of on-farm hygiene
practices, milk quality and udder health (Hutchison et al. 2005). The
microbial flora in raw milk can be measured, or at least closely estimated
by the standard plate count (SPC) method. Despite some limitations
(mentioned below), the SPC method is accepted as a standard method by
international organizations such as the American Public Health Association
(APHA), the International Dairy Federation (IDF) and the International
Standardization Organization (ISO). The sampling, storage, transport
and mixing of the milk sample(s) to be used for SPC analyses as well as
the conduct of the SPC method are very critical in obtaining meaningful
counts and internationally accepted protocols should be strictly followed
(International Standardization Organization 2003, 2008, 2010). The SPC
method has certain advantages. It does not require sophisticated equipment,
costly consumables or highly educated personnel. However, the personnel
should be careful, meticulous and well-trained. Raw milk is subjected
to successive 10-fold serial dilutions with the use of appropriate sterile
diluents (e.g., quarter-strength Ringer’s solution, 0.1% peptone water).
The diluents used for the dilution process must provide adequate osmotic
strength to prevent bacterial lysis. The ten-fold serial dilutions of milk
are then pour-plated into standard methods agar (SMA) and incubated
aerobically at 30°C for 72 hr. SMA is non selective. Plates containing 10–300
(IDF) or 25–250 (APHA) colonies are used to calculate the SPC, i.e., the total
number of colony-forming units of bacteria, yeasts and molds per mL of
milk. The cfu/mL value is thus based on the number of microorganisms
that can grow and produce visible colonies in the agar plate after the fore
mentioned time, temperature and atmospheric conditions of incubation. The
SPC method is also used routinely in dairy- and other food microbiology
teaching laboratories, as it is very practical for conveying the necessary
scientific principles and rationale to the undergraduate dairy science/
microbiology student. On the other hand, the SPC technique is very tedious
both in terms of conduct as well as with respect to colony counting. This
method is also prone to certain limitations. Bacterial cells in raw milk (and
other foods) often form clumps; each such clump will give rise to a single
colony after incubation of inoculated plates. In addition, in cases where two
or more bacterial cells lie in close proximity in the agar upon solidification,
they will give rise to a single colony after incubation. Injured cells may
not form (visible) colonies during the time frame of incubation whereas,
depending on their spatial distribution in the agar milieu (e.g., towards the
surface as opposed to towards the bottom of the petri dish), some of the
strictly anaerobic bacteria present in raw milk will most likely not grow.
Hence, viable but non-culturable microorganisms are not counted. For all
the aforementioned reasons, the SPC method might underestimate the total
number of viable microorganisms present in raw milk. Most importantly,
the method is impractical for the dairy industry because of the prohibitive
length of time necessary to obtain results. Additional colony count methods
that are more suitable for routine testing (i.e., routine methods) have been
developed and used, such as the plate loop method (Thompson 1960) and
the spiral plate method (Gilchrist et al. 1973).
An alternative approach to determine the bacterial counts of raw milk
is the total microscopic count method. This method relies on counting
stained bacteria on a special glass slide on which a known volume of milk is
applied (typically 0.01 mL), following sample treatments to fix the sample.
Several optical fields are counted using bright field illumination (Hill 1991).
The method is relatively fast, yet tedious to the operator, particularly when
a high number of samples must be examined. Another limitation is the
inability to discriminate between living and dead bacteria, i.e., the method
estimates total bacterial counts as opposed to total viable counts.
Additional available direct or indirect methods for assessing the
bacteriological content of raw milk are available and their advantages
and disadvantages are described in comprehensive reviews (O’Toole
1983, International Dairy Federation 1991a, Vasavada 1993, Suhren and
Reichmuth 2000). Today, however, most milk processors rely on direct,
automated, rapid (ca. 10 min), and high-throughput (up to 200 milk samples
per hr) enumeration methods for determining total bacterial counts in
incoming raw milk. These automated bacterial cell counters operate by
degradation and separation (via centrifugation) of milk somatic cells and
milk particles and constituents that are similar in size to bacterial cells,
followed by fluorescent staining and counting of individual bacterial cells
using flow cytometry. The results of automated analyzers are expressed as
IBC/mL (Individual Bacterial Counts per mL), rather than CFU/mL. IBC
estimates can be converted to CFU values via specific algorithms obtained
through correlation analyses between the two parameters (Cassoli et al.
2007). Several parameters of automated flow-cytometry based bacterial
counters have been evaluated (Lachowsky et al. 1997, Bolzoni et al. 2000,
2001). Manufacturers of automated, high-throughput milk analyzers
continuously come up with more advanced and more reliable models. Thus,
the interested user should consult the most updated relevant technical/
performance sheets. In recent years, automatic flow-cytometry based milk

analyzers have been evaluated for their reliability to enumerate bacteria in
ewes’ (Tomáška et al. 2006) and goats’ milk (Sierra et al. 2009, Ramsahoi et
al. 2011). Finally, it should be emphasized that although the determination
of the total viable counts in raw milk provides an estimate of overall milk
quality for regulatory purposes, it does not provide any indication regarding
the presence, types or levels of pathogenic microorganisms in raw milk.
Microbial Groups Associated with Raw Milk

A great variety of bacterial and fungal species have been isolated from raw
milk worldwide. As mentioned in the previous section, these contaminating
organisms can originate from the lactating animal, the housing/milking
environment, the animal feed or even humans. Both non-pathogenic and
pathogenic microorganisms can therefore be present in freshly milked raw
milk (Desmasures et al. 1997a, Uraz and Çitak 1998, Hassan and Frank 2011,
Vacheyrou et al. 2011, Amagliani et al. 2012, Delavenne et al. 2012, Hill et
al. 2012, Jackson et al. 2012).
Although total viable counts and SCC in raw milk are useful indicators
of milk quality (udder health and milking hygiene), some milk processors
perform supplementary microbiological tests on incoming raw milk as
indicators of milk production conditions. Such tests include the laboratory
pasteurization count (LPC) also called the thermoduric count, the coliform
count (CC) and the preliminary incubation count (PIC). The output of these
testing procedures can help identify and eliminate, or at least minimize
sources of raw milk contamination. The LPC estimates the number of
thermoduric bacteria left in milk after simulating batch pasteurization
(62.8°C for 30 min). LPC is used mainly as an indicator of milking equipment
sanitation and proper maintenance. The CC is obtained by plating milk on
selective media that allow the growth of coliform bacteria. In raw milk from
farm animals free from coliform mastitis, coliform counts have been used
as indicators of the degree of fecal contamination of raw milk. However,
the identification of coliform bacteria of non-fecal origin (environmental
coliforms) has questioned the usefulness of the CC as an indicator of
direct fecal contamination of raw milk. The PIC is obtained after holding
the milk at 12.8°C for 18 h prior to plating. This incubation period at a
relatively low temperature allows the multiplication of bacterial groups that
can proliferate at low temperatures. The comparison of the PIC with the
SPC of the unincubated sample indicates the level of contamination with
psychrotrophic bacteria. Elevated psychrotrophic counts in raw milk are
associated with improper cleaning or sanitizing procedures and/or poorly
cleaned refrigerated bulk tanks. Although no regulatory standards exist
for these supplementary microbiological tests, the procedures used for the
analyses must be standardized to ensure accuracy of the results (Murphy

and Boor 2000, Davidson et al. 2004). Some studies have further looked
into possible associations or correlations both among different milk quality
indicators, as well as among milk quality indicators and farm management
practices (Boor et al. 1998, Pantoja et al. 2009, Elmoslemany et al. 2009,
2010). In general, these different milk quality indicators are not strongly
correlated. A detailed study by Martin et al. (2011) aimed at evaluating the
ability of currently applied raw-milk microbiological tests to predict the
quality of commercially pasteurized fluid milk (2% fat). Raw milk samples
taken from silo tanks just prior to pasteurization were examined using
a variety of tests commonly applied to raw milk, including SCC, SPC,
psychrotrophic bacterial count, ropy milk test, CC, PIC, LPC, and spore
pasteurization count. Raw milk was pasteurized using four different time-
temperature combinations, ranging from 76.7°C for 25 s up to 80.3°C for
33s. Pasteurized milk samples were subjected to microbiological (SCC, CC)
and sensory evaluation analyses at several time points post-pasteurization
up to the end of shelf-life (21 days). The authors reported poor correlations
(typically R2 < 0.45) between the results from the raw-milk tests and the
results from tests used to evaluate pasteurized milk quality, suggesting the
need for new tests that measure the specific biological barriers that limit
the shelf-life and quality of pasteurized milk.
In scientific publications pertaining to dairy science and technology,
the microorganisms present in raw milk are usually classified in different
groups/types bearing distinct implications to different aspects of milk
and dairy products’ hygiene, processing or quality. These groups of
microorganisms are discussed in the following sections.
Bacteria
There are hundreds of different bacterial genera known to date and likely
thousands of different species. Different bacterial genera can differ in their
structural or physiological properties. For instance, most bacterial genera
can be classified into Gram-positive or Gram-negative based on structural
differences in their bacterial cell envelopes. In addition, differences in their
physiological responses to oxygen, osmotic pressure, acidity or temperature
as well as in their biosynthetic and metabolic activities are further used
to classify bacteria into groups of special interest in food microbiology.
Research in bacteriology over the last three decades or so has revealed that
bacteria are quite versatile and adaptive organisms towards various forms
of environmental stress (cold, osmotic, acid or heat stress). These adaptation
mechanisms can be quite elaborate and include both physiological and
genetic responses.
Gram-negative bacteria
Gram-negative bacteria of concern in raw milk include genera, species or

serotypes that are pathogenic to humans, as well as genera or species that
negatively affect the quality of milk and dairy products. Some of the genera
of Gram-negative bacteria that have been isolated with various frequencies
from raw milk include Acinetobacter, Aeromonas, Campylobacter, Citrobacter,
Enterobacter, Escherichia, Flavobacterium, Klebsiella, Moraxella, Pseudomonas,
Salmonella, Serratia, Yersinia and Xanthobacter (Jayarao and Wang 1999,
Jackson et al. 2012). Pathogenic Gram-negative bacteria are discussed in
a later section. Gram-negative bacteria associated with milk spoilage can
either belong to the group of “coliforms” or consist of non-coliform bacteria.
Coliforms are defined as Gram-negative bacteria that can ferment lactose
with the production of gas within 48 h of incubation at 32 or 35°C (Davidson
et al. 2004). The four principal genera of coliform bacteria are Escherichia,
Enterobacter, Citrobacter and Klebsiella. The non-coliform, Gram-negative
group consists of a large, heterogeneous group of bacterial genera that
include Acinetobacter, Aeromonas, Flavobacterium, Moraxella, and Pseudomonas.
Among these, Pseudomonas spp. have been studied extensively because they
are notorious spoilage agents of milk and dairy products. Jayarao and Wang
(1999) tested raw bulk tank milk from 131 dairy producers in the US for
the presence and levels of coliforms and other non-coliform Gram-negative
bacteria. The results of their study suggested that the populations of both
coliforms and non-coliforms vary considerably and include a wide variety
of Gram-negative species, even species that had not been associated with
raw milk at the time. Coliforms were detected in ca. 62% of the samples
with populations ranging from 0 to 4.7 log10 cfu/mL, with an average of 3.4
log10 cfu/mL. Non-coliforms were present in ca. 76% of the samples with
populations ranging between 0 and 6.2 log10 cfu/mL (average 4.8 log10 cfu/
mL). Pseudomonas was the most prevalent genus, as it accounted for ca. 50%
of the total isolates and for ca. 74% of the non-coliforms.
Spore-forming bacteria
Spore-forming bacteria are a group of microorganisms that exhibit variable

phenotypic characteristics with respect to oxygen requirements and
temperature growth range and optima. Two main genera of spore-forming
bacteria are of interest to dairy microbiologists, i.e., the Gram-positive
bacteria belonging to the genera Bacillus (aerobic or facultative anaerobic)
and Clostridium (strictly anaerobic). These bacteria form endospores,
thick-walled structures that are released from vegetative cells upon cell
lysis. Endospores contain less moisture than the corresponding vegetative
cells and are significantly more resistant to disinfectants, heat or other
environmental stresses compared to their vegetative counterparts because

of their relatively dehydrated state and distinct structural and physiological
properties (Leggett et al. 2012).
Spore-forming bacteria are ubiquitous in the environment (soil, animal
feces, silage, and bedding materials) and as such, contamination of raw
foods, including raw milk is not an infrequent event (Postollec et al. 2012).
The aerobic spore-forming bacteria belonging to Bacillus spp. and related
genera have been studied extensively by dairy microbiologists as they are of
great importance both in terms of quality (spoilage) and safety (De Jonghe
et al. 2010). Members of the B. cereus group (Bartoszewicz et al. 2008) as well
as those of the B. subtilis group and B. licheniformis are commonly present in
raw milk. Other aerobic sporeformers isolated from raw milk belong to the
genera Paenibacillus, Oceanobacillus, Brevibacillus, Lysinibacillus, Ureibacillus,
Ornithinibacillus and Sporosarcina (De Jonghe et al. 2008, Coorevits et al.
2008). Spore counts in raw milk are generally low (usually up to ca. 103/
mL) (Lukasova et al. 2001, te Giffel et al. 2002, Coorevits et al. 2008) and
can be influenced by environmental, housing and feeding factors within
dairy farms. The type of bedding material, the degree of contamination of
the teats with soil and the milking equipment (Christiansson et al. 1999,
Magnusson et al. 2007) or persistence strategies of spore-forming bacteria,
such as the ability to adhere to stainless steel and form biofilms (Shaheen
et al. 2010) can influence aerobic spore counts in milk.
Most of the endospores of spore-forming bacteria can survive milk
pasteurization, and, upon certain conditions, they can germinate and grow
during product fermentation or storage. In addition, some species such as
B. sporothermodurans produce highly heat-resistant endospores, which can
survive even UHT processing of milk (Pettersson et al. 1996, Klijn et al.
1997, Scheldeman et al. 2006). Heat-resistant aerobic spore-forming bacteria
have been isolated from various sites within dairy farms (animal feed and
milking equipment) including raw milk (Scheldeman et al. 2005).
Some aerobic spore-formers are known to cause food spoilage via
the production of extracellular enzymes (proteases, lipases) and/or food
poisoning though the production of toxins (food intoxications). B. cereus is
probably the best known example, a frequent spoilage bacterium in (dairy)
foods and the causative agent of two food poisoning syndromes (one of the
emetic and one of the diarrheal type). Bacillus spp. present in raw milk other
than B. cereus (Coorevits et al. 2008) have shown to possess spoilage potential
or being capable of toxin production (McKillip 2000, De Jonghe et al. 2010).
Paenibacillus is a genus of facultative anaerobic, endospore-forming
bacteria, originally included in the genus Bacillus and then reclassified as
a separate genus (Ash et al. 1993). Paenibacillus spp. are now recognized
as significant psychrotolerant spoilage organisms of pasteurized milk
(Huck et al. 2007). In an extended pasteurized milk survey in the US, many
genera of Gram-negative and Gram-positive bacteria (both Bacillus spp. and

Paenibacillus spp.) were isolated from milk from all geographical regions
sampled. Regarding the Gram-positive spore-formers, however, the authors
recorded a shift in the predominant population of endospore-forming
spoilage bacteria from Bacillus spp. to Paenibacillus spp. over the products’
shelf-life. Hence Paenibacillus spp. were more frequently isolated towards
the end of the shelf-life of high-temperature, short-time (HTST) pasteurized
milk (i.e., beyond 14 d of storage at 6°C). The authors suggested that the low
rate of isolation of Paenibacillus spp. during the early post-pasteurization
days indicate that these bacteria may be present in low numbers in raw
milk; they are nonetheless capable of growing at low temperatures to levels
that limit the shelf-life of HTST pasteurized milk (Ranieri and Boor 2009). A
great diversity of Bacillus spp. and Paenibacillus spp. exist in the dairy farm
environment and can therefore contaminate raw milk. Aerobic endospore-
forming bacteria are also isolated throughout the fluid milk processing line,
i.e., from raw milk through packaged products (Scheldeman et al. 2004,
Huck et al. 2007, 2008, Ivy et al. 2012).
The presence of thermophilic bacilli in dairy products undergoing
intense heat treatment (such as milk powder) is usually the result of selection
by the conditions (high temperatures) employed during manufacture.
These bacteria can grow in sections of dairy plants where temperatures
reach 40–65°C and readily form biofilms. Obligate thermophiles grow
only at elevated temperatures (ca. 40–68°C) and therefore have limited
potential to lead to spoilage at the usual (room or refrigeration) storage
temperature of dairy products. Anoxybacillus flavithermus and Geobacillus
spp. are examples of obligate thermophilic bacilli (Burgess et al. 2010). The
facultative thermophilic bacilli, on the other hand, are members of the genus
Bacillus (e.g., B. licheniformis, B. coagulans) and, depending on the strain,
are capable of growing at both mesophilic and thermophilic temperatures.
These facultative thermophilic bacilli can cause spoilage of pasteurized
milk, cream or UHT milk. A comprehensive review on thermophilic bacilli
and their importance in dairy processing, including their food spoilage
potential, enumeration and identification methods has been published by
Burgess et al. (2010).
Clostridium spp. are anaerobic spore-formers and, similar to aerobic
spore-forming bacteria, they are widespread in the (dairy) environment.
Clostridia have been isolated from several sources in dairy farms such as
soil, silage, forage, hay and raw milk, with silage identified as a major source
of raw-milk contamination. Frequent raw-milk clostridial isolates include
C. disporicum, C. tyrobutyricum and C. sporogenes (Julien et al. 2008, Cremonesi
et al. 2012). Garde et al. (2011) detected lactate-fermenting clostridial spores
in 97% of the raw ovine milk samples examined. The Most Probable Number
(MPN) counts ranged from 0.36 (detection limit of the MPN method)
to 240 spores/mL with most of the milk samples having a spore count
between 1 and 10 spores/mL. Spores of clostridia that germinate and grow
fermentatively (converting lactic acid to butyric acid, carbon dioxide and
hydrogen) towards the later stages of cheese maturation are the causative
agents of the “late blowing” defect in cheeses (butyric acid fermentation)
with long ripening times (Cocolin et al. 2004). C. tyrobutyricum is considered
the primary cause of “late blowing” although additional species of clostridia
such as C. sporogenes may play secondary roles in cheese defects (Klijn et
al. 1995, Le Bourhis et al. 2007, Garde et al. 2011).
Clostridium spp. are also important in dairy microbiology as they are
involved in food poisoning episodes. C. perfringens food poisoning is usually
linked with spore germination and proliferation of vegetative cells after
temperature abuse of heat-treated foods. Other pathogenic clostridia include
C. botulinum, the causative agent of food-borne botulism, C. baratii and
C. butyricum. C. butyricum has been implicated in outbreaks of food-borne,
type-E botulism (Meng et al. 1997, Peck 2009). It has been reported that
clostridial proliferation in freshly produced raw milk is improbable due to
its highly positive Eh value (Goudkov and Sharpe 1965). Nonetheless, the
botulinum neurotoxin is the most toxic substance known to date and the
(raw) milk supply has been considered as a likely target for terrorist attacks
in terms of deliberate contamination with botulinum toxin. C. botulinum
neurotoxins type A and B and their corresponding complexes are inactivated
by at least 99.99% and 95%, respectively during raw milk pasteurization at
72°C for 15 s (Weingart et al. 2010).
Psychrotrophic bacteria
As mentioned previously, upon withdrawal from the mammary gland,

raw milk contains microorganisms at low concentrations, even under ideal
milking conditions. If raw milk is to be collected by the dairy industry for
further processing, it needs to be cooled and maintained at low temperatures
in order to prevent microbial growth. Therefore, refrigeration is applied
during the time interval between milking at the farm and processing at the
dairy plant as a means to keep microbial counts to low levels and prevent
milk microbiological deterioration. Depending on the circumstances, this
time period can vary from less than 24 hr to up to 2 or more days given
that: a) raw-milk pick-up from the farms is not always done on a daily
basis, particularly for small-sized cow farms or small-ruminant farms, and
b) upon arrival to the dairy plant, raw milk is stored in silos for several
hours (e.g., for quality control checks and in cases of late-evening deliveries)
before processing.
The rates of enzymatic reactions are reduced at low temperatures and, in
general, the growth of mesophilic microorganisms is significantly retarded
or even inhibited at low temperatures (e.g., refrigeration). The principle

physiological mechanisms responsible for microbial growth retardation/
inhibition at low temperatures involve cold-induced physicochemical
changes in the cell membrane lipid bilayer, protein misfolding and ribosome
instability. The refrigerated storage of raw milk, however, selects for
microorganisms that are capable of acclimation and proliferation at low
temperatures. Such microorganisms possess the genetic background and
concomitant physiological mechanisms enabling adaptation and proliferation
under chill stress (Jones and Inouye 1994, Graumann and Marahiel 1996,
Berry and Foegeding 1997, Beales 2004). Mesophilic microorganisms that are
also capable of proliferation at refrigeration temperatures (0–7°C) are called
psychrotrophic. Psychrotrophic organisms are characterized by temperature
growth optima and maxima in the mesophilic temperature range, yet
they are able to withstand and proliferate, albeit slower, at refrigeration
temperatures. Therefore psychrotrophic microorganisms possess a selective
advantage over other raw-milk microorganisms which are unable to grow
during cold storage of milk. Hence, upon prolonged storage of raw milk
at refrigeration temperatures, psychrotrophic microorganisms gradually
increase in numbers and, depending on the length and temperature of cold
storage, they can eventually become the dominant microbial group of raw
milk (Celestino et al. 1996).
Among other microbial groups of raw milk, most probably
psychrotrophic microorganisms cause the greatest concern to the dairy
industry nowadays, both in terms of quality and safety. Psychrotrops that
contaminate raw milk during milking originate from the farm environment
(e.g., soil or contaminated rinsing or cooling water), or from poorly cleaned
and sanitized milking equipment (Thomas 1966, Christiansson et al. 1999).
Psychrotrophic microorganisms can consist of molds, yeasts and bacteria
(Cousin 1982). Psychrotrophic bacteria belonging to different genera have
been isolated from raw or heat-treated milk. These include both Gram-
negative (e.g., Pseudomonas, Aeromonas, Serratia, Acinetobacter, Alcaligenes,
Achromobacter, Enterobacter, Flavobacterium) and Gram-positive (e.g.,
Bacillus, Clostridium, Corynebacterium, Microbacterium, Micrococcus) bacteria
(Cousin 1982). Pseudomonas spp. are probably the best known and most
studied among the Gram-negative psychrotrophic organisms. Growth of
pseudomonads in raw milk occurs from the beginning of the dairy chain
(farm tank), under both optimal and suboptimal storage temperature
conditions (De Jonghe et al. 2011). Raw-milk isolates belonging to several
additional bacterial genera (mainly Gram-negative), however, have been
recently identified as psychrotrophic and some also possess lipolytic and/
or proteolytic traits, which may vary at the species or even strain level
(Munsch-Alatossava and Alatossava 2006, Martins et al. 2006, Hantsis-
Zacharov and Halpern 2007, Ercolini et al. 2009, Nörnberg et al. 2010).
The results of different studies focusing on psychrotrophs in raw milk

are not directly comparable, as differences in the relative representation
and populations of different psychrotrophs have been noted among
studies, depending on factors such as milking practices, raw-milk storage
temperatures and holding times and milk type (e.g., bovine vs. ovine)
(Sanjuan et al. 2003). Different studies, however, help portray the often
underestimated variety of raw-milk microorganisms capable of growing
at refrigeration temperatures. In addition, the presence of unidentified
psychrotrophic bacterial isolates has been recently reported in raw milk
(Hantsis-Zacharov and Halpern 2007). It has been also demonstrated
that several Gram-negative proteolytic psychrotrophic bacteria isolated
from raw milk are capable of production of acylated homoserine lactones
in vitro. It has been speculated, therefore, that quorum sensing mechanisms
may be involved in the spoilage potential of such bacteria (Pinto et al. 2007)
and a review of the literature suggests the involvement of quorum sensing
mechanisms in spoilage of different food commodities (Ammor et al. 2008).
In order to control the microbiological quality of raw milk, several treatments
have been proposed and implemented, such as thermization (typically
heating milk at 65°C for 10–20 s), addition of CO2, or microfiltration (Roberts
and Torrey 1988, Champagne et al. 1994, Singh et al. 2012).
The dynamics and shifts in the bacterial populations of raw milk
during refrigerated storage were studied using molecular methods
(Lafarge et al. 2004, Rasolofo et al. 2010, Raats et al. 2011). Using Temporal
Temperature Gel Electrophoresis (TTGE) and Denaturing Gradient Gel
Electrophoresis (DGGE) analyses, Lafarge et al. (2004) showed that a 24-h
storage of raw cows’ milk at 4°C results in considerable evolution of certain
psychrotrophic bacterial populations, including pathogenic psychrotrophs.
The authors also noted considerable variation in the bacterial dynamics
between milk samples, indicating that the presence of different species
and/or strains in raw milks prior to refrigeration may significantly affect
their microbial balance during refrigerated storage. Rasolofo et al. (2010)
studied the bacterial dynamics in raw milks using a combination of
culture-dependent and molecular methods. Milk samples that had been
treated by either addition of CO2, thermization or microfiltration were
monitored over 7 days of storage at 4 or 8°C. Dominant bacterial species in
untreated, CO2-treated and thermized milk samples at day 3 belonged to
the genera Staphylococcus, Streptococcus, Clostridium, Aerococcus, Facklamia,
Corynebacterium, Acinetobacter and Trichococcus. Dominant bacterial genera
detected in micro-filtered milk were Stenotrophomonas, Pseudomonas and
Delftia. Pseudomonas spp. dominated the bacterial population of untreated,
CO2-treated and micro-filtered milk samples at day 7. Staphylococcus spp.
and Delftia spp. were the dominant bacterial genera in thermized milk.
Raats et al. (2011) demonstrated that considerable evolution of bacterial
communities occurs during cold storage of raw milk, but the taxonomic
diversity decreased with storage time as a consequence of some microbial
populations’ dominance during refrigeration. In their study, raw milk
samples were collected from bulk tanks of dairy farms and silo tanks of an
associated industrial processing plant. According to their analyses Gram-
positive bacteria (Bacilli, Clostridia, and Actinobacteria) prevailed in the
milk from the farm tanks during cold incubation, whereas milk samples
from the dairy plant were dominated by Gram-negative species belonging
to Gammaproteobacteria, especially Pseudomonadales.
Reduced quality of raw and processed milks is frequently a consequence
of the proliferation and metabolic activities of psychrotrophic bacteria.
Although the Gram-negative psychrotrophs are effectively destroyed by
pasteurization, some species of psychrotrophic bacteria can produce lipolytic
and proteolytic enzymes during growth in raw milk. These enzymes are
either not inactivated, or only marginally affected by the time/temperature
schemes used in the dairy industry for the manufacture of processed dairy
products (e.g., pasteurization, UHT processing). These lipases and proteases
can lead to the development of off-odors in raw milk. Additionally, being
heat-stable, they retain activity after the heat treatment of milk and therefore
gradually degrade milk proteins and lipids, leading to noticeable off-odors
and reducing dairy products’ shelf-life (Griffiths et al. 1981, Sørhaug and
Stepaniak 1997, Nörnberg et al. 2010). Therefore numerous published
manuscripts have emphasized the importance of the initial microbiological
quality of raw milk and the importance of the rapid and efficient cooling
of milk upon milking in terms of milk and dairy products’ quality (Banks
et al. 1988, Griffiths et al. 1987, 1988, Zeng et al. 2007).
As stated above, some aerobic spore-forming bacteria belonging to the
genus Bacillus (e.g., B. cereus) are psychrotrophic (Grosskopf and Harper
1974). Psychrotrophic strains of aerobic spore-formers are a major concern
to the dairy industry because they are able to survive milk pasteurization
and can subsequently proliferate in dairy products such as pasteurized
milk stored at low temperatures, often leading to spoilage (Meer et al.
1991). For instance, in the absence of post-pasteurization re-contamination
of milk by Gram-negative psychrotrophic bacteria (Eneroth et al. 2000),
the shelf-life of pasteurized milk largely depends on the presence (levels)
and spoilage potential of psychrotolerant spore-formers (Fromm and Boor
2004). Psychrotrophic aerobic spore-forming bacteria may be also significant
in terms of food safety. Psychrotrophic strains of B. cereus producing
enterotoxin have been identified (Van Netten et al. 1990). Also, the ability of
certain B. cereus strains to produce toxins in milk stored at 8°C under aerated
conditions has been demonstrated (Christiansson et al. 1989). Other well-
known psychrotrophic pathogens (Schofield 1992) that have been isolated
from raw milk are Listeria monocytogenes, Yersinia enterocolitica and Aeromonas
hydrophila. L. monocytogenes and Y. enterocolitica have been identified as the

causative agents in food-borne outbreaks associated with the consumption
of contaminated raw milk, raw-milk cheeses or dairy products that had
been contaminated post-pasteurization (Tacket et al. 1984, Lundén et
al. 2004). Amongst these pathogens, undoubtedly, L. monocytogenes is of
greatest concern for the dairy industry and its cold-adaptive mechanisms
have been studied in detail (Angelidis et al. 2002, Tasara and Stephan 2006).
The non-proteolytic group II Clostridium botulinum are psychrotrophic.
The reported C. botulinum outbreaks associated with dairy products are
rare, yet large, and have been associated with both commercial and home-
prepared dairy products. However, the role of the psychrotrophic group II
C. botulinum in dairy outbreaks is unclear due to the lack of epidemiological
data (Lindström et al. 2010).
A great number of comprehensive review articles on psychrotrophic
microorganisms of concern to raw milk and processed milk products can
be found in the literature. These review articles cover aspects pertaining
to the types (genera, species) of relevant organisms, their growth potential
and growth kinetics at refrigeration temperatures, their spoilage potential
through production of enzymes, public health considerations, their negative
effects and methods of control in raw milk and dairy products. Whereas
some of the information presented in older reviews may be outdated (e.g.,
due to the re-classification of certain microbial species to new or different
genera), older review articles offer a nice historical overview of approaches
practiced over the years to isolate, identify and characterize psychrotrophs
and of approaches used or proposed in order to minimize their negative
effects in raw milk and dairy products. Only a few of the many published
review articles on psychrotrophs are cited here (Cousin 1982, Champagne
et al. 1994, Shah 1994, Sørhaug and Stepaniak 1997).
Thermoduric bacteria
The term “thermoduric” is used to denote bacteria (Gram-positive

or Gram-negative) that are usually isolated from raw milk after
pasteurization. Some authors use the term thermoduric to denote countable
survivors following milder heat treatments such as milk thermization
(e.g., 60–65°C for ca. 10 s). In official textbooks, thermoduric bacteria
are defined as “microorganisms (vegetative cells or spores) that survive
pasteurization treatment” (Frank and Yousef 2004). Thermoduric bacterial
counts are estimated by heating milk at 62.8°C ± 0.5°C for 30 min to simulate
batch pasteurization and survivors are counted using the SPC method. The
presence of thermoduric bacteria in sufficiently high numbers in raw milk will
result in small, though significant surviving populations after pasteurization.
In the absence of post-treatment contamination, the microorganisms that
survive pasteurization can affect the shelf-life of pasteurized milk. This is

particularly the case for psychrotrophic thermoduric bacteria, such as those
belonging to the genus Bacillus (Collins 1981).
Although some dairy microbiology textbooks include only a few, yet
diverse bacterial genera under this category (Bacillus spp., Clostridium spp.,
Corynebacterium spp., Kocuria spp., Lactobacillus spp., Micrococcus spp.,
Microbacterium spp., Rothia spp.), it appears that members of additional
bacterial genera could potentially meet the definition of “thermoduric”.
For instance, members of the genera Staphylococcus spp., Pseudomonas
spp., and Moraxella spp., have been isolated from raw milk samples after
heat treatment at 80°C for 10 min, i.e., a heat treatment more intense than
laboratory pasteurization (Coorevits et al. 2008). Ranieri and Boor (2009)
characterized the bacterial isolates obtained from 2% fat pasteurized milk
samples processed at 18 fluid milk processing plants, representing five
geographical regions across the US. Overall, 21 different bacterial genera
were identified by 16S rDNA sequencing. The most frequently isolated
Gram-positive genus was Bacillus; Paenibacillus was the second most
frequently isolated Gram-positive genus. However, additional Gram-
positive bacteria identified were Staphylococcus, Leuconostoc, Enterococcus,
Streptococcus, Brevibacillus, Corynebacterium, Lactococcus, Microbacterium,
Micrococcus, and Oceanobacillus. Pseudomonas was the most frequently
isolated Gram-negative genus. Other Gram-negative bacteria identified
belonged to the genera Acinetobacter, Yersinia, Enterobacter, Shewanella,
Aeromonas, Flavobacterium, Pantoea, Sphingobacterium and some isolates
belonged to the Enterobacteriaceae family (Ranieri and Boor 2009). It was
not possible, however, to distinguish how many of these isolates were post-
pasteurization contaminants.
As previously discussed, in the absence of post-pasteurization
contamination, thermoduric spore-forming psychrotrophs (e.g., members of
the genera Bacillus and Paenibacillus) often limit the shelf-life of refrigerated
pasteurized milk.
Lactic acid bacteria
Lactic acid bacteria (LAB) constitute one of the main groups of raw-milk
microorganisms. The most common source of LAB in raw milk is the
udder skin and teat canal, but LAB also colonize animal sites such as the
vagina or the intestine. The term often used to denote LAB present in
raw milk is autochthonous, or non-starter LAB, to distinguish them from
the starter LAB, i.e., the known and characterized strains of LAB that are
deliberately added to milk for the manufacture of fermented dairy products.
Traditionally, the manufacture of fermented dairy products relied upon
the presence and acidification activity of LAB present in raw milk. This
practice is still used nowadays for the manufacture of some artisanal dairy
products. The biopreservation effect of LAB relies on the fermentation
of the major milk sugar (lactose) and the production of organic acids
with a concomitant reduction of milk pH and therefore the creation of an
acidic environment, unfavorable for the proliferation and/or survival of
pathogenic microorganisms. The rate of acid production as well as the
final pH is of vital importance for the safety of fermented dairy products.
The production of several antimicrobial compounds such as bacteriocins,
which are active against several food-borne pathogens, also contributes to
the bio-preservative effects of LAB. Nowadays a vast majority of fermented
dairy products is produced using well-defined (mixtures of) LAB strains.
Most LAB do not grow at refrigeration temperatures and souring of raw
milk is highly unlikely under adequately refrigerated conditions. Elevated
titratable acidity in milk nowadays is rare and usually an indication of
raw-milk temperature abuse in the farm or during transport. In certain
instances, the presence of adventitious LAB or the use of inappropriately
selected (e.g., overactive) starter LAB, can lead to quality defects in
fermented dairy products (e.g., bitterness, production of gas, or extreme
acidity). On the other hand, underactive LAB, due to the presence of
antibiotic residues or bacteriophages in raw milk, can delay or even halt
the fermentation process.
LAB are usually classified based on: a) their optimum growth
temperature into mesophilic (around 30°C) and thermophilic (around 43°C),
and b) fermentation reactions/metabolic pathways into homofermentative
(lactic acid is the primary and most abundant end-product of lactose
fermentation) and heterofermentative (other compounds in addition to
lactic acid are produced in significant amounts, such as acetic acid, or
carbon dioxide). Raw-milk LAB belong to all major LAB genera (Lactococcus,
Lactobacillus, Streptococcus, Leuconostoc, Pediococcus, Enterococcus). There are
probably hundreds of published articles on the isolation, physiological and
technological characterization of LAB isolated from raw bovine, caprine or
ovine milk and raw-milk cheeses and one such study is cited here (Franciosi
et al. 2009). Details on LAB used in the manufacture of dairy products are
presented in Chapter 4 of this book.
Mastitis-causing organisms
Mastitis (intra-mammary infection, IMI) is one of the costlier diseases of

lactating animals. Mastitis alters milk composition, reduces milk secretion
and has serious animal-welfare, economic and possibly public health
implications. Depending on its clinical manifestation (clinical or subclinical),
severity (e.g., acute, sub-acute, chronic), the epidemiology of the primary
pathogen (contagious or environmental) and the specific etiology, mastitis

results in variable degrees of animal suffering, losses in milk yield, increases
of milk SCC and animal culling. Mastitis can be of infectious or traumatic
etiology but the vast majority of cases are of infectious nature. Mastitis-
causing pathogens include bacteria, fungi, viruses or algae. Bacterial
pathogens (including mycoplasms) are responsible for the vast majority
of infectious mastitis cases in lactating ruminants.
Contagious bovine mastitis is typically spread between cows during
milking. The primary reservoir of contagious pathogens is the infected cows’
udder quarter(s). Contagious mastitis is usually caused by Staphylococcus
aureus, Streptococcus agalactiae and Mycoplasma spp. Environmental mastitis
is usually the result of infection with coliform bacteria (Escherichia coli,
Klebsiella spp., Enterobacter spp.), non-coliform Gram-negative bacteria such
as Serratia spp., Pseudomonas spp., Proteus spp., Gram-positive bacteria such
as Enterococcus faecium and E. faecalis, some Bacillus spp. and streptococci
other than Str. agalactiae (i.e., “environmental streptococci” such as
Str. uberis). Prototheca spp. are unicellular algae rarely reported to infect
the mammary gland of dairy cattle (Marques et al. 2010). Environmental
mastitis pathogens originate from the farm environment (e.g., bedding
material). The etiology, control/prevention and treatment of the various
types of bovine mastitis have been reviewed by several experts in the field
(Watts 1988, Cullor 1993, Fox et al. 2005, Contreras and Rodríguez 2011,
Fox 2012, Hogan et al. 2012, Schukken et al. 2012).
Staphylococcus spp. are the most prevalent pathogens responsible
for IMIs in small ruminants. Other pathogens such as Streptococcus
spp., members of the Enterobacteriaceae family, Pseudomonas aeruginosa,
Mannheimia haemolytica, Mycoplasma spp., Corynebacteria and fungi can
cause IMI in small ruminants, albeit at a lower incidence (Contreras et
al. 2007). Coagulase-negative staphylococci (CNS) are the most prevalent
pathogens causing subclinical mastitis in dairy ruminants. CNS can also
produce persistent subclinical mastitis and significantly increase the
SCC of milk. Contreras et al. (2007) have summarized the etiological,
epidemiological and control aspects of mastitis in small ruminants.
The role of viruses and viral infections in bovine mastitis has been
reviewed by Wellenberg et al. (2002). At least four bovine pathogenic
viruses have been isolated from the milk of cows with clinical mastitis
(bovine herpesvirus 1, bovine herpesvirus 4, foot-and-mouth disease virus
and parainfluenza 3 virus). The authors concluded that viral infections can
play a direct or more likely an indirect (e.g., via immune suppression, teat
skin or mammary tissue lesions predisposing the animals to bacterial IMIs)
role in the etiology of bovine mastitis and that the role of viruses warrants
further investigation.
Human pathogenic bacteria
The intestinal tract of lactating mammals is the source of several microbial

species that can be pathogenic for humans. These pathogenic organisms are
therefore frequently present in the environment of lactating animals as well
as on the animal hide and udder skin and can contaminate raw milk during
milking under non-hygienic conditions. Additionally, vegetative cells and
spores of spore-forming bacterial pathogens and molds can be found in
the environment and their transfer to raw milk can be airborne or via dirt.
The lactating udder of animals with clinical or sub-clinical mastitis as well
as biofilms formed at the inner surfaces of milking and storage equipment
can also constitute sources of pathogenic microorganisms.
The consequences of human illnesses due to food-borne pathogens are
very severe (Hoffmann et al. 2012). Judging from their involvement in the
reported food-borne outbreaks, processed milk and dairy products appear
to maintain a good safety record among other food categories (http://
www.cdc.gov/foodsafety/outbreaks/multistate-outbreaks/outbreaks-list.
html). The consumption of unpasteurized milk and dairy products made
with raw milk, however, is not advisable (Leedom 2006, Cavirani 2008,
LeJeune and Rajala-Schultz 2009, Oliver et al. 2009, Giacometti et al. 2012,
Langer et al. 2012). In the US for instance, between 1973 and 1992, 46 raw-
milk associated outbreaks were reported to the Centers for Disease Control
and Prevention, with a median number of people becoming ill in these
outbreaks equal to 19 (Headrick et al. 1998). Nonetheless, in some States
as well as EU Member States the sale of raw milk for human consumption
is allowed, e.g., in delicatessen stores or via self-service automatic vending
machines. There are dozens if not hundreds of published articles dealing
with the prevalence of specific pathogens or groups of pathogens in raw
milk worldwide, and numerous pathogenic organisms have been found
in or associated with raw milk over the years. The complete listing of
these pathogens or a thorough description of their incidence in raw milk,
epidemiological spread, symptoms and pathogenesis in humans, methods of
intervention for prevention of milk contamination or pathogen inactivation,
or listing of the relevant outbreaks (raw-milk outbreaks or outbreaks from
the consumption of dairy products made with unpasteurized milk) would
be unfeasible to cover in one chapter. Furthermore, due to differences in the
sampling and analytical protocols used in the pathogen prevalence studies it
is not possible to report “average” pathogen-specific prevalence estimates in
raw milks. The reader is therefore more suited to consult recently published
comprehensive review articles on these subjects. For instance, Oliver et
al. (2005, 2009) reviewed the food-borne pathogens in milk and the dairy
farm environment and have commented on their food safety and public
health implications; the potential food safety hazards associated with the
consumption of raw milk are addressed and data on the prevalence of food-
borne pathogens in raw milk and raw milk-borne disease outbreaks in the
US from 2000 until 2008 are summarized. In addition, under the section
“Pathogens in milk”, the second edition of the encyclopedia of dairy sciences
(Fuquay et al. 2011) contains chapters dedicated to 14 pathogenic bacterial
families, genera or species commonly associated with raw milk (Bacillus
cereus, Brucella spp., Campylobacter spp., Clostridium spp., Coxiella burnetii,
Escherichia coli, Enterobacteriaceae, Enterobacter spp., Listeria monocytogenes,
Mycobacterium spp., Salmonella spp., Shigella spp., Staphylococcus aureus and
Yersinia enterocolitica). The chapters have been written by highly-respected
experts in the field and contain updated information on pathogens’
characteristics, physiology, resulting milk-borne illness, toxins, outbreaks,
incidence in dairy products including raw milk, potential sources and
suggested control measures at the farm. Pathogens in dairy products are
also covered in Chapter 3 in this book. The remaining of this section focuses
on specific (emergent?) bacterial agents with known or suspected public
health importance, recently shown to be occasionally present in raw milk.
Mycobacterium avium subsp. paratuberculosis (MAP) is the causative
agent of paratuberculosis (Johne’s disease), a chronic debilitating disease
(infectious enteritis with cachexia) affecting ruminants and other animals
worldwide. Johne’s disease has been reported in many countries worldwide.
Across the EU there are many published studies on the prevalence of MAP
in cattle and a smaller number of studies on sheep and goats (Nielsen and
Toft 2009). Infected animals are shedding the organism periodically in both
feces and milk (Streeter et al. 1995). The presence of MAP in milk for human
consumption poses concerns due to its possible association with Crohn’s
disease in humans (Grant 2005). The occurrence of MAP in bulk tank milk
and individual milk samples at cattle dairy farms worldwide was reviewed
by Okura et al. (2012). The authors reported a considerable variation of MAP
in bulk tank milk and individual cows’ milk. The overall MAP apparent
prevalence and 95% CI based on PCR and culture were summarized to 0.1
(0.04–0.22) or 10% on a per cent basis in bulk tank milk samples, and 0.2
(0.12–0.32) or 20% on a per cent basis in individual milk samples.
Milk pasteurization is a heat treatment process for a given period of
time that has been designed to inactivate the most heat-resistant, non-
spore-forming pathogenic bacteria that may be present in raw milk, i.e.,
Mycobacterium bovis and Coxiella burnetti, the causative agents of tuberculosis
and Q-fever in humans, respectively. The legal minimum time/temperature
combination for pasteurized milk is the heating of milk at 72°C for 15 s
(HTST) or 63°C for 30 min [low-temperature, long-time pasteurization
(LTLT)] (Commission Regulation (EC) No. 853/2004). There are several
published reports indicating survival of low numbers of MAP after
pasteurization of either artificially contaminated or naturally contaminated
milk. Therefore it has been suggested that, depending on the initial MAP
load of raw milk, low numbers of MAP may occasionally survive HTST
pasteurization (Grant 2006, Van Brandt et al. 2011b). Review articles have
been published summarizing the available (and often contradictive) data
regarding the prevalence of MAP in raw and pasteurized milks and other
dairy products, as well as the effects of pasteurizing treatments on MAP
survival in milk (Eltholth et al. 2009, Gill et al. 2011). Only a few studies
have been published on the behavior of MAP during the production and
storage of fermented milk products (cheddar cheese, yogurt, acidophilus
milk and kefir). It appears that the type of fermented dairy product, the type
of starter culture, the stage of MAP inoculation (pre- vs. post-fermentation
inoculation), the MAP strain, and the pH of the finished product affect the
(extent of) survival of MAP (Donaghy et al. 2004, Van Brandt et al. 2011a,
Klanicova et al. 2012). More studies are warranted, however in this field.
Helicobacter pylori is the causative agent of gastric ulcers and other
pathogenic conditions in humans. The presence of H. pylori in a small
fraction of raw cows’ milk samples examined was demonstrated via
fluorescence in situ hybridization (Angelidis et al. 2011), although its
presence had been proposed/suspected by the findings of earlier studies
based on PCR (Fujimura et al. 2002, Quaglia et al. 2008). It has been
hypothesized that sheep may constitute a reservoir of H. pylori (Dore et al.
2001). At present, the risk of human H. pylori infection via consumption of
contaminated raw milk or dairy products manufactured with contaminated
raw milk remains unknown.
Arcobacter spp. are Gram-negative, spiral-shaped bacteria belonging
to the family Campylobacteraceae. Arcobacter spp. have been repeatedly
isolated from feces of livestock animals including cows, whereas only
a few investigations have reported their presence in feces of goats and
sheep (Van Driessche et al. 2003, De Smet et al. 2011). Arcobacter spp. are
considered emerging waterborne and food-borne human pathogens with
infection symptoms similar to campylobacteriosis. Consumption and
handling or raw or undercooked meats (mainly poultry) are potential
sources of infections in humans (Lehner et al. 2005). The potential role of
Arcobacter spp. in human disease, however, needs further evaluation. A few
studies from different countries have reported the isolation of Arcobacter
spp. at different frequencies (5.8–46%) from raw bovine milk (Scullion et
al. 2006, Shah et al. 2012).
Generalizations in food microbiology/food safety should be avoided
or at least made with caution. Nonetheless, from a practical point of view
if raw milk is to be used for the manufacture of processed milks or other
dairy products whose technology involves a heat treatment step equal to
or more intense than that of pasteurization, the main food-safety concerns
ought to be the potential presence of S. aureus enterotoxins, mycotoxins

and possibly MAP.
Fungi (Yeasts and Molds)
Yeasts and molds are eukaryotic microorganisms and their differentiation

is often difficult due to their structural transitions during reproduction
and/or environmental/growth conditions. Molds usually grow by forming
elongated filaments (hyphae) resulting in the formation of mycelia. Molds
are strictly aerobic organisms, whereas yeasts can grow both in the presence
and absence of oxygen. Yeasts and molds are special in the sense that they
can grow in environments characterized by high osmolality, increased
acidity or low temperature. Fleet (1990) has summarized some of the
properties of yeasts that are important for their growth and predominance
in dairy products. These are the fermentation or assimilation of lactose,
the production of extracellular proteolytic and lipolytic enzymes, the
assimilation of lactic and citric acid, their potential for growth at low
temperatures and their tolerance of elevated osmotic strength (high salt
concentrations). Yeast populations in raw milk are typically less than
103 cfu/mL and, unless bacterial growth is inhibited by the presence of
antibiotic residues, they usually do not grow during refrigerated storage
of milk, as they are overgrown by psychrotrophic bacteria (Cousin 1982).
Yeasts may develop in spoiled raw milk as secondary flora, when bacterial
growth has ceased and the pH of milk has dropped significantly.
Compared to the bacterial diversity, very few studies have looked
into the fungal diversity of raw milks. The study of Vadillo Machota
et al. (1987) is one of the few studies in the literature dedicated solely
to the examination of yeasts and molds in raw milk. In this study, the
examination of 103 tank milk samples in Spain revealed more than twenty
different fungal genera, the most frequently isolated being Geotrichum,
Cladosporium, Penicillium, Aureobasidium and Aspergillus. More recent
studies have shown that common mold genera isolated from raw milk
include Penicillium spp., Aspergillus spp., and Eurotium spp. (Vacheyrou
et al. 2011), whereas common yeast genera/species include Candida spp.,
Kluyveromyces spp., as well as Debaryomyces hansenii, Rhodotorula spp. and
Cryptococcus spp. (Callon et al. 2007, Mallet et al. 2012). The analysis of
raw milk with molecular, DNA-based methods can help identify a wider
variety of yeast species. For instance, Cocolin et al. (2002) studied the yeast
biodiversity in raw cows’ milk by using a) traditional culture methods,
and b) PCR to amplify a region of the 26S rRNA gene from a DNA pool
extracted directly from raw milk, followed by DGGE. The resulting bands
were extracted and sequenced to identify yeast isolates. The combination
of PCR-DGGE led to the identification of a number of additional yeast
species, not isolated via the traditional cultural approach. Chen et al. (2009)
reported on the biodiversity of yeasts in raw milk from dairies in China.
The authors identified 11 different species of yeasts using an integrated
approach including phenotypic and molecular (RAPD-PCR analysis and
partial sequencing of the 26S rDNA) methods. A recently published article
reported the results of a Canadian survey aiming on characterizing the
fungal microflora of raw cows’ milk (111 samples) and raw-milk cheeses
collected from 19 dairy farms (Lavoie et al. 2012). Molecular identification
analyses of the isolates using the ITS1-5.8S-ITS2 rDNA region showed that
almost two-thirds of the fungal isolates were yeasts that could be assigned
to 37 species/11 genera and the remaining isolates were molds that could
be assigned to 33 species/25 genera. Debaryomyces hansenii was the most
abundant among the yeast species (21% of the 339 yeast isolates) and was
detected in the milk of 14 from the 19 farms. Candida was the most abundant
(44% of the isolates) and most diverse (11 species) genus. Other frequently
identified yeast genera were Cryptococcus spp. (10.9% of the yeast isolates)
and Pichia spp. (8% of the yeast isolates). Eurotium was the most abundant
mold genus (about one-quarter of the mold isolates) and was isolated
from the milk of 13 from the 19 farms. Lichtheimia was the second most
abundant mold genus (13.3% of the mold isolates) and was isolated from
nine farms. The authors argued that only a fraction of the fungal species
may have been actually identified. Nonetheless, the findings of their study
indicate that the fungal profile of milk differs from farm to farm. In France,
Delavenne et al. (2011) evaluated the fungal diversity in a smaller collection
of raw milk samples (nine), which also included goat and ewe samples,
in addition to cow milk samples. Following DNA extraction from milk
samples, a semi-nested PCR method was used to amplify the ITS1 region of
fungal DNA and PCR products were analyzed by ion-pair, reverse-phase,
denaturing, high-performance liquid chromatography (D-HPLC). Fractions
of each peak were retrieved and sequenced. The approach enabled the
identification of 27 fungal species (18 yeast species belonging to 9 genera,
Candida, Cryptococcus, Debaryomyces, Geotrichum, Kluyveromyces, Malassezia,
Pichia, Rhodotorula and Trichosporon and 9 mold species belonging to 7
genera, Aspergillus, Chrysosporium, Cladosporium, Engyodontium, Fusarium,
Penicillium and Torrubiella). The authors reported the highest fungal diversity
in cow milk with a total recovery of 23 different species (five mold and 18
yeast), whereas only six (one mold and five yeast) and seven (one mold
and six yeast) species were recovered in goat and ewe milk, respectively.
The most common fungal species among the nine milk samples were
G. candidum, K. marxianus and Candida spp. such as C. parapsilosis (Delavenne
et al. 2011). In Italy, Corbo et al. (2001) analyzed 26 samples of raw cow,
ewe, goat and water buffalo milk for the presence of yeasts using cultural
and biochemical methods and reported the isolation of 36 yeast species.
The most frequently occurring yeasts belonged to the species Trichosporon

cutaneum (15.2%), Candida catenulata (10.5%), Yarrowia lipolytica (8.6%),
C. zeylanoides (4.8%) and C. sake (4.8%).
Yeasts and molds are of interest to dairy microbiologists for several
reasons (Rohm et al. 1992, Jakobsen and Narvhus 1996, Leclercq-Perlat
2011). On the positive side, some species are used in dairy fermentations.
An example is the use of yeasts residing in kefir grains for the production
of Kefir, a fermented dairy product that undergoes a mixed lactic and
alcoholic fermentation (Wang et al. 2012). Another example is the use
of molds for the ripening of certain types of cheeses, e.g., the use of
Penicillium roquefortii for the interior mold-ripened cheese Roquefort and
P. camemberti for the surface mold-ripened cheese Camembert. On the other
hand, being ubiquitous organisms (air, soil), yeasts and molds can easily
contaminate raw milk. The contamination of dairy products with yeasts or
molds is often the reason leading to quality defects and/or public health
concerns. For instance, the accidental yeast contamination followed by
their proliferation during the manufacture or ripening of dairy products
leads to the generation of off-odors, as a result of extensive proteolysis of
milk proteins and/or lipolysis of milk fats, respectively, or causes bloating
of cheese containers due to the production of carbon dioxide. The surface
contamination of yoghurt by yeasts or molds often leads to visible surface
growth and product rejection. Yeast spoilage is a problem primarily in
fermented milks and cheeses and the main defects caused by spoilage yeasts
are the development of fruity or bitter flavors, discolorations and swelling
of products or product containers due to gas production. Some of the yeast
strains isolated from raw milks are able to grow at refrigeration temperatures
and also able to produce proteinases and phospholipases when incubated
in laboratory media under refrigeration temperatures (Melville et al. 2011).
Molds can act as spoilage agents mostly in cheeses, yogurt and
sweetened condensed milk and spoilage is typically the result of airborne
post-pasteurization contamination. In addition, some mold species are
known to produce mycotoxins. Aflatoxins (AFs) are mycotoxins produced
by some strains of the mold species Aspergillus flavus, A. parasiticus and
A. nominus. From a public health perspective, AFs are probably the most
important mycotoxins because of their highly toxic chronic or acute effects
on human health; AFs are potent human carcinogens (Pitt 2000, Fujimoto
2011) and can be present in raw milk usually as a result of mold-growth and
AF production in animal feedstuffs. There are four main AFs: AFB1, AFB2,
AFG1 and AFG2. AFB1 can be produced in animal feeds during production
or storage. Upon ingestion by lactating animals the AFB toxins are converted
to the AFM metabolites in the liver and ca. 0.9% of ingested AFB1 is found in
the milk as the hydroxylated metabolite AFM1 (Tabata 2011). The occurrence
of AFM1 in milk, especially bovine milk, is of particular concern for public
health because of the importance of cows’ milk as a foodstuff for children

and adults. In the EU, the maximum limit for AFM1 in milk is set at 0.05
µg/Kg (Commission Regulation (EC) No 1881/2006). Chromatographic
and ELISA methods are used for the determination of the AF content of
raw milk (Tabata 2011). AFs are particularly heat tolerant. Although the
results of studies on AFM1 degradation during heat processing of milk and
dairy products are not always consistent, most studies indicate that AFM1
is not appreciably altered during the time/temperature combinations used
in milk processing (pasteurization, sterilization). Hence, the presence of
AFs in raw milk poses a severe risk to public health. Therefore raw milk
containing AFs in levels exceeding the legal limits should never be used or
further processed for animal or human consumption. Numerous surveys
are published each year worldwide conveying the results of investigations
regarding the presence and/or quantitative determination of AFs in raw
milk or other dairy products (Galvano et al. 1996, Prandini et al. 2009). The
construction and use of a vaccine for lactating dairy cows for the prevention
of AFB1 carry over in milk has been reported (Polonelli et al. 2011).
Viruses
Human pathogenic viruses
There are many pathogenic viruses for humans known to be transmitted via
consumption of contaminated foods, and according to a literature review
by Newell et al. (2010) food-borne viruses belong to at least 11 known viral
families. Contamination of foods with most of these food-borne viruses often
results from non-hygienic food-handling practices by human carriers. In
addition, the actual involvement of foods in viral food-borne outbreaks is
very difficult to approximate, because in many cases, following the initial
food-borne incident (“seeding event”), viruses easily spread from one
infected individual to another, without involvement of contaminated food
sources. Among food-borne viruses, noroviruses and the hepatitis A virus
have been the causative agents in well-documented causes of food-borne
illness (Koopmans and Duizer 2004). Cliver (1997) stated that “all known
food-borne viruses except the agent of tick-borne encephalitis are human
specific and transmitted by a fecal-oral cycle”.
There are some publications dated back in the seventies about the
incidence/contamination of raw milk with animal viruses (zoonotic or
not) and human viral outbreaks that have occurred as a consequence
of consumption of contaminated milk or raw-milk cheeses (Kefford et
al. 1979). It should be stressed, however, that in most cases the origin of
viral contamination of raw milk in these instances was external, i.e., from
environmental sources such as contaminated water or human carriers, and
most of the implicated viruses (poliomyelitis virus or “infectious hepatitis”,

now called hepatitis A) were of human origin. More recently, Wellenberg
et al. (2002) reviewed the role of viruses and viral infections in bovine
mastitis. Animal viruses that have been isolated from raw bovine milk are
the bovine herpesvirus 1, the bovine herpesvirus 4, the foot-and-mouth
disease (FMD) virus, and the parainfluenza 3 virus. The authors concluded
that viral infections can play a direct or indirect role in the etiology of
bovine mastitis. It has been hypothesized that bovine herpesvirus 4 could
represent a danger for human health. The virus has been shown to replicate
in permissive human cells and protect non-permissive, persistently infected
cells from apoptosis (Gillet et al. 2004). A subsequent study demonstrated
that pasteurization was sufficient to completely inactivate the infectivity
of 3.0 × 106 plaque-forming units of bovine herpesvirus 4 per mL of milk
(Bona et al. 2005).
The literature pertaining to the incidence of zoonotic viruses in raw milk
is scant. Some animal-origin viruses that are pathogenic to humans can be
found in the raw milk of infected lactating mammals. Milk from rabid cows
can contain the rabies virus (Lyssavirus), one of the members of the viral
family Rhabdoviridae. In the US, two incidents of potential mass exposures
to rabies through drinking unpasteurized milk have been reported (Centers
for Disease Control and Prevention 1999). Tick-borne encephalitis is a
zoonotic, potentially lethal neurological viral infection usually transmitted
to humans by bites of ticks (Ixodes ricinus and Ixodes persulcatus) (Mansfield
et al. 2009). The tick-borne encephalitis virus (TBEV) is a member of the
genus Flavivirus. The TBEV can be found in the milk of cows and goats with
encephalitis and consumption of unpasteurized milk from TBEV infected
animals may constitute a secondary means of transmission to humans
(Mansfield et al. 2009, Cisak et al. 2010, Caini et al. 2012). FMD affects all
cloven-hoofed animals and is probably the most contagious among animal
diseases. It is caused by an Aphthovirus of the family Picornaviridae. FMD
can be rarely transmitted to humans in close contact with infected animals
(Bauer 1997, Prempeh et al. 2001). The FMD virus is secreted into the milk of
infected animals before the onset of clinical signs. Therefore the movement
of contaminated milk during FMD outbreaks can contribute to the spread
of the disease among susceptible animals (Donaldson 1997). Literature data
on human infections by consumption of contaminated raw milk seem to
be limited to a report (cited by Bauer 1997) describing the self-infection of
veterinarians who deliberately drank raw milk from infected cows in the
19th century. Current minimum pasteurization standards of milk may not
be adequate to completely eliminate FMD virus (Tomasula and Konstance
2004). Rift Valley Fever (RVF) is a viral zoonosis that affects sheep, goats,
buffalos and cattle. RVF virus is a Phlebovirus of the family Bunyaviridae.
Humans get infected by mosquito bites, especially during periods of
intense epizootic activity, i.e., during ongoing epidemics among animals.

It has been stated that “Drinking raw, unpasteurized milk from infected
animals can also transmit RVF” (Balky and Memish 2003). However, to
my knowledge there are no published reports documenting milk-borne
RVF infections in humans. Foamy viruses are a subfamily of retroviruses.
Cows infected with bovine foamy virus (BFV) shed BFV into the milk
(Romen et al. 2007). Humans can be infected by simian foamy viruses. I
am not aware of any published studies pertaining to the likelihood of BFV
transmission to humans. Whether additional zoonotic animal viruses exist
with the potential of milk-borne transmission to humans appears to be
unsubstantiated at present.
Lactic bacteriophage
Strains of Streptococcus thermophilus and species of the genera Lactococcus,

Lactobacillus, Leuconostoc and Pediococcus are widely used by the dairy
industry as starter cultures for the manufacture of a variety of fermented
dairy products. Bacteriophage are “bacteria-eating” viruses (viruses that
infect bacteria) that require bacterial hosts to propagate. Bacteriophage
are ubiquitous in nature and are found in all bacterial ecosystems.
Bacteriophage specific for dairy starter cultures, i.e., phage attacking and
inactivating LAB (lactic bacteriophage) have been recognized as a significant
and often persistent problem for the dairy industry. Lactic bacteriophage
are often present in raw milk. The presence of lactic bacteriophage in
the milk used for milk fermentations (e.g., cheesemaking) leads to loss
of fermentative capacity associated with starter culture lysis, which can
significantly retard or even halt the fermentation process. Despite the
development of a variety of counter-measures, such as the application
of starter culture rotation schemes, improved sanitation strategies, and
the use of bacteriophage-resistant starter strains, phage contamination
during dairy products’ manufacture continues to be the leading cause of
failed or retarded fermentations. The dairy environment frequently serves
as a phage reservoir, especially the incoming milk and lysogenic starter
cultures. The technological importance of phage in the dairy industry
has been reviewed by Lyne (2011). Sturino and Klaenhammer (2004) have
reviewed the life cycles of bacteriophage as well as the defense strategies
used by the dairy industry aiming at protecting starter cultures against
phage-related problems.
Indigenous (Natural) Antimicrobial Agents of Raw Milk

Raw milk contains several indigenous antimicrobial agents. Probably the
best known antimicrobial agents are the immunoglobulins (i.e., pathogen-
specific antibodies), the non specific defense agents lactoferrin and lysozyme
and the enzyme lactoperoxidase (International Dairy Foundation 1991b,
Korhonen et al. 2000). Several peptides and other organic compounds (e.g.,
free fatty acids) in milk have shown to possess antimicrobial properties. In
addition, a wide variety of bacteriocins with variable antimicrobial spectra
can be produced by LAB. In recent years, the presence of additional host-
defense related minor proteins and peptides has been documented in cow’s
milk (Hettinga et al. 2011, Wheeler et al. 2012).
Bovine milk contains low levels of lysozyme (ca. 0.1 µg/mL) but its
concentration increases during mastitis (1–2 µg/mL). The antibacterial
role of lysozyme relies on the cleavage of the glycosidic bond between
N-acetylmuramic acid and N-acetylglucosamine in bacterial cell-wall
peptidoglycan (International Dairy Federation 1991b, Benkerroum 2008).
Lactoferrin is a whey protein with iron chelating properties. Its principle
antimicrobial action relies on depriving bacteria of iron, which is an
essential element for bacterial growth. Its concentration in bovine milk
(ca. 0.2 mg/mL) can increase several-fold during mastitis (International
Dairy Federation 1991b). Recent studies have attributed additional beneficial
properties to lactoferrin such as anti-cancer, immunomodulatory and
antioxidant properties (García-Montoya et al. 2012). Lactoperoxidase (LP),
i.e., the milk peroxidase, is one of the most abundant enzymes in bovine milk
constituting ca. 0.5% of the whey proteins and one of the three components
of the LP system of raw milk. LP is an oxidoreductase which catalyses the
oxidation of thiocyanate ions (SCN-) that are present in milk as a result of the
cows’ diet into hypothiocyanate ions (SCNO-) at the expense of hydrogen
peroxide (H2O2). Hypothiocyanate ions and other intermediates are the
reactive products of the LP system of milk and are potent antimicrobials.
The LP activity and thiocyanate content in raw milk can be affected by
factors such as the individual animal, the lactating species, the animal feed,
the time after milking and the stage of lactation (Althaus et al. 2001, Fonteh
et al. 2002, Yaqub et al. 2012). The hydrogen peroxide needed to activate
the LP system can be generated by the LAB of milk or by an indigenous
enzymatic system (xanthine oxidase/hypoxanthine). Most typically, H2O2
is provided exogenously in order to activate the LP system and thus help
preserve raw milk in situations where refrigerated storage of raw milk
between milking and processing is not possible (Haddadin et al. 1996). The
concentrations of the components of the LP system, the extent and range
of its antimicrobial action and its applications in milk and dairy products
have been presented in related review articles (Wolfson and Sumner 1993,
Kussendrager and van Hooijdonk 2000, Seifu et al. 2005).
The raw-milk natural antimicrobial compounds exert a bacteriostatic
effect for a time period following milking, the duration of which may
depend on several factors including storage temperature. For instance, the
duration of the antibacterial effect following activation of the LP system

was found to be inversely related to the storage temperature of milk
(International Dairy Federation 1988). The raw-milk natural antimicrobial
compounds are inactivated at variable degrees upon milk pasteurization or
more intense heat treatments. LP is relatively heat-stable and its inactivation
has been reported to start at 70°C; its heat stability depends on pH, being
less heat-stable under acidic conditions (Kussendrager and van Hooijdonk
2000). The inactivation of LP after heat treatment of milk at 85°C for 20 s is
used as an index for the determination of milk that has undergone high-
temperature processing (Extended Shelf-Life milk).
Concluding Remarks
The determination of the microbiological flora of raw milks has been the
subject of scientific research for more than one century. Depending on the
nature of the organisms sought, traditionally milk samples (diluted or
undiluted) are plated onto agar media (selective or nonselective) and are
incubated (aerobically, under microaerophilic or even anaerobic conditions)
at temperatures thought or known to be optimum for the growth of the
target organism(s). Enrichment steps in semi-selective or selective broths are
frequently used to enable preferential proliferation of the target organism(s)
against the background microbial flora of raw milk. Other approaches and
steps such as filtration and centrifugation have been used as a means of
bacterial concentration, assisting subsequent detection steps. Following
the isolation of any given microorganism, further identification strategies
include the direct observation under the microscope (wet mounts and
stained-preparations) in which characteristics such as the size, morphology
and motility of the microorganism can be evaluated, as well as the presence
of specific structures (e.g., capsules, flagella, spores). Additional laboratory
tests are then conducted to narrow down the identity of unknown isolates,
such as biochemical and serological tests. Biochemical assays most often
target enzymatic activities that rely on the possession and expression of
specific genes by the isolate. Biochemical tests include the utilization of
sugars, the determination of proteolytic and lipolytic traits, types of energy
metabolism, physiological growth boundaries (e.g., temperature, pH,
osmolarity), tolerance to antimicrobial agents and other enzymatic activities.
The methods for bacterial identification and enumeration based on
culture, microscopy and biochemistry have supported dairy microbiology
for decades and have provided invaluable service to the scientific
community and public health agencies. However, the phenotypic culture-
based identification approaches are often hampered by difficulties.
The morphology and/or motility of certain bacterial species under the
microscope may vary depending on temperature and/or other culture
conditions (e.g., osmolarity). The number of biochemical tests necessary

for the identification of microbial isolates to the species level, can in some
instances be very high, with a concomitant requirement in growth media,
reagents, incubation times and labor. In addition, culture and phenotypic
(biochemical) identification methods often fail to make reliable distinctions
between isolates belonging to the same species because of variable
expression of phenotypic characteristics or because of ambiguous or
“intermediate” biochemical results. Routine bacteriological culture fails to
identify viable, yet unculturable organisms or fastidious microorganisms
requiring specific growth factors.
During the last decades the application of PCR revolutionized many
aspects in microbiology by permitting rapid and selective cloning of specific
target DNA (or RNA) regions among a heterogeneous collection of DNA
(or RNA) sequences. The ability to amplify specific genetic fragments was
successfully paired with other advances in nucleic acid research such as the
ability for detailed sequencing, specific labeling, specific cutting (restriction)
and analysis via electrophoretic separation. These and other principles have
been used as the basis for the development of specialized genomic-based
methods for microbial identification. Hence, in recent years researchers
have progressively started to rely on molecular identification methods.
These methods are generally faster, more sensitive and more robust. The
application of molecular approaches has helped identify microbial species of
technological, spoilage, food-safety, or clinical importance that were either
present in very low numbers, uncultivable, or previously not associated with
raw milk. Most often bacterial identification methods rely on unraveling
the sequence of the gene encoding the 16S ribosomal subunit (16S rRNA),
whereas the 26S rRNA is the most common target for eukaryotic cell
identification. The 16S rRNA gene possesses conserved regions found in
all prokaryotes as well as regions whose sequence is hyper-variable and in
most cases specific enough to make identifications to the species or even the
sub-species level. The sequencing of the gene encoding the RNA-polymerase
beta-subunit is another alternative target.
Numerous molecular approaches (both culture-dependent and culture
independent) have been developed for the analysis of the microbial
composition of raw milk and raw-milk cheeses and only a few recent
examples are cited here (Callon et al. 2007, Giannino et al. 2009, Ajitkumar et
al. 2012, Bhatt et al. 2012, Deperrois-Lafarge and Meheut 2012). The choice of
a specific technique and approach depends upon the aim of the investigation
and in particular on whether the research aims on elucidating the general
microbial diversity of an ecosystem, identifying specific microorganisms,
or both. In addition, molecular techniques can provide semi-quantitative
or quantitative output. Quigley et al. (2011) have presented an extensive
review of DNA-based technologies and molecular approaches that are
available for the analysis of the microbial composition of raw milk and raw
milk cheeses, covering technical aspects, advantages and disadvantages.
Most experts nowadays seem to agree that it is probably most beneficial
to apply polyphasic approaches, i.e., both culture-dependent and culture
independent methods when assessing the diversity of the raw milk
microbiota.
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CHAPTER 3
Dairy Pathogens:
Characteristics and Impact
Photis Papademas* and Maria Aspri
INTRODUCTION
Milk and dairy products can be an ideal medium for the growth of a variety
of microorganisms, both pathogenic and spoilage. Even though milk
and milk products are amongst the safest food worldwide and account
only a small percentage of all food-borne diseases, they have an inherent
potential for causing illness as they are potentially the source of a very
broad range of microbial, chemical, and physical hazards. The presence of
food-borne pathogens in milk and milk products is due to direct contact
with contaminated sources in the dairy farm environment, to excretion
from the udder of an infected animal and during processing and handling.
Some important pathogens for milk and milk products include
Salmonella spp., Listeria monocytogenes, Staphylococcus aureus, Campylobacter
spp., pathogenic Escherichia coli, Cronobacter sakazakii and will be discussed
in detail in this chapter.
Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of

Technology, P.O. Box 50329, 3603, Limassol, Cyprus.
* Corresponding author: [email protected]
Figure 1. Sources of Contamination of Milk and Dairy Products (After: Oliver et al. 2005)
Listeria monocytogenes
Introduction
Listeria monocytogenes, has been recognized as a significant food-borne

pathogen, and has become one of the most studied microorganisms in the
last decades (Ryser and Marth 2004). The organism was first identified as a
causative agent for animal illness in 1926, when Murray, Webb, and Swann
isolated L. monocytogenes as the cause of a septicemic disease affecting
rabbits and guinea pigs in their laboratory at Cambridge in England. This
strain was named Bacterium monocytogenes, as it was observed to infect
monocytes of the blood.
Characteristics
According to Bergey’s Manual of Systematic Bacteriology (1994) Listeria genus

includes: L. monocytogenes, L. ivanovii, L. innocua, L. seeligeri, L. welshimeri
and L. gray. L. monocytogenes is pathogenic for humans and animals, and
L. ivanovii is mainly pathogenic for animals, primarily sheep. Other species
are considered to be non-pathogenic. It has thirteen serotypes, but the most
common causes of disease are 1/2a, 1/2b, and 4b.
L. monocytogenes is a gram-positive, micro-aerophilic, non–spore-
forming cocoid to rod shape, measuring 0.4–0.5 µm in diameter and 0.5–2
µm in length. The tumbling motility by means of peritrichous flagella is
characteristic of Listeria and often used as a conventional marker for Listeria
identification. The degree of motility is temperature dependent, for example
Dairy Pathogens: Characteristics and Impact 71
Table 1. Pathogens associated with milk and dairy products.
Milkborne Pathogens
Organism Disease Characteristics Associated Foods
Salmonella spp. Typhoid like fever, headache, Raw milk, dairy products
abdominal pain, diarrhea, Others: Eggs, meat fish,
nausea, vomiting, constipation, poultry, shrimp, salad dressings
malaise, chronic symptoms (especially home-made that
(arthritis) contain raw eggs)
Listeria Nausea, vomiting, diarrhea, Fresh soft cheeses, raw milk,
monocytogenes fever, influenza like symptoms, inadequately pasteurised milk,
meningitis, encephalitis. In Others: Ready to eat deli-meals,
pregnant women can lead to hot dogs, raw and smoked fish
spotaneous abortion, premature
delivery, or still birth
Staphylococcus Nause, vomiting, abdominal Milk and dairy products Others:
aureus cramps, retching, prostration Meat and meat products, salads
(chicken, potato, egg and
makaroni), cream pastries
Campylobacter jejuni Diarrhea, abdominal pain Raw milk Others: Raw
and cramps, fever, vomiting, and undercooked poultry,
headache contaminated water, shellfish
Bacillus cereus Diarrheal toxin: Watery diarrhea, Milk, dairy products Others:
abdominal cramps, nausea Meats, vegetables, rice, pasta,
Emetic toxing: nausea and soups, fish
vomiting, diarrhea may be
present
Escherichia coli Watery diarrhea, abdominal Raw milk Others: Contaminated
cramps, low-grade fever, nausea, water, undercooked ground beef,
malaise raw apple juice and cider, alfalfa
sprouts, cut melons
Cronobacter sakazakii Meningitis, Enteritis Powdered Infant formula
Mycobacterium paratuberculosis, fever, chills, Raw milk, pasteurised milk (few

avium ssp. weight loss cells might survive)
paratuberculosis
(MAP)
Shigella spp. Abdominal pains and cramps, Milk and dairy products
diarrhea, fever, vomiting, stools Others: bakery products (cream-
may contain blood, pus or mucus filled pastries), sandwitches, raw
vegetables and salads (potato,
tuna, chicken, macaroni)
Yersinia Fever, abdominal pain, diarrhea Raw milk, Others: oysters and
enterocolytica and and vomiting fish, Meat
Y. pseudoturbeculosis
it shows mobility when growth temperature is between 20 and 25°C but

reduces on 37°C. L. monocytogenes is not a fastidious organism and grows
well in most common nutrient media, including brain heart infusion broth
(BHI), trypticase-soy broth with 0.6% yeast extract (TSBYE), Luria broth
(LB), etc. L. monocytogenes is catalase positive, oxidase negative and ferments
rhamnose, dextrose, esculin, and maltose, but does not ferment xylose and
manitol. These biochemical properties are used for the differentiation of
L. monocytogenes from other members of the genus Listeria.
The organism’s ability to grow and reproduce in harsh conditions makes
it a food-borne pathogen of great concern. Optimum growth temperature is
30–37°C, although L. monocytogenes grows reasonably well at temperatures
over a wide range of temperature from 0–42°C. The growth rate reduces
as the growth temperature decreases. The pH range for growth is 5–9 with
an optimum pH of 6–8. However, these values depend on the acidulant,
strain, and temperature. L. monocytogenes is also quite salt tolerant being
able to grow in 10% sodium chloride. The organism is ubiquitous in the
environment. It has been isolated from fresh and salt water, soil, sewage
sludge, decaying vegetation and silage.
Food-borne Illness
Listeria monocytogenes is the causative agent for the disease Listeriosis,

one of the most important foods borne illness of humans. Listeriosis is a
zoonotic illness that affects both animal and humans and is transmitted via
three main routes: direct contact with animals, cross-infection of new-born
babies in hospital and food-borne infection (Ryser and Marth 2004). The
latter two sources result in the majority of listeriosis cases in humans. As
previously noted, L. monocytogenes is widely distributed in the environment.
Thus humans can come into contact with this pathogen through a variety
of sources including animals, meats, milk, dairy products, seafood, and
plants as well as insects, air, dust, dirt, feces and other humans. Also, it
can be transmitted by cross-infection of newborn babies in hospitals and
via food vehicles.
Pregnant women, newborns, elderly and immunocompromised
individuals are more susceptible to the disease and experience a more severe
illness. Listeriosis is a very serious and often fatal infection with a fatality
rate that often reaches as high as 30–40%, even though the morbidity of
listeriosis is relatively low (Liu 2006).
Symptoms of listeriosis range from flu-like vomiting and diarrhea to
septicemia, meningitis and meningoencephalitis. Listeriosis refers to the
more serious life-threatening illness while gastroenteritis is the mild illness
experienced by healthy adults.
In pregnant women, it most commonly features as an influenza-like
illness with fever, headache and occasional gastrointestinal symptoms, but
there may be associated transplacental foetal infection which can result
in abortion, still birth, premature labour or birth of a severely ill infant.
L. monocytogenes is one of the few bacteria capable of crossing the placenta

and gain direct access to the fetus. Newborn babies may also acquire
infection after birth from the mother or from other infected infants.
Listeriosis in the newborn can be an early-onset syndrome, which occurs at
birth or shortly afterwards, or a late-onset disease appearing several days to
weeks after birth. Early-onset illness results from utero infection, possibly
through the aspiration of infected amniotic fluid, and is characterised
by pneumonia, septicaemia and widely disseminated granulomas. In
immunocompromised and elderly adults, the illness typically involves
infection of the tissues surrounding the brain (meningitis) and infection of
the bloodstream (septicemia). Healthy adults are thought rarely to suffer
from listeriosis. However, high levels of this organism can cause symptoms
in healthy individuals that are similar to flu-like symptoms of vomiting,
nausea and diarrhea.
The infective dose (ID) of L. monocytogenes is very unclear. The
minimum dose of bacteria required to cause clinical infection depends on
several factors that include the virulence mechanism of the microorganism,
immune status of the host, the concentration of the pathogen in the
contaminated food, as well as the food and the amount of food consumed.
Based on documented outbreaks of listeriosis, foods that contained around
102–104 cfu/g have been responsible in various outbreaks (Ooi and Lorber
2005).
Detecting L. monocytogenes
There is a variety of conventional and rapid methods currently available

for the detection and identification of L. monocytogenes in food samples
and specimens from animal listeriosis. The most commonly used culture
reference methods for the detection of Listeria in foods are the ISO 11290
standards (International Organization for Standardization 1996). In the
United States of America (USA) two main standards are used as reference
methods to isolate L. monocytogenes from foods. One of the protocols was
developed by the US Food and Drug Administration (FDA) to isolate
Listeria spp. from dairy products, seafood, and vegetables (Hitchins 2003).
The US Department of Agriculture (USDA) developed another method
to isolate the organism from meat and poultry products as well as from
environmental samples (USDA 2013). All of the methods require an
enrichment step to increase the viable cell count to detectable numbers.
Enrichment steps are followed by plating onto agars containing selective/
differential agents (Jeyaletchumi et al. 2010). Oxford agar was the medium
of choice of the International Organization for Standardization (ISO) until
1998, when it was replaced by PALCAM agar. In 2004, it was replaced by
a chromogenic agar, ALOA (Agar Listeria Ottaviani Agosti), which allows
the differentiation of L. monocytogenes and L. innocua. The detection in the

first two plating media is based on the hydrolysis of aesculin which does
not differentiate between species. In ALOA, the detection is based on the
phosphatidylinositol phospholipase C enzyme (PI-PLC) activity present in
L. monocytogenes and in L. ivanovii, and b-glucosidase activity detected by
a chromogenic substrate (Gasanov et al. 2005).
The confirmation of suspected colonies can be performed by testing a
limited number of biochemical markers such as hemolytic activity (CAMP
test) and sugar fermentation patterns. Figure 2 summarises the ISO 11290
method for the detection of L. monocytogenes. Rapid methods for the
identification of L. monocytogenes include enzyme-linked immunosorbent
assay (ELISA) kits, nucleic acid assay kits, and Polymerase chain reaction
(PCR) (Jemmi and Stephan 2006). For a detailed account of the molecular
methods employed for the identification of bacteria in dairy products see
Chapter 6.
Sample
Primary Enrichment Secondary Enrichment
Enrichment of Listeria in sample In Fraser Broth

with Fraser Broth Incubation: 35 or 37ºC, 48 +/– 2 hours
Incubation: 30ºC, 24+/– 2 hours
Plating
Streaking of enriched cultures on Oxford Agar plate

Incubation: 35ºC, 24 hours
Observe for black colonies at 24 and 48 hours
Streaking of enriched cultures on PALCAM Listeria Selective
Incubation: 30 and 35 or 37ºC 24–48 hours
Observe for green colonies with black halo at 24 and 48 hours
Purification
Select 5 presumptive colonies for confirmation. If well separated colonies

are not available, streak one colony on Tryptone Soya Yeast Extract Agar
Confirmation
Purified colonies are confirmed with standard tests:
• Gram staining
• Motility test
• Carbohydrate fermentation test
• D-hemolysis and CAMP test (lysis tests)
Figure 2. ISO 11290 for detection of L. monocytogenes in food samples (After: Scharlau 2007).
Color image of this figure appears in the color plate section at the end of the book.
Association with Milk and Milk Products
Dairy products such as raw and pasteurized milk and soft cheeses have
been associated with a number of major outbreaks of listeriosis (Kasalica
2011), since Listeria population may be extremely acid and salt resistant.
L. monocytogenes’ association with soft cheeses is due to the cheese
ripening process. It survives poorly in unriped soft cheeses such as cottage
cheese but well in products such as Camembert and Brie (especially if
made from raw milk). During this ripening process microbial utilization
of lactate and release of amines increase the surface pH allowing Listeria
spp. to multiply. Hard cheese such as Parmesan (Parmigiano Reggiano)
does not favour growth (Yousef and Marth 1990) while in white-brined
cheeses, i.e., Feta, the acidic environment of the cheese (pH 4.6) will inhibit
the growth of Listeria spp. but the pathogen will survive even for 90 days
(Papageorgiou and Marth 1989). It was also reported by Belessi et al. (2008)
that the growth of L. innocua in Feta cheese was assisted by the presence
of fungi. The findings indicated that the growth of fungi on the surface of
Feta cheese and yogurt may compromise the safety of these products by
enhancing survival of the bacterium. Particularly, when fungi increase the
pH of Feta cheese, L. innocua demonstrates better survival and prolonged
storage may raise concerns for the development of acid-resistant Listeria
populations.
In high-scalding cheeses, i.e., Mozzarella, Halloumi the bacteria will not
survive the stretching/cooking temperatures. For fresh Halloumi cheese
(pH 6.2) post-scalding contamination may result in a favorable environment
for Listeria spp. growth therefore, careful handling of the cheese is crucial.
Gougouli et al. (2008) showed that in ice cream the freezing conditions
did not cause a severe stress in L. monocytogenes cells capable of leading
to a significant “additional” lag phase during the subsequent growth
of the pathogen at chilling conditions. Additionally they observed that
under freezing conditions, no significant changes in the population of the
pathogen were observed throughout a 90-d storage period for either of the
inoculum levels tested. The above statements highlight the importance of
post-pasteurisation hygiene conditions to be employed in all dairy factories.
Salmonella spp.
Introduction
Salmonella are one of the most important food-borne pathogens and have
been recognized for over 100 years as the cause of illnesses ranging from
mild to severe food poisoning (gastroenteritis), and even more severe
typhoid (enteric fever), paratyphoid, bacteraemia, septicaemia and a variety
of associated longer-term conditions (sequelae).
Characteristics
Salmonella is facultative anaerobe, gram negative flagellated rod-shaped

bacterium belonging to the family of Enterobacteriacae. The size of the
rods is about 2–3 x 0.4–0.6 µm in size. They are non-spore forming, oxidase
negative and catalase positive and most members of the genus are motile
by peritrichous flagella. They are generally: able to reduce nitrate to nitrite,
able to grow on citrate as sole carbon source, capable of producing acid and
gas from glucose, able to produce hydrogen sulfide on triple sugar iron and
decarboxylate lysine and ornithine, and able to hydrolyze indole and urea.
Strains of Salmonella are antigenically distinguishable by agglutination
(formation of aggregates/clumping) reactions with homologous antisera
and the combination of antigens possessed by each strain, referred to as the
antigenic formula, is unique to each Salmonella serotype. These are classified
in two species, S. enterica and S. bongori. S. enterica is further divided into
six subspecies, Salmonella enterica subsp. arizonae, Salmonella enterica subsp.
diarizonae, Salmonella enterica subsp. enterica, Salmonella enterica subsp.
houtenae, Salmonella enterica subsp. indica, and Salmonella enterica subsp.
salamae (Tindall et al. 2005).
Optimum growth for salmonellae occurs at 35–37°C (mesophilic), but
they can grow at temperature from 5°C to 46°C, depending on serotype
(El-Gazzar and Marth 1992). Although optimal growth pH is between 6.5
and 7.5, salmonellae can grow in more acidic environments (pH 4). They
do not require sodium chloride for growth, but can grow in the presence
of 0.4 to 4%. They are sensitive to heat and often killed at temperature
of 70°C or above (pasteurization temperature). They require high water
activity (aw) between 0.99 and 0.94 but can survive very well in dry foods
(aw < 0.2) (Pui et al. 2011).
Food-borne Illness
Illness caused by Salmonella serotypes known as salmonellosis. Infection

is initiated by consumption of raw or undercooked contaminated animal
food (common source of infection for humans) or water containing fecal
material. In addition to transmission by food, Salmonella can also be spread
via the environment in a number of ways, indirectly infecting humans and
other animals, as it can survive for long periods of time under both wet and
dry conditions. Transmission can also occur through direct contact with ill
or in apparently infected farm animals or ill pets such as cats and dogs.
The outcome of this infection largely depends on the serotype and type of
host. Serotypes Typhimurium and Enteritidis can cause disease in humans,
cattle, poultry, sheep, pigs, horse and wild rodents.
There are two clinical types of human salmonellosis: enteric fever

(a severe, life-threatening illness) following infection with S. typhi or paratyphi
and the more common food-borne illness syndrome caused by nontyphoid
Salmonella species. In both cases, the responsible microorganisms enter the
body via the oral route.
Salmonellosis generally is a self-limiting acute gastroenteritis similar to
intestinal influenza, and is believed to be grossly underreported. Onset of
non-typhoidal salmonellosis typically occurs 12 to 36 hours after ingestion
of the contaminated food characterized by nausea and vomiting; symptoms
which tend to subside within a few hours. Symptoms include nausea,
vomiting, chills, fever, abdominal pain or cramps, and headache, is typically
followed by diarrhea (El-Gazzar and Marth 1992). The illness usually lasts 4
to 7 days and most persons recover without antibiotic treatment. The elderly,
infants and those with underlying chronic illness or immuno-compromised
individuals are more likely to have a severe illness.
In some cases septicemia can occur as a complication of gastroenteritis
which can be fatal in immunocompromised hosts. Prolonged septicemic
infections can result in localized tissue and organ infections, especially
in those previously damaged or diseased. The severity and duration of
symptoms depends upon the concentration of the pathogens in the food,
the physiological composition and the type of the food consumed the
susceptibility of the host, and the virulence of the pathogen. As few as 15
cells can cause illness.
S. typhi is responsible for bacteremia-related enteric fever referred to as
typhoid fever. Onset typically occurs within 8 to 15 days, and sometimes
as long as 30 to 35 days. Symptoms include fever, headache, malaise,
anorexia, and congestion of the mucous membranes, especially of the upper
respiratory tract. The high mortality rate of S. typhi (10%) compared to other
Salmonella spp. can be reduced with the prompt administration of antibiotics.
Detecting Salmonella spp.
Culture and colony counting methods, Polymerase Chain Reaction (PCR)

as well as immunology-based methods are the most common tools used for
pathogens detection including Salmonella detection. They involve counting
of bacteria, DNA analysis and antigen-antibody interactions, respectively.
These methods are often combined together to yield more robust results.
Detection of Salmonella in foods by conventional culture methods
consist of four steps 1) non selective pre-enrichment, 2) selective enrichment
in different media, 3) plating on selective and indicative media, and 4)
confirmation through biochemical and serological tests (ISO 6579: 2002).
The culture method is time-consuming and labor intensive, requiring a
minimum of 4–6 days.
Pre-enrichment in a non selective medium needed to increase the

recovery of sub lethally damaged cells of salmonella. Buffered peptone
water and lactose broth are two of the most widely used media for pre-
enrichment.
The second step is enrichment in selective media. This increases
the number of salmonella to a level where detection on selective agar
plates is possible, while at the same time restricted the growth of other
microorganisms present by selective agents in the media. The most
commonly used media for selective enrichment are Rappaport-Vassiliades
soy broth, selenite cysteine broth, and tetrathionate broth. Modified
semisolid Rappaport-Vassiliades is another selective media particularly
useful for detecting salmonella in feces and environmental samples.
From the selective enrichment broths, cultures are streaked on selective
solid media in order to obtain isolates colonies. Most standard methods
recommend employing two media in parallel. Commonly employed media
include brilliant green, xylose lysine Tergitol-4, bismuth sulfite, Hektoen
enteric, and xylose lysine deoxycholate agars. Recently, chromogenic agars
have also been used.
Finally, presumptive Salmonella colonies from selective media are
subcultured on nonselective plates in order to get well-isolated colonies
that can be used for confirmation by biochemical and serological analysis.
Most common biochemical reactions for salmonella confirmation include
triple sugar iron, mannitol, urea, ornithin decarboxylase, and lysine
decarboxylase. A serological verification by determining the antigenic
composition is performed. The O and H antigens are determined by
agglutination testing using polyvalent antisera. Miniaturized tests such as
API 20 E (bioMerieux) and BBLTM EnterotubeTM II (BD Diagnostics) have
been developed for confirmation and they present an efficient and labour-
saving alternative. Figure 3 summarises the International Organization for
Standardization method for the detection of Salmonella.
Following confirmation of the identity of Salmonella, it is important for
food surveillance purposes and the investigation of outbreaks, to subtype or
‘fingerprint’ Salmonella serotypes. There are a variety of techniques available
now that can allow confident traceability of strains in factory environments.
These include biotyping, serotyping (including variation in H antigens),
phage typingantibiotic resistance patterns (resistotyping), various molecular
typing methods including pulsed-field gel electrophoresis (PFGE) and
ribotyping.
Outbreaks of human salmonellosis have been linked to the consumption of

unpasteurized milk or milk products. In addition, inadequate pasteurizarion
Figure 3. ISO 6579 Detection of Salmonella in Food (After: Scharlau 2007).
or post process contamination have occasionally resulted in milk and milk

products that tested positive for Salmonella spp. Entry of Salmonella spp.
via contaminated raw milk into dairy food processing plants can lead to
persistence of this pathogen in biofilms, and subsequent contamination
of processed milk products and exposure of consumers to the pathogen.
Presumably, the largest outbreak of Salmonellosis ever reported in the
USA occurred in Illinois (Ryan et al. 1987), where an antimicrobial-resistant
strain of Salmonella typhimurium yielded 16,000 culture-confirmed cases
traced to two brands of pasteurized 2% milk produced by a single dairy
plant. The authors reported that the study of stored isolates showed it had
caused clusters of salmonellosis during the previous ten months that may
have been related to the same plant, suggesting that the strain had persisted
in the plant and repeatedly contaminated milk after pasteurization.
Staphylococcus aureus
Introduction
Staphylococcus aureus was first described in 1880 by the Scottish surgeon,

Sir Alexander Ogston from pus in a knee joint abscess. The first description
of food-borne illness dates back 1884, when a spherical organism in cheese
caused a large food-poisoning outbreak in Michigan in the United States.
Since then we know that S. aureus is a very frequent microorganism in
humans and animals with approximately 20–50% of persons being long-
term carriers, mainly as a part of the normal skin flora and in anterior nares
of the nasopharynx, but also the throat and hair.
Characteristics
Staphylococcus aureus subsp. aureus (S. aureus) belongs to the genus

Staphylococcus and to the family Staphylococcaceae, which includes the lesser
known genera of Gemella, Jeotgalicoccus, Macrococcus, and Salinicoccus. The
name of the organism is derived from Greek words staphyle (a bunch of
grapes) and coccus (grain or berry).
S. aureus are spherical, coccus shaped Gram positive bacteria of
0.5–1.5 µm in diameter that appear in pairs, short chains, or bunched, grape-
like clusters. They are non motile, non-spore forming facultative anaerobes
which ferment most of the sugars except raffinose and salicin producing
lactic acid during fermentation. They are catalase and coagulase positive
and oxidase negative.
In general, S. aureus is mesophile with growth temperature range from
7 to 48°C, with an optimum temperature for growth of 35°C. The pH range
for growth is between 4.5 and 9.3, with the optimum between pH 7.0 and
7.5 (Singh and Prakash 2010).
With regard to water activity (aw), the staphylococci are unique in being
able to grow at lower levels than other non-halophilic bacteria. Growth has
been demonstrated at as low as 0.8 Aw under ideal conditions. These low
Aw conditions are too low for the growth of many competing organisms.
Thus, it is highly tolerant to salts and sugars and has historically been
responsible for food-borne disease outbreaks illnesses from contaminated
hams (salt) and pies (sugar).
Food-borne Illness
Staphylococcal food poisoning, also known as staphyloenterotoxicosis or

staphyloenterotoxemia, is the name of the condition caused by the ingestion
of SEs (Le Loir et al. 2003). Staphylococcal food poisoning occurs not as the
result of the ingestion of the organism itself, but through ingestion of one
or more of the 14 staphylococcal enterotoxins (SE A-N) produced by some
strains of S. aureus. With 30–50% of the population carrying S. aureus in
their nostrils and on skin and hair, foods can become contaminated quite
readily prior to or following heat treatment during processing and handling.
Staphylococcal enterotoxins are heat-stable exoproteins consisting
from 236 to 296 aminoacids with a molecular mass of 25–35 kDa. Upon
hydrolysis, 18 amino acids are present, mostly aspartic acid, glutamic acid,
lysine and tyrosine.
There are five different types of classical enterotoxins (SEA-SEE) which
are distinct in antigen reaction. Recently, new types of enterotoxins and
enterotoxin-like types (SEG-SEV) have been described in S. aureus. There
are several enterotoxins but only, SE A, B, C1, C2, C3, D, E, G, H, I, and J
have been identified (Argudin et al. 2010). The most common toxin and
the one that usually involved in staphylococcal outbreaks is Enterotoxin
A and data from staphylococcal outbreaks indicate that less than 1 µg of
toxin can result in illness.
The onset of symptoms including nausea, vomiting, retching,
diarrhea, and abdominal cramps, normally develops within 1 to 6 hours
following ingestion of the contaminated food. A toxin dose of less than
1.0 µg in contaminated food will produce symptoms of staphylococcal
intoxication. This toxin level is reached when S. aureus populations
exceed 106 cfu. However, in highly sensitive people a dose of 100–200 ng
is sufficient to cause illness. Depending on individual susceptibility to the
toxin, the amount of toxin ingested and the general health of the affected
individual, symptoms may also include headaches, cold sweats, and rapid
pulse, transient changes in blood pressure, prostration and dehydration.
Recovery generally takes one to two days rarely resulting in complications
or hospitalization.
Detecting S. aureus
The most successful and widely used medium for S. aureus is called
Baird Parker. This contains tellurite, glycine and lithium chloride as
selective agents, pyruvate and egg yolk to assist the recovery of damaged
cells. Reduction of the tellurite by S. aureus gives characteristic shiny,
jet-black colonies which are surrounded by a zone of clearing resulting
from hydrolysis of the egg yolk lipovitellenin and often with an inner
cloudy zone due to the precipitation of fatty acid salts. The appearance
of the colonies on Baird-Parker agar gives a presumptive identification of
S. aureus which is confirmed through the production of coagulase (Asperger
and Zangerl 2003).
Molecular techniques based on the polymerase chain reaction (PCR)
have been developed using specific primers directed against a variety of
sequences in the S. aureus genome including genes for thermostable nuclease
and enterotoxins. There are also kits available for detection based on nucleic
acid hybridization. Enterotoxins can be detected based on immunological
methods following extraction from the food matrix, and there are numerous
kits available using maenzyme linked immunoassay or latex agglutination.
Outbreaks of staphylococcal poisoning have been linked to milk and milk

products for over 100 years with S. aureus emerging as a major milk-borne
pathogen. Milk and dairy products constitute 1–9% (mean 4.8%) of all
S. aureus outbreaks in Europe. Most outbreaks linked to the use of raw
milk due to mastitic dairy cows. Although S. aureus inactivated by
standard heat treatments, food-borne outbreaks related to pasteurized
products occur as the result of staphylococcal enterotoxin production
prior to heat treatment or as a result of post-pasteurization contamination.
An excellent, comprehensive report published by the European Food
Safety Authority (EFSA 2003) describes the Staphylococcal enterotoxins
(SE) in dairy products. The report states that the conditions favouring SE
production in milk and dairy products are (a) Liquid milk is an excellent
medium for the growth and SE production (favourable pH, water activity
and nutrients) and at temperatures 7–43°C, numbers of enterotoxinogenic
S. aureus may increase rapidly even when the initial levels are low, (b) in
cheese manufacturing enterotoxinogenic S. aureus can multiply and produce
enterotoxin during the first stages of production when the pH of the curd
is higher than 5.0 and the competing LAB bacteria have not reach high
number. This favorable for multiplication of S. aureus period extends, in the
different types of cheeses from several (5–10) to 48 hours at the maximum
(c) experiments for enterotoxins production in pasta filata cheeses, internal
mould ripened cheeses and processed cheeses failed to prove the presence
of enterotoxin whatever the size of the inoculum of enterotoxinogenic
S. aureus was used, (d) on the contrary, whey cheeses and imitation cheeses
appears to be a favourable environment for growth of S. aureus and even
small inoculum (10²–10³ cfu/g) can result in enterotoxin production, (e) In
cream production critical stage is considered the period of cream ripening
if the process is conducted in favorable for S. aureus growth, and (f) in milk
powder and ice cream production critical stages are the pre-drying and pre-
freezing periods in which S. aureus can multiply if temperature is favorable.
Cronobacter sakazakii
Introduction
Cronobacter spp. (Enterobacter sakazakii) is an opportunistic food-borne

pathogen that has been linked with serious infections in infants through
dried infant milk formula, which causes bacteraemia and meningitis and is
associated with necrotizing enterocolitis (NEC). Most cases of Cronobacter
infections have been detected among newborn and very young infants. The
first reported cases attributed to this organism occurred in 1958 in England
and resulted in the death of two infants (Gurtler et al. 2005).
Characteristics
It is a member of the family Enterobacteriaceae, and belongs to the genus

Cronobacter. This organism was known as yellow pigmented Enterobacter
cloacae until 1980 after which it was renamed as E. sakazakii (Farmer et
al. 1980). The new name E. sakazakii was proposed based on differences
between E. sakazakii and E. cloacae in DNA-DNA hybridization, some
biochemical traits, production of yellow-pigmented colonies and antibiotic
susceptibility. Recently, E. sakazakii was reclassified as a genus Cronobacter
compromising five spp.: C. sakazakii, C. malonaticus, C. tuicensis, C. muytjensii
and C. dublinensis (Iversen et al. 2008).
C. sakazakii is a Gram-negative rod bacterium of 0.3 to 10 µm long by
1.0 to 6.0 µm wide that are motile by peritrichou flagellated. It is facultative
anaerobic, oxidase negative, nonspore forming, nonacid-fast bacterium.
The temperature growth range of Cronobacter is 6–47°C with an optimal
range of 37–43°C (Iversen and Forsythe 2003). However, some strains are
inhibited at temperatures above 44°C and some strains have the ability to
grow at 5°C. In the Enterobacteriaceae family, Cronobacter spp. is amongst
the most thermo-tolerant. Cronobacter spp. have the ability to survive in
acidic environments with pH levels as low as 3. Of the members of the
Enterobacteriaceae family, Cronobacter spp. also appears to be well adapted
to dry stress.
Food-borne Illness
C. sakazakii is an emerging human pathogen with mortality ranges from

40 to 80% among infected infants, and those who survive the infection
usually develop irreversible neurological sequelae. C. sakazakii has been

implicated in severe forms of neonatal infections, such as meningitis,
bacteraemia, sepsis and necrotizing enterocolitis. C. sakazakii can cause
also systemic respiration, cardiovascular and neurologic symptoms such as
destruction of the frontal lobes of the brain, seizures, spastic quadriplegia,
hypothermia, fever, Cheyne-Stokes respirations, bradycardia, poor feeding,
irritability, jaundice, grunting respirations, instability of body temperature,
hemorrhagic cerebral necrosis, meningo encephalitis, necrotic softened
brain, cyst formation, liquefaction of cerebral white matter and severe
neurologic complications (Arsalam et al. 2013). Low birth-weight neonates
45 (< 2.5 Kg) and infants < 28 days of age are at higher risk compared to
more mature infants (Iversen and Forsythe 2003).
Sources of contamination include floor drains, air, vacuum, canister,
broom bristles, room heater, electrical control box, transition socks
and condensate in a dry product processing plant (Shaker et al. 2007).
Cronobacter sakazakii infections are treated with antibiotics such as
carbapenems or antipseudomonal penicillins (i.e., mezlocillin, piperacillin,
piperacillin/tazobactam, ticarcillin, and ticarcillin/clavulanate) (Dumen
2010).
Detecting C. sakazakii
The isolation of C. sakazakii, from dried foods requires a series of steps to

resuscitate stressed cells that would otherwise not be cultured. Methods
for isolation and enumeration of C. sakazakii from powdered formula have
improved in the last years. These methods include the ISO/TS 22964:2006,
the US-FDA(2002), and the revised method of the US-FDA which combines
both a PCR assay and two newly developed chromogenic agars for detection
(Yan et al. 2012).
Table 2 compares the available methods for the detection of C. sakazakii.
According to US Food and Drug Administartion (FDA) protocol enrichment
of the PIF samples in water overnight is required. This is followed by a
second enrichment step in Enterobacteriaceae enrichment (EE) broth for up
to 24 h. Then samples are plated on violet red bile glucose agar overnight and
presumptive colonies purified onto TSA. The resulting yellow-pigmented
colonies are selected for biochemical tests using analytical profile index
(API) 20E test strips. On the other hand, the International Organization
for Standardization method consists of pre-enriching the PIF samples in
buffered peptone water at 37°C overnight. Then the samples are enriched
in modified lauryl sulfate (addition of vancomycin) at 44°C overnight.
Following the second enrichment, the samples are plated on to chromogenic
agar and incubate at 25°C for a period of 48–72 h.
Table 2. Protocols for detection of C. sakazakii.
Procedure FDA (Original) ISO 22964 FDA (revised)

Pre-enrichment Make 1:10 (w/v) of sample in Make 1:10 (w/v) of sample in BPW, Make 1:10 (w/v) of sample in BPW,
distilled water, incubate overnight incubate at 37°C for 18 ± 2 h incubate at 36°C for 6 h
at 36°C
Selective Transfer 10 ml pre-enrichment to Transfer 100 µl pre-enrichment to 10 ml
Enrichment 90ml EE broth, incubate overnight mLST/Vancomycin medium incubate at
at 36°C 44°C for 24 ± h
Selection/Isolation Make an isolation streak and Streak from the cultures mLST/ Centrifuge 40 ml samples, 3000 g x 10
spread plate from each EE broth Vancomycin medium one loopfull on the min and resuspend pellet in 200 µl PBS;
onto VRBG agar, incubated chromogenic agar and incubate at 44°C Spread 100 µl onto chromogenic media and
overnight at 36°C for 24 ± 2 h incubate overnight at 36°C
Confirmation Pick 5 presumptive positive Select five typical colonies and streak on Pick two typical colonies from each
colonies and streak onto TSA and TSA agar, incubate at 25°C for 48 ± 4 h chromogenic media and confrmed with
incubate overnight at 25°C real-time PCR, API20E , Rapid ID 32E
Identification Yellow colonies are confirmed Select one colony from each TSA plate
with the API20E test kit for biochemical characterization
Detection time 5 6 3
(days)
Currently molecular detection methods such as polymerase chain

reaction (PCR), real-time PCR, immunoassays and DNA microarray-based
assays are used for its identification.
C. sakazakii is considered a ubiquitous microorganism it can be found in a

wide variety of foods and in waters and several areas including hospitals
and houses. While this organism has been isolated from a variety of foods
such cheese, fermented bread, tofu, sour tea, cured meats, minced beef
and sausage meat, food factories and environments, most infections are
associated with rehydrated infant milk formula (RIMF). Powered infant
milk formula (PIMF) is basically a non-sterile product which can be, once
rehydrated, a good medium for microorganisms. Therefore the presence
of C. sakazakii in PIMF is mainly due to post-processing, environmental
contamination, the addition of contaminated ingredients during powder
production or is due to colonization by C. sakazakii of utensils such as bottles,
brushes and spoons used in PIMF preparation (Shaker et al. 2007).
Bacillus cereus
Introduction
There are several Bacillus species that have been responsible for food-borne
illness, although the only one that is frequently involved is Bacillus cereus.
Hauge (1955) was the first to establish B. cereus as a food poisoning organism.
Characteristics
The Bacillus cereus group is a very homogeneous cluster within the genus
Bacillus and consists of six species: B. cereus, B. thuringiensis, N. anthracis,
B. mycoides, N. pseudomycoides and B. weihenstephanensis (Ehling-Schulz et
al. 2004). B. cereus is a Gram-positive, catalase positive motile, facultative,
aerobic sporeformer. Its cells are rod-shaped, hence the name “Bacillus”
(“rod”). The name “cereus” (“wax”) was given because its colonies have
a waxy appearance on agar plates. Dimensions of vegetative cells are
typically 1.0–1.2 µm in diameter by 3.0–5.0 µm in length, with rounded or
square ends and are often arranged in pairs or chains (Arnesen et al. 2008).
The temperature range for the growth of B. cereus has been reported to
be between 8–55°C with the optimum being in the range of 28–35°C. The
range of pH for growth of B. cereus has been reported to be 4.9–9.3 with the
optimum being 6.0–7.0, and a water activity (aw) greater than 0.94.
Food-borne Illness
Bacillus cereus is an opportunistic pathogen commonly isolated from soil

and causes two types of food poisoning syndromes, emesis and diarrhea
(Granum and Lund 1997).
The diarrheal type is caused by heat-labile enterotoxins produced
by B. cereus in the intestine after ingesting large numbers of cells and is
mostly associated with proteinaceous foods such as meat. Three different
enetrotoxins have been characterised, namely haemolysin BL (Hbl), non-
haemolytic enetrotoxin (Nhe) and cytotoxin K (CytK) (Lund et al. 2000).
It is characterised by an incubation period of 8–16 h before the onset of
watery diarrhea, abdominal pain and occasionally nausea and vomiting.
Generally, the symptoms resolve within 12–24 hours, however in some rare
cases symptoms perpetuate and can eventually lead to death.
The emetic syndrome on the other hand is caused by consumption
of pre-fromed toxin mostly in farinaceous food items, particularly fried
or cooked rice and pasta (Ehling-Schulz et al. 2004). In most outbreaks,
these foods are stored after preparation, in conditions (room temperature)
that allow rapid growth and toxin production. The emetic syndrome is
caused by cereulide, a small cyclic non-ribosomally synthesized heat-stable
dodecadepsipeptide. The syndrome is characterised by an incubation period
of 0.5–5 h and is accompanied by symptoms such as vomiting, nausea,
malaise and occasional diarrhea. Generally, the symptoms are relatively
mild and disappear within 24 hours.
Usually it takes 105–107 cells in total for the diarrheal syndrome and
10 –108 cells per gram of food to cause the emetic syndrome (Ceuppens et
5
al. 2013). The wide range of infective dose is partly due to the consumption
of spores which can survive the acidic environment in the stomach and
partly due to the ability of different strains to produce different amounts
of enterotoxins. Therefore, any food containing more than 103 B. cereus per
gram cannot be considered completely safe for consumption.
Detecting B. cereus
Selective media is primarly used to isolate B. cereus by direct plating. MYP

(Mannitol-yolk-polymyxin) and PEMBA (Polymyxin-pyruvate-egg yolk
mannitol-bromothymol blue agar) are the most widely used selective media
for isolating B. cereus. Both media are based on the diagnostic features of
B. cereus of lecitithin hydrolysis and inability to ferment mannitol. B. cereus
forms blue and pink colonies on PEMBA and MYP respectively, surrounded
by a halo of lecithin hydrolysis. Polymyxin acts as the selective agent by
inhibiting the growth of competitive organisms (Bottone 2010). In addition a

chromogenic agar BCM developed which relies on the phosphotidylinositol
phospholipase C hydrolyase enzyme can be used for the detection of
B. cereus.
Presumptive colonies are confirmed by biochemical tests; include
anaerobic utilization of glucose, Voges-Proskauer, L-tyrosine decomposition,
nitrate reduction and growth in 0.0001% lysozyme.
Commercial kits (immunoassays) are available for the detection of the
diarrheal enterotoxin (Andersson et al. 2004). The Oxoid Bacillus cereus
RPLA enterotoxin detection kit (Oxoid Ltd., Basingstoke, UK) detects the
HblC (L2) component, whereas the Tecra Bacillus diarrheal enterotoxin
visual immunoassay kit (Bioenterprises pty, Australia) mainly detects the
NheA protein. Neither assay is quantitative.
In the dairy industry, B. cereus is an important cause of microbe-related

problems. It is practically impossible to completely avoid the presence of
B. cereus in raw milk samples, because B. cereus spores are ubiquitously
present in the farm environment: in soil, on cattle-feed and in cattle-faeces.
From these sources, the spores easily contaminate the udders and the raw
milk. Furthermore, B. cereus spores are very hydrophobic and attach to the
surfaces of processing equipment of the dairy industry. Spores attached to
processing equipment may germinate, multiply and re-sporulate, resulting
in a continuous source of contamination. In addition, its spore-forming
and psychrotrophic properties and the insufficiency of pasteurization to
kill the spores enable B. cereus to grow and produce toxins in pasteurized
milk at refrigeration. Besides causing food-borne illness, B. cereus is also
responsible for the spoilage of pasteurized milk and its products resulting
in off-flavors, sweet curdling and bitter cream.
Campylobacter spp.
Introduction
Campylobacter spp. has been known as a veterinary problem from the early
twentieth century. Campylobacter jejuni subsp. jejuni (referred to as C. jejuni
throughout this chapter) is a major cause of bacterial human gastroenteritis
in both developed and developing nations. It is one of the most important
milk-borne pathogens that has come to rival or even surpass Salmonella
as an etiological agent of human gastroenteritis worldwide.
Characteristics
The name Campylobacter is derived from the Greek word “Kampylos”,

which means curved. There are 11 species in the genus Campylobacter
including C. fetus, C. hyointestinalis, C. sputorum, C. jejuni, C. coli, C. lari,
C. upsaliensis, C. mucosalis, C. concisus, C. curvus, and C. rectus and belongs
to the delta-epsilon group of proteobacteria, which also comprises
Helicobacter, Arcobacter, Sulfurospirillum , the species Bacteroides ureolyticus
and Wolinella.
C. jejuni cells are pleomorphic and can be curved, spiral or occasionally
straight rods (splender vibiod cells) that are 0.2 to 0.8 µm wide and 0.5 to
5 µm long, which form an “S” or a “V” shape when two or more bacterial
cells are grouped together. They are gram negative and non sporeforming.
The cells are highly motile and have a corkscrew-like motion by means
of an unsheathed polar flagellum at one or both ends of the cell. C. jejuni
are microaerophilic, requiring an oxygen concentration of 3–15% and
capnophilic with a carbon dioxide concentration of 3–5%. They are catalase
and oxidase positive and urease negative. Temperature range for growth is
37–42°C, with an optimum temperature of 41.5°C, but they cannot survive
cooking or pasteurization temperature (Silva et al. 2011). The organism is
sensitive to heat, drying and acidic conditions (pH of less than 4.9 with
optimal growth at pH 6.5 to 7.5) and salinity.
Food-borne Illness
C. jejuni is estimated to be responsible for 90% of campylobacteriosis cases

in humans, and C. coli for the remaining 10% (Janseen et al. 2008). C. jejuni
infection is generally sporadic and in contrast to infections caused by other
food-borne pathogens such as Salmonella and E. coli O157, outbreaks are
rarely reported. The reported outbreaks are usually associated with drinking
contaminated/ untreated water, consumption of raw milk and contaminated
food and through contact with domestic animals.
In addition, C. jejuni infection is linked to traveller’s diarrhea. In
temperate regions there is a peak in the number of cases in summer and
to a lesser extent autumn and spring with the most at risk groups in both
developed and developing countries being children, the elderly and the
immunocompromised.
The most common symptoms are acute gastroenteritis, cramping
abdominal pain, fever, and more rarely, vomiting and headaches (Hariharan
et al. 2004). Diarrhea often develops shortly after onset of abdominal pain
and varies from mild, non-inflammatory, watery symptoms to severe and
bloody. In otherwise healthy individuals, infection last for approximately
4 days with an incubation period of 1–10 days. The infection dose for
humans may be as few as 500–10000 cells depending on vehicle of ingested

materials, virulence of the strain and the susceptibility of the individual.
Normally the C. jejuni infection is a self-limiting disease, but in severe cases,
macrolide antibiotic (erythromycin) or fluoroquinolones (ciprofloxacin) is
the choice of treatment.
Detecting Campylobacter spp.
Campylobacter spp. is typically fragile bacteria that are difficult to culture

and maintain in laboratory due to the special requirements for growth
temperature and gaseous environment.
For food and other samples where a low number/injured Campylobacter
spp. cells are suspected to be present in a highly mixed background flora,
an enrichment step is necessary prior to isolation on selective agar plates.
There are many different enrichment broths, some of the most frequently
employed enrichment broths are Bolton, Preston, Park-Sanders and Exeter.
Since Campylobacter is sensitive towards peroxides, radical scavengers like
horse/sheep blood and charcoal are often included in these enrichment
broths, as well as growth promoting reagents like ferrous sulphate, sodium
metabisulphite and sodium pyruvate (FBP) (Silva et al. 2011). Media
commonly used with antibiotics include cefoperazone, amphotericin
B, polymixin B, cycloheximide, rifampicin, trimethoprim lactate and
vancomycin. Furthermore, culturing is performed at approx. 42°C in a
microaerobic atmosphere (5% O2, 10% CO2, and 85% N2).
Following enrichment, or directly from samples with presumed high
numbers of Campylobacter, the samples are spread on selective agar plates.
Some of the most common ones are: modified charcoal cefoperazone
deoxycholate (mCCDA), Skirrow, Karmali, Preston, Abeyta-Hunt-Bark
(AHB), Campy-cefex and Butzler. According to ISO 10272, two selective
agars with different selective principles must be used in parallel in order
to increase the yield. Agar plates are incubated at 42°C for 24–48 h in
a microaerophilic atmosphere and confirmatory tests on characteristic
colonies. Typical colonies on selective agar media are smooth, convex and
glistening with a distinct edge or flat, translucent, shiny, and spreading
with an irregular edge. They are colorless to light cream or grayish with
diameter range from pinpoint to 5 mm.
Identification of Campylobacter spp. presumptive colonies is performed
by sub-culturing five colonies from selective media onto non-selective
media. These are examined microscopically regarding their morphology
and motility. Furthermore, a number of tests can be performed to confirm
the identification and determine the species; growth at 25, 37 and 42°C,
catalase, oxidase, glucose utilisation, and hippurate hydrolysis (Levin 2007).
Regarding milk samples The United States Food and Drug

Administration (US-FDA) recommends a method for isolation of
Campylobacter spp. (Fig. 4). Many rapid methods have been developed
for isolation and detection of Campylobacter in recent years, such as latex
agglutination test, polymerase chain reaction (PCR) technique, enzyme-
linked immunosorbent assay (ELISA) and immunomagnetic separation
(IMS) method.
Figure 4. Procedure for Isolation and Identification of Campylobacter spp. from Milk (After:
United States Food and Drug Administration).
Reported cases of campylobacteriosis (Heuvelink et al. 2009, Longenberger

et al. 2013) are linked with the consumption of raw milk rather than
processed dairy products. Both studies stress the point that Campylobacter
jejuni identified as the causal microorganism is isolated from raw milk
therefore, dairy establishments that sell raw milk directly to the consumer
should be aware of this pathogen.
On the other hand, El Sharoud (2009) reported low prevalence of

Campylobacter in Egyptian dairy products (raw milk and fresh Domiati
cheese) but a higher than expected survival rate. This suggests that this
food-borne strain of C. jejuni may develop adaptive strategies that aid
survival under food preservation conditions, which contradicts with what
is known about this pathogen as a stress-sensitive organism.
Yersinia spp.
Introduction
The genus Yersinia is named after Alexander Yersin, a Swiss bacteriologist

who first isolated the plaque bacillus in Hong Kong in 1894, while
investigating a catastrophic epidemic of bubonic plaque that killed an
estimated 60000 people.
Characteristics
Y. enterocolitica are included in the genus Yersinia, which are classified into
the family Enterobacteriaceae, a group of gram-negative, oxidase-negative,
catalase positively, non spore-forming and facultatively anaerobic bacteria.
Y. enterocolitica are rods or coccobacilli of 0.5–0.8 x 1–3 µm in size. Of the 11
species, only 3 species of Yersinia are recognised to be pathogenic to humans,
the plague bacillus, Yersinia pestis, and two that cause gastroenteritis;
Y. enterocolitica and Y. pseudotuberculosis (Bottone 1997). Strains belonging
to Y. enterocolitica are urease positive and can be differentiated from
Y. pseudotuberculosis with a positive result for fermentation of sucrose, and
negative reactions for rhamnose and melibiose fermentation. Most of the
strains are motile at 25°C but non-motile at 37°C. It can be isolated from
many sources, such as food, drinking water, sewage, environmental water
and human clinical samples with a world-wide distribution.
Y. enterocolitica can grow over a pH range of 4–10, generally with an
optimum pH of 7–8 depending on the acidulant used, environmental
temperature, composition of the medium, and growth phase of the
bacteria. It is a psychotropic bacterium therefore it has the ability to grow at
temperatures below 4°C, but the optimum growth temperature is 28–30°C.
Food-borne Illness
Human clinical infections with this species ensue after ingestion of the
microorganisms in contaminated food or water or by direct inoculation
through blood transfusion (Sabina et al. 2011). Infection with Y. enterocolitica
can cause a variety of symptoms depending on the age of the person
infected. Infection with Y. enterocolitica occurs most commonly in young

children, under seven years old and the infection is more common in the
winter (Bottone 1997).
It is a self limiting enterocolitis with an incubation period of 1–11 days.
Common symptoms include diarrhea and/or vomiting, fever and acute
abdominal pain caused by mesenteric lymphadenitis, and it is often clinically
indistinguishable from acute appendicitis. Sometimes post-infections, more
specifically extra-intestinal sequelae, such as reactive arthritis, erythema
nodosum, erythema multiforme, scarlatiniform exanthemata and septicemic
types deserve particular clinical attention.
Detection
Methods to isolate Yersinia from food are problematic due to competition

and over-growth by food microflora. Enrichment procedures usually
exploit the psychrotrophic character of Y. enterocolitica by incubating at
4ºC for 7–21 days (Fredriksson-Ahomaa and Korkeala 2003). The most
commonly used enrichment media are phosphate buffered saline (PBS) or
phosphate-buffered saline with sorbitol and bile salts (PSB) or tryptone soya
broth (TSB). The disadvantage of cold enrichment for prolonged periods
can increase the isolation rate other psychotrophics organism present in
the sample.
Y. enterocolitica grows in most routine media including blood, chocolate,
MacConkey (MAC), in which they produce colorless colonies as it can’t
ferment lactose, and Salmonella-Shigella (SS) agar, but these media are low
selective, as Y. enterocolitica strains grow slowly and of overgrowth by other
enteric bacteria. However, the best results for the selective isolation of
Y. enterocolitica from food samples give the Cefsulodin-Irgasan-Novobiocin
(CIN) agar. In addition to the antibiotics, the medium contains deoxycholate
and crystal violet as selective agents and mannitol as a fermentable carbon
source. After incubation at 28ºC for 24 hours Y. enterocolitica forms colonies
with a deep red centre (bull’s eye) with a sharp border surrounded by a
translucent zone.
Confirmation of identity of Y. enterocolitica may be made using
commercial identification kits. Rapid methods for detection of Y. enterocolitica
have also been developed such as polymerase chain reaction (PCR) methods.
Y. entercolitica has been isolated from raw cow’s milk in Mexico City
(Bernardino-Varo et al. 2013) while other authors (Ackers et al. 2000)
have reported that the same pathogen was associated with pasteurized
bottled milk sold from a local dairy. This incident was connected to
postpasteurization contamination, indicating the need for controls and
training/awareness of dairy farmers. Tacket et al. (1984) stated that
outbreaks of enteric disease caused by pasteurized milk are rare; although
the ability of Y. enterocolitica to grow in milk at refrigeration temperatures
makes pasteurized milk a possible vehicle for virulent Y. enterocolitica.
Shigella spp.
Introduction
The genus Shigella was first reported by the Japanese microbiologist Kiyoshi
in 1898. It was classified into four species based on the O antigen; Shigella
dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei which were
also known as Shigella subgroups A, B, C and D respectively.
Characteristics
Shigella is a genus of gammaproteobacteria and belongs to the family

of Enterobacteriaceae. They share common characteristcis and genetic
relatedness with members of the genus Escherichia and in particular with
enteroinvasive E. coli.
It is a small, non-spore forming, gram negative rod with a diameter
of 0.3 to 1 µm and a length of 1 to 6 µm. It is catalase positive, oxidase
negative, facultative anaerobe and is non-motile because of the absence
of H antigens. It is non-capsulated and possesses the K and O antigens.
O antigen (somatic antigen) is useful in serological identification to classify
the four species. K antigen is the capsule antigen which occasionally
interferes with O antigen determination. The Shiga toxin, also called
as verotoxin, is produced by Shigella dysenteriae type 1. The toxin has a
molecular weight of 68 kDa. It is a multisubunit protein made up of an
A subunit (32 kDa), responsible for toxic action of the protein and five
molecules of the B subunit (7.7 kDa), responsible for binding to a specific cell
receptor. Shigella is a typical mesophile and is able to grow at temperatures
ranging from 12°C to 48°C (optimum 37°C), at a pH range of 5.0 to 7.3
(Zaika and Philis 2005).
Food-borne Illness
Shigella spp. are intracellular bacterial pathogens that inhibit the

gastrointestinal tract of humans and are the causative agent of shigellosis
(Niyogi 2005). Shigellosis is a global human health problem that is estimated
to affect 80–165 million individuals annually. It is the most important

cause of bloody diarrhea worldwide, especially in developing countries
with poor hygienic conditions (Jennison and Verma 2004). Shigellosis is
exclusively a human disease and is usually acquired through contaminated
food and water sources. Shigella infects via the oral-faecal route and
infection is transmissible with as few as a 100 microorganisms, partly due
to the ability of the bacterium to survive the highly acidic environment
of the stomach such as gastric secretions. Shigellosis is also frequently
acquired from consumption of raw vegetables harvested in fields where
sewage was used as fertilizer, as well consumption of oysters that have
been contaminated with sewage. Transmission also occurs by accidentally
drinking contaminated water in the swimming pools.
The most common symptoms of shigellosis range from water diarrhea
to severe dysentery. Severe dysentery is characterized by fever, abdominal
pains, nausea, vomiting and acute permanent bloody and mucoid diarrhea
(Phalipon and Sansonetti 2007). The condition may be asymptomatic in
some cases or severe, especially in children. Symptoms can last from 3 to
14 days.
In the absence of effective treatments, patients with shigellosis can
develop secondary complications such as septicemia and pneumonia.
The effectiveness of the transmission of Shigella is due to the fact that
Shigella is highly infectious, the 10 to 200 organisms that are sufficient to
cause infection is significantly lower than that reported for most other
enteric pathogens such as for Vibrio spp. and Salmonella spp. which require
at least 104 to 105 organisms to cause infection.
Detecting Shigella spp.
Due to the lack of interest in Shigella as food-borne pathogens, laboratory

protocols for the isolation and identification are undeveloped. A
pre-enrichment step in Selenite-F(SF), or Tetrathionate (TT) broth or gram-
negative broth is required for the isolation of Shigella.
The common selective/differential agar media used for the recovery
of Shigella are MacConkey (MAC), Xylose Lysine Deoxycholate (XLD),
Hektoen (HEK) and Salmonella-Shigella (SS) and Deoxycholate Citrate Agar
(DCA). It has typical nonlactose fermenting characteristic colonies in lactose
enriched media such as on MAC, DCA and SS agar. Shigella is resistant to
bile salts and this characteristic is usually useful in the selective media.
Colonies on the MacConkey and DCA agar appear to be large, 2 to 3 mm in
diameter, translucent and colourless (non-lactose fermenting). Whereas, on
the XLD agar, colonies appear to be much smaller (1 to 2 mm diameter) and
red in colour as lysine is decarboxylated producing alkaline end products
which raises the pH and cause the agar to turn into deep red colour.
Figure 5. Detection of Shigella spp. in Food Samples (After: AES Chemunex 2008).
Shigella spp. is better related to raw milk but in developing countries

where hygiene and sanitation facilities are often poor the pathogen may
contaminate food products too. The recent findings from a survey carried
out in Egypt (Ahmed and Shimamoto 2014) from 1600 food samples (50:50
dairy and meat) showed that Shigella spp., along with E. coli 0157:H7 and
S. enterica serovar typhimurium, were isolated at a rate of 1.7%, 3.4% and
4.3%, respectively. The results may show a low prevalence but since the
bacterium may cause dysentery it should not be underestimated.
Escherichia coli
Introduction
Escherichia coli, originally called “Bacterium coli commune”, was first isolated
from children feces in 1885 by the German bacteriologist Theodor Escherich.
Nowadays it is the one of the most understood bacteria and is a common
inhabitant of the gastrointestinal tract of humans and animals.
Characteristics
Escherichia coli are Gram-negative, non-sporeforming bacilli and belong

to the family of Entrobacteriacae. They are approximately 1.1–1.5 µm in
wide and 2.0–6.0 µm in length and occur as single straight rods. E. coli is
a facultative anaerobe, catalase positive, oxidase negative and is capable
of reducing nitrates to nitrites. When growing fermentatively on glucose
or other carbohydrates, it produces acid and gas (mainly H2 and CO2).
The classic differential test in order to separate E. coli from Shigella and
Salmonella is the ability of E. coli to ferment lactose, which the latter two
genera fail to do.
Most E. coli strains are typical mesophiles, capable of growing over
a wide range in temperature from 7–50°C with an optimum temperature
of 37°C. E. coli can grow within a pH range of 5.5–8.0 with best growth
occurring at neutrality.
Food-borne Illness
Even though E. coli is a group of harmless bacteria that are most often
used as indicator organisms for fecal contamination and breaches in
hygiene, there are some strains that have acquired virulence factors
that has allowed them to cause serious disease especially diarrhea.
Pathogenic Escherichia coli is categorized into the following six categories
according to their virulence properties, clinical manifestations, pathogenic
mechanisms, clinical syndromes and O:H serogroups: enteropathogenic
E. coli (EPEC), enterotoxigenic E. coli (ETEC), enterohaemorrhagic E. coli
(EHEC), enteroinvasive E. coli (EIEC), diffusely adherent E. coli (DAEC),
and enteroaggregative E. coli (EAEC) (Jafari et al. 2012).
Enteropathogenic E. coli (EPEC)
EPEC were the first group of E. coli recognised as a causative agent of

diarrheal illness in humans. EPEC are a major cause of acute or chronic
enteritis and infant diarrhea in developing countries where sanitation and
water quality may be poor Other symptoms include vomiting, fever, malaise
and dehydration and appear usually 12–36 h after ingestion of the organism.
Enterotoxigenic E. coli (ETEC)
ETEC were first recognised as a causative agent of diarrhea in the 1960s

and 1970s. ETEC strains produce toxins which are heat-labile (LT) and/
or heat-stable (ST) and they are responsible for secretory diarrhea in both
humans and animals. Illness caused by ETEC usually occurs 12 and 36 h after
ingestion of the organism and usually persists for 2–3 days and symptoms
include acute watery diarrhea that may be mild and of short duration or
more severe such as cholera characterized by watery stools accompanied by
vomiting and severe stomach pains. The infective dose of ETEC for adults
has been estimated to be at least 108 cells; but the young, the elderly and
the infirm may be susceptible to lower levels.
Enterohaemorrhagic E. coli (EHEC)
EHEC strains are implicated in food-borne diseases principally due to

ingestion of uncooked minced meat and raw milk. They are more commonly
referred to as verocytotoxigenic (VTEC) as they produce a toxin which has
a cytotoxic effect on vero cell lines. These strains produce shiga like toxins.
E. coli O157:H7 is the prototype of this group. The symptoms of infection
from this group of organisms includes watery diarrhea which may develop
into bloody diarrhea (haemorrhagic colitis), severe abdominal pain and
Haemolytic uraemic syndrome (HUS), a cause of acute renal failure, and
thrombotic thrombocytopenic purpura (TTP) (Kaper 1998). The pathogen
has an incubation period of 1–14 days with illness duration of 5–7 days.
The infectious dose for O157:H7 is estimated to be 10–100 cells; but no
information is available for other EHEC serotypes (Teunis et al. 2004).
Enteroinvasive E. coli (EIEC)
EIEC (enteroinvasive E. coli) cause a broad spectrum of human’s diseases.

They are biochemically, genetically and pathogenetically closely related
to Shigella spp. However the infective dose appears to be higher than that
of Shigella and required at least 106 cells to cause illness in health adults
EIEC cause invasive inflammatory colitis and dysentery with a clinical

presentation (blood and mucous stools accompanied by fever and severe
cramps). Other symptoms are headache, fever, and cramping. Onset of
illness occurs about 24 h post ingestion.
Diffusely adherent E. coli (DAEC)
DAEC are a major cause of urinary tract infections worldwide, but its role
as a causative agent of diarrhea is not completely understood.
Enteroaggregative E. coli (EAEC)
EAEC is the most recently identified and described diarrhoeagenic E. coli

(Huang et al. 2006). This bacterium was described in 1987, and identified
in a child from Chile with persistent diarrhea (Nataro 2005). EAEC cause
watery diarrhea by adhering to the epithelium of terminal ileum and
colon in a characteristic aggregative pattern followed by a damage/
secretion stage.
Detecting E. coli
Pathogenic E. coli represents a phenotypically diverse group of pathogens

and no single method or approach can be used to detect or isolate all of the
pathotypes of concern. Therefore different methods have been developed
for the detection and isolation of certain pathotypes.
Pathogenic E. coli strains that ferment lactose and are not adversely
affected by elevated temperatures (e.g., 44°C) can be isolated using standard
procedures for E. coli. Most pathogen detection in foods is performed on
a 25 g sample which is enriched in 225 ml of a suitable enrichment broth.
In the Food and Drug Administration Bacteriological Analytical Manual
(FDA BAM) method the recommended procedure for pathogenic E. coli is
to pre-enrich the sample in 225 ml brain heart infusion (BHI) broth at 35°C
for 3 hours to facilitate resuscitation of sub-lethally injured cells. The entire
pre-enrichment is transferred to 225 ml of tryptone phosphate (TP) broth
and incubated at 44°C for 20 hours, after which time an aliquot of enriched
broth is plated onto eosin-methylene blue (EMB) agar and MacConkey
agar plates. These are incubated at 37°C for 24 hours. Further identification
includes biochemical tests, serotyping and examination for key virulence
associated genes.
Another biochemical feature of E. coli is β-glucuronidase activity,
therefore a fluorogenic or chromogenic glucuronide is incorporated into a
conventional media and enzyme activity is detected by the production of

colour or flurorescence.
Regarding the isolation of E. coli 0157 the reference method ISO
16654:2001 is used. This method is based on an enrichment step in modified
TSB with novobiocin (mTSBn) at 41.5°C for an initial period of 6 hours and
then for a further period of 12 to 18 hours. This is followed by a separation
and concentration step and then isolation on chromogenic media such as
CT-SMAC (Farrokh et al. 2013).
Shiga toxin producing E. coli (STEC) are an important cause of human food-
borne outbreaks. The consumption of raw milk dairy products may be an
important route of STEC infection. There are 2 STEC strains of serotypes
O157:H7 and O26:H11, and Miszczycha et al. 2014, reported that the survival
of E. coli O26:H11 was 13 times greater than that of E. coli O157:H7 at the
end of digestion in contaminated raw milk cheeses. E. coli O157:H7 has also
been isolated from dairy farms, even though a survey in Italy reported from
Conedera et al. 2004 showed that none of the dairy products collected (2948
including mozzarella cheese, pasteurized and raw bovine and ovine milk)
had tested positive for VTEC E. coli 0157:H7. A very comprehensive review
on Shiga-toxin-producing Escherichia coli (STEC) and their significance in
dairy production has been published by Farrokh et al. (2013), where the
authors review the survival of STEC during cheese making and in particular
the factors affecting the growth of STEC during cheesemaking (i.e., salt,
pH, temperature, water activity), behaviour of STEC during cheesemaking.
As a conclusion, the International Dairy Federation in its IDF Factsheet
(2012) states that in the processing industry, milk pasteurization is
recommended, since pasteurization will eliminate pathogens including
STEC. Good hygienic practices at the processing facility are also to be
applied in order to prevent post-pasteurization contamination. At farm
level, strict hygiene practices should be followed during the production and
transport of raw milk. Additional hygiene practices are to be implemented
in the production of raw milk cheese and other dairy products.
Mycobacterium spp.
Introduction
Mycobacterium species are a large, varied group of organisms, which

include some significant pathogens (such as M. tuberculosis, M. bovis,
M. avium subsp. paratuberculosis, and M. leprae) along with many free-
living non-pathogens. Mycobacterium tuberculosis, the causative agent of

tuberculosis (TB, also known as the “white plague”), was identified by
Robert Koch in 1882. Mycobacterium avium subsp. paratuberculosis (MAP)
causes paratuerculosis in animals (Johne’s disease) and could have a role
in human diseases like Crohn’s (Marchetti et al. 2013). Though it requires
a suitable cell medium for growth, MAP can survive in the environment
well beyond its presence in an animal host, creating drastic problems for
farmers trying to eradicate Johne’s disease from their herds.
Characteristics
The genus Mycobacterium is a member of the Actinobacteria. MAP, an

obligate intracellular pathogen, is a small (0.5 x 1.5 µm), rod shaped bacteria
which grow in circular colonies reaching about 1–2 mm in diameter. They
are usually found to be off-white or yellow depending on the medium, and
have the ability to adapt easily into many environments. A common feature
of mycobacteria is the chemical composition of their cell walls which have
a high lipid content, that avidly retains Carbol fuchsin dye even in the
presence of acidic alcohol (acid fast staining) (Glickman and Jacobs 2001) .
Food-borne Illness
MAP and its association (or not) to human Crohn’s Disease is a debate
which started in 1984, when a Mycobacterium similar to the agent of
Johne’s disease was isolated and cultivated from the intestinal tissue of three
patients with Crohn’s disease. Crohn’s disease is a chronic inflammatory
bowel disease which currently is of unknown cause. The most widely
accepted hypothesis is that Crohn’s disease occurs as a result of an abnormal
immune response within the gastrointestinal mucosa. Grant (2006) reports
that transmission to humans via consumption of animal-derived foods is a
distinct possibility as milk, other dairy products, beef and water have been
identified as possible food vehicles of transmission. To date, viable MAP
has been cultured from raw cows’, sheep and goats’ milk, retail pasteurized
cows’ milk, and some retail cheeses in several countries during recent
studies. MAP has not been isolated from retail beef to date, although limited
testing has been carried out.
The public health consequences, if any, of low numbers of viable MAP
being periodically consumed by susceptible individuals are uncertain.
Moreover, an association between MAP and Crohn’s disease is not proven,
but neither can it be discounted on the basis of current evidence.
Detecting Mycobacterium aviums subsp. paratuberculosis
It is generally accepted that molecular techniques (i.e., PCR basis) should

be employed for the detection of MAP from a wide range of samples, while
antibodies aginst MAP could be tested by commercially available ELISA
tests for indirect confirmation test. Liapi et al. (2013) examined 192 MAP
ELISA-positive sheep and goats from Cyprus using faecal culture and
genotype MAP isolates using IS1311 PCR and restriction endonuclease
analysis (IS1311 PCR-REA) with HinfI restriction enzyme. The same
methods, i.e., ELISA and RT-PCR, were also used by Marchetti et al. (2013) in
order to quantify MAP in bulk tanker milk (BTM). Botsaris et al. (2010) also
reported rapid methods for the determination of MAP in milk and cheese.
Marchetti et al. (2013) reported that the transmission of MAP to humans

most likely occurs via contaminated milk and milk products. MAP can
survive low-temperature holding (63°C for 30 min) and high temperature-
short time (HTST) (72°C for 15 sec) pasteurization, some surveys showed the
presence of MAP in commercially pasteurized milk purchased at retail. The
effect of homogenisation of heat inactivation of MAP in milk was studied
by Hammer et al. (2014) showing that none of the 3 homogenization modes
applied showed a statistically significant additional effect on the inactivation
of MAP during HTST treatment.
Dairy Outbreaks
Dairy products have frequently been implicated in food poisoning
outbreaks. There are two main reasons for that; one is the fact that the gross
chemical composition for most of the dairy products favors the growth and
survival of microorganisms (i.e., high protein content, presence of lactose,
available moisture—water activity, aw), and second, the sheer volumes of
dairy products consumed by almost all population groups making dairy
products a very good vehicle for microorganisms. Fortunately, processing of
milk and milk products (i.e., pasteurization, ultra high temperature, drying,
evaporating, filtrating of bacteria, fermenting/acidification) minimizes risks
either by posing hurdles for bacteria to grow (low pH), decreasing available
water (lowering water activity), or by destroying bacteria (temperature).
Nonetheless, milk and other dairy products (i.e., cheese) could also be
consumed raw (according to the provisions of legislation EC 853/2004);
therefore strict hygienic conditions must apply; at farm, during milk
transportation, during manufacture at the factory and at retail level in order
to avoid food poisoning outbreaks. Table 3 below summarizes recent alert
cases as reported by the Rapid Alert System for Food and Feed (RASFF),
European Union concerning milk and milk products and certain common
pathogens.
Langer et al. (2012) published a study (results are shown in Table 4)
where they describe the outbreaks related to dairy products both raw
and pasteurized in the USA between the years 1993–2006. They reported
that the causative agent was identified for all 73 outbreaks involving raw
dairy products; all were caused by bacteria. One outbreak was caused by
Campylobacter spp. and shigatoxin producing Escherichia coli. Among the
remaining 72 outbreaks, 39 (54%) were caused by Campylobacter spp., 16
(22%) by Salmonella spp., 9 (13%) by shiga-toxin producing E. coli, 3 (4%)
by Brucella spp., 3 (4%) by Listeria spp., and 2 (3%) by Shigella spp. Among
the 30 outbreaks involving pasteurized dairy products for which the
Table 3. Milk and milk products involved in recent alert cases (RASFF, EU).
Pathogen Milk and Milk No. of cases Severity Notes

products 2013–03/2014
Listeria Cheese (Raw or 25 alert Most cases involved
monocytogenes pasteurized) sheep or goat raw milk
cheeses from France
Salmonella enteritidis Cheese 1 alert
Salmonella spp. Pasteurised cow 2 alert
milk cheese,
Raw milk
cheese
E. coli (shigatoxin Raw milk 5 alert Country of origin: France
producing) cheeses
Table 4. Characteristics of disease outbreaks after consumption of dairy products 1993–2006,

United States.
Product Total Associated Associated Associated deaths

Outbreaks illnesses hospitalizations
Raw
Fluid milk 46 930 71 0
Cheese 27 641 131 2
Total 73 1,571 202 2
Pasteurized
Fluid milk 10 2,098 20 0
Cheese 38 744 17 1
Total 48 2,842 37 1
All dairy 121 4,413 239 3
After: Langer et al. (2012)

causative agent was reported, 13 (44%) were caused by norovirus, 6 (20%)

by Salmonella spp., 4 (13%) by Campylobacter spp., 3 (10%) by Staphylococcus
aureus, and 1 (3%) each by Clostridium perfringens, Bacillus cereus, Listeria
spp., and Shigella spp.
A total of 48 reported outbreaks involved pasteurized dairy products.
The source of contamination was reported for 7 (14%) of these outbreaks,
of which at least 4 (57%) probably resulted from post-pasteurization
contamination by an infected food handler.
More recent reports (Food safety News 2012) state that in the USA
between the years 2010–2012; 24 raw dairy outbreaks were reported with
309 illnesses and no deaths (22 fluid raw milk, 2 aged raw milk cheeses); 2
pasteurized dairy outbreaks with 39 illnesses and no deaths; 1 pasteurized
Mexican-style cheese sporadic illness and no deaths; 2 queso fresco
Mexican-style cheese outbreak with 67 illnesses and no deaths and finally
3 sporadic illnesses and hospitalizations from illegal Mexican-style cheese
with no deaths. The major pathogens involved in most of the cases stated
above were: L. monocytogenes, E. coli O157:H7, C. jejuni, and S. aureus. A
comprehensive study carried out by de Buyser et al. (2001) reported the
implication of milk and milk products in food-borne disease in France
and other industrialsed countries. Four etiologic agents were considered:
Salmonella spp., S. aureus, L. monocytogenes, and pathogenic E. coli and the
results showed that in France, out of 69 reported outbreaks for which the
implication of milk and milk products was considered as confirmed because
the etiologic agent was isolated from food; 48% of the food vehicles were
from raw milk, but for 51%, the type of milk was not specified. A majority
of these outbreaks were traced to cheeses made from raw or unspecified
milk. S. aureus was by far the most frequent pathogen associated with
these outbreaks, followed by Salmonella spp. In England and Wales, the
situation seems different because the most frequently reported outbreaks
were salmonellosis associated with the consumption of raw milk.
Microbiological Sampling Plans

Foodstuffs should not contain micro-organisms or their toxins or metabolites
in quantities that present an unacceptable risk for human health. The
safety of foodstuffs is mainly ensured by a preventive approach, such as
implementation of good hygiene practice (a prerequisite programme) and
application of procedures based on hazard analysis and critical control point
(HACCP) principles. Testing against the criteria as set down in Regulation
(EC) No 2073/2005 should be undertaken when validating or verifying the
correct functioning of systems in place. In addition, food business operators

(FBOs) should determine shelf-life by a strict testing programme to ensure
that the criteria are met over the entire intended shelf-life of the product.
Article 4 of Regulation (EC) No 852/2004 places an obligation on FBOs
to comply with microbiological criteria for foodstuffs. 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. The producer or manufacturer of a food product has to decide
whether the product is ready to be consumed as such, without the need to
cook or otherwise process it in order to ensure its safety and compliance
with the microbiological criteria.
In ensuring compliance with the relevant microbiological criteria set
out in Annex I to Regulation (EC) No 2073/2005, the FBOs at each stage of
food production, processing and distribution, including retail, must take
measures, as part of their procedures based on HACCP principles together
with the implementation of good hygiene practice, to ensure the following:
a) that the supply, handling and processing of raw materials and
foodstuffs under their control are carried out in such a way that the
process hygiene criteria are met,
b) that the food safety criteria applicable throughout the shelf-life of
the products can be met under reasonably foreseeable conditions of
distribution, storage and use.
The Commission Regulation (EC) No 2073/2005 and its amendments;
Commission Regulation (EC) 1441/2007 and 365/2010 on microbiological
criteria for foodstuffs for Milk and Milk products state the following:
Food category: Pasteurized milk and other pasteurized liquid dairy
products# | Micro-organisms: Enterobacteriaceae | n = 5 | c = 0 | m = <
1/ml, M = 5/ml| Analytical reference method: ISO 21528–2 | Stage where
the criterion applies: End of the manufacturing process | Action in case
of unsatisfactory results: Check on the efficiency of heat-treatment and
prevention of recontamination as well as the quality of raw materials.
Food category: Cheeses made from milk or whey that has undergone heat
treatment | Micro-organisms: E. coli* |n = 5 |c = 2 | m= 100 cfu/g | M=1000
cfu/g | Analytical reference method ISO 16649–1 or 2 | Stage where the
criterion applies: At the time during the manufacturing process when the
#
The criterion does not apply to products intended for further processing in the food industry.
* E. coli is used here as an indicator for the level of hygiene.
E. coli count is expected to be highest** | Action in case of unsatisfactory

results: Improvements in production hygiene and selection of raw materials.
Food category: Cheeses made from raw milk | Micro-organisms: Coagulase-
positive staphylococci |n = 5 | c = 2 | m = 104 cfu/g | M = 105 cfu/g |
Analytical reference method: EN/ISO 6888–2 | Stage where the criterion
applies: At the time during the manufacturing process when the number
of staphylococci is expected to be highest | Action in case of unsatisfactory
results: Improvements in production hygiene and selection of raw materials.
If values > 105 cfu/g are detected, the cheese batch has to be tested for
staphylococcal enterotoxins.
Food category: Cheeses made from milk that has undergone a lower heat
treatment than pasteurization+ and ripened cheeses made from milk or
whey that has undergone pasteurization or a stronger heat treatment +|
Micro-organisms : Coagulase-positive staphylococci | n = 5 |c = 2 | m = 100
cfu/g | M = 1000 cfu/g | Analytical reference method: EN/ISO 6888–1 or 2
Food category: Unripened soft cheeses (fresh cheeses) made from milk or
whey that has undergone pasteurization or a stronger heat treatment+|
Micro-organisms: Coagulase-positive staphylococci |n = 5 | c = 2 | m =
10 cfu/g | M = 100 cfu/g | Analytical reference method: EN/ISO 6888–1
or 2 | Stage where the criterion applies: End of the manufacturing process
| Action in case of unsatisfactory results: Improvements in production
hygiene. If values > 105 cfu/g are detected, the cheese batch has to be tested
for staphylococcal enterotoxins.
Food category: Butter and cream made from raw milk or milk that has
undergone a lower heat treatment than pasteurization | Micro-organisms:
E. coli* | n = 5 | c = 2 | n = 10 cfu/g | n = 100 cfu/g | Analytical reference
method: ISO 16649–1 or 2 | Stage where the criterion applies: End of
the manufacturing process | Action in case of unsatisfactory results:
Improvements in production hygiene and selection of raw materials |.
Food category: Milk powder and whey powder# | Micro-organisms:
Enterobacteriaceae | n = 5 | c = 0 | m,M = 10 cfu/g## | Analytical reference
method: ISO 21528–1 | Stage where the criterion applies: End of the
manufacturing process | Action in case of unsatisfactory results: Check
** For cheeses which are not able to support the growth of E. coli, the E. coli count is usually
the highest at the beginning of the ripening period, and for cheeses which are able to
support the growth of E. coli, it is normally at the end of the ripening period.
+
Excluding cheeses where the manufacturer can demonstrate, to the satisfaction of the
competent authorities, that the product does not pose a risk of staphylococcal enterotoxins.
#
The criterion does not apply to products intended for further processing in the food industry.
##
m = M.
on the efficiency of heat treatment and prevention of recontamination |

Micro-organisms : Coagulase-positive staphylococci | n = 5 | c = 2 |m =
10 cfu/g | M = 100 cfu/g | Analytical reference method: EN/ISO 6888–1
or 2 | Stage where the criterion applies: End of the manufacturing process
| Action in case of unsatisfactory results: Improvements in production
hygiene. If values > 105 cfu/g are detected, the batch has to be tested for
staphylococcal enterotoxins. |.
Food category: Ice cream++ and frozen dairy desserts | Micro-organisms:
Enterobacteriaceae |n = 5 | c = 2 | m = 10 cfu/g | M = 100 cfu/g | Analytical
reference method: ISO 21528– 2 | Stage where the criterion applies: End
of the manufacturing process | Action in case of unsatisfactory results:
Improvements in production hygiene |.
Food category: Dried infant formulae and dried dietary foods for special
medical purposes intended for infants below six months of age | Micro-
organisms: Enterobacteriaceae |n = 10 | c = 0 | m, M+ = Absence in 10 g
| Analytical reference method: ISO 21528– 1 | Stage where the criterion
applies: End of the manufacturing process | Action in case of unsatisfactory
results: Improvements in production hygiene to minimise contamination$|.
Food Category: Dried follow-on formulae| Micro-organisms:
Enterobacteriaceae |n = 5 | c = 0 | m, M+ = Absence in 10 g | Analytical
reference method: ISO 21528– 1 | Stage where the criterion applies: End
of the manufacturing process | Action in case of unsatisfactory results:
Improvements in production hygiene to minimise contamination|.
Food Category: Dried infant formulae and dried dietary foods for special
medical purposes intended for infants below six months of age| Micro-
organisms: Presumptive Bacillus cereus |n = 5 | c = 1 | m = 50 cfu/g, M
= 500 cfu/g | Analytical reference method: EN/ISO 7932 | Stage where
the criterion applies: End of the manufacturing process | Action in case of
unsatisfactory results: Improvements in production hygiene. Prevention of
recontamination. Selection of raw material|.
n = number of units comprising the sample; c = number of sample units
giving values between m and M.
++
Only ice creams containing milk ingredients.
+
m=M.
$
Parallel testing for Enterobacteriaceae and E. sakazakii shall be conducted, unless a
correlation between these micro-organisms has been established at an individual plant
level. If Enterobacteriaceae are detected in any of the product samples tested in such a plant,
the batch has to be tested for E. sakazakii. It shall be the responsibility of the manufacturer
to demonstrate to the satisfaction of the competent authority whether such a correlation
exists between Enterobacteriaceae and E. sakazakii.
Interpreting the Legislation

As an example: in the food category cheeses made from raw milk the sample
is made up of 5 units (n), the maximum number of sample units giving
values between m and M is 2(c). The batch is rejected if 1 unit gives values
higher than M and if c is surpassed.
It is recomended that the reader, with regards to EU legislation, should

seek for current updates in the European Community’s website or webpages
such as Food Safety Ireland (www.fsai.ie).
Food Safety Management Systems and the Dairy Industry
The implementation of Food Safety Management Systems (FSMS) in the

dairy industry is of paramount importance if we are to produce dairy
products that are safe for consumption. The ISO/TS 22002–1:2009 specifies
detailed requirements to be specifically considered in relation to ISO
22000:2005, i.e. a) construction and layout of buildings and associated
utilities; b) layout of premises, including workspace and employee facilities;
c) supplies of air, water, energy, and other utilities; d) supporting services,
including waste and sewage disposal; e) suitability of equipment and its
accessibility for cleaning, maintenance and preventive maintenance; f)
management of purchased materials; g) measures for the prevention of
cross-contamination; h) cleaning and sanitizing; i) pest control; j) personnel
hygiene. These aspects together with the required on-farm practices are
discussed in Chapter 7.
Conclusions
Dairy pathogens are of paramount importance as they can cause major food
scares to the consumers. The fact that dairy products are widely consumed,
daily, in many forms by the majority of the world’s population including
special groups (i.e., infants, the elderly, and the immune-compromised)
makes their strict control inherent. We should also be aware of emerging
dairy-related pathogens (i.e., Mycobacterium avium subsp. paratuberculosis)
and assess their impact to human health, apart from the obvious food
poisoning. Surveys from underdeveloped countries as well as from the
developed part of the world should continue to yield results in order to
enhance our understanding on dairy pathogens that up to now may not have
been given the attention they required. Finally, the acquired knowledge on
prevalence and impact of dairy pathogens in the food chain would enable
the scientific community to make informed decisions regarding issues such
as the direct selling (or not) of raw milk, from animal species.
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CHAPTER 4
Dairy Starter Cultures

Thomas Bintsis1,* and Antonis Athanasoulas2
INTRODUCTION
Fermented milk products, such as yogurt and cheese, appeared in human
diet about 8000–10000 years ago. Up to the 20th century, milk fermentation
remained an unregulated process, and, the discovery and characterization
of lactic acid bacteria (LAB) have changed the views on milk fermentation.
In the early 1960s, commercial starter culture companies developed
the production technology to freeze-dry liquid cultures and produce
concentrated frozen starter cultures for the direct inoculation of bulk starter
tanks at the dairy. In the beginning of 1980s Chr. Hansen released the first
Direct-Vat-Set (DVS) culture comprising defined single strains. Today,
several commercial starter companies offer an extensive range of frozen
and freeze-dried concentrated cultures for direct inoculation, eliminating
the need for use of bulk starters, and thus, propagation problems. In fact,
these fermentation businesses, together with probiotic products, represent
a total global market value of over 100 billion Euros (de Vos 2011).
While the traditional method for the manufacture of fermented dairy
products was the “inoculation” of the milk with a sample of a previous
day product, i.e., back-slopping. This method had certain drawbacks and
is not used anymore, except for some home-made products. There was a
significant microbial variability both in the milk and the natural starter,
hence a great fluctuation in the quality of the product. Thus, the substitution
1
PhD, Dairy Science, 25 Kappadokias St. 55134 Thessaloniki, Greece.
2
Starter Cultures Technical Manager, TYRAS SA 5th klm Trikala-Pili, 42100, Trikala, Greece.
Dairy Starter Cultures 115
of the back-slopping with a selected starter culture was conceived a necessity

very early. Nowadays, since the production of dairy products is automated
and requires large quantities of milk, and total control of the process, the use
of commercial starter cultures has become an integral part of a successful
production of any fermented dairy product.
Bacterial starter cultures are defined as “prepared cultures that contain
one or several strains of microorganisms at high counts (in general more
than 108 cfu/ml or 108 cfu/g of viable bacteria) being added to bring about
a desirable enzymatic reaction (e.g., fermentation of lactose resulting in acid
production, degradation of lactic acid to propionic acid or other metabolic
activities directly related to specific product properties” (International
Organization for Standardization/International Dairy Federation 2010).
The main function of the starter is to produce lactic acid, that is, the
acidification of milk. Besides, the acidifying bacteria contribute to the flavour,
texture and nutritional value of the fermented foods, through production
of aroma components, production or degradation of exopolysaccharides,
lipids and proteins, and the production of nutritional components such
as vitamins. In addition, they contribute to the inhibition of adventitious
organisms and pathogens.
The process for the production of starter cultures has been reviewed by
Høier et al. (2010) and Tamime (2002), and a typical process may include
the following steps:
a) handling of inoculation material,
b) preparation of media,
c) propagation of cultures in fermenters under pH control,
d) concentration,
e) freezing,
f) drying, and
g) packaging and storage.
Classification of Starter Cultures

Starter cultures used by the dairy industry can be divided into two broad
groups: mesophilic and thermophilic. Mesophilic starters have an optimum
temperature for growth at 30°C and are used in the production of most
cheese varieties (soft and semi-hard), while thermophilic have an optimum
temperature at 37°C and are used in the production of yogurt and hard and
semi-hard cheeses with high cooking temperatures (Table 1).
The mesophilic cultures are divided into LD cultures and O cultures.
LD cultures contain citrate-fermenting bacteria (L = Leuconostoc species
and D = Lc. lactis subsp. lactis biovar. diacetylactis), which produce aroma
and CO2 from citrate. The O cultures contain only acid-producing strains,
Table 1. Types of starter cultures and species of lactic acid bacteria used in some dairy products.
Type of Species Product

culture
Mesophiles
Type O Lactococcus lactis subsp. lactis Cheddar, White-brined
Lactococcus lactis subsp. cremoris cheeses, Cottage, Raclette,
Edam, Compté, St. Paulin.
Type LD Lactococcus lactis subsp. lactis Gouda, Tilsitter, Blue
Lactococcus lactis subsp. cremoris cheeses, Camembert and
Lactococcus lactis subsp. lactis biovar. diacetylactis other mould ripened
Leuconostoc mesenteroides subsp. cremoris cheeses, Sour cream, Butter.
Thermophiles
Streptococcus thermoplillus Fermented milks,
Mozzarella, Emmental,
Compté, Asiago, Sarde.
Streptococcus thermoplillus Yogurt, Fermented milks,
Lactobacillus delbreuckii subsp. bulgaricus Mozzarella, Feta, White-
brined cheeses.
Lactobacillus helveticus Emmental, Compté,
Lactobacillus delbreuckii subsp. lactis Beaufort, Asiago, Sarde.
Mixed
Lactococcus lactis subsp. lactis Cheddar, Colby, Chester,
Lactococcus lactis subsp. cremoris Leicester, Gouda,
Streptococcus thermoplillus Manchego.
Lactococcus lactis subsp. lactis White-brined cheeses
Lactococcus lactis subsp. cremoris
Streptococcus thermoplillus
Lactobacillus delbreuckii subsp. bulgaricus
Lactobacillus casei subsp. casei Yakult
Lactobacillus acidophilus Acidophilus milk
and produce no gas. The L cultures and D cultures also exist, but are only
used to a minor degree in the cheese industry. Traditional, mesophilic O
cultures are used in many cheeses, where the main focus is on a rapid
and consistent acidification of the milk. The LD cultures are used in most
continental semi-hard and soft cheeses (Table 1).
Starters are also subdivided into defined- and mixed-strain cultures.
Defined-strain cultures are pure cultures with known physiological
characteristics and technological properties. These consist of 2–6 strains,
used in rotation as paired single strains or as multiple strains and enable
industrial-scale production of high quality products. Mixed-strain cultures
contain unknown numbers of strains of the same species and may also
contain bacteria from different species or genera of LAB (Sheehan 2007). For
a detailed classification of starter cultures see Tamime (2002) and Parente
and Cogan (2004).
An authoritative list of microorganisms with a documented use in food

was established as a result of a joint project between the International Dairy
Federation (IDF) and the European Food and Feed Cultures Association
(EFFCA) (Bourdichon et al. 2012a). The “2002 IDF Inventory” listed 82
bacterial species and 31 species of yeast and molds whereas the 2012 IDF-
EFFCA updated inventory contains 195 bacterial species and 69 species of
yeasts and molds (Bourdichon et al. 2012b).
Adjunct Cheese Cultures
Adjunct cultures are defined as any cultures that are deliberately added at
some point of the manufacture of cheese, but whose primary role is not acid
production (Chamba and Irlinger 2004). The application of adjuncts could
be seen as an attempt by the cheese-maker to add back to the product, in
a controlled manner, some of the biodiversity removed by pasteurization,
improved hygiene and the defined strain starter system. Adjuncts can be
added in the milk or at a later stage of the cheese-making, e.g., in the salting
brines (Bintsis et al. 2002).
During cheese ripening, the starter bacteria and most other organisms
in the curd die; they autolyse and release intracellular enzymes as ripening
progresses (Khalid and Marth 1990a). On the other hand, the nonstarter
lactic acid bacteria (NSLAB) persist and can grow from levels of 102 to 104
cfu/g to ~108 cfu/g after manufacture, where they can have a significant
impact on flavour, either positively or negatively (Khalid and Marth 1990,
Cogan et al. 2007). The role of the secondary micro-flora is evident in certain
cheese varieties, e.g., “eye” formation due to fermentation of lactate by
propionibacteria in Swiss-type cheeses (i.e., Emmental, Gruyère de Comte)
and neutralization of the pH of the surface of a cheese due to metabolism
of lactate by yeasts and moulds in mould- and smear-ripened cheeses
(i.e., Camembert and Limburger) (Dezmazeaud and Cogan 1996). Penicillium
camemberti cultures are commercially available for the manufacture of
mould-ripened soft cheeses (i.e., Brie and Camembert), and Penicillium
roqueforti for blue-veined cheeses (i.e., Danish Blue, Stilton, Gorgonzola
and Roquefort). In addition, Geotrichum candidum are used in mould-
ripened cheeses since its deacidification activity stimulates the growth of
moulds on the cheese surface, and yeasts such as Debaryomyces hansenii,
Kluyveromyces lactis, Candida utilis, Saccharomyces cerevisiae and Rhodosporium
infirmominiatum are used in bacterial smear cheeses to stimulate the growth
of smear bacteria, due to their deacidification potential, and for their
debittering effect, due to their aminopeptidase activity (Chamba and Irlinger
2004). For the manufacture of bacterial smear-ripened cheeses, cultures of
Brevibacterium linens and Brevibacterium casei are available, since they belong
to the typical surface bacterial flora of such cheeses and contribute to the
formation of aromatic sulphur compounds (Bockelmann 1999).
In addition, it has been suggested that addition of the adjunct cultures
may be an indirect solution for controlling the growth of non-desirable
bacteria in cheese (Crow et al. 2001, Di Cagno et al. 2003). The term
“protective cultures” has been applied to microbial food cultures exhibiting
a metabolic activity contributing to inhibiting or controlling the growth
of undesired microorganisms in food (European Food and Feed Cultures
Association 2011). These undesired microorganisms could be pathogenic
or toxigenic bacteria and fungi but spoilage causing species may also be
included.
The technological properties of a number of Lactobacillus strains isolated
from cheese have been studied, including milk acidification kinetics and
proteolytic properties, as well as tolerance to salts and phage resistance
(Briggiler-Marco et al. 2007). For example, Lactobacillus casei I90 and
Lactobacillus rhamnosus I91 with low acidifying activity were found to be
appropriate for use as adjunct cultures as they did not alter the composition
of the cheese products and improved their sensory attributes. Interestingly,
the contribution of Lactobacillus paracasei subsp. paracasei and Lactobacillus
plantarum to proteolysis of Cheddar cheese appears to be mainly at the
level of free amino acids (i.e., due to exopeptidase activity) (Lynch et al.
1999). Thus, depending on the biochemical activities of the selected strains,
together with the rate of autolysis and/or release of enzymes, the water-
soluble peptides can be further degraded to small peptides and free amino
acids. Khalid and Marth (1990) emphasized the role of the proteolytic
and lipolytic activities in the development of cheese flavour and Peterson
and Marshall (1990) concluded that the high proteolytic and peptidolytic
activities of lactobacilli have an influence on the extent of proteolysis, but the
growth of heterofermentative strains have been associated with undesirable
flavours in Cheddar cheese.
In order to become an appropriate adjunct culture, it has been suggested
that a microorganism needs to be able to:
1. reach and maintain high levels of cell density during ripening,
2. cause no defect in the product, and
3. impact positively on the overall quality of the cheese.
Probiotic cultures have been extensively used during the last 10 years in
the dairy industry. Probiotic bacteria are “live microorganisms which when
administered in adequate amounts confer a health benefit on the host” (Food
and Agriculture Organization/World Health Organization 2001). Recently,
the addition of probiotic bacteria to cheese has been increasing applied (Ross
et al. 2002, Karimi et al. 2011, Kasimoglu et al. 2004, Souza et al. 2008, Rodgers
2008, Ong et al. 2006, Gardiner et al. 2002, Cruz et al. 2009, Araujo et al. 2010).
EU Regulation EC No. 1924/2006 on nutrition and health claims made on

foods has led to increased focus on the clinical documentation available for
probiotic strains (EU 2006). It is generally assumed that in order to provide
a beneficial health effect, the probiotic bacteria must be viable at the time
of consumption and remain viable throughout the gastrointestinal tract.
Some of the health benefits from probiotic bacteria include: (a) improving
intestinal tract health, (b) enhancing the immune system, (c) synthesising
and enhancing the bioavailability of nutrients, (d) reducing symptoms of
lactose intolerance, (e) decreasing the prevalence of allergy in susceptible
individuals, and (f) reducing risk of certain cancers (Parvez et al. 2006).
Genetics
LAB used for starter cultures in dairy products belong to a number
of bacterial genera including Lactococcus, Streptococcus, Lactobacillus,
Pediococcus, and Leuconostoc, all members of the Firmicutes. Moreover, some
probiotic cultures are mostly members of the genus Bifidobacterium, which
also produce lactic acid as one of their major fermentation end-products,
however, from the taxonomical point of view, they are members of the
Actinobacteria. LAB have either homofermentative metabolism, which
mainly produce lactic acid, or heterofermentative metabolism, which, apart
from lactic acid, yield a variety of fermentation products such as acetic acid,
ethanol, carbon dioxide and formic acid (Hutkins 2001).
The genetics of the LAB used as starter cultures in the dairy industry
have been reviewed (Broadbent 2001, Callanan and Ross 2004, Klaenhammer
et al. 2002, Morelli et al. 2004, Mills et al. 2010) and an overview is presented
below. In addition, complete genome sequences of a number of LAB have
been published, and an updated review of the available nucleic acid
databases is published every year in the Nucleic Acid Research (Galperin
and Fernandez-Suarez 2012).
Lactococcus spp.
Lactococci are mesophilic LAB that were first isolated from green plants
(Klaenhammer et al. 2002). These bacteria, previously designated as the
lactic streptococci (Streptococcus lactis subsp. lactis or S. lactis subsp. cremoris)
were placed in this new taxon in 1987 by Schleifer (Klaenhammer et al.
2002). Lactococci are selected for use as starters based on their metabolic
stability, their resistance to bacteriophage, and their ability to produce
unique compounds—often from amino acid catabolism. Lc. lactis subsp.
lactis form one of the main constituents in starter cultures (Table 1) where
their most important role lies in their ability to produce acid in milk and
to convert milk fat and protein into flavour compounds.
To date, the complete genome sequences of three lactococcal strains have
been published (Mayo et al. 2008); Lc. lactis subsp. lactis IL1403 (Bolotin et al.
2001), Lc. lactis subsp. cremoris MG1363 and NZ9000 (Wegmann et al. 2007,
Linares et al. 2010) and Lc. lactis subsp. cremoris SK11 (Makarova et al. 2006).
There are noticeable differences between strains, e.g., the chromosome
of Lc. lactis subsp. lactis MG1363 is 160 kb larger than that of Lc. lactis subsp.
lactis IL1403 and has an average Guanine + Cytosine (G+C) content of 35.8%,
and thus, encodes more proteins (Table 2).
Lc. lactis subsp. cremoris strains are preferred over Lc. lactis subsp. lactis
strains because of their superior contribution to product flavor via unique
metabolic mechanisms (Salama et al. 1991). With the knowledge of the
complete genome sequences, Lc. lactis subsp. cremoris was found to contain
greater genome sizes than Lc. lactis subsp. lactis IL1403 (approximately 2.37
Mb), with Lc. lactis subsp. cremoris MG1363 containing the largest genome
size of approximately 2.53 Mb, followed by Lc. lactis subsp. cremoris SK11
with a genome size of 2.44 Mb (Mills et al. 2010). Approximately 85% DNA
sequence identity was observed between the coding domains of Lc. lactis
subsp. lactis IL1403 and Lc. lactis subsp. cremoris MG1363, whereas 97.7%
identity was observed between Lc. lactis subsp. cremoris MG1363 and
Lc. lactis subsp. cremoris SK11 (Wegmann et al. 2007). Interestingly, a
complete set of competence genes was observed on the Lc. lactis subsp. lactis
IL1403 genome, indicating that the strain may have the ability to undergo
natural DNA transformation (Mills et al. 2010).
Many of the traits in lactococci which render these microorganisms
suitable for dairy fermentations are in fact encoded on plasmids (Mills et
al. 2006, McKay 1985). Traits such as lactose utilization, casein breakdown,
bacteriophage resistance, bacteriocin production, antibiotic resistance,
Table 2. Comparison of characteristics of sequences for Lc. lactis subsp. lactis MG1363 and
Lc. lactis subsp. lactis IL1403.
Lc. lactis subsp. lactis Lc. lactis subsp. lactis

MG1363 IL1403
Size (Mb) 2.53 2.37
No of ORFs ~2500 ~2310
Total GC% 35.8 35.4
GC% ORFs 36.7 36.1
No phage genes ~200 293
Phage DNA (kb) 134 293
IS elements 92 52
After: Kok et al. (2005)

resistance to and transport of metal ions, and exopolysaccharide (EPS)

production have all been associated with extra-chromosomal plasmid DNA.
Plasmids isolated from lactococci range in size from 3 to 130 kb, have a
G+C content of 30–40% and vary in function and distribution, with most
strains carrying between 4 and 7 per cell (Davidson et al. 1996). Plasmids are
commonly exchanged between strains via conjugation (McKay 1985, Dunny
and McKay 1999) and with the chromosome by insertion sequence (IS)
elements (Hughes 2000). Presumably, these exchanges and rearrangements
mediate rapid strain adaptation and evolution but also add to the instability
of important metabolic functions (Klaenhammer et al. 2002).
Streptococcus thermophilus
Streptococcus thermophilus is the second most commercially important

starter culture. S. thermophilus is used, along with Lactobacillus spp., as a
starter culture for the manufacture of several important fermented dairy
foods, including fermented milks, yogurt, Feta and Mozzarella cheeses
(Table 1). Although research on the physiology of S. thermophilus has
revealed important information on some of these properties, including sugar
and protein metabolism, polysaccharide production, and flavor generation,
only recently has the genetic basis for many of these traits been determined.
To date, three complete genome sequences have been published for
S. thermophilus (Mayo et al. 2008); S. thermophilus CNRZ1066, S. thermophilus
LMD-9 and S. thermophilus LMG18311. The genome of S. thermophilus is
1.8 Mb, making it among the smallest genomes of all LAB. Although a
moderate thermophile, it is phylogenetically related to the more mesophilic
lactococci and has a comparable low G+C ratio between 36.8 and 39%
(Table 3). Moreover, S. thermophilus is related to human pathogenic strains
of streptococci such as Streptococcus pneumoniae, Streptococcus pyogenes and
Streptococcus agalactiae (Hols et al. 2005). However, the most important
pathogenic determinants are either absent or present as pseudogenes, unless
they encode basic cellular functions (Bolotin et al. 2004). S. thermophilus has
therefore diverged from its pathogenic relatives to occupy the well-defined
ecological niche of milk (Mills et al. 2010). Pastink et al. (2009) compared,
using a genome-scale metabolic model of S. thermophilus LMG18311
with those of Lc. lactis subsp. lactis and reported the minimal amino acid
auxotrophy (only histidine and methionine or cysteine) of S. thermophilus
and the broad range of volatiles produced by the strain compared to
lactococci. The unique pathway for acetaldehyde production, which is
responsible for yogurt flavour, was also identified in S. thermophilus.
Unlike Lactococcus spp., plasmids are thought to play a relatively
insignificant role in S. thermophilus, reported to be found in about 20–59% of
Table 3. Genomes of lactic acid bacteria used as starter cultures in dairy industry.
Genus Species Strain Size (Mb) %GC

Lactococcus lactis subsp. Lactis IL1403 2.3 35.4
lactis subsp. cremoris MG1363 2.6 37.1
lactis subsp. cremoris SK11 2.3 30.9
Streptococcus thermophilus CNRZ1066 1.8 39.0
thermophilus LMD-9 1.8 36.8
thermophilus LMG18311 1.9 39.0
Lactobacillus Rhamnosus HN001 2.4 46.4
delbrueckii subsp. bulgaricus ATCC11842 1.8 50.0
gasseri ATCC33323 1.8 35.1
acidophilus NCFM 2.0 34.7
johnsonii NCC533 2.0 34.6
plantarum WCFS1 3.3 44.5
helveticus CNRZ32 2.4 37.1
Pediococcus Pentosaceus ATCC25745 2.0 37.0
Leuconostoc Mesenteroides ATCC8293 2.0 37.4
Propionibacterium Freudenreichii ATCC6207 2.6 67.4
Bifidobacterium Longum NCC2705 2.3 60.1
After: Klaenhammer et al. (2002)
strains examined (Madera et al. 2003, Turgeon et al. 2004, Hols et al. 2005).
Streptococcal plasmids are generally small, ranging in size from 2.1 to 10
kb and encode few industrially useful phenotypic traits, which include
low molecular weight, heat-stress proteins and specificity subunits of
bacteriophage-resistant restriction modification (R⁄M) systems (O’ Sullivan
et al. 1999) (Solow and Somkuti 2000, El Demerdash et al. 2006).
Lactobacillus spp.
The genus Lactobacillus encompasses a large number of different species that

display a relatively large degree of diversity. Similar to S. thermophilus, the
lactobacilli also belong to the thermophilic group of LAB starter cultures.
Lactobacilli commonly used for dairy fermentations include L. delbreuckii
subsp. bulgaricus, L. delbreuckii subsp. lactis, L. helveticus and L. acidophilus
(Thunell and Sandine 1985). To date a number of complete genome
sequences are available for lactobacilli (Mayo et al. 2008), including the
starter strains L. delbreuckii subsp. bulgaricus ATCC11842 (van de Guchte et
al. 2006), L. delbreuckii subsp. bulgaricus ATCC BAA-365 with genome sizes
of ~1.8 Mb (Makarova et al. 2006) and L. helveticus DPC4571 with a genome
size of ~2.0 Mb (Callanan et al. 2008).
L. plantarum has one of the largest genomes known among LAB
(Chevalier et al. 1994, Daniel 1995). The circular chromosome of L. plantarum
WCFS1 consists of ~3.3 Mb with an average G+C content of 44.5%, and is
among the largest of lactic acid bacteria. In addition, the strain harbours
three plasmids of ~36 kb (G+C 40.8%), 2.3 kb (G+C 34.3%), and ~1.9 kb
(G+C 39.5%), respectively (Klaenhammer et al. 2002).
Lactobacillus rhamnosus is one of the few species of Lactobacillus that
have been used as probiotic organisms in functional foods. A strain of
L. rhamnosus, designated HN001, has been identified that has both flavour
enhancing and probiotic attributes, therefore, it can be used as an adjunct
during cheese manufacture to reduce adventitious microflora, accelerate
cheese ripening, and improve cheese flavour (Klaenhammer et al. 2002).
Lactobacillus johnsonii strains have been mainly isolated from the
feces of humans and animals (Johnson et al. 1980, Fujisawa et al. 1992),
suggesting that these bacteria constitute part of the natural intestinal flora.
L. johnsonii La1 (formerly L. acidophilus La1) has been extensively studied
for its probiotic properties and is commercialized in the LC1 fermented
milk products (Klaenhammer et al. 2002). La1 shows immunomodulatory
properties (Link-Amster et al. 1994, Schiffrin et al. 1995) and antimicrobial
properties (Bernet-Camard et al. 1997, Felley et al. 2001, Pérez et al. 2001).
L. helveticus is quite closely related (< 10% sequence divergence) to
L. amylovorus, L. acidophilus, L. delbrueckii, L. acetotolerans, L. gasseri, and
L. amylophilus (Schleifer and Ludwig 1995). The genome size of L. helveticus
has been determined to be 2.4 Mb by PFGE (Klaenhammer et al. 2002).
Approximately 40 chromosomal genes and four plasmids have been
sequenced from L. helveticus. L. helveticus is a component of “thermophilic”
starter cultures used in the manufacture of a number of fermented dairy
products (Hassan and Frank 2001) and grows on a relatively restricted
number of carbohydrates that includes lactose and galactose and typically
requires riboflavin, pantothenic acid and pyridoxal for growth (Hammes
and Vogel 1995).
The L. delbrueckii species contains three subspecies, L. delbrueckii subsp.
delbrueckii, L. delbrueckii subsp. lactis, and L. delbrueckii subsp. bulgaricus.
L. delbrueckii subsp. bulgaricus grows on a relatively restricted number of
carbohydrates and typically requires pantothenic acid and niacin (Hammes
and Vogel 1995).
Phylogenetically, L. delbrueckii subsp. bulgaricus is closely related to
L. amylovorus, L. acidophilus, L. helveticus, L. acetotolerans, L. gasseri, and
L. amylophilus (Schleifer and Ludwig 1995). The G+C ratio of L. delbrueckii
subsp. bulgaricus (49–51%) is somewhat higher than that found among other
species (34–46%) within this phylogenetic tree (Hammes and Vogel 1995).
The genome size of L. delbrueckii subsp. bulgaricus has been determined to
be 1.8 Mb by PFGA (Klaenhammer et al. 2002).
Very few chromosomal genes (< 15) have been sequenced from
L. delbrueckii subsp. bulgaricus, however the complete sequence of a small
cryptic plasmid and the partial sequence of a bacteriophage are known.
Gene transfer systems for L. delbrueckii subsp. bulgaricus include two

conjugation-based gene transfer systems (Thompson et al. 1999) and
electrotransformation (Serror et al. 2002).
Pediococcus spp.
Phylogenetically, Pediococcus and Lactobacillus are related and form a super-

cluster; all species of Pediococcus fall within the Lactobacillus casei–Pediococcus
sub-cluster. However, morphologically, they are distinct since they form
tetrads via cell division in two perpendicular directions in a single plane.
Pediococcus can be described as the only acidophilic, homofermentative, LAB
that divide alternatively in two perpendicular directions to form tetrads
(Simpson and Taguchi 1995).
Pediococcus pentosaceus can be isolated from a variety of plant materials
as well as bacterial-ripened cheeses and is a typical component of the NSLAB
of most cheese varieties during ripening (Beresford et al. 2001) and has been
suggested as an acid producing starter culture in the dairy fermentations
(Caldwell et al. 1996, 1998).
Strains of P. pentosaceus have been reported to contain between three
and five resident plasmids (Graham and McKay 1985). Plasmid-linked traits
include the ability to ferment raffinose, melibiose, and sucrose, as well as, the
production of bacteriocins (Daeschel and Klaenhammer 1985, Gonzalez and
Kunka 1986). Plasmids can be conjugally transferred between Pediococcus
and Enterococcus, Streptococcus, or Lactococcus and electrotransformation has
been utilized to introduce plasmids into pediococci, including P. pentosaceus
(Klaenhammer et al. 2002).
Leuconostoc spp.
Leuconostoc mesenteroides is a facultative anaerobe requiring complex growth

factors and amino acids (Garvie 1986). Most strains in liquid culture appear
as cocci, occurring singly or in pairs and short chains; however, morphology
can vary with growth conditions; cells grown in glucose or on solid media
may have an elongated or rod-shaped morphology. Cells are Gram-positive,
asporogenous and non-motile. Although L. mesenteroides is commonly
found on fruits and vegetables, it has been extensively used as an industrial
dairy starter culture (Table 1). Under microaerophilic conditions, has a
heterofermentative reaction. Glucose and other hexose sugars are converted
to equimolar amount of D-lactate, ethanol and CO2 via a combination of
the hexose monophosphate and pentose phosphate pathways (Garvie
1986). Other metabolic pathways include conversion of citrate to diacetyl
and acetoin and production of dextrans and levan from sucrose (Cogan et
al. 1981). Viscous polysaccharides produced by L. mesenteroides are widely

recognized as causing product losses and processing problems in the
production of sucrose from sugar cane and sugar beets (Klaenhammer et
al. 2002).
Brevibacterium spp.
The Brevibacterium genus is a heterogeneous mixture of coryneform

organisms that have particular application to industrial production of
vitamins, amino acids for fine chemical production, and are commonly
used in cheese production (Rattray and Fox 1999). This genus contains
9 species from diverse habitats, such as soil, poultry, fish, human skin, and
food. While Brevibacterium linens is phenotypically similar to Arthrobacter
globiformis, cellular pigmentation, cell wall composition, DNA/DNA
hybridization and 5S RNA analysis show that Brevibacterium is distinctly
different (Park et al. 1987). PFGE analysis indicates that diversity within the
species is related to polymorphisms in the 16S rRNA genes with genome
sizes that range from 3.2 and 3.9 Mb (Lima and Correia 2000).
B. linens is a non-motile, non-spore forming, Gram-positive coryneform
that tolerates high salt concentrations (8–20%) and is capable of growing
in a broad pH range (5.5–9.5), with an optimum of pH 7.0, without being
a fast acid-producer. B. linens has been found to produce extracellular
protease (Rattray et al. 1997), high levels of volatile compounds from
amino acid catabolism (Ferchichi et al. 1985, Ummadi and Weimer 2001)
and bacteriocins (Valdes-Stauber and Scherer 1996).
Propionibacterium spp.
Propionibacteria are high G+C Gram-positive bacteria belonging to the

class of Actinobacteria that prefer anaerobic growth conditions and have
a peculiar physiology (Axelsson 2004). They produce propionate as
their major fermentation product. Propionate fermentation yields more
energy and, consequently, biomass than any other anaerobic microbial
fermentation. Furthermore, propionibacteria utilize polyphosphate and
pyrophosphate instead of ATP for several energy-dependent reactions
and their metabolism is tuned to synthesize high levels of porphyrins, in
particular B12 (Mills et al. 2010).
Propionibacteria have long been employed in the production process
of Swiss-type cheeses for which they are indispensable for the typical “eye”
formation and production of characteristic taste components (Guinee and
O’Callanhan 2010).
Bifidobacterium spp.
Species of the genus Bifidobacterium are Gram positive bacteria, strictly

anaerobic, fermentative rods, often Y-shaped or clubbed at the end and
contain DNA with a relatively high G+C content (Table 2). Bifidobacteria
have been shown to be the predominant species in the gastrointestinal tract
of infants, and represent the third most numerous species encountered in
the gastrointestinal of adult humans, considerably out-numbering other
microbial groups. The role of these bacteria in human health has stimulated
significant interest in the dairy industry, and has highlighted the position
of these bacteria in the development of functional foods, used as adjunct
cultures.
Despite growing consumer interest, key aspects regarding Bifidobacterium
species, such as metabolic activities (particularly relating to catabolism of
prebiotics) and physiology are still poorly understood. The determination
of the complete genome of the B. breve strain NCIM 8807 (Klaenhammer et
al. 2002), later designated as B. breve UCC 2003 (Sheehan et al. 2007), was
undertaken as a first step towards the molecular analysis of a probiotic
Bifidobacterium species. The genus is comprised of 31 characterized species,
11 of which have been detected in human feces (Tannock 1999). B. longum
is often the dominant species detected in humans and is one of the few
species to regularly harbor plasmids. It is a leading member of the probiotic
bacteria, and numerous studies have suggested its potential health benefits
(Ballongue 2004). These include modulation of the host immune system,
resistance to infectious diseases, and control of inflammatory bowel disease
and prevention of colorectal cancer (Crittenden 2004). The potential health
benefits attributed to the B. longum species clearly illustrate that this
species possesses many very interesting characteristics. It is anticipated
that identification and functional analysis of the genetic determinants
involved in these activities will strengthen the evidence for the involvement
of B. longum in these significant health benefits. It should be noted that
the selection of suitable strains for probiotic purposes is very difficult as
inherent characteristics of strains of B. longum that are necessary for its
survival and competition in the human large intestine are currently very
poorly understood (O’Sullivan 1999).
Identification and Typing Methods

Traditionally, LAB species have been identified on the basis of cell
morphology, analysis of fermentation products and associated enzyme
activities, and the ability to utilize various carbohydrate substrates
(Axelson 1998). The application of these approaches in the classification
and identification of LAB has been the subject of several reviews (Lane et
al. 1985, Axelson 1998, Hammes and Vogel 1995, Tannock 1999). In general,
phenotypic methods suffer from a lack of reproducibility generated by
conditions of culture related to different laboratories, and are often unable
to distinguish between closely related strains (Albuquerque et al. 2009,
Huys et al. 2006, Schleifer et al. 1995).
Genus to Species Identification
The use of nucleic acid probes, that is fragments of a single-stranded nucleic

acid that will specifically bind (hybridize) to complementary regions of
a target nucleic acid has been extensively used for the identification of
LAB (Ben Amor et al. 2007). Analysis of nucleic acids provides a basis for
identification methods that are reproducible from one laboratory to another
(Bintsis et al. 2008).
The application of 16S or 23S rRNA targeted probes is one of the most
reliable approaches. Today, with the availability of rapid and automatic DNA
sequencing technology, direct sequencing of the 16S rRNA gene has emerged
as the most powerful and relatively easy one-step method for classification
of bacteria (Axelsson 2004). Bacterial rRNA subunit sequences, namely
16S rRNA genes, intergenic spacer 16S–23S rRNA and 23S rRNA genes,
have been widely used over the years to study bacterial phylogeny (Rudi
et al. 2007, Myers et al. 2006, Eom et al. 2007). With automated sequencing
systems and convenient direct PCR sequencing methods, it has become an
easy task to determine the 16S rRNA sequence from any bacterium in a short
time. The low variability of 16S rRNA genes from closely related species,
the intra-genomic variability among the different chromosomal 16S rRNA
gene copies (Coenye and Vandamme 2003, Morandi et al. 2005), and the
failure to identify a collective conserved region for the design of universal
primers (Baker et al. 2003) are the main reported limitations.
Genotypic typing: strain identification
Typing methods refer to methods capable of discriminating microorganisms

at or near the strain level. These methods are generally applied after
the previous identification of the target microorganisms in which clonal
discrimination resolution is required. They have been routinely used in
epidemiological studies (Schleifer et al. 1995).
The most powerful of these are genetic-based molecular methods known
as DNA fingerprinting techniques, e.g., pulsed-field gel electrophoresis
(PFGE) of rare-cutting restriction fragments, ribotyping, randomly amplified
polymorphic DNA (RAPD), and amplified fragment length polymorphism
(AFLP), which have been applied extensively for the infraspecific
identification and genotyping of LAB and bifidobacteria isolated from
fermented food products as well as from the human gastrointestinal tract

(McCartney 2002). Basically, these methods rely on the detection of DNA
polymorphisms between species or strains and differ in their dynamic range
of taxonomic discriminatory power, reproducibility, ease of interpretation,
and standardization.
Ribotyping is a variation of the conventional PFGE analysis and the
probes used in ribotyping vary from partial sequences of the rDNA genes
or the intergenic spacer regions to the whole rDNA operon (O’ Sullivan
1999). Ribotyping has been used to characterize strains of Lactobacillus spp.
and Bifidobacterium spp. from commercial products as well as from human
fecal samples (Giraffa et al. 2000, Zhong et al. 1998). However, ribotyping
provides high discriminatory power at the species and subspecies level
rather than on the strain level. PFGE was shown to be more discriminatory
in typing closely related L. casei and L. rhamnosus as well as L. johnsonii
strains than either ribotyping or RAPD analysis (Tynkkynen et al. 1999,
Ventura et al. 2002).
Randomly amplified polymorphic DNA arbitrary amplification, also
known as RAPD, has been widely reported as a rapid, sensitive, and
inexpensive method for genetic typing of different strains of LAB and
bifidobacteria. RAPD profiling has been applied to distinguish between
strains of Bifidobacterium spp. and between strains of the L. acidophilus group
and related strains (Tynkkynen et al. 1999, Roy et al. 2000, Torriani et al.
1999). Several factors have been reported to influence the reproducibility and
discriminatory power of the RAPD fingerprints, i.e., annealing temperature,
DNA template purity and concentration, and primer combinations.
Metabolism of Starter Cultures and Flavor Development

The three main pathways which are involved in the development of flavor
in fermented dairy products are glycolysis (fermentation of lactose), lipolysis
(degradation of fat) and proteolysis (degradation of caseins) (Law 1999,
Smit et al. 2005, Tamime and Robinson 1999). Lactate is the main product
generated from the metabolism of lactose and a fraction of the intermediate
pyruvate can alternatively be converted to diacetyl, acetoin, acetaldehyde
or acetic acid (some of which can be important for typical yogurt flavours).
The contribution of LAB to lipolysis is relatively little, but proteolysis is the
key biochemical pathway for the development of flavour in fermented dairy
products (Souza et al. 2001). Degradation of caseins by the activities of rennet
enzymes and the cell-envelope proteinase and peptidases yields small
peptides and free amino acids, the latter of which can be further converted
to various alcohols, aldehydes, acids, esters and sulphur compounds for
specific flavour development (Tamime and Robinson 1999, Smit et al. 2005).
Lactose Metabolism
LAB need a sugar for energy production and subsequent growth. In dairy
products, the sugar is lactose—a disaccharide composed of glucose and
galactose. Transport of lactose into the cell is carried out using a translocation
system involving the phospoenol pyruvate phosphotransferase (PTS) in
lactococci, while S. thermophilus use a proton motive force (PMF) (Cogan
and Hill 1993). Lactose fermentation by LAB has been reviewed by Broome
et al. (2002), Cogan and Hill (1993), Cocaign-Bousquet et al. (1996), Poolman
(2002), and Tamime and Robinson 1999.
After transporting into the cell, lactose is fermented with one of the
four pathways as shown in Fig. 1. For example, in lactococci the tagarose
pathway is followed and lactose transport and the enzymes for the pathway
are plasmid encoded (Crow et al. 1983). Galactose is only metabolized by
L. helveticus and some strains of L. delbrueckii subsp. lactis (Gal+) and probably
leuconstoc via the Leloir pathway. Glucose-6-P is metabolized by the
glucolytic pathway in the lactobacilli and by the phosphoketolase pathway
in leuconstoc. L-lactate is generally the sole product of fermentation, but
when LAB are grown on galactose, maltose or low levels of glucose other
products are formed, form pyruvate metabolism (Fig. 2).
Citrate is present at a low concentration in milk and is metabolizes by
Leuconostoc spp. and some strains of Lc. lactis subsp. lactis (citrate-utilizing,
Cit+) to CO2 which is responsible for “eye” formation in some cheeses
(Parente and Cogan 2004). In addition, other important aroma compounds
are produced in fermented milks, cheese and butter (Fig. 3). Cit+ strains of
Lc. lactis subsp. lactis contain a plasmid which encodes the transport of
citrate. Citrate metabolism has been reviewed by Hugenholtz (1993).
The presence of a citrate permease is essential for the metabolism of
citrate. The citrate permeases of both Lc. lactis subsp. lactis and Leuconostoc
spp. were found to be pH dependant and their highest acidity was between
pH 5.0 and 6.0. The citrate inside the cell is converted to oxaloacetate, by the
enzyme citrate lyase, and then oxaloacetate is decarboxylated to pyruvate.
In lactococci, pyruvate is then converted to acetate, diacetyl, acetoin,
2, 3-butanediol and CO2. The enzyme pyruvate formate lyase is able to
convert pyruvate to formate, acetate, acetaldehyde and ethanol under
anaerobic conditions and at high pH (> 7.0). Under aerobic conditions
and at pH 5.5 to 6.5, pyruvate can be converted to acetate, acetaldehyde,
ethanol and the minor products acetoin, diacetyl and 2, 3-butanediol via the
multienzyme pyruvate dehydrogenase complex (Fig. 3). In Leuconostoc spp.,
the pyruvate produced from citrate is converted to lactate, although at low
pH and in the absence of glucose (or lactose) Leuconostoc spp. will produce
diacetyl and acetoin. Acetate is also formed via the heterofermentative
metabolism of lactose during co-metabolism with citrate (Broome et al.
2002).
Lactose
Lactose 6-P Lactose
Galactose 6-P Glucose Galactose Glucose

ATP ATP ATP
ADP
Galactose 1-P
ADP ADP
Tagarose 6-P Glucose 6-P Glucose 1-P Glucose 6-P

ATP NAD+
ADP NADH
Fructose 6-P 6-Phosphogluconate
Tagarose 1,6-biP ATP NAD+
ADP NADH
Fructose 1,6-biP Ribulose 5-P CO2
Xylulose 5-P
Pi
Glyceraldehyde 3-P
Pi NAD+ Acetyl-P
CoAsh
NADH
1,3-diPhosphoglycerate Pi
ADP Acetyl-CoA
NADH
ATP
3-Phosphoglycerate CoAsh NAD+
Acetaldehyde
NADH
2-Phosphoglycerate NAD+
Ethanol
Phosphoenolpyruvate
ADP
ATP
Pyruvate
NADH
NAD+
Lactate
Tagarose Glycolytic Leloir Phosphoketolase

pathway pathway pathway pathway
Figure 1. Lactose metabolism pathways in lactic acid bacteria (After: Cogan and Hill 1993,
Hutkins 2001, Tamime and Robinson 1999).
Over the last 15–20 years numerous attempts have been made to change
metabolite production in LAB, via metabolic engineering, from lactic acid
to production of other flavour compounds—usually by removing lactate
NAD+ NADH
Pyruvate Acetyl-CoA
NADH NADH ADP
C
á -Acetolactate
ATP
NAD+ NAD+
Diacetyl Acetoin Lactate Ethanol Acetate
Figure 2. Pyruvate metabolism in lactic acid bacteria (After: Cogan and Hill 1993, Hutkins
2001, Tamime and Robinson 1999).
Citrate
Acetate
Oxaloate
CO2
Pyruvate
TPP
α-Acetolactate
CO2 O2
Acetaldehyde-TPP
CO2
CO2
2,3-Butanediol Acetoin Diacetyl
NAD NADH + H NAD NADH + H
Figure 3. Citrate metabolism in lactic acid bacteria (After: Cogan and Hill 1993, Hutkins 2001,
Tamime and Robinson 1999).
dehydrogenase (LDH), the enzyme directly responsible for reduction of

pyruvate to lactate (Hugenholtz 2008, Hugenholtz et al. 2000). The relative
simplicity of Lc. lactis subsp. lactis sugar metabolism via the pyruvate
pathway together with the availability of the complete genome sequence
(Bolotin et al. 2001) makes this bacterium a top model for the study of
LAB. The metabolic re-routing of sugar metabolism has been extensively
reviewed (Hugenholtz and Kleerebezem 1999, Hugenholtz and Smid
2002, Kleerebezem and Hugenholtz 2003). Initial metabolic engineering of
Lc. lactis has focussed primarily on the rerouting of pyruvate metabolism.
Sugar metabolism was diverted towards the production of α-acetolactate,
the precursor of diacetyl, by either disruption of lactate dehydrogenase
(LDH) or by the nisin inducible expression system (NICE), through the
overproduction of NADH oxidase. By combining the latter strategy with

disruption of the gene encoding α-acetolactate decarboxylase, high diacetyl
production from glucose and lactose was achieved.
Protein Metabolism
LAB are fastidious microorganisms and are unable to synthesize many

amino acids, vitamins and nucleic acid bases (Parente and Cogan 2004).
Depending on the species and the strain, LAB require from 6 to 14 different
amino acids (Chopin 1993, Kunji et al. 1996). Since free amino acids in milk
are limited and amino acids are present as protein components, the growth
of LAB requires the hydrolysis of milk proteins.
The hydrolysis of peptides to free amino acids and the subsequent
utilization of these amino acids is a central metabolic activity in LAB
(Christensen et al. 1999), and proteolysis has been identified as the key
process influencing the rate of flavour and texture development in most
cheese varieties and have been reviewed (Souza et al. 2001, Upadhyay et al.
2004, Law 1999) and the catabolism of amino acids has been reviewed by
Curtin and McSweeney (2004). The degradation of milk proteins to peptides
is catalysed by proteolytic enzymes present in LAB (Christensen et al. 1999,
Thomas and Mills 1981), and peptides are then further hydrolysed by
exopeptidases and endopeptidases to small peptides and amino acids (Fox
and Wallace 1997). The main peptidases derived from lactic acid bacteria
used as dairy starter cultures are shown in Table 4.
There are several reports in the literature on the biochemical
characterization of peptidolytic and aminopeptidase activities from a
number of LAB (Atlan et al. 1990, Bockelmann et al. 1991, Bintsis et al.
2003, Booth et al. 1990, El Abboudi et al. 1992, Khalid and Marth 1990b).
However, since the genomes of many LAB have been sequenced, a thorough
comparative analysis of their proteolytic systems has been advanced at a
genome scale (Liu et al. 2010, Varmanen et al. 2000, Vesanto et al. 1995). It
has been found that the proteolytic activity of the LAB is chromosome linked
and the number of plasmids have been detected in most of the strains is
very limited. Thus, the gene encoding the cell surface endopeptidase from
L. delbrueckii subsp. bulgaricus has been sequenced and a comparison of
DNA sequences for the cell surface endopeptidases of L. delbrueckii subsp.
bulgaricus and lactococci showed little genetic homology (Gilbert et al.
1996). In addition, the LAB genomes in the L. acidophilus group encode a
relatively higher number and variety of proteolytic system components.
Some enzymes are only found in a few LAB strains, such as the cell-wall
bound proteinase (PrtP). PrtP was only found on the chromosome of
L. acidophilus, L. johnsonii, L. bulgaricus, L. casei, L. rhamnosus and
S. thermophilus strain LMD9, as well as on the plasmid of Lc. lactis
Table 4. Some peptidases derived from lactic acid bacteria used as dairy starter cultures .
Peptidase
Peptidase Substrate
Substrate hydrolyzed
Aminopeptidases
PepA Glu/Asp (X)n
PepC X (X)n
PepL Leu X
PepN X (X)n
PepP X Pro (X)n
PepX X Pro (X)n
Dipeptidases
PepV X X
PepD X X
Tripeptidase T (PepT) X X X
Proiminopeptidase (PepI) Pro X (X)n
Prolidase (PepQ) X Pro
Prolinase (PepR) Pro X
Endopeptidases
PepF (X)n X X X (X)n
PepO (X)n X X (X)n
PepE (X)n X X (X)n
PepG (X)n X X (X)n

After: Hutkins (2001), Christensen et al. (1999)
subsp. cremoris SK11 (Liu et al. 2010). Members of both the PepE/PepG
(endopeptidases) and PepI/PepR/PepL (proline peptidases) superfamilies
are absent in lactococci and streptococci. On the other hand, many of the
peptidases, such as aminopeptidases PepC, PepN, and PepM, and proline
peptidases PepX and PepQ are present in all LAB genomes, usually with
one gene per genome, and seem to be essential for bacterial growth or
survival (Liu et al. 2010).
The formation of amino acids from the proteolysis of cheese caseins
is very important for the development of flavour during the maturation
process. Free amino acids contribute directly to the cheese flavour (Seth
and Robinson 1988) and, additionally, they serve as substrates for other
flavour-generating compounds (Fox and Wallace 1997). In addition,
undesirable bitter peptides may lead to the formation of off-flavours, and
the ability of the micro-flora to hydrolyse bitter peptides is equally important
(Arora and Lee 1990). The conversion of free amino acids in more specific
flavour components is the next, and much more complicated, process in
overall flavour generation. The principal compounds formed are amines,
other amino acids, α-keto acids and sulphur compounds. In lactococci,
transamination in which the α-amino group of the amino acid is transferred
to a keto acid acceptor appears to be the first step in the degradation of
aromatic and branched chain amino acids. One of the key amino acids
in the formation of flavour compounds is methionine. This amino acid
is transaminated using α-ketoglutarate as the amino group acceptor to
form 4-methylthio-2-oxobutyrate which appears to be degraded either
nonenzymically or enzymically to form methanethiol. Methanethiol in
turn is the precursor to a number of potential flavour compounds such as
dimethyl sulphide, dimethyl disulphide and S-methylthioesters (Broome
et al. 2002).
Overproduction of specific amino acid-converting enzymes in starter
bacteria, such as cystathione-β-lyase involved in methionine- and cysteine-
metabolism has had little effect on the ripening in cheese (Fernandez et al.
2002), while increased production of α-ketoglutarate and overproduction
of aminotransferases gave a clear stimulation of the flavour formation by
LAB (Rijnen et al. 1999, 2000). The limited impact of metabolic engineering
in this area is mainly due to the lack of descriptive, and predictive, models
for overall amino acid metabolism in the LAB, which could guide the
different metabolic engineering strategies. Genomics techniques, however,
have been very productive in this area with the development of different
high throughput analyses for amino acid-converting enzymes and DNA
microarrays for analysis of the presence and expression of relevant genes
in different Lc. lactis strains (Hugenholtz 2008).
Lipid Metabolism
The enzymatic metabolism of milk fat is limited during the manufacture

of fermented dairy products. The degradation of milk fat releases free fatty
acids and glycerol, monoacylglycerides or diacylglycerides. However,
certain free fatty acids are essential flavor compounds in certain cheeses
(e.g., caprine milk cheeses). In addition, they react with alcohols or free
sulphydryl groups to form esters and thioesters, respectively, or act as
precursors of a number of other flavour compounds, such as lactones (Fox
and Wallace 1997). Esterase activity has been detected in various lactobacilli
(Khalid et al. 1990), and esters contribute to the characteristic flavor of Swiss-
type cheeses (Fox and Wallace 1997) and Feta cheese (Bintsis et al. 2003).
Exopolysaccharide Production
Extracellular polysaccharides (EPSs) are produced by a variety of
bacteria and are present as capsular polysaccharides (CPS and LPS)
bound to the cell surface, or are released into the growth medium
(Hassan 2008). These polymers can consist of a single type of sugar
(homopolysaccharides) or are regular, repeating units consisting of different
sugars (heteropolysaccharides). EPSs play a major role in the production of
yogurt, cheese, fermented cream and milk-based desserts (Jolly et al. 2002)
where they contribute to texture, mouth-feel, taste perception and stability
of the final products (Hassan 2008). Capsular EPSs form capsules around
the cell wall and are not secreted into the medium. Unattached EPSs are
secreted outside the cell wall of the producer and are responsible for the
“ropy” phenotype observed in fermented milks. The genetic machinery
responsible for EPSs production in lactococci is generally encoded on
large operons containing more than 10 genes. As the producing strains
are food-grade, the EPSs are also considered food-grade. In addition, it
has been suggested that these EPSs or fermented milks containing these
EPSs are active as prebiotics (Gibson and Robertfroid 1995), cholesterol-
lowering (Nakajima et al. 1992) and immunomodulants (Hosono et al. 1997).
EPS-producing strains of S. thermophilus and L. delbreuckii subsp. bulgaricus
have been shown to enhance the texture and viscosity of yogurt and to
reduce syneresis (Hassan et al. 2003, Doleyres et al. 2005). Broadbent et al.
(2003) extensively reviewed the topic of EPS production in S. thermophilus,
providing a detailed overview of its biochemistry, genetics and applications.
With regard to the genetic machinery encoding EPS production in
S. thermophilus, it has been suggested that a dozen or more unique EPSs
gene clusters may occur (Rallu et al. 2002). These clusters range in size from
15 to 20 kb and are chromosomally encoded.
Bacteriophage Resistance
Bacteriophages (phages) are viruses infecting bacteria and multiply in
the bacterial host cells resulting in cell lysis (i.e., virulent phages). The
word originates from the Greek word “φάγω”, which means “eat”, so
bacteriophages are the viruses that attack bacteria and they are the main
reason for slow or faulty fermentations during dairy-products making.
They cause no harm to human beings, animals or plants and cannot
cause any human diseases. With a size in the range of 0.1–0.2 µm, phage
are considerably smaller than bacteria. Therefore they can be transferred
through air and can thus be present in all production areas (Chr. Hansen
2002). The general morphology of a bacteriophage consists of a head and
protruding tail. The hexagonal head is a protein envelope. Inside this
envelope is the genetic information (DNA or RNA) of the phage. Under
the head, the collar and the tail are located through which the DNA is
injected into the bacterium. At the end of the tail, a baseplate and tail fibres
are located. They will read and recognise the host specific receptors on the
bacterial cell wall (Chr. Hansen 2002).
Over the years different methods have been proposed to classify
bacteriophages, but they were not accepted universally. However, a recent
approach to bacteriophage taxonomy, which is accepted universally, has
identified three groups known as bacteriophage families, namely the
Myoviridae, Podoviridae and Siphoviridae (Tamime et al. 1999). Based on
morphological distinctions, there are three main types of bacteriophages:
morphotypes A, B, and C, with the B or Siphoviridae group being the most
common type that infect lactic acid bacteria. The group B phages have long
tails with heads that have either a small isometric (B1 sub-group), prolate
(B2), or elongated (B3) shape. The lactococcal phages generally belong to
the B1 or B2 group, whereas the phages that infect S. thermophilus are all in
the B1 group (Hutkins 2001).
While total loss of the final product as a result of phage infection
is infrequent nowadays, phage infection continues to result in quality
defects that affect the flavour, texture and even safety of dairy products
and so continues to receive attention in both research and manufacturing
communities. Phage resistance mechanisms are probably best characterized
in lactococci. Indeed, within lactococci there are four main cellular defences
which interfere with different stages of the phage lytic cycle, namely,
adsorption inhibition (Ads), injection blocking, R⁄M and abortive infection
(Abi). The Ads phenotype prevents the attachment of the phage particle
to the cell surface and can often be induced through the generation of
bacteriophage insensitive mutants (BIMs) (Mills et al. 2007). In Lactococcus
spp., this has been attributed to nonspecific point mutations in chromosomal
genes encoding cell receptor sites by masking of receptors through,
for example, polysaccharide production (Forde and Fitzgerald 1999).
Potentially, one of the most exciting breakthroughs in phage resistance
research recently is the study of S. thermophilus BIMs. Interestingly, within
streptococci the CRISPR loci have recently been shown to play a role in BIM
formation. These loci consist of highly conserved repeats of approximately

21–48 base pairs, which are separated by variable sequences of constant and
similar length, called spacers of approximately 20–58 base pairs (Horvath et
al. 2008). It has been proposed that these spacers function as small interfering
RNAs resulting in termination of the phage lytic cycle. A recent comparative
study of the CRISPR loci in LAB genomes resulted in the identification of
multiple CRISPR families within Bifidobacterium and Lactobacillus as well
as Streptococcus, and similar CRISPR loci were found in distant organisms,
suggesting that these loci have evolved independently in select lineages,
partly due to selective pressure from phage predation (Horvath et al. 2009).
Exploitation of the CRISPR system offers many opportunities for strain
improvement such as strain typing, engineered defence against phage,
selective silencing of endogenous genes (Sorek et al. 2008), as well as
development of intelligent starter rotation strategies through exploitation
of the diversity introduced into an otherwise homogeneous population
(Mills et al. 2009).
Constant exposure to phage has resulted in the evolution of a diverse
range of defence mechanisms in lactococci, including a large range of
different Abi systems. This mechanism comes into play after injection of
phage DNA and includes a broad range of defences which can interfere with
genome replication, transcription, translation and packaging or assembly
of phage particles. This interruption of phage development leads to the
release of few or no phage and to the death of the infected cell, thus further
propagation of phage is prevented and the bacterial population survives
(Chopin et al. 2005). To date, of the 21 Abis’ identified in Lactococcus spp.
most are plasmid encoded and range from AbiA to AbiV. The phenotype is
most often conferred by a single gene, although there are a few exceptions
where the involvement of two genes has been proposed (AbiE, G, L and T).
Protein homology has rarely been observed between lactococcal Abis and no
homology with known proteins has been found, preventing any prediction
being made on their mode of action (Chopin et al. 2005).
Bacteriophages enter the dairy industry through raw milk, and thus, it
is a difficult task to eliminate entry of phages into the dairy plant, because
raw milk continually enters the facility. Phages are disseminated throughout
the dairy plant by aerosol and human carriers (Hassan and Frank 2001).
They multiply at incredible rates; in the time it takes for one “non-infected”
bacterium to produce four new bacteria by two generations of fission, one
bacteriophage has grown to a total of 22,500 phages (Tetrapak 1995). So
it is obvious that it is absolutely impossible not to have phages in a dairy
industry. Instead, in everyday life we have to learn how to coexit with them.
Currently, although the commercial starter cultures are phage
insensitive when launched in the market, it is usually possible to detect
bacteriophage after a period of time due to the rapid evolution of the
phage. During cheese production, problems with phage attack against

Lc. lactis spp. are most common, followed by problems with phage attacking
S. thermophilus. Phages attacking Lactobacillus spp. and Leuconostoc spp. in
starter cultures represent only a minor problem during dairy fermentations
(Høier et al. 2010). Practically, it is impossible to keep bacteriophages out
of industry production but there are a lot of preventive measures (i.e., use
of commercial phage insensitive starters and avoid use of bulk starters,
use of sterile air in the headspace of the incubation tanks, use of defined-
strain starter cultures with different phage-host spectra within a carefully
designed rotation scheme) that can be applied in order to control the phage
proliferation.
Bacteriocin Production
Bacteriocins are polypeptides synthesized ribosomally by bacteria and can
have a bacteriocidal or bacteriostatic effect on other bacteria (McAuliffe
et al. 2001, Ross et al. 2002). In general, bacteriocins lead to cell death by
inhibiting cell wall biosynthesis or by disrupting the membrane through
pore formation (Twomey et al. 2002). Bacteriocins are therefore important in
food fermentations where they can prevent food spoilage or the inhibition
of food pathogens. Although different classification schemes have been
proposed (Klaenhammer 1993, Nes et al. 1996, Kemperman et al. 2003), the
bacteriocins of LAB have been classified into two major classes. Bacteriocins
are classified into either lanthionine-containing bacteriocins (class I)
(lantibiotics) or the non-lanthionine-containing bacteriocins (class II). The
class II family is relatively diverse in terms of sequence variations as well as
in modes of action, and has therefore been divided further into subgroups
(Diep et al. 2009). Lantibiotics contain post-translationally modified amino
acids such as lanthionine, β-methyllanthionine and the dehydrated residues
dehydroalanine and dehydrobutyrine. The best known lantibiotic is nisin,
which has gained widespread application in the food industry and is used
as a food additive in at least 50 countries, particularly in processed cheese,
dairy products and canned foods (Delves-Broughton et al. 1996). Another
useful bacteriocin in terms of starter culture improvement is lacticin 3147,
encoded on the 60.2 kb plasmid, pMRC01, and this plasmid has been
transferred to over 30 different lactococcal hosts (Ryan et al. 1996, Coakley et
al. 1997). Lacticin 3147 has been shown to be effective in many food systems
for the control of food spoilage or pathogenic bacteria (McAuliffe et al.
1999, Ross et al. 1999, Morgan et al. 2001, O’Sullivan et al. 2006). Recently,
Streptococcus macedonicus ACA-DC 198, was found to produce a lantibiotic
named macedovicin, which inhibits a broad spectrum of lactic acid bacteria,
several food spoilage species (e.g., Clostridium spp.) and oral streptococci
(Georgalaki et al. 2013); Streptococcus macedonicus ACA-DC 198 has been
isolated from Greek Kasseri cheese and produce the lacticin 481 lantibiotic
macedocin (Georgalaki et al. 2002).
Bacteriocin-producing LAB have also been applied to improve the
flavour and quality of fermented foods through two strategies; by using
bacteriocin-producing LAB to control adventitious microbial populations
(Ryan et al. 1996) and secondly by using bacteriocin-producing LAB as cell
lysis-inducing agents to increase the rate of proteolysis in cheese (Garde
et al. 2002, Oumer et al. 2001). Many strategies have been developed for
the detection of antibacterial production by microorganisms including
phenotypic methods using indicator strains (Morgan et al. 2000) and liquid
chromatography/mass spectrometry techniques (Zendo et al. 2008). The
detection and identification of bacteriocins in producing strains has also
been greatly aided by PCR amplification of putative bacteriocin genes using
specific primers designed to known bacteriocins (Trmcic et al. 2008) and by
in silico searching amongst bacterial DNA sequences (Draper et al. 2009).
Safety Aspects of Genetic Engineering

A complete understanding of the metabolic potential of a LAB, established
through knowledge of gene function, pathway reconstruction and
prediction of phenotype through metabolic models, is a powerful tool
in developing metabolic engineering strategies (de Vos and Hugenholtz
2004). Both directed and uncontrolled (i.e., occurring during random
mutagenesis) genetic alterations result in a change of the genetic code
of the microorganism that may affect the transcription and translation
processes and, consequently, may influence metabolic processes in the
cell. The nature of the DNA modification could dictate the necessity for
proceeding to a safety assessment procedure. Interestingly, self-cloning
genetic modifications are considered as GMOs, whereas, uncontrolled
genetic modifications (e.g., mutations via IS) together with conjugation
and transduction are not.
Spontaneous mutations may occur in LAB by natural events such as
IS elements (de Visser et al. 2004), radiation, erroneous DNA replication or
transcription, and other factors. For example, in L. bulgaricus a spontaneous
IS element-mediated deletion of the lacZ gene altered lactose metabolism
resulting in limited fermentation capacity, and thus, manufacture of a high
quality (Mollet and Delley 1990). The level of such mutations depends upon
the growth conditions, and by the screening of natural isolates of LAB,
strains with improved fermentation characteristics can be selected (Sybesma
et al. 2006). The frequency of mutations can be further increased by exposing
LAB to mutagenic conditions such as UV light (Bintsis et al. 2000).
For controlled genetic modification, a variety of techniques have
been developed to generate genetic modified starters, such as cloning
systems, chromosome modification systems and expression systems (de

Vos 2001). The most popular transformation system for generating directed
genetic alterations in LAB is electroporation with self-replicating vectors.
Alternative systems are conjugation and transduction (Gasson 1990).
Targeted gene replacement or removal and inactivation of genes can
also be applied via (non-replicating) vectors using the natural event of
crossing-over during cell division and DNA replication (Leenhouts et
al. 1996). Compared to the use of replicating vectors in genetic modified
starters that result in new or enhanced cellular behaviour, the deletion of
genes after double cross-over events (by using a non replicating plasmid)
does not result in the addition of any DNA to the genetic content of the cell
(Sybesma et al. 2006).
A specific aspect related to the application of vectors in industrial strain
improvement is the use of selection markers. These markers should be
carefully selected and must be food-grade. For example, antibiotic resistance
markers cannot be used and strains carrying transferable antibiotic
resistance genes, such as enterococci should not be used as starters.
Certain targeted modifications of the genetic content of DNA may
occur via conjugation and transduction (Gasson 1990). These processes are
considered natural events, and thus, bacteria that are modified by using
these transfer systems are not considered as GMOs.
Starter cultures are substances used in foods and in the United States
are regulated according to the Food Drug and Cosmetic Act, in which the
status of Generally Recognized As Safe (GRAS) was introduced (Food and
Drug Administration 2012). Accordingly, a GRAS substance is generally
recognized, among qualified experts, as having been adequately shown
to be safe under the conditions of its intended use. Starter cultures are an
integral part of traditional fermented foods and, as a significant number
of people have consumed these foods for many centuries before 1958, the
fermenting microorganisms of these products can be said to be GRAS
(Bourdichon et al. 2012c).
In the European Union, starter cultures are considered ingredients
and must satisfy the legal requirements of food legislation. The regulation
EC 258/97 amended in regulation EC 1829/2003 lay down the framework
for use of genetically modified food and feed (EC 2003). According to
Sybesma et al. (2006), the legislation regarding the use of GMOs is not yet
completely clear on a number of important scientific matters. The legislation
predominantly focuses on the methodology rather than on the end product
and hence, holds too strongly to the definition of GMOs. Consequently,
organisms in which the genetic material has been altered by recombinant
DNA techniques in a way that does occur naturally, for instance by point
mutations or small deletions, are considered to be GMOs.
Since LAB are widely used as starter cultures for fermentation in the
dairy, meat and other food industries and have a long history of use by man
for food production and preservation, they are considered as food-grade
microorganisms.
The European Food Safety Authority (EFSA) has recently introduced the
“Qualified Presumption of Safety” (QPS) for a premarket safety assessment
of microorganisms used in food and feed production (European Food
Safety Authority 2006). QPS is applicable to food and feed additives, food
enzymes and plant protection products. The QPS system was proposed to
harmonize approaches to the safety assessment of microorganisms across
the various EFSA scientific panels. The QPS approach is meant to be a fast
track for species for which there is a sufficient body of knowledge that all
strains within a species are assumed to be safe.
In the QPS approach, safety assessment will depend upon the body of
knowledge available for a given microorganism. For some bacterial groups
(e.g., most lactobacilli, including those dominating natural cultures such
as L. helveticus and L. delbrueckii, or S. thermophilus) the identification at
genus/species level and the lack of acquired antibiotic resistance will be
sufficient to assure a safety status (European Food Safety Authority 2006),
while identification and characterization at strain level should be required
for some microbial groups, such as for example enterococci (European Food
Safety Authority 2010).
The QPS list covers only selected groups of microorganisms which
have been referred to EFSA for a formal assessment of safety (European
Food Safety Authority 2006, Leuschner et al. 2010). Seventy-nine species of
microorganisms have so far been submitted to EFSA for a safety assessment
(Pedersen et al. 2005); the list is updated annually (European Food Safety
Authority 2010). It should be noted that the absence of a particular organism
from the QPS list does not necessarily imply a risk associated with its use.
Individual strains may be safe, but this cannot be ascertained from the
existing knowledge of the taxonomic unit to which it belongs. Another
reason for a species not being on the list could be that EFSA has not been
asked to assess the safety of any strains of the species.
Specifications
Commercial starter cultures are supplied either in a frozen or freeze-dried
form and a food safety management system based on the principles of
HACCP is applied in the manufacturing procedure in order to assure the
stability and safety of the product (Tamime 2002). The required specifications
are presented in Table 5.
Table 5. Specifications for starter cultures.
Type of criterion Contaminanta Unit Liquid and Dry

frozen
Process hygiene Non-lactic acid bacteriab CFU/g < 500 < 500
Yeasts and moulds CFU/g <1 < 10
Enterobacteriaceae CFU/g <1 < 10
Coagulase-positive CFU/g <1 < 10
staphylococci
Food safety Salmonella spp. Absence in 1 g Absence Absence
Listeria monocytogenes Absence in 1 g Absence Absence
a
Contaminants can be tested in process environment and in process or products samples. The
set-up of environmental samples compared to process or product samples shall be based on
HACCP principles and justified against the specifications shown in the Table above.
b
This criterion is only relevant as a contaminant in cultures containing only lactic acid bacteria.
After: International Organization for Standardization and International Dairy Federation (2010)
The safety of starter cultures has been advanced through molecular

methods for their identification and taxonomy and modern methods are
used to assure safety during manufacture (Hansen 2002).
Labeling shall be in accordance with national legislation, where
applicable. The following items should appear on the starter culture product
label:
a) name of product,
b) type of product (e.g., mesophilic culture) or bacterial composition in
accordance with international scientific nomenclature,
c) type of products (e.g., freeze-dried, concentrated),
d) net contents which may be indicated in one of the following units:
grams, milliliters, units,
e) name and address of the manufacturer, packer, distributor, importer,
exporter or vendor,
f) country of manufacture (optional),
g) code and lot identification,
h) expiry date (month and year), and
i) storage conditions.
In addition, technical data should accompany the product, including:
a) application areas of use, b) instructions for use (inoculation rate,
incubation temperature, etc.), c) composition (types of bacteria, type of
culture, etc.), and d) certificate of analysis, certificate of compliance or
similar.
Conclusions
Starter cultures are a part of a rapidly developing field, and the results from
research in the last 15 years, since the discovery of the complete genome
sequence of Lc. lactis subsp. lactis IL1403 by Bolotin et al. (2001), have
led to the development of commercial starters with desirable properties.
Advances in the genetics, molecular biology, physiology, and biochemistry
of LAB have provided new insights and applications for these bacteria in
the dairy industry. On the other hand, the constant risk of bacteriophage
attack justifies the continuous need to search for new strains for improved
processes.
Dairy industry is now capable of producing safe and nutritious
products with different flavours, sometimes with special health-promoting
properties, which satisfy the demands of all consumer and market niches,
and resemble the characteristics of the traditional products. In addition, the
use of selected strains of given species with known metabolic properties
and high technological performances has improved the total quality
control of the manufacturing process. Interestingly, the potential intended
and unintended effects and related risks of using genetic modified starter
cultures can be predicted in an accurate way and verification is feasible.
This combined with profound safety assessments, such as QPS, ensures
safety for consumers and safety for the environment.
These developments could lead to novel functional foods, produced
from processes with well-characterized metabolic pathways, illustrating
new possibilities for the application of these food-grade microorganisms.
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CHAPTER 5
Application of Probiotics in
the Dairy Industry: The Long
Way from Traditional to Novel
Functional Foods
Adele Costabile1,* and Simone Maccaferri2
INTRODUCTION
Dairy products have always been very important functional foods, and
they currently represent an important sector in the “health and wellbeing”
industry.
Dairy foods are generally divided in three categories (Saxelin 2008):
• Basic products (milk, fermented milks, cheeses, ice creams, etc.).
• Value-added products, in which the milk composition has been
modified for enhancing some of the basic properties ascribable to the
food. Examples of value-added products are low-lactose or lactose-free
dairy products, sodium-reduced or calcium-enriched milk products,
among others.
• Functional dairy products with proven health benefit. These kinds
of products are based on the enrichment of the dairy food with a
functional component. Most commonly, functional dairy foods are
1
Research Fellow, Department of Food and Nutritional Sciences, The University of Reading,
PO Box 226, Whiteknights, Reading RG6 6AP.
2
Department of Pharmacy and Biotechnology, University of Bologna Via Belmeloro 6, 40126
Bologna, Italy.
enriched with probiotics, but prebiotics-enriched dairy products also

fall in this category. According to the most commonly acknowledged
definition, probiotic foods are food products which contain one, or
a combination of probiotic ingredients in a adequate matrix and in
sufficient concentration, so that after their ingestion, the postulated
effect is obtained and is beyond the usual nutrient suppliers (de Vrese
et al. 2001).
In this chapter, we will explore the different applications of functional
dairy products based on the utilization of probiotics, with discussion on
the advancement of the research in this field, the main typologies of dairy
probiotic foods and their related technological characteristics, health claims,
and requirements to be met for commercialisation .
Notably, the dairy sector is the largest functional food market,
accounting for nearly 33% of the broad market (Granato et al. 2010). To
date, different functional dairy products containing probiotics have been
developed, evaluated and commercialised these include: yogurts and
fermented milks, cheeses, yog-ice creams, cheese-based dips, probiotic
fermented lactic beverages, and dairy desserts (Di Criscio et al. 2010).
Dairy Products
Yogurt and Fermented Milks
Yogurt and fermented milks are considered to be the main carrier for the
delivery of probiotics in the dairy industry. In recent years, since many
consumers’ associate yogurt—especially probiotics yogurt, with good
health, per capita consumption of these dairy products has increased
drastically (Hekmat and Reid 2006).
Traditionally, yogurts are prepared through a fermentation procedure
of milk by specific pure cultures of lactic acid bacteria, such as Streptococcus
thermophilus and Lactobacillus bulgaricus. However, even if the simple and
standardized procedure at the basis of the yogurt production is widely
established and commonly utilized, many factors influence the probiotics
viability and its efficacy in promoting human health. Indeed, probiotic
cell survival during the product’s shelf life is a striking factor influenced
by culture conditions, strain selection, level of inoculation, medium
composition, the interactions among starter and probiotics species, final
acidity, availability of nutrients and sugars, and storage and logistics
conditions along the distribution chain (Talwalkar and Kailaspathy 2004,
Dave and Shah 1997, Donkor et al. 2006, Vinderola et al. 2002, Ranadheera
et al. 2010, Plessas et al. 2012).
Dairy Probiotics and Novel Functional Foods 157
For instance, while studying the survival of a Bifidobacterium strain in

a yogurt matrix, it has been demonstrated that the concentration of milk
fat is negatively correlated with the viability of probiotic cultures, thus
resulting in higher fat concentration leading to sensible inhibitory effects
(Vinderola et al. 2000). Similarly, a correlation has been shown between
post storage pH in yogurts, presence of specific fruit pulp and the viability
of probiotics. Mixed berry and passion fruits led to lower levels of viable
probiotics lactobacilli in the tested yogurts with respect to plain-yogurt,
whilst mango and strawberry had an enhancing activity. Therefore, taking
into account the fruit mixtures as a paradigm of a single, simple exogenous
factor influencing the probiotics viability in the food matrix, the importance
of carefully studying the single environmental contributions which
might reduce the viability of probiotic cultures, is easily understandable
(Kailasapathy et al. 2008).
Probiotic Cheeses
Cheeses are food products characterized by an intrinsic versatility in

relation to the incorporation of probiotic bacteria, and their suitability for
the delivery of probiotics in all age groups, from children to the elderly.
Indeed, the consumption of cheese has increased in many countries during
the past decade (da Cruz et al. 2009) and the design of novel probiotics
foods should take this kind of product into account.
In fact, cheeses are considered much more advantageous for delivering
viable probiotics rather than products of fermentation, as yogurt or fermented
milk beverages. In particular, cheeses are commonly characterized by an
higher pH, a higher buffering capacity, solid consistency and an higher fat
content, thus resulting in greater protection to the probiotic cells during
storage and passage through the upper gastrointestinal tract (Plessas et al.
2012). The higher pH is reflected in a more favourable environment for the
viability of probiotic bacterial cells, as well as the fact that the higher fat
content exerts a protective function against the peptic enzymes.
Similar to other dairy products, the probiotic bacteria included in cheese
products mainly belong to the Lactobacillus and Bifidobacterium genera.
Furthermore, some probiotics strains of Enterococcus and Propionibacterium
have been characterized for their utilization in cheese manufacturing
(Stanton et al. 1998).
To date, several studies are aimed at developing different types of
probiotic cheeses, using cheeses such as Cheddar, Crescenza cheese, cottage
cheese and fresh cheese (as reported in Table 1, modified from Plessas et
al. 2012).
Table 1. Probiotics in several cheese types.
Cheese Type Included Probiotics

Cheddar L. salivarius, L. paracasei, L. acidophilus, L. casei, B. lactis,
B. longum, B. infantis
Gouda L. acidophilus, Bifidobacterium spp.
Fresh cheese L. acidophilus, L. casei, B. longum, B. bifidum
Feta cheese L. casei
Crescenza cheese B. bifidum, B. longum
Probiotic Ice-creams
Ice cream is a broad category including several related products, such as

plain ice-cream, reduced- or low-fat ice-cream, puddings, variegated ice
cream, mousse, frozen yogurt and sorbet (Cruz et al. 2009). These products
have demonstrated a great potential as vehicles for probiotic cells, both
for the socio-economics reasons underpinning the easy distribution of
ice creams along the entire life span and for scientific and technological
reasons. In fact, the ice-cream matrix, which is composed by milk proteins,
fat and lactose, among others ingredients, represents a good probiotic
micro-environment. Furthermore, ice cream is considered a suitable and
supportive environment for acting as a probiotic carrier because of its lower
storage temperature, and the strict maintenance of the cold chain and a
minor risk of temperature abuse during frozen storage (Cruz et al. 2009).
All of these factors lead to a higher viability in the high concentration of
probiotics at the time of consumption.
However, since frozen storage temperature significantly affects
different characteristics of lactic acid bacteria, as acid development and
proteinase activity, a robust analysis aimed at assessing the efficacy of the
supplemented probiotics in the small intestine must be undertaken. Indeed,
it has been recently demonstrated that same probiotic strains incorporated
in frozen food products exert better viability and activity during shelf-life,
in comparison to non frozen foods (Heenan et al. 2004). At the same time,
the damage to probiotic cells caused by freezing and thawing, as well as
mechanical stresses that accompany manufacturing must be taken into
account on a case by case basis, in order to avoid lower viability and negative
functional effects on the consumer (Ranadheera et al. 2010).
While the incorporation of probiotics in cheeses has a longer history, the
development of ice cream under the concept of functional food is a relatively
new idea. To date, a limited number of studies have been conducted, and
most of the research has been performed in order to bypass the technological
hurdles for the incorporation of probiotic bacteria into the ice-cream, in order
to lead to satisfactory products, both in terms of palatability and sensorial

characteristics, as well as in term of functionality.
Different Lactobacillus spp. (L. johnsonii La1, L. rhamnosus GC) have
been evaluated for their ability to easily survive as a probiotic supplement,
without influencing the physical properties of the ice-creams (Alamprese et
al. 2002, Alamprese et al. 2005). The utilization of well-evaluated probiotic
strains, as well as the employment of the most appropriate technological
methodologies, has been demonstrated to lead to the production of probiotic
ice-creams with a good sensory acceptance (Vardar and Oksuz 2007).
Micro-biotechnology Aids the Development of Better Probiotic

Dairy Products
A dairy probiotic product must include a suitable level of probiotic cells
in order to exert a health-promoting activity (usually 106–107 CFU/g). At
the same time, the included probiotics must not produce an unfavourable
flavour, in order to gain a wide sensory acceptance by consumers. For
example, probiotic bifidobacteria are known to be extensive acetate and
lactate producers, which are potentially beneficial short-chain fatty acids
but, however they are characterized by a very pungent off-flavour, which
might require the concomitant utilization of flavouring agents, as well as
additional synthetic molecules or natural products.
The evolution of knowledge in the field of nano- and micro-
biotechnology in the food sciences are tremendously impacting the
possibilities to promote a greater viability and to aid in avoiding the
utilization of flavouring agents. One of the most effective strategies is
represented by the addition of microencapsulated cells of probiotic cultures
to dairy food products. Nowadays, different food products containing
microencapsulated probiotics are marketed in the EU and US market.
Mainly, they are dairy products, such as ice cream, fermented milk or
chocolate snacks (Burgain et al. 2011).
Microencapsulation is the envelopment of small solid particles,
including bacterial cells, in a coating. In the recent years, the incorporation of
natural ingredients, polyphenols, volatile additives, and bacteria in a small
capsule, in order to create a preserved and stable environment, protected
from any potential exogenous factor influencing the health effects of the
encapsulated agent.
Whilst allowing the protection of these bacteria during their transit in
the upper gastrointestinal tract, to entrap probiotic microorganisms in a
microcapsule is further useful in order to prevent interfacial inactivation,
stimulation of production and excretion of secondary metabolites and
their continuous utilization. Microparticles used in the dairy industry are
water-insoluble, in order to maintain their structural integrity and are thus

produced in order to allow progressive liberation of the probiotic cells
during their presence in the large intestine (Ding and Shah 2007, Nazzaro
et al. 2012). The most commonly used polymers used in the food industry
to produce probiotic microcapsules—characterized by being GRAS,
biocompatible and deriving by natural products—are chitosan, alginate,
carrageenan, whey protein, pectin, poly-L-lysine and starch (Nazzaro et
al. 2012).
Evaluation of the Probiotics to be Included in Dairy Products:

Safety and Functional Assessments
The exertion of widely acknowledged probiotics characteristics is pivotal
for promoting the health characteristics ascribable to the plethora of dairy
products to date which have been commercialised for restoring a number
of conditions described previously in this chapter.
In order to be beneficial to human health, probiotics must fulfil several
criteria. The joint Food and Agriculture Organisation of the United Nations/
World Health Organisation Expert Consultation established guidelines
for the Evaluation of Health and Nutritional Properties of Probiotics in
Food (Food and Agriculture Organisation of the United Nations/World
Health Organisation 2001), with the aim of identifying and establishing
the minimum requirements needed for the definition of probiotics (Fig. 1).
STRAIN IDENTIFICATION
Molecular taxonomic identification
using 16S rRNA
SAFETY ASSESMENT
Pre-requisite in order to in depth study
the activity and the efficacy of the
probiotics
FUNCTIONAL EVALUATION
In vitro and animal models for
assessing main probiotic traits
EFFICACY EVALUATION
In vivo human study for substantiating
health effect
Figure 1. Scheme of the guidelines for the Evaluation of Health and Nutritional Properties of
Probiotics in Food (Food and Agriculture Organisation of the United Nations/World Health
Organisation 2001) (After: Collado et al. 2009).
Strain Identification
A probiotic products correct identification must be fully qualified in

relation to the incorporated bacterial species. Molecular taxonomy tools,
based on the 16S rRNA sequencing, must be used in order to ensure the
most reliable and univocal identification of the probiotic strain, which
will be futher characterized for its safety and functional activities. Indeed,
strain identification is of crucial importance in order to link a probiotic
strain to a specific health effect (Gueimonde and Salminen 2006, Collado
et al. 2009). Whilst the importance of correct taxonomic identification is an
established concept, described and taken into account by European QPS
(Qualified Presumption of Safety) strategy, the source of the probiotic strain
has lately been debated. Indeed, even if one of the main recommendations
is that probiotic strains have to be autochthonous of the ecosystem where
they will be a part once ingested (Kõll et al. 2010), the probiotic potential
of bacteria from other ecosystems would not be a priori to be excluded. For
this reason, the number of studies on fermented foods (e.g., sourdoughs,
cheeses, yoghurt and pickled vegetables) as alternative sources of novel
probiotic candidates is increasing (Cammarota et al. 2009, Vitali et al. 2012).
Safety Assessment
This is considered an essential phase in the selection and evaluation of

probiotics for their utilization in the dairy industry, since probiotics strains
must be safe for human consumption. Commonly utilized lactobacilli and
bifidobacteria have a long history of safe use, and they are categorized as
“generally recognized as safe” (GRAS). However, for every bacterial strain
in use, some evaluation procedures must be undertaken. In particular,
the probiotic strains considered as candidates for use need a series of
in vitro analysis to verify the absence of beta-hemolytic activity and other
harmful enzymatic activities, such as beta-glucosidase, N-acetyl-beta-
glucosaminidase and beta-glucuronidase. Finally, since the importance of
the upsurge of antibiotic resistance bacteria, from a medical perspective,
it is mandatory to include the susceptibility to the main antibiotics as a
requisite for the safety of a probiotics strain before it is commercialised
(Gueimonde and Salminen 2006).
Functional Activity
Tolerance to gastrointestinal conditions
Maintaining proper viability along the entire gastrointestinal tract is

important to ensure the optimal functionality of probiotics. After ingestion,
probiotic bacteria must be able to overcome the acidic environment of the

stomach and not be altered by the bile secretion in the duodenum. For this
reason, in vitro digestion methods have been employed to evaluate the
ability of probiotic strains to survive the passage through the gastrointestinal
tract. These experimental approaches are mainly based on screening for acid
pH tolerance and bile effects (Gueimonde and Salminen 2006).
Adhesion
The adhesion to the intestinal mucosa is often regarded as a prerequisite for

the colonisation of the gut lumen, and is an important probiotic trait related
to the ability of the strain to cross-talk with the immune system exerting
a beneficial immune modulation. Different in vitro cell models have been
developed for evaluating the adhesion of probiotic strains, mainly based
on the utilization of HT-29 and Caco2 cells. Adhesion is one of the most
strain-dependent physiological characteristics, since the adhesion levels of
the probiotic strains show a great variability amongst genus and species
(Candela et al. 2008, Collado et al. 2005, Maccaferri et al. 2012).
Antimicrobial substances
The capability of probiotic bacteria to produce antimicrobial metabolites

is of immense interest for enhancing the ecological characteristics of the
environment in which the probiotic is meant to exert its activity. Two groups
of antimicrobial or bacteriostatic substances have been described so far:
i) low molecular mass compounds, as organic acids, which are characterized
by a broad spectrum of action; ii) antimicrobial proteins, as bacteriocins,
which have an higher molecular mass and which have a relatively higher
specificity against groups of microorganisms (Collado et al. 2009, Chen and
Hoover 2003). The utilization of probiotics- or LAB-produced bacteriocins
is of particular interest for the food and dairy industry. In fact, bacteriocins
may be considered natural preservatives used to antagonize the growth of
undesired microorganisms in foods to enhance food safety and to extend the
product shelf life (Chen and Hoover 2003, Schillinger and Holzapfel 1996).
Immune modulation
Probiotics can interact with mucosa-associated lymphoid tissues and bind

to epithelial surface receptors, inducing humoral and cellular immune
responses. The establishment and maintenance of a well-balanced ratio
between pro- and anti-inflammatory cytokines are crucial for human
health. Therefore, study of the dynamic cytokine modulation elicited by
a microorganism is a hot topic in the selection of novel probiotic strains.

A wide strain-specific variation in the immune responses stimulated by
probiotics has been described, and several in vitro cell models have been
developed to evaluate their immunomodulatory effects (Delcenserie et
al. 2008). Even if these cellular models lack the complexity of the human
immune system, they aid in clarifying the mechanisms involved in different
means of bacterial sensing by human colonocytes and immunocompetent
cells (Boirivant and Strober 2007). Several in vitro and in vivo studies
demonstrated the two main effects of probiotics on host immunity:
(i) strengthening of the immunological barrier by stimulating the
development and maintaining the state of alert of the innate and adaptive
immune system, and (ii) decreasing of the immune responsiveness to
unbalanced inflammatory conditions. Both of these health-promoting
activities are accomplished through an effective modulation of the balance
of pro- and anti-inflammatory cytokine production (Vanderpool et al.
2008). Many probiotic species have been demonstrated to share a relatively
common immune pattern, such as a reduction in Th2 cytokines (i.e., IL-4,
IL-5, IL-6, IL-10, and IL-13) or a shift toward Th1-mediated immunity (i.e.,
IL-2, TNF-α, and IFN-γ production).
The Health-Promoting Probiotic Activities: Facts, Trends and

Scientific Substantiation
Increasing evidence is supporting the importance of the role of diet and
nutritional status among the most important modifiable determinants of
human health, through a plethora of presumptive mechanisms among which
intestinal microbiota-mediated processes are thought to be essential.
The concept of functional foods, which provide additional health
benefits and may reduce the risk of disease and/or promote general
well-being, is steadily growing (Granato et al. 2010). Among the several
functional foods marketed worldwide, probiotics represent the most
widely established and studied. Therefore, it is not surprising that the dairy
sector, which is strongly linked to probiotics, holds the largest share in the
functional food market, accounting for nearly one third of the market. It
has been reported that the consumer market for probiotics foods is > 1.4
billion Euros in Western Europe (Saxelin 2008).
The majority of probiotic products currently marketed contain species
of Lactobacillus and Bifidobacterium, which are the main bacterial genera
characterized as probiotics (Wassenaar and Klein 2008), accordingly with
the definition of Food and Agriculture Organisation of the United Nations/
World Health Organisation . Probiotics are defined as “live microorganisms
which when administered in adequate amounts confer a health benefit on
the host” (Food and Agriculture Organisation of the United Nations/World

Health Organisation 2001). To be considered as probiotics, microorganisms
should fulfill the following criteria: i) being non-pathogenic and non-toxic;
ii) being able to survive through the GIT; iii) being stable during the intended
product shelf life and contain an adequate number of viable cells to confer
health benefit to the host.
Besides the most established bifidobacteria and lactobacilli, other
lactic acid bacteria (LAB) with probiotic properties are Enterococcus faecalis,
E. faecium, Sporolactobacillus inulinus. Similarly, non-LAB probiotics or
nonlactic microorganisms with putative probiotic traits have been described
in literature, as Propionibacterium freudenrichii, Saccharomyces cerevisiae,
S. boulardii, Kluyveromyces marxianus, Lactococcus lactis and Lecuonostoc
mesenteroides. Table 2 reports the most commonly studied probiotic strains
in humans in vivo studies for the characterization of their health-promoting
activities (Boyle and Tang 2006, Senok et al. 2005, Shah 2007).
Conversely, L. delbrueckii spp. bulgaricus and S. thermophilus are found
in a number of preparations with presumptive health-promoting activities,
such as traditional yogurt, but since these microorganisms are not expected
to survive and grow in the host’s intestinal tract, they are not commonly
classified as probiotics (Senok et al. 2005).
To date, probiotics microorganisms are mainly incorporated into
dairy products, such as cheese, yoghurt, ice cream and dairy desserts,
in order to be included as part of the normal Western diet. Generally,
high concentrations of viable microorganisms (about 107–10 9 CFU/g)
are included into alimentary probiotics formulation and are required
in order to provoke the desired health-promoting effect (Ranadheera
et al. 2010).
Probiotics have been demonstrated to exert health promoting effects
through several proposed mechanisms (Fig. 2), which rely on microbe-gut
epithelium, microbe-immune system and microbe-microbe interactions.
Table 2. Most commonly used probiotic species.
Lactobacillus spp. Bifidobacterium spp. Other bacterial genera Probiotic yeasts

L. acidophilus B. bifidum E. coli Nissle S. cerevisiae
L. casei B. breve E. francium S. boulardii
L. crispatus B. infantis E. faecium K. marxianus
L. fermentum B. lactis E. faecalis
L. gasseri B. longum P. freudenrichii
L. johnsonii B. adolescentis L. lactis
L. paracasei B. essensis L. mesenteroides
L. plantarum P. acidilactici
L. reuteri
L. rhamnosus
L. helveticus
SCFAs production
Enhancement of
barrier function
Improved lactose
tolerance and lowering Suppression of
of cholesterol levels pathogenic bacteria
infection
Probiotics effects on the

Immune human health Increased
modulation mucin
production
Stabilization of
Improvement of gut microbiota Production of
mineral absorption and composition antimicrobials/
production vitamins/ bacteriocins
micronutrients
Figure 2. Effects of probiotics on the human health. Microbe-microbe interactions are

represented in light blue, microbe-host interactions are represented in orange; interactions
leading to effects directed either to host and microbes are represented in pink.
These mechanisms include: i) SCFAs production and enhancement of the

barrier function of the intestinal epithelium; ii) suppression of growth
and binding of pathogenic bacteria; iii) increased mucin production; iv)
induction of antimicrobial and heat-shock protein production; v) alteration
of the immune activity of the host through modulation of host signaling
pathways; vi) improvement in absorption of minerals and production of
vitamins/micronutrients; vii) reduction of cholesterol and improvement
of lactose tolerance (Aragon et al. 2010, De Vrese et al. 2001, Ventura et
al. 2009, Kumar et al. 2011, Thomas and Versalovic 2010). Furthermore,
probiotics can alter colonic fermentation and stabilize the symbiotic
microbiota (Spiller 2008), improving the dynamic interplay between the
resident bacterial community and the host.
Application of Probiotics to Promote Human Health
Many studies indicate probiotics as promising in the treatment of irritable

bowel syndrome, allergies and maintenance of remission in inflammatory
bowel diseases (Floch et al. 2011, Preidis and Versalovic 2009).
Probiotics in Irritable Bowel Syndrome
Irritable bowel syndrome (IBS) is a very common functional gastrointestinal

disorder defined by the coexistence of abdominal discomfort or pain
associated with alterations in bowel habits. Nowadays, the estimated
prevalence of IBS in industrialised countries is of 10–15% and, despite its
prevalence and impact on quality of life, few therapies have been found to
be effective for treating IBS. Different studies indicated that the aetiology of
IBS is most likely multifactorial, due to abnormalities in intestinal motility,
visceral hypersensitivity, altered brain-gut interaction, food intolerance,
unbalanced gut microbiota composition, and persistence of chronic low-
grade inflammatory conditions (Brenner et al. 2009).
Due to the effects in modulating the immune function, motility,
secretion and gut sensation, probiotics have been suggested to have the
potential to exert a beneficial role in managing IBS symptoms (Camilleri
2008). Furthermore, the utilization of novel “omics” approaches, as
metabolomics, suggested that IBS patients could be characterized by a
potential dysregulation in energy homoestasis and liver function, resulting
in unbalanced levels of serum glucose and tyrosine, which may be improved
through probiotics supplementation (Hong et al. 2011).
Recently, Clarke et al. 2012 performed an extensive review of relevant
literature describing the clinical trials for the assessment of the efficacy of
probiotics based containing LAB in the management of IBS symptoms; 42
clinical trials have to date been reported in literature. In general, clinical trials
involving LAB have reported improvement in abdominal pain, discomfort,
abdominal bloating and distension as their main endpoints. Furthermore,
beside these more robust endpoints, benefits over placebo have been also
indicated in several clinical trials using global quality of life indices.
The most convincing evidence supporting the beneficial role of
bifidobacteria in IBS management came from trials involving B. bifidum
3562, which have been demonstrated as effective in reducing abdominal
pain/discomfort and bloating, as well as an improved composite severity
score both in two trials (O’Mahony et al. 2005, Whorwell et al. 2006).
B. bifidum 3562 has recently been demonstrated to promote immunoregulatory
responses (Koniecza et al. 2012) and to induce an increase in plasma levels
of tryptophan, a precursor of serotonin which is known to play a role as
neurotransmitter in the brain-gut axis (Desbonnet et al. 2008). Another
Bifidobacterium strain, B. bifidum MIMb75 have been demonstrated to
significantly reduce pain/discomfort, bloating and global IBS symptoms
(Guglielmetti et al. 2011).
While few studies investigated bifidobacteria, a larger number of
clinical trials aimed at evaluating the efficacy of Lactobacillus strains in
IBS. To date, L. acidophilus SDC 2012, 2013 (Sinn et al. 2008), L. paracasei
B2106 (Andriulli et al. 2008) and L. plantarum 299V (Niedzieilin et al.

2001) have been demonstrated to reduce abdominal pain/discomfort and
L. plantarum 299V was further demonstrated to improve the overall IBS
symptoms (Niedzieilin et al. 2001). A broad number of studies investigated
the mechanistic insight at the basis of the probiotic activity of Lactobacillus
strains. Notably, it has been demonstrated that L. acidophilus can produce
visceral analgesic effects by up-regulating the expression of opioid and
cannabinoid receptors in colonic epithelial cell line and in vivo in murine
models (Rousseaux et al. 2007). Lactobacillus paracasei was shown to attenuate
gut muscle hypercontractility in animal models of post-infectious IBS (Verdu
et al. 2008). Furthermore, certain Lactobacillus species and strains affected
epithelial integrity of colonic epithelial cell monolayers as measured by
trans-epithelial electrical resistance (Parassol et al. 2005).
In addition to monostrain probiotic formulations, multispecies
probiotics have been evaluated for their efficacy in ameliorating from IBS
symptoms. Indeed, it has been recently indicated that probiotic mixtures
appear to show greater efficacy than single strains, including strains that are
components of the mixtures themselves (Chapman et al. 2011). However, it
is still unclear whether this difference is due to the synergistic interactions
between strains.
One of the best characterized multispecies probiotics is VSL#3, a mixture
containing strains of three lyophilised species, Bifidobacterium, Lactobacillus
and Streptococcus, which was found to be effective at alleviating abdominal
pain and reducing bloating in adults and children suffering from IBS, as
well as in improving their quality of life (Guandalini et al. 2010, Kim et al.
2003, 2005, Michail and Kenche 2011). Kajander et al. (2005) demonstrated
that a probiotic mixture containing L. rhamnosus GG, L. rhamnosus LC705,
B. breve 99 and P. freudenreichii ssp. shermani JS was effective in improving a
composite IBS symptom score, which included abdominal pain, distension
and flatulence.
Dairy probiotics other than bacterial strains have also been suggested
to be useful in IBS management, even if they are not yet widely in use. S.
boulardii, a species of yeast which has been described as a biotherapeutic
agent able to decrease the expression of inflammation-associated cytokine
IL-8, IL-6, IL-1b, TNF-α and IFN-γ (Zanello et al. 2009), improved the
quality of life of IBS patients better than a placebo but was not a superior
substitute for individual IBS (Choi et al. 2011). Similarly, K. marxianus B0399,
which has very recently been demonstrated to be a probiotic strain with
immune-modulatory activity and able to impact on the composition and
functional activity of the human gut microbiota (Maccaferri et al. 2012),
in combination with B. animalis subsp. lactis Bb12 was demonstrated to be
effective in reducing abdominal pain and bowel movements abnormalities
in IBS patients (Lisotti et al. 2011).
Probiotics in Inflammatory Bowel Disease
Inflammatory bowel disease (IBD) is a complex and heterogeneous group

of pathological conditions that involves an interaction between genetic,
immunologic and environmental factors. An altered composition of the
gut microbiota, defective clearance of bacteria and enhanced mucosal
uptake, resulting in increased immune stimulation are characterizing
of IBD (Packey and Sartor 2009, Ewaschuk et al. 2006). From a clinical
perspective, IBD includes ulcerative colitis (UC) and Crohn’s disease (CD).
Whilst corticosteroids, mesalazine and antibiotics are the most commonly
used therapeutic approaches to treat IBD, different probiotic trials are
substantiating the potential beneficial role of probiotics in UC, CD and
pouchitis. Notably, a broad array of studies have been performed in order
to indicate the role of probiotics in alleviating particular symptoms of each
of the three pathological conditions (Haller 2010, Ewaschuk et al. 2006).
Probiotic microorganisms used in clinical trials aimed at inducing or
maintaining remission of CD included S. boulardii (Guslandi et al. 2000),
L. johnsonii LA1 (Marteau et al. 2006, Van Gossum et al. 2007) and
L. rhamnosus GG (Prantera et al. 2002). Guslandi et al. (2000) demonstrated
that S. boulardii was more effective than mesalazine in maintaining clinical
remission of CD. Successively, Vilela et al. (2008) demonstrated that
S. boulardii was effective acting through the improvement of the CD-like
abnormal intestinal barrier function. McCarthy et al. (2001) demonstrated
that oral administration of L. salivarius UCC118 was decreasing disease
activity in a cohort of patients suffering from mild to moderately active
CD. Conversely, clinical trials studying the efficacy of L. rhamnosus GG and
L. johnsonii LA1 did not find differences among probiotics treatment and
placebo in maintaining clinical or endoscopic remission of CD.
Unlike the paucity of data and human studies in CD, a larger number
of trials reported the effects of probiotics treatments in UC. In particular,
two large studies demonstrated that E. coli Nissle 1917 was equivalent to
mesalazine in maintaining remission in patients with UC (Rembacken et
al. 1999, Kruis et al. 2004). The potential clinical relevance of E. coli Nissle
1917 was first demonstrated in several animal models in which probiotics
resulted in a significant reduction in the secretion of pro-inflammatory
cytokines and other markers of intestinal inflammation as well as improved
histological findings (Kamada et al. 2005). Furthermore, E. coli Nissle 1917
was demonstrated to strengthen the mucosal barrier through the induction
of epithelial B-defensin 2, a human antimicrobial peptide, and zonula
occudens-2, a prominent member of the tight junctions, and to induce a
potent anti-inflammatory response (Schlee et al. 2007, Zyrek et al. 2007,
Helwig et al. 2006).
The use of LAB as unique probiotic strains did not show relevant results
in the management of UC. Zocco et al. (2006) did not show any efficacy of
probiotic L. rhamnosus GG in maintaining UC remission, whereas Fujimori
et al. (2009) demonstrated that the utilization of B. longum was effective
in improving general IBS symptoms only when administered within a
synbiotic formulation.
In addition to a single strain probiotics formulation, multispecies
probiotics were investigated and tested in clinical trials. Sood et al. (2009)
demonstrated that VSL#3 probiotic mixture was effective in inducing UC
remission in adults suffering from mild to moderate UC, whereas Tursi
et al. (2010) demonstrated a significant clinical response in the group of
UC patients subjected to probiotics therapy. Recently, Miele et al. (2009)
further demonstrated that VSL#3 was useful in the maintenance of UC
remission. Notably, the clinically active probiotic combination VSL#3 has
been demonstrated to exert a potent induction of IL-10 by intestinal and
blood dendritic cells and inhibited generation of pro-inflammatory Th1
cells (Hart et al. 2004).
Probiotics in Allergies and Atopic Dermatitis
In the last decades, a rapid rise in the prevalence of allergic and autoimmune
disorders has been observed (Bach 2002). In particular, atopic dermatitis
(AD) is the most common chronic inflammatory skin disease in infancy.
Allergic diseases are generally associated with an imbalance in the TH1/TH2
cytokine response and with stimulation of IgE and IgA synthesis (Winkler
et al. 2007). Since different probiotic strains have been demonstrated to
induce a reduction in TH2 cytokines (i.e., IL-4, IL-5, IL-6, IL-10, IL-13) or a
shift towards TH1-mediated immunity (i.e., IL-2, TNF-α, IFN-γ production)
(Vanderpool et al. 2008, Thomas and Versalovic 2010), a potential role for
probiotics in the management of allergies and AD could be suggested.
Most studies assessing probiotic effects in the treatment of allergic
diseases have focused on AD with or without associated food allergies in
infants and children. Studies aimed at investigating the role of probiotics
in adult-type AD are less frequent. Trials evaluating probiotic efficacy have
investigated on either the treatment or prevention of AD and, to date, only
a few rigorous randomized controlled studies have been performed.
The majority of studies have evaluated probiotic formulations
containing Lactobacillus species, alone or in combination with Bifidobacterium
species. The primary outcome of these trials was the change in the score
for the evaluation of AD severity (SCORAD).
First pioneering studies from Majamaa and Isolauri (1997) and Isolauri
et al. (2000), performed in small numbers of children who were administered
with L. rhamnosus GG, demonstrated that probiotic treatment resulted in
a rapid significant improvement in SCORAD with respect to placebos. In

contrast to these initial studies, more recent and larger trials have failed to
confirm beneficial effects of probiotics in the treatment of AD.
Recently, three meta-analyses summarized and evaluated, with
some controversial conclusion, the clinical efficacy of probiotics in AD
management (Boyle et al. 2008, Lee et al. 2008, Michail et al. 2008). Lee
et al. (2008) and concluded that current evidence is more convincing for
the efficacy of probiotics in the prevention rather than in the treatment of
paediatric AD. Boyle et al. (2008) indicated that probiotics do not appear
to be effective for the treatment of AD and that no sufficient evidences are
reported to support their use for this condition. Vice versa, Michail et al.
(2011) demonstrated that, even if the utilization of probiotics significantly
reduced the SCORAD severity index score, it was associated with modest
clinical effects and rapidly discontinued after the cessation of probiotic
treatment.
Similar controversial findings were provided more recently by
other clinical trials. A longitudinal study by Gore et al. (2012) performed
administering L. paracasei CNCM I-2116 and B. lactis CNCM I-3446 to infants
with AD aged 3–6 months demonstrated no beneficial effects of probiotics in
the treatment of AD. Furthermore, probiotics did not affect the progression
of AD from age 1 to 3 years.
Conversely, Drago et al. (2011) demonstrated that L. salivarius LS01
provoked a significant improvement in SCORAD index in a double-blind
placebo-controlled study on a cohort of 38 adult patients with AD. Similar
results were found by Moroi et al. (2011), who administered L. paracasei
K71 to 34 adult-type AD subjects, demonstrating a significant decrease
of skin severity scores after treatment. Whilst not taking into account
clinical parameters, Roessler et al. (2012) demonstrated that a probiotic
mix containing L. paracasei Lpc-37, L. acidophilus 74-2 and B. animalis subsp.
Lactis DGCC420 lowers the genotoxic potential of faecal water in adult AD
patients.
Taken together, research on the efficacy of probiotics in the management
of AD, especially concerning prevention, has to be sustained in order
to translate promising mechanistic insights related to their immune
modulation and anti-genotoxic activity into meaningful health claims.
Besides the utilization of probiotics to manage AD in infants, children
and adults, increasing evidence is supporting the administration of
probiotics already during pregnancy and within the first months of life, in
order to reduce the risk of developing AD. Doege et al. (2012) performed a
meta-analysis on the randomised, double-blind, placebo-controlled trials
aimed at assessing the impact of probiotics intake during pregnancy on the
development of AD in children. Doege et al. 2012 concluded that probiotics
significantly reduce the risk of development of AD, in particular when
Lactobacillus strains were administered. Conversely, no significant effects

of mixed probiotics formulation including lactobacilli and bifidobacteria
were demonstrated.
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CHAPTER 6
Application of Molecular
Methods for Microbial
Identification in Dairy Products
Paul A. Lawson1,* and Dimitris Tsaltas2
INTRODUCTION
Although traditional approaches such as cultivation, physiological and chemo
taxonomic methods are the cornerstone of the isolation and characterization
of individual organisms and complex communities; molecular methods
have made, and continue to make, incredible contributions to the study of
microbial diversity. A major advantage of molecular methods is the ability
to process large numbers of samples simultaneously and have been termed
high-throughput methods. Principle investigators and students alike,
therefore, favor such methods due to the huge amount of data that can be
generated in a relatively short period of time in a cost-effective manner.
Indeed many would argue that molecular methods have surpassed the
more traditional methods, but this viewpoint is unwise. No one method
can answer all questions and even the most powerful approaches such as
genome analysis must be complemented by physiological investigations to
provide a comprehensive polyphasic approach (Rainey 2011). For example,
a gene may well be present in the genome but is it expressed at all, and if
1
Department of Microbiology and Plant Biology, University of Oklahoma, OK 73019, USA.
2
Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University
of Technology, P.O. Box 50329, 3603 Limassol, CYPRUS.
* Corresponding author
so, when? These questions are particularly important when considering

the microbial community as a whole. Therefore, it is essential that high-
throughput molecular methods be used in tandem with more traditional
methods for a comprehensive investigation of microorganisms present and
their potential roles in food spoilage as food pathogens, food additives,
etc. With respect to food-borne organisms and the associated pathogens, in
addition to cultivation and enzyme-linked immunosorbent assay (ELSA)
three main approaches using molecular tools may be employed. The first
are methods based on the polymerase chain reaction (PCR) (Hayden 2004)
with Escherichia coli (Tsai et al. 1993, Naravaneni and Jamil 2005) Salmonella
(Rahn et al. 1992) Shigella (Frankel et al. 1990) Yersinia (Ibrahim et al. 1992)
Vibrio cholera (Shangkuan et al. 1995), Vibrio parahaemolyticus (Tada et al.
1992) Vibrio vulnificus (Brauns et al. 1991), Listeria monocytogenes (Simon et
al. 1996), and Staphylococcus aureus (Wilson et al. 1991). A further refinement
was the introduction of Real-Time PCR that is now the most commonly
used technology for quantification of specific DNA fragments (Wittwer and
Kusukawa 2004). The amount of product synthesized during the PCR is
measured in real time by detection of the fluorescent signal produced as a
result of specific amplification. The PCR methods are rapid and sensitive,
but care should be taken with appropriate controls as false-positive and
false-negative results can lead to misleading conclusions.
The second and most recent group is the microarray-based techniques,
which is an extension of checkerboard hybridization methods. These
methods allow for the simultaneous identification of the increasing number
of food-borne pathogens worldwide in a single reaction (Sergeev et al. 2004).
The basic idea is that many selected probes are attached spot wise in an
array format to a solid surface, and each spot contains numerous copies
of a probe. The array is subsequently hybridized with DNA isolated from
the sample of interest labeled with fluorescence. During the hybridization
phase, the labeled fragments will bind to the spotted probes based on
DNA complementarity. As a high-throughput method, microarray-based
techniques have some advantages, such as informative, highly repeatable,
and potential to combine detection, identification, and effect quantification
of unlimited number of food-borne pathogens in a single experiment.
The third approach is the direct sequencing of genes that are then used
in a phylogenetic analysis. The most extensively used for this purpose is
the 16S rRNA gene (Ludwig et al. 2011). This technology can be used for
the rapid identification of pure culture isolates or for a community wide
approach where all the 16S genes present are sequenced from a single DNA
sample (Quigley et al. 2012b). Innovations to automated DNA sequencing
and Next Generation Sequencing are occurring at an almost unbelievable
rate (Loman et al. 2012) and in addition to other hardware improvements,
the development of computer software to handle huge amounts of data
Application of Molecular Methods for Microbial Identification in Dairy Products 179
generated by genomic and proteomic approaches facilitated the expansion

of these technologies away from the specialist research laboratories to many
clinical and diagnostic facilities. In particular the Basic Local Alignment
Search Tool (BLAST) (Altschul et al. 1990), provide a rapid mean of
sequence identification, coupled with sequence alignment programs such
as CLUSTAL (Thompson et al. 1994) and MEGA (Tamura et al. 2007). The
Chunlab (http://www.chunlab.com/), and EzTaxone [http://eztaxon-e.
ezbiocloud.net/(Kim et al. 2012)] also have an excellent suit of user-friendly
software packages that are now widely used. Subsequent manipulation
of data and the construction of phylogenetic trees have contributed to
the proliferation of genomic based methods. Although some components
such as the hardware for the aforementioned methods can be beyond the
financial capabilities of many laboratories, an increasing phenomenon is
the “out-sourcing” of these methods to university or commercially based
facilities. Indeed, the past decade has seen a large number of biotechnology
companies offering high-throughput and rapid processing of bacterial and
fungal isolates or extracted DNA submitted and within days a result is
returned normally in electronic format. However, although this allows rapid
and cost efficient identifications with a reduced need for specialist training,
care is still required for the correct and accurate analysis of data received.
Without a doubt, out-sourcing of this type has facilitated the proliferation
of these powerful molecular methods into many laboratories dealing with
food microbiology, often confirming the results of strains identified by more
classical methods including miniaturized biochemical/enzymatic kits that
are extremely useful in the diagnostic laboratory.
Another very strong driving force for the development and deployment
of molecular methods to characterize the microbial flora of foods is the
denomination of Protected Designation of Origin (PDO) that is well
documented through links among the areas of origin, the procedures and
the final products. Sensorial and texture characteristics of dairy products,
are not only attributed to the microbial population of lactic acid bacteria
and enterococci (Randazzo et al. 2009). Therefore, characterizing the
microbial population of fermented products contributes understanding
the biochemical “evolution” of these products and provides information on
their identity (for PDO purposes) and technological development. Recently
O’Sullivan and coworkers (2013) have reviewed nucleic acid based methods
investigating the microbial related cheese quality defect problems, showing
another very useful contribution by these methods. In addition, they have an
extensive reference to next generation sequencing, giving good introductory
information for its use in food microbiology. Neviani and coworkers (2013)
have also reviewed the case of Grana Padano and Parmigiano Reggiano
cheeses research, via molecular methods done over the last decade. In the
same context Rodrigues and coworkers (2012) reviewed the analytical
strategies for characterization and validation of functional dairy foods. The

probiotic content leading to the transformation of milk to a variety of healthy
products (yogurt, cheese, dairy drinks, etc.) via glycolysis, proteolysis and
lipolysis can very well be characterized using molecular methods in parallel
with analytical chemistry and classic microbiology ones.
It is beyond the scope of this chapter to describe in detail all molecular
methods but it will outline some of the most useful molecular approaches
applied to dairy microbiology. A basic knowledge of microbiology and
molecular biology will be assumed, for each method discussed, a short
introduction outlining the principle will be given and extensive use of
references will provide the reader with additional resource material.
Experience shows that in addition to access to these methods it is extremely
beneficial to visit colleagues where such methods are running routinely
in order for a smooth transition to their introduction into the laboratory.
The text will focus on the methods that are the most accessible to majority
of laboratories with a modest budget. Table 1 shows the methods and
principles behind those that will be presented in this chapter.
Table 1. Description and principles of molecular-based methods.
Method Principle
16S rDNA gene sequencing Sequence determination of the 16S rDNA gene that contains
both conserved regions to allow sequence alignment and
variable regions that allow diagnostic identification and
provide evolutionary relationships.
Random Amplified Employs short arbitrary primers and low stringency
Polymorphic DNA (RAPD) hybridisation to randomly amplify DNA fragments which
are separated on agarose to give a fingerprint pattern.
Denaturing or Temporal Small PCR amplicons (distinguished by differences in their
Temperature Gradient Gel DNA sequences) are separated from a low to high gradient.
Electrophoresis (DGGE or DGGE uses a chemical gradient (urea or formamide)
TTGE) while TTGE has a temperature gradient and a constant
concentration of denaturing agents.
Real-Time PCR (qPCR) Uses species-specific primers to target a gene/organism. A
fluorescent probe or dye is used to monitor the amplification
of the target DNA in real time enabling quantification of a
target organism.
Terminal Restriction Fluorescent, end labeled, PCR products are digested with
Fragment Length restriction endonucleases and separated by electrophoresis.
Polymorphisms (tRFLP) The end-labelled terminal restriction fragments are
compared with DNA size standards. Different groups have
a difference in the number and location of restriction sites
giving rise to different fragment lengths.
Intergenic Transcribed Analyses the bacterial ITS region located between the 16S
Spacer Analysis (ITS) and 23S ribosomal genes. Allows the differentiation between
strains of the same species or closely related species.
When choosing a technique, one must first address the question of

what information we wish to derive from the sample being investigated.
For instance, do we wish to study the general microbial diversity of an
ecosystem (Duthoit et al. 2003, Callon et al. 2007, Bonetta et al. 2008) or
identify specific microorganisms present (Delbes and Montel 2005) or both
(Martin-Platero et al. 2009)? Our endeavor is to answer these questions in
good faith, accurately and directly.
Genomic Methods
Isolation of DNA
The recovery of good quality DNA is important to the outcome of most

DNA-based molecular approaches but is especially important in culture-
independent methods. It is crucial that DNA extracted from a mixed
community is truly representative of all the organisms present and is of
sufficient quality and concentration. In addition to insufficient or preferential
cell lysis, the presence of compounds such as fats, carbohydrates, proteins
and salts that may inhibit downstream manipulations should be eliminated
(Wilson 1997). However, many laboratories now use a range of commercially
available DNA extraction kits that have mainly overcome many earlier
problems. Added advantages are the elimination of harmful chemicals
such as phenol and little required prior knowledge of complex molecular
methods. However, the classical phenol/chloroform is useful as it introduces
the novice to the basic principles of the extraction process and it has the
benefit of the observation of DNA during the extraction process unlike kit
systems.
In the case of dairy products it is a common problem acquiring non
degraded and inhibitor free DNA as described, tested and reviewed by
Pirondini and coworkers (Pirondini et al. 2010). Similarly Quigley with
coworkers (2012a) have compared five commercial kits and two in-house,
concluding that best results were obtained by the two commercial (solid-
phase/column extraction—Milk Bacterial Isolation Kit from Norgen Biotek
and Power Food Microbial DNA Isolation Kit from MoBio Laboratories) and
an in-house method based on liquid-liquid extraction with purification steps
using phenol-chloroform and ethanol. The authors favour the use of the
Power Food Microbial DNA Isolation Kit from MoBio Laboratories because
it is faster and less laborious being in a ready-to-use format. Conclusively,
we can suggest that before the use of commercial kits for DNA extraction
from raw milk, but not cheeses, one should consider the multifactorial
environment of the fermented product and tests should be made in each
case before the final decision is made.
Table 2. Commonly used DNA extraction methods.
Method Extraction type Principle

In-House Lytic Method Liquid–liquid extraction Cell lyses using, chaotropic
(Quigley et al. 2012a) agents, enzymes and mechanical
force. Purification relies on using
phenol-chloroform and ethanol
purification.
QIAamp®DNA stool mini Solid-phase/column Cell lyses using chaotrophic
kit (Qiagen Ltd.) extraction agents, detergents, proteinase K
and heating, uses an exclusive
adsorption resin to remove
impurities. DNA purification
uses a silica-gel membrane.
Chemagic Food Basic kit Mobile solid-phase/ Cell lyses using chaotrophic
(Chemagen magnetic bead extraction agents and RNase A. Magnetic
BiopolymerTechnologie) beads as solid-phase for binding
target DNA.
Wizard®Magnetic DNA Mobile solid-phase/ Cell lyses using chaotrophic
Isolation kit (Promega Inc.) magnetic bead extraction agents and RNase A. DNA
bound and purified using
magnetic beads as solid-phase
support.
FastPrep®Kit Solid-phase/column Cell lyses using chaotropic
(MP Biomedicals) extraction buffers. DNA is purified by
a silica-based GeneClean®
procedure.
PowerFood™ Microbial Solid-phase/column Cell lyses based on chaotrophic
DNA Isolation kit extraction agents, mechanical lyses and
(MoBio Laboratories Inc.) inhibitor removal technology.
DNA binding is based on silica
membrane spin column.
Guanidine Thiocyanate Liquid–liquid extraction Cell lyses using detergents,
method (Duthoit et al. chaotrophic agents, mechanical
2003) lysis plus heat. DNA extraction
using phenol-chloroform and
ethanol purification.
DNA extraction—Protocol 1
1. Resuspend cells (1–2 loopfuls or approximately 1 ml of actively growing

culture) in 500 µl TES buffer (0.05 M Tris, 0.05 M NaCl, 0.005 M EDTA,
pH 8.0). Add 5 µl lysozyme (10 mg/ml), incubate at 37°C for 15–30
min.
(N.B. Some cells are easier to break open and therefore the lysozyme
step may be omitted. However if the cells prove to be resistant the
lysosyme concentration and incubation time can be increased. If
problems persist an alternative enzyme such as lysostatin or mutolysin
may be used).
2. Add 8 µl each of Proteinase K and RNase (10 mg/ml), mix. Incubate for
1h at 65°C. (The increased temperature inhibits non-specific nucleases,
which would otherwise degrade the DNA).
3. Add 120 µl 10% SDS and return immediately to the 65°C water-bath
for a further 10 min. When the cells lyse the solution may become clear.
4. Remove from water-bath and leave to cool. Add an equal volume of
phenol/chloroform mix until emulsion forms. Centrifuge on low-speed
setting 1500–6500 rpm for 10 min.
5. Three layers should form, a lower solvent layer, an upper aqueous
layer containing the DNA and a layer of protein/cell debris separating
the two other layers. Carefully take off the top layer into a clean
microcentrifuge tube using a wide bore blue tip (i.e., cut with scissors
and smooth with bunsen flame). If the solution is too concentrated
the solution may be very cloudy. Add TES until the solution clears
and repeat the phenol/chloroform step until no more protein material
remains.
6. To the DNA solution add 2.5 volumes of 100% ethanol (–20°C). Mix
gently, DNA strands should start to precipitate. Centrifuge on high-
speed for 5 min. Air-dry for 20 min (or speed vacuum for 5 min)
and resuspend in 50–500 µl (depending on the yield) sterile water
or TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). Keep the solution
concentrated and fairly viscous (Even if no DNA is seen, continue with
the protocol as very small amounts of DNA can be amplified by PCR).
7. Run 5 µl of the DNA solution on a 0.8% agarose gel with 1 µg lambda
standard to get an approximate concentration. Usually very high
quality DNA is seen as a tight band almost as good as the Lambda
standard. Make a dilution 10 ng/µl for PCR.
DNA extraction—Protocol 2 (for PCR)
Although the first step for many molecular methods is the extraction of high
quality DNA that requires the lysis of bacterial cells prior to downstream
manipulations, for amplification of genes by PCR, a simple boiling method
is often satisfactory.
1. Colonies are suspended on 100 µl of sterile water and incubated at
95–100ºC for 10 min.
2. Centrifuge at 12000 rpm for 5 min to remove cell debris, transfer
supernatant to a clean tube making sure none of the cell pellet is
disturbed.
(If cells prove to be more recalcitrant to lysis, a simple lysis buffer (0.5
M NaOH, 0.05 M sodium citrate) may be used. After 5 min incubation
at room temperature, samples are pelleted and the supernatant
transferred to a clean tube and treated as above).
Many research groups also apply the so called “Colony PCR” method
where very small amount of cells are picked up via a pipette tip and
immediately transferred to the PCR reaction mix. Although it is a very
successful technique for the most common bacteria, special care and practice
is required in order to acquire very small amount of cells.
DNA extraction—Protocol 3 (Quigley et al. 2012a)
1. DNA is isolated by resuspending pellet obtained from 1 ml milk or

1 ml homogenized cheese in 500 µl of breaking buffer for enzymatic
lysis (20 mmol l–1 Tris HCl (pH8), 2 mmol l–1 EDTA, 2% Triton X100,
50 µg ml–1 lysozyme, 100 U mutanolysin) and incubated at 37°C for
1 h.
2. Protein digestion is then performed by adding 250 µg ml–1 proteinase
K and incubating at 55°C for 1 h.
3. The suspension is transferred to a 2 ml microcentrifuge tube containing
0.3 g zirconium beads and shaken for 90 sec in a bead beater, twice
and centrifuged at 12,000 g x 10 min.
4. The supernatant is transferred to a clean tube and combined with equal
volume of phenol:chloroform:isoamylalcohol (25:24:1), mixed gently
and centrifuged at 12,000 g x 2 min.
5. The top aqueous phase is transferred to a clean tube and one-tenth the
volume of 3 mol l–1 sodium acetate and 2 volumes of 100% ice cold
ethanol are added. The suspension is mixed gently and stored at –20°C
overnight.
6. The sample is centrifuged at 14,000 g x 10 min, the supernatant
removed and the pellet is washed with 70% ice cold ethanol followed
by centrifugation at 12,000 g x 5 min and the pellet dried.
7. The pellet is re-suspended in 100 µl TE buffer.
Polymerase Chain Reaction (PCR)
PCR is a method for synthesizing multiple copies of (amplifying) a specific

piece of DNA. DNA polymerase copies strands of DNA (Mullis and Faloona
1987). Four basic components are required:
• A DNA template containing the target sequence that is to be amplified.
For pathogen detection this sequence must be highly specific to the
organism concerned, often a single gene, such as a virulence gene or
the 16S rRNA gene.
• Primers—a pair of short single-stranded DNA sections, which are
exactly complementary for specific parts of the target sequence.
• A heat-stable DNA-polymerase enzyme, usually Taq polymerase from

a thermophilic bacterium, which catalyzes the reaction.
• Free nucleotides that are used as the building blocks for multiple copies
of the DNA template.
A number of companies now offer “Mastermix” kits where all the
necessary components are supplied and only the DNA is required.
The first stage in the PCR process is to raise the temperature to about
90–95ºC. This causes the double stranded DNA to denature, or melt, into
single strands. The temperature is then reduced to about 50–65ºC to allow
the two primers to bind, or anneal, at specific points on the single-stranded
DNA of the target sequence. Finally, the temperature is raised to 70–74ºC
and the DNA-polymerase enzyme catalyzes the duplication of the target
sequence, starting at the annealed primers on each single strand, in a
process known as extension. This, results in two double-stranded DNA
fragments that are identical copies of the original target sequence. The
temperature cycling process is then repeated a number of times, typically
25–35, creating a theoretical doubling of the number of copies of the target
sequence at each cycle. This gives an exponential increase in target DNA
concentration and produces sufficient DNA for reliable detection from a
single target sequence in a few hours.
Multiplex PCR is also an approach of many molecular microbiology
publications and is of interest to molecular food microbiology as well
(del Rio et al. 2007, Senan et al. 2008, Cremonesi et al. 2011, Pal et al. 2012,
Bottari et al. 2013). Similarly, in a study analyzing the bacterial population,
structure and dynamics of treated and untreated cold stored milk,
Rasolofo and coworkers (2010) followed via real time PCR Staphylococcus
aureus, Aerococcus viridans, Acinetobacter cslcoaceticus, Streptococcus uberis,
Corynebacterium variabile and Pseudomonas fluorescens. In order to better
approach this issue, a careful optimization using appropriate control
organisms should be undertaken to resolve potential problems such as
unspecific amplifications and/or primer competition.
An extensive list of primers for a variety of PCR based analysis methods
is presented together with other analytical advances in food microbiology
in the review by Juste and coworkers (2008).
Real Time PCR
Rapid, sensitive and accurate methods for microbial identification have been
expanded with the introduction of real time PCR or also called quantitative
real time PCR. As a procedure, real time PCR follows the same principle
of PCR with the difference of detecting the end product of the reaction
in real time using fluorescent dyes (Mackay 2004). These dyes are either
nonspecific DNA intercalating or sequence specific DNA probes labeled with

fluorescent reporter molecules. Very recently Boyer and Combrisson (2013)
have reviewed the opportunities of quantitative PCR in dairy microbiology.
Using DNA intercalating dyes live and dead cells can also discriminated
(Moreno et al. 2006, Nocker and Camper 2006, 2009, Nocker et al. 2006,
2007a, 2007b, 2010, Kramer et al. 2009, Rodrigues et al. 2012).
Real Time PCR with dsDNA binding dyes
Fluorescent DNA binding dyes bind to all double stranded DNA thus DNA
increased product during PCR leads to increased fluorescence intensity
measured at each cycle. The most commonly used dye is SYBR Green and
is sold either alone or incorporated in master mixes by a wide range of
companies.
Reactions are prepared as usual with the addition of the dye and runs
are performed on special PCR instruments carrying UV or LED lamps for
excitation of the dye and sensitive detectors/cameras for the measurement
of emitted light.
Some major pitfalls the reader should have in mind and seriously consider
are the following:
• Poor Primer Design. The use of primer design software is strongly
recommended. Most primer design software includes adjustable
parameters for optimal primer design. These parameters consider
primer melting temperature (Tm), complementarity, and secondary
structure as well as amplicon size. The primer melting temperature
(Tm) of each PCR primer should be between 58–60°C and the Tm
of both primers should be within 1°C. Regions of low-complexity
sequence can be problematic in designing a unique primer. The
best option would be to select an alternative region and if that is
not possible, choosing longer primer sequences with higher Tm or
optimization of the thermal cycling protocol may be necessary to help
reduce nonspecific binding. Designing primers that generate very long
amplicons may lead to poor amplification efficiency. Ideally, amplicon
length should be 50 to 150 bases for optimal PCR efficiency. In cases
in which longer amplicons are necessary, optimization of the thermal
cycling protocol and reaction components may be necessary.
• Poor Quality DNA. Degraded or impure DNA can limit the efficiency
of the sensitive PCR reaction and reduce yield. It not uncommon to
have a positive PCR reaction and negative real time PCR reaction using
the same set of primers. Residues of cell (proteins, polysaccharides,
etc.) and phenol and/or salts from DNA isolation method used are
common PCR inhibitory substances.
• Incorrect Concentration of Primers. Primers should be reconstituted

into working stock concentrations accurately. It is important to take
into account the volumes that will be routinely pipetted and for this
reason in-house or commercial master mixes should be considered
when setting up real-time PCR assays. A common range of working
stock concentrations for primers is 10–100 µM.
• Baseline, Threshold, Efficiency and Standard Curves. To obtain
accurate threshold cycle (Ct) values, the baseline needs to be set two
cycles earlier than the Ct value for the most abundant sample. For real-
time PCR data to be meaningful, the threshold should be set when the
product is in exponential phase. Typically this is set at least 10 standard
deviations from the baseline. The efficiency (Eff) of the reaction can be
calculated by the following equation:
Efficiency = 10 (–1/slope) – 1
The efficiency of the PCR should be 90–110% and could be affected
by a number of variables. These factors can include length of the
amplicon, secondary structure, primer design, etc. Since real time PCR
can provide quantitative meaningful results, standard curves should
be prepared. The standard curve should extend above and below the
expected abundance of target.
Real Time PCR with fluorescent reporter molecules
Fluorescent reporter probes will detect only DNA containing the probe
sequence. This significantly increases specificity because detection and
quantification does not take place in non-specific DNA amplification. In
addition fluorescent probes labeled with different color flurophores can
be used in multiplex assays detecting several genes in one reaction. The
reactions are as those commonly use in normal PCR. Typical probes used
in many laboratories are TaqMan probes which are hydrolysis probes.
TaqMan probes consist of a fluorophore covalently attached to the 5’–end
of the oligonucleotide probe and a quencher at the 3’–end. Several different
fluorophores (FAM, TET, HEX, Cy3, Cy5, JOE, VIC, ROX, and Texas Red)
and quenchers (TAMRA, MGB) are available. The quencher molecule
quenches the fluorescence emitted by the fluorophore when excited by the
cycler’s light source. As long as the fluorophore and the quencher are in
proximity, quenching inhibits any fluorescence signals. During the reaction,
primers and probe anneal to DNA target and once polymerase reaches the
probe its exonuclease degrades the probe releasing the fluorescent reporter
from the quenching molecule also attached to the probe and as a result
fluorescence is detected.
Similarly to Real Time PCR with dsDNA binding dyes, there are
additional major pitfalls that the reader should consider for Real Time PCR
with fluorescent reporter molecules:
• Poor probe design. TaqMan® probe Tm should be ~10°C higher than
the primer Tm.
• Concentration of probes is incorrect. A common range of working stock
concentrations for probes is 2–10 µM.
• Ordering a probe labeled with a dye not calibrated or supported on
the real-time PCR instrument being used.
• Combination of flurophores and quenchers. A well-chosen combination
of flurophores and quenchers is very important for maximum
fluorescence, minimum background, maximum signal to noise ratio
and maximum sensitivity (Marras 2006). Good advice for compatibility
of instruments and the combination of fluorophores/quenchers is
usually provided on instrument and molecular probes manufactures
websites.
Dead or Live Discrimination
Viability assessment has been well explored using propidium iodide,

propidium monoazide (PMA) staining (Nocker et al. 2007a, 2009, Kramer et
al. 2009, Boyer and Combrisson 2013, O’Sullivan et al. 2013). Accumulating
data support better results from propidium monoazide due to smaller
probability to enter intact cell membranes as well as new applications
of these type of dyes (Nocker et al. 2009, 2010). Elizaquivel et al. (2014)
have recently reviewed the developments in the use of viability dyes and
quantitative PCR in the food microbiology field. The article concludes
that novel approaches, assessing metabolic activity towards preferential
detection of viable cells could complement the use of viability dyes.
Methods with Electrophoretic Output
Restriction enzyme based
Restriction Enzyme Analysis—Pulsed Field Gel Electrophoresis (REA-PFGE).

Restriction enzyme analysis via pulsed field gel electrophoresis (REA-
PFGE) is performed by rare cutting endonucleases (SmaI, ApaI, NotI, and
SalI). The approximate number of fragments ranges from 10 to 30 for
the corresponding genome sizes. Pulsed field gel electrophoresis uses a
periodically reoriented electric field that helps large DNA molecules to
move in agarose gels (usually 1%). Generally, analysis using one restriction
enzyme provides good and reliable differentiation as demonstrated with
Lactobacillus in olive fermentations using ApaI (Argyri et al. 2014, Blana et

al. 2014). However, Vancanneyt and coworkers (2006) suggested the use of
2–3 restriction enzymes for strains of Lactobacillus.
Although low in cost (except initial equipment investment), REA PFGE
is time consuming and therefore tends to be used as a supplementary
technique for the purpose of confirming or improving results (Coppola et
al. 2008). More information on PFGE can be found on PulseNet International
www.pulsenetinternational.org. Finally, statistical analysis is required post
electrophoresis for clustering purposes. Software like GelCompar (Applied
Maths, Sint-Martens-Latem, Belgium, http://www.applied-maths.com/
gelcompar-ii) is required for clustering analysis. Sample preparation
and handling is one of the most important parameters the reader should
consider. This is because high molecular weight DNA is easily cleaved
through shearing and imparts very high solution viscosity. For these
reasons, DNA samples for PFGE are generally prepared by embedding in
gel medium as “gel plugs”. Cellular source material is suspended in low
gelling agarose and the gel suspension is poured into molds. All subsequent
manipulations (cell lysis, DNA purification and restriction digestion) are
performed by diffusing reagents into the resultant gel plugs. The processed
gel plugs are then carefully loaded into wells of an agarose gel used for
PFGE.
Restriction Fragment Length Polymorphism (RFLP). During Restriction
Fragment Length Polymorphism analysis, the DNA sample is digested by
restriction enzymes and the resulting restriction fragments are separated
according to their sizes by gel electrophoresis. RFLP analysis was the first
DNA profiling technique with widespread application due to low cost.
Modifications of RFLP are PCR-RFLP, ARDRA-PCR (Amplified Ribosomal
DNA Restriction Analysis) and ISR-RFLP-PCR (Intergenic Spacer Region
Restriction Fragment Length Polymorphism PCR). The initial step of DNA
amplification via PCR and the following digestion is common in all cases. In
ARDRA and ISR-RFLP-PCR we use, in particular, ribosomal DNA regions as
have been used by Aquilanti and coworkers (2006) and others [(Moschetti
et al. 1998), computerized databases with LAB fingerprints from (Blaiotta
et al. 2002, Chan et al. 2003, Fortina et al. 2003, Mora et al. 2003, Moreira
et al. 2005)].
It is pertinent to note the possibility of over or under estimation of the
microbial community in the dairy environment since every organism shows
different cell lysis resistance, genome size and GC content and as a result
leading to differential amplification (Sanchez et al. 2006a, 2006b).
Similar to this method but with added features is Terminal Restriction
Fragment Length Polymorphism (T-RFLP) that is based on variation of the
16S rRNA gene. This technique has been used for both characterizations
of microbial populations and structure and their dynamics (Rademaker
et al. 2005, 2006, Sanchez et al. 2006a). Analysis is based on the restriction
endonuclease digestion of fluorescently end labeled PCR products using
a genetic analyzer and appropriate software (Applied Biosystems 2005).
PCR Based Methods
Randomly Amplified Polymorphic DNA (RAPD)
Randomly amplified polymorphic DNA technique is PCR based where

arbitrary primers (8–12 nucleotides) with low stringency hybridization
are randomly amplifying DNA fragments separated via polyacrylamide
gel electrophoresis. RAPD is an inexpensive and powerful typing method
for many bacterial species including microbial populations from the dairy
environment (Corroler et al. 1998, Baruzzi et al. 2000, Suzzi et al. 2000,
Albenzio et al. 2001, Bouton et al. 2002, Mannu and Paba 2002, Andrighetto
et al. 2004, Psoni et al. 2006, Sanchez et al. 2006a, Aquilanti et al. 2007,
Ercolini et al. 2009).
Reproducibility of RAPDs is slightly problematic but as long as there
is adequate experience, optimization and standardization, the technique
is very useful and cost effective. In particular all PCR related parameters
(primer to template ratio, DNA polymerase and MgCl2 concentration, and
thermal cycles) are required to be optimized.
Other limitation of RAPDs are that there is no possibility to distinguish
from single copy to multiple copies of the targeted locus, while co-dominant
RAPD markers observed as different-sized DNA segments amplified from
the same locus, are detected rarely. Mismatches between the primer and
the template may result in the total absence of PCR product as well as in a
decreased amount of product.
Alternative methods to proceed after RAPDs is the development
of locus-specific, co-dominant markers, isolation of bands, cloning and
sequencing. From the acquired sequence new, longer and specific primers
are designed and we proceed with the so called Sequenced Characterized
Amplified Region Marker (SCAR) (NCBI 2014).
Extensive lists of primers used for RAPDs in dairy microorganisms
can be found in Coppola et al. (2008). In these lists the reader can also
find primers for yeast characterization (see also recent publication by
Fadda et al. 2010). Finally, Moschetti and coworkers (1998) demonstrated
how statistical analysis of the results can allow grouping of Streptococcus
thermophilus strains. Most recent application of RAPDs in conjunction with
16S rDNA pyrosequencing was described by Cruciata et al. (2014). From
this work it is apparent than when new species or strains appear, an array
of techniques (at least two) are categorically required in order to increase
confidence or results.
Amplified Fragment Length Polymorphism (AFLP)
Amplified Fragment Length Polymorphism uses the combination of

restriction enzymes and PCR. Restriction enzymes are used to digest
genomic DNA, followed by ligation of adaptors to the sticky ends of the
restriction fragments. A subset of the restriction fragments is then selected
to be amplified by using primers complementary to the adaptor sequence,
the restriction site sequence and a few nucleotides inside the restriction
site fragments. The amplified fragments are separated and visualized on
denaturing polyacrylamide gels or via automated capillary sequencing
instruments.
The digestion is performed with two different restriction endonucleases,
one with an average cutting frequency and a second with higher cutting
frequency (EcoRI – MseI, TaqI). Following digestion, double-stranded
nucleotide adapters are usually ligated to the DNA fragments serving
as primer binding sites for PCR amplification. The use of PCR primers
complementary to the adapter and the restriction site sequence yields strain-
specific amplification patterns (Amor et al. 2007, Goering 2013).
AFLP has mostly been employed in clinical studies, but its successful
application for strain typing of the Lactobacillus acidophilus group, L. johnsonii
isolates and L. plantarum group, has been reported (Gancheva et al. 1999,
Torriani et al. 2001, Ventura and Zink 2002). Improvements in AFLP include
the use of multiple enzyme adapters and fluorescent labeled primers thus
increasing throughput.
Analysis software such as GeneScan (Applied Biosystems, Foster City,
CA, USA) may be used for compiling data, analysis and presentation.
Worth mentioning is that the resulting data are not scored as length
polymorphisms, but instead as presence-absence polymorphisms. The AFLP
method is time consuming and laborious, requiring either good quality
large polyacrylamide gels or expensive equipment such as sequencing
instruments and for these reasons is not suggested unless experience is
already established.
Denaturing Gradient Gel Electrophoresis (DGGE)
Denaturing gradient gel electrophoresis is a technique using a chemical

gradient to denature the sample as it moves across an acrylamide gel.
Denaturing agents (usually urea and/or formamide) are capable of inducing
DNA to melt at various stages. As a result of this melting, the DNA can be
analyzed for single components, even those as small as 200–700 base pairs.
In practice, in DGGE, the DNA is subjected to increasingly denaturing
conditions causing most fragments to melt in a step-wise process. In this
way we may discriminate differences in DNA sequences or mutations of
various genes. By placing two samples side-by-side on the gel and allowing
them to denature together, we can easily see even the smallest differences
in two samples or fragments of DNA. Main disadvantages to this technique
are the small reproducibility due to difficulties preparing the acrylamide
gels and the difficulties handling and preparing polyacrylamide. These
problems are partially addressed by TGGE/TTGE (Temperature Gradient
Gel Electrophoresis or Temporal Temperature Gel Electrophoresis), which
uses temperature, rather than chemical, gradient to denature the sample.
In this case the denaturing agent is added at a constant concentration.
DGGE has been used by an extensive number of dairy microbiologists
worldwide Randazzo et al. 2002, 2009, Chen et al. 2008, Dolci et al. 2008a,
2010, Gala et al. 2008, Nikolic et al. 2008, Rantsiou et al. 2008b, Van Hoorde
et al. 2008, Alegria et al. 2009), and the same happened for TGGE (Henri-
Dubernet et al. 2004, 2008, Abriouel et al. 2008).
Resolution problems shown by all electrophoretic methods could be
resolved by the addition of a GC-clamp to one of the primers increasing
resolution when using DGGE (Sheffield et al. 1989, Cocolin et al. 2001, Chen
et al. 2008). Also, fragments with identical migration are strongly suggested
to be analyzed by sequencing since closely related species might co-migrate
(Ogier et al. 2004, Parayre et al. 2007, Giannino et al. 2009, Masoud et al.
2011). Confirmatory results may be acquired via DNA sequencing of the
same or other appropriate PCR products (Chen et al. 2008, Masoud et al.
2011).
In order to reveal metabolically active microbiota of dairy products
scientists have used RNA processed through reverse transcription and
analyzed also by PCR-DGGE (Randazzo et al. 2002, Florez and Mayo 2006,
Rantsiou et al. 2008a, Masoud et al. 2011). In a recent attempt by Porcellato
et al. (2012a, 2012b) combining DGGE with high resolution melt analysis of
DGGE bands, the authors compared reference strain bands with unknown
bands, concluding in high accuracy results of identification.
Single Strand Conformation Polymorphism-PCR (SSCP-PCR)
Single stranded conformation polymorphism method uses an acrylamide

gel or a capillary electrophoresis for the separation of denatured PCR
products. Microbial communities have been analyzed by amplifying the V4
region of 18S rRNA for yeasts and the V2 and V3 regions of 16S rRNA for
bacteria. Overall SSCP follows DGGE/TTGE methods in applications and
could be characterized as a good profiling method for microbial populations
but is not the most appropriate for identification of microbial species due to
co-migration of amplicons of different species (Saubusse et al. 2007, Coppola
et al. 2008, Verdier-Metz et al. 2009). RNA based SSCP profiles have also
been used for characterizing sensorial properties of dairy products (Giraffa

and Neviani 2001, Duthoit et al. 2005).
Multi Locus VNTR Analysis (MLVA)
Multiple Locus VNTR Analysis is a method for genetic analysis that takes
advantage of the polymorphism of tandemly repeated DNA sequences.
VNTR stands for Variable Number of Tandem Repeats. This method is
well known in forensic science since it is the basis of DNA fingerprinting in
humans. When applied to bacteria, it contributes to forensic microbiology
through which the source of a particular strain might eventually be traced
back. For this reason it has been extensively used for pathogenic bacteria
(Stefano et al. 2008, Chen et al. 2011, Radtke et al. 2012, Seale et al. 2012,
Tilburg et al. 2012). In MLVA a number of well-selected and characterized
(in terms of mutation rate and diversity) loci are amplified by polymerase
chain reaction (PCR) so that the size of each locus can be measured. From
this size, the number of repeat units at each locus can be deduced. Repeat
unit sizes and repeat sequences can vary when multiple loci are examined
in a number of different isolates of an individual microbial species. It has
been documented on many occasions that the number of repeat units
per locus is a strain-defining parameter. Consequently, there is isolate
specificity in the number of repeats per locus, when different strains of a
given bacterial species are compared. The resulting information is a code
which can be easily compared to reference databases (http://www.mlva.
eu/). MLVA had limited use in dairy microbiology up to now (Diancourt et
al. 2007, Matamoros et al. 2011) but has found extensive use in pathogenic
and food spoilage microorganisms (Duffy 2009). Well-designed multiplex
PCR primers producing MLVA banding patterns in lactic acid bacteria or
other dairy microflora microorganisms will provide ample new data. A
cornerstone for the adoption of this method will be the development of open
access reference online databases with MLVA patterns of such organisms.
Variable Tandem Repeats can be found using online tools; Tandem
Repeats Finder [http://tandem.bu.edu/trf/trf.html (Benson 1999)] or the
Microorganism Tandem Repeat Database [http://minisatellites.u-psud.fr/
GPMS/(Denoeud and Vergnaud 2004)].
DNA Sequence Based Methods
Ribosomal DNA sequencing for identification and classification of

prokaryotes and fungi
Without doubt the single most important advance both in the identification
of individual species/strains and in the characterization of communities
within complex ecosystems is the application of 16S rRNA sequencing.

Since the work of Woese and Fox (1977) most community surveys are
focusing on RNA genes and intergenic spacers. For the microbial world
16S, 23S and 5S rRNA genes (bacteria) and 18S, 28S and 5.8S rRNA genes
(fungi) are sequenced and catalogued in online databases. Ribosomal RNA
genes show extremely high sequence homogeneity within species and this
is because of concerted evolution thus repeated rRNA genes are treated as
one locus. As a result rRNA genes are used in phylogeny studies and species
identification (Ludwig et al. 2011, Rainey 2011). However, the 16S rRNA
gene has a number of weaknesses in that recent speciation events cannot be
recognized resulting in a lack of resolution between closely related species.
However, this can be overcome by utilizing alternative chronometers or a
number of house-keeping genes (De Vos 2011).
Bacterial 16S rRNA genes comprise nine hypervariable regions
(V1–V9) exhibiting significant sequence diversity among species (Baker et
al. 2003). V3 region has mostly been used, although different sets of primers
provide different areas to be analyzed within it. In fungi the rRNA genes
are showing reduced taxonomic resolution while the internal transcriber
spacers (ITS) provide the analyst with a higher discriminatory power. The
ITS region is located between the 18S (also called Small Subunit—SSU) and
the 28S rRNA genes. 25–28S regions are also called Large Subunit—LSU.
The 5.8S rRNA gene splits the ITS region into two parts; ITS1 and ITS2. In
an excellent review of the culture independent methods for microbial ID in
cheeses Jany and Barbier (2008) recommend using other targets in addition
to ITS when analyzing cheeses microbial communities because the dairy
environment hosts many difficult to discriminate genuses. Schoch and
coworkers (2012) concluded that among the regions of the ribosomal cistron,
the internal transcribed spacer (ITS) region has the highest probability of
successful identification for the broadest range of fungi, with the most
clearly defined barcode gap between inter- and intraspecific variation. The
LSU had superior species resolution in some taxonomic groups, such as
the early diverging lineages and the ascomycete yeasts, but was otherwise
slightly inferior to the ITS. The SSU has poor species-level resolution in
fungi. The authors are clearly proposing that ITS should be adopted as the
primary fungal barcode marker. Other genes for fungal IDing are CO1, RPB1,
EF-1α, BenA and GPD (Berbee et al. 1999, Einax and Voigt 2003, James et al.
2006, Seifert et al. 2007, Seifert 2009).
The progression from RNA cataloging whereby short oligonucleotide
sequences were laboriously generated to produce the first phylogenetic
classification frameworks to the use of almost full length 16S rRNA gene
sequences via the use of reverse transcriptase sequencing of rRNA is well
documented (Collins et al. 1991, Weisburg et al. 1991, Williams et al. 1991).
Further advances came with the incorporation of the direct sequencing of
PCR-generated DNA amplicons, automated DNA sequencing and ever-

improving computer software have made the use of 16S gene sequencing
routine for many laboratories. Indeed, there appears to be no end to technical
improvements with next generation sequencing methods being reported
almost before the previous advance reaches the majority of laboratories.
Technically, PCR reactions are performed using universal primer sets
amplifying V1–V2 or V1–V3 hypervariable segments of the 16S rRNA
gene (Hunt et al. 2011, Ercolini et al. 2012). Primers used to amplify the 16S
rRNA genes and internal sequences used to derive the entire sequence are
given in Table 3. Although the primers are designed towards conserved
regions within the 16S molecule, variation between taxa (especially at the
5’ end of the molecule) is sometimes observed and a number of alternative
primers are provided. For rapid screening often only a single primer (R536)
which covers a number of diagnostic variable regions is required for a
rapid identification. Sequences that may be candidates for novel taxa may
be subjected to a full phylogenetic analysis. Work presented by Kumar
et al. (2011) using pyrosequencing, concluded that averaging V1–V3 and
Table 3. Shows commonly used 16S rRNA primers for amplification and complete 16S gene
sequencing. The numbering refers to the positions of the 16S rRNA of Escherichia coli (Brosius
et al. 1978).
Primer Sequence Target E. coli position

GM3Fa AGAGTTTGATCCTGGC Bacteria 8–24
F27b AGAGTTTGATCCTGGCTCAG Bacteria 8–27
F399b ACTGCTGCCTCCCGTAGGAG Bacteria 361–342
R536b CAGCAGCCGCGGTAATAC Bacteria 518–536
F786b GATTAGATACCCTGGTAG Bacteria 786–803
R947b TTCGAATTAAACCACATGC Bacteria 965–947
GM4Ra TACCTTGTTACGACTT Bacteria 1492–1507
R1542b AAGGAGGTGATCCAGCCGCA Bacteria 1542–1522
A2Fac TTCCGGTTGATCCYGCCGGA Archaea 7–26
A2Fbd TTCCGGTTGATCCTGCCGGA Archaea 7–26
A3Fae TCCGGTTGATCCYGCCGG Archaea 8–27
A109Ff ACKGCTCAGTAACACGT Archaea 109–128
Ab127Rg CCACGTGTTACTSAGC Archaea 112–127
A348Rh CCCCGTAGGGCCYGG Archaea 335–349
A934Rg GTGCTCCCCCGCCAATTCCT Archaea 915–934
A1098Fc GGCAACGAGCGMGACCC Archaea 1098–1114
A1115Rc GGGTCTCGCTCGTTG Archaea 1100–1114
fD1i AGAGTTTGATCCTGGCTCAG Bacteria 8–17
rD1i AAGGAGGTGATCCAGCC Bacteria 1540–1524
a
(Muyzer et al. 1995), b(Hutson et al. 1993), c(Reysenbach and Pace 1995), d(Lopez-Garcia et al.
2001), e(McInerney et al. 1995), f(Whitehead and Cotta 1999), g(Achenbach and Woese 1995),
h
(Barns et al. 1994), i(Weisburg et al. 1991, Ercolini et al. 2009)
V7–V9 regions provides similar results to Sanger sequencing while allowing

significantly greater depth of coverage than the Sanger method.
The explosion of microbial identification due to modern genomic tools
such as high-throughput capillary and next generation sequencing created
an era of “omics” and “microbiomes”. Today a huge number of publication,
almost on a monthly basis, demonstrate the communities of microorganisms
that share our body space, soil, plants, animals, inert surfaces and our
food matrixes. The term “microbiome” was originally coined by Joshua
Lederberg (Lederberg and Mccray 2001), who argued the importance of
microorganisms inhabiting the human body in health and disease. It appears
with the development of the tools and the metagenomic approach that the
unknown microcosm simply knows no boundaries.
Commercial kits and services now make the use of this powerful tool
accessible to most laboratories. However, one such protocol for a commonly
used kit and apparatus is given below. It is advisable to use sterile, UV
treated bench space and irradiate all tubes, tips, pipettes for 10 min in order
to eliminate any possible contamination of extraneous DNA which will
be easily amplified from the universal primers. If possible avoid bringing
amplified PCR products into the PCR setup area.
Protocol—DNA Sequencing with BigDye Terminators Ver 3.1 [Recourse

material: (Alcorn and Anderson 2004, Kolbert et al. 2011)]
Sequencing Reactions
For each reaction add the following:
• Template DNA (see below) 2 to 5 µl
• Primer (1.6 µM) 2.0 µl
• Sequencing dilution buffer or TM (2.5X) 2.0 µl
• BigDye Mix 0.65 µl
• Water to total 10.0 µl
Mix well and spin briefly.
PCR Program
Use hot lid
Initial denaturation 96°C for 30 sec
45 cycles of:
96oC for 10 sec
55°C for 15 sec
60°C for 4 min
Reactions are light sensitive and will degrade. Wrap in foil or put in
box to limit effects of light on samples.
DNA template quantities:

PCR product
500–1000 bp: 25–35 ng (typically 2–3 µl)
1000–2000 bp: 40–70 ng (typically 3–4 µl)
>2000 bp: ~70–200 ng (4–6 µl depending on concentration).
Removal of Unincorporated Dye-Terminators
Unincorporated dye can cause serious problems with peak detection and
must be removed.
1. Briefly spin plates/tubes at 1000 rpm to bring down any condensation.
Remove mat/tape/lid from sequencing reaction plate/tube.
2. Add 20 µl 95% ethanol/3M sodium acetate solution (19:1) to each 10
µl reaction. Note: the final concentration of ethanol should be 60%.
3. Seal with mat/tape (or cap tubes) and mix by vortexing.
4. Let sit for 20 min at room temperature to precipitate. Note: less than
15 min may result in lost signal.
5. Place plate in support rack and spin the centrifuge with plate rotor at
3600×g for 30 min (similarly for tubes).
6. Fold two paper towels to size of plate. Carefully remove covering
and invert plate onto paper towel. Then insert towel-side-down in
centrifuge. Spin at 900 rpm for 1 min.
7. Wash pellets by adding 100 µl of 70% ethanol.
8. Spin in support rack at 3600×g for 10 min, and repeat step 6.
9. Dry by leaving plate uncovered on bench-top for 25 min, or until no
trace of ethanol remains. Pellets will not be visible. Cover for storage.
Prepare Samples for Capillary Sequencer
1. Add 20 µl Hi-DiTM (deionized Formamide) to each sample.
2. Cap tubes or cover plates. Vortex thoroughly, and centrifuge briefly if
there are droplets on tube walls.
3. Heat for 3 min at 95°C in a thermal cycler, then chill samples in a cold
block or on ice for 3 min.
4. Vortex.
5. Spin briefly in a centrifuge to bring down any condensate.
6. Run on ABI 3130xl with 36 cm capillary array using POP7 Polymer for
46 min at 8.5 kV and an injection time of 18 sec at 1.2 kV.
Whether using in-house or commercial services, data is normally
retrieved in an electronic format and electropherograms can be viewed using
a number of programs for each of the commonly used operating systems
(4 Peaks by A. Griekspoor and Tom Groothuis, mekentosj.com for Macintosh
and Chromas Litehttp://technelysium.com.au/for Windows). Although
trimming of terminal sequence data and the combination of sequences

to form a complete sequence may be automated; it is essential to check
this data or manually edit such data. Analysis of sequences and accurate
identification depends on the use of high quality data.
MicroSeq®
The MicroSeq® Full Gene 16S rDNA and 500 16S rDNA Bacterial
Identification Kit provide all the reagents necessary for determining the
sequence of the 16S rDNA or a part of it. The resulting DNA sequence is
analyzed and compared to a library of 16S rDNA bacterial gene sequences
using MicroSeq® ID Analysis Software and the MicroSeq® ID 16S rDNA
Full Gene Library. MicroSeq® ID Analysis Software enables you to analyze
sequences obtained with any of the MicroSeq® 500 16S rDNA Bacterial
Identification Sequencing Kit, MicroSeq® Full Gene 16S rDNA Bacterial
Identification Sequencing Kit and the MicroSeq® D2 rDNA Fungal
Sequencing Kit. The MicroSeq® ID 16S rDNA Full Gene Library (v1.0)
includes over 1200 validated 16S rDNA sequences. Fungal identification,
Applied Biosystems offers MicroSeq® D2 rDNA Fungal Identification
Sequencing Kit which contains reagents for amplifying and sequencing
the D2 expansion segment region of the nuclear large-subunit (LSU)
ribosomal RNA gene. Variation within this region is sufficient to identify
most organisms at the species level. More than 1070 validated nuclear large-
subunit (LSU) ribosomal RNA gene sequences are included in the library.
Data Analysis
For rapid comparisons with reference sequences a number of DNA

databases are used for identification purposes. BLAST [http://www.ncbi.
nlm.nih.gov/BLAST), FASTA (http://www.ebi.ac.uk/Tools/sss/fasta/
nucleotide.html, http://fasta.bioch.virginia.edu/fasta_www2/fasta_list2.
shtml, http://www.genome.jp/tools/ fasta/(Pearson and Lipman 1988)]
and the Ribosomal Database Projects [http://rdp.cme.msu.edu (Maidak et
al. 1999, Cole et al. 2009)], Chunlab (http://www.chunlab.com/), EzTaxon-
e[http://eztaxon-e.ezbiocloud.net/(Kim et al. 2012)] and RIDOM [http://
www.ridom-rdna.de (Harmsen et al. 1994, 2002)]. Searches are returned
from a search and alignment algorithm typically as a series of pair-wise
alignments of decreasing similarities. Identical or similar sequences
corresponding to known species can then be compared with phenotypic,
biochemical and chemo taxonomic information available. Sequences with
no close matches (3% sequence dissimilarity is often used as a cutoff point)
may represent novel species and a full phylogenetic reconstruction may
be performed.
Partial or full sequences derived from the use of multiple primers

(Table 3) should be subjected to quality checking of the raw data. Sequences
can then be aligned and phylogenetic reconstructions performed using a
number of software programs such as MEGA, SeqTools or on-line tools
such as those provided by the Ribosomal Database Project.
For many laboratories a result showing the nearest relatives and a
percent sequence similarity may be sufficient. The organism from which
the sequence was derived can then be compared with biochemical and
phenotypic data. When the sequence is returned with a low percentage, this
may indicate an organism may have been isolated that represents a novel
taxon at either the genus or species level or, on occasion, higher taxonomic
levels. A number of databases were established in the 1980s and 90s (EMBL,
Genbank, RDP) but one must be aware that little quality checking was
performed and erroneous data is still present. More recent databases such as
the SILVA [http://www.arb-silva.de (Pruesse et al. 2007)] and Greengenes
[http://greengenes.lbl.gov (DeSantis et al. 2006)] have proved to be very
useful and are based more on taxonomic frameworks.
For additional reference material and more detailed explanation of
methods and computer software discussed, the reader is directed to the
following references (Lepp and Relman 2011, Ludwig et al. 2011). Such
programs require investment of time to become fully acquainted with their
capabilities, each have excellent help and tutorial sections; many individuals
now prefer the on-line analysis tools where software pipelines allow the
user to more easily navigate programs for phylogenetic analyses.
Phylogenetic analysis
A phylogeny is an estimate of the evolutionary relationships between

taxa or genes. This phylogeny is based on incomplete information with
no direct information from the past and with real evolutionary processes
not completely known. The most popular methods include distance
methods, maximum parsimony, and maximum likelihood. Algorithms
attempt to convert molecular data consisting of variables (A, T, C, and
G) into continuous variable represented by a branch length. Therefore
“phylogenetic inferences” are only a “best estimate” of evolutionary history
and a number of methods have been developed, each with advantages
and disadvantages. Phylogenetic relationships are typically presented
as radial dendograms commonly referred to as “trees” (Fig. 1). In this
format, sequences are linked to internal branching nodes via vertical and
horizontal lines, the vertical lines only give structure to the tree whereas
the horizontal lines represent the phylogenetic distance often denoted as
a percent or number of nucleotides substituted per 100 bases. In order to
provide some confidence to the robustness of tree topologies generated,
Abiotrophia defectiva ATCC 49176T (D50541)

98
Eremococcus coleocola CCUG 38207T (Y17780)
Facklamia hominis CCUG 36813T (Y10772)
Globicatella sanguinis NCFB 2893T (S50214)
Dolosicoccus paucivorans CCUG 39307T (AJ012666)
Ignavigranum ruoffiae CCUG 37658T (Y16426)
100 Aerococcus urinae NCFB 2893T (M77819)
Aerococcus viridens ATCC 11563T (M58797)
100 Alkalibacterium iburiense JCM 12662T (AB188091)
Alkalibacterium psychrotolerans JCM 12281T (AB125938)
98 Alkalibacterium olivoapovliticus WW2-SN4aT (AF143511)
Allofustis seminis CCUG 45438T (AJ410303)
1% Atopostipes suicloacale PC79T (AF445248)
92 Alloiococcus otitis NCFB 2890T (X59765)
Dolosigranulum pigrum NCFB 2975T (X70907)
Figure 1. Phylogenetic associations constructed using the neighbor-joining method.
resampling procedures are used. This Bootstrapping, put simplistically,

generates 100–1000 slightly different data sets presenting different orders
of sequences added to the algorithm and a consensus tree is generated. The
values obtained (often given at the branching nodes, Fig. 1) represent the
percent of how often two sequences (or clusters of sequences) are obtained
by that particular method. Given the shortcoming of all the models used it
is encouraged that at least two methods be compared.
It is not appropriate to discuss the details of each method and the
models of evolution and it can be overwhelming for a novice when faced
with different alignment programs, phylogenetic algorithms and models;
indeed this is why many now prefer the online-tools that take the uploaded
sequence and with a few key strokes a phylogenetic tree is presented to the
user! However, it is important for individuals to understand these programs
and the underlining principles employed. The reader is encouraged to
consult the following references (Swofford et al. 1996, Hall 2004, Lepp and
Relman 2011, Ludwig et al. 2011).
Next generation sequencing in dairy microbiology
Although sequencing can also be performed via classic Sanger method

(Rasolofo et al. 2010) and is often preferred for very sensitive analysis or
checking sequences determined using high through-put methods, Next
Generation Sequencing (NGS) methods are rapidly gaining popularity due
to their shear capacity to generate a huge amount of data in one operational
run (Loman et al. 2012). Total isolated DNA is amplified using 16S rDNA
primers and cloned on appropriate vector (pGEM-T-Easy, Promega, USA).
Inserts can be sequenced either by previously used primers or vector
universal primers in a bidirectional way. NGS techniques offer three major
advantages in dairy microbiology research. The first is to provide a thorough
examination of the biodiversity of a sample using universal primers and the

second relies on the thoroughness of the technique translating into analysis
of more sequences increasing, in this way, the capacity to observe less
abundant bacterial phylotypes. The third advantage is that sequence data
and analysis help elucidating the molecular basis of how microorganisms
respond to the food substrate and microenvironment (Solieri et al. 2013). In
recent years an increasing number of research groups incorporate NGS in
their dairy ecosystem investigations (Alegria et al. 2012, Ercolini et al. 2012,
Lusk et al. 2012, Masoud et al. 2012, Quigley et al. 2012b, Ercolini 2013).
The major difference between capillary based sequencing and NGS is
that one to ninety six samples can be analyzed simultaneously in the first,
while millions of sequences can be analyzed by the second. In addition NGS
technology does not require the generation of vector based libraries so it
is free from cloning associated biases. On the other hand, NGS technology
is computationally demanding which translates to increased cost of
instrumentation and requirement of bioinformatics specialists/competent
staff (Ercolini 2013).
As mentioned earlier, the elucidation of microbiomes in a variety of
live and inert systems where life exists, has been a subject of frequent
discussions over the last three years (2011–2013) (Bokulich and Mills
2013, Castro-Carrera et al. 2014). From Soggiu et al. (2013) we receive the
discussion about the milk and cheese microbiome for safety and quality of
dairy products and their reasons have led to dozens of published work on
the microbiomes of PDO dairy products; it is of unprecedented scale as is
the ease of acquiring them.
The application of metagenomics through an NGS platform is also of
much interest. The term metagenomics was introduced by Handelsman
and coworkers (1998) while referring to the study of genetic material
recovered directly from environmental samples. This broad field may also
be referred to as environmental genomics, ecogenomics or community
genomics. Instead of solely targeting the 16S rDNA genes that is normally
undertaken, all genes present in a habitat are now being sequenced due to
the huge capacity of NGS methods.
Single locus sequence typing
Single locus sequence typing has been developed by the use of 16S rRNA
gene sequence analysis and later via the use of more discriminatory
genes such as elongation factor Tu gene (tuf) (Jian et al. 2001), DNA repair
recombinase (recA) (Ventura and Zink 2003), chaperonin Hsp60 (Cpn60)
(Blaiotta et al. 2008), RNA polymerase β subunit (rpoB) (Rantsiou et al. 2004),
and β subunit of DNA gyrase (gyrB) (Itoh et al. 2006).
Multi locus sequence typing
Although single locus sequence typing (De Vos 2011) hasn’t been extensively
used in dairy microbiology, multilocus sequence typing (MLST) has become
a popular approach in dairy fermented products for the characterization
of the microflora. The robustness of MLST derives from the advantage that
sequence data are far less ambiguous and easier to record and interpret than
band patterns produced from all other electrophoresis based techniques
(Spratt 1999).
PCR amplification and sequencing of internal regions of multiple
housekeeping genes (usually seven) assigns numeric allelic designations
and the individual strains are characterized by a seven digit MLST sequence
type. Using algorithm we may identify a parent as the one with the greatest
number of single locus variants. Various tools exist in the internet for
graphical representation of clonal complexes which group a minimum of
5–6 of the 7 allelic designations.
Databases of MLST information are these days easily accessible offering
portability of the data and facilitating global usage of them (http://www.
mlst.net/databases/default.asp, http://pubmlst.org/databases/,http://
www.pasteur.fr/recherche/genopole/PF8/mlst/, http://cge.cbs.dtu.dk/
services/MLST/). Increased cost and time associated with sequencing is
of limited importance since the introduction of chip based sequencing
methods. An MLST analysis for Lactococcus lactis subsp. lactis and cremoris
genotypes gave deeply branched trees (Fernandez et al. 2011). The genes
used were atpA, pheS, rpoA, bcaT, pepN and pepX. For PCR conditions the
reader should refer to Rademaker et al. (2007) and data analysis can be
performed using MEGA software (Tamura et al. 2007).
Whole genome sequence typing
Since the introduction of next generation sequencing approaches we are

witnessing a tremendous race of competition for the fastest and cheapest
production of genomes (Loman et al. 2012). We are currently heading
to the 1000 euros genome sequence and it becomes obvious that we can
also compare whole genome sequences with the same cost and easiness.
Whole genome sequencing is of course the ultimate molecular typing
approach and is following similar evolutionary speeds as informatics
and bioinformatics (Chun and Rainey 2014). Common conflicting reports
such as those published for the 2011 German E. coli outbreak that claimed
different origins of the strain (Mellmann et al. 2011, Rasko et al. 2011) are
most likely attributed to lagging bioinformatics tools for the interpretation
of these “new” type of data.
Whole genome sequence techniques offer two major advantages in dairy

microbiology research. The first is to provide a thorough examination of the
total sequence of an organism, identifying all interesting genetic variation
that explains unique physiological attributes useful for the development
of new dairy products or the characterization of old ones. In addition any
questionable genetic material (antibiotic resistance genes, pathogen related
genes etc.) may be avoided. Second, whole genome sequencing provides
ample amount of data, useful for future phylogenetic studies in a cost and
time effective manner (Prajapati et al. 2011, 2012, Papadimitriou et al. 2012).
Fluorescent In Situ Hybridization—FISH

Fluorescent in situ hybridization is a single cell analysis determining spatial
arrangement and semi quantitative information. Fixed cells are hybridized
with a fluorescently labeled DNA probe and visualized by epifluorescent
microscopy. The samples can be isolated bacteria or food samples
appropriately sectioned for microscopical observation (Juste et al. 2008,
Rantsiou et al. 2008b). Studies by Ercolini and coworkers (2003a), (2003b)
and Coppola and coworkers (2008) gave very interesting insights into the
spatial arrangement of LABs and their possible role in the development of
microenvironments within the cheese matrix. FISH is not an appropriate
method for mapping total diversity as there are practical limits to how
many probes can be used simultaneously.
Of a similar approach and use is the combination of fluorescently
labeled cells and flow cytometry where quantitative results at the species
level can be combined by exploring the physiological state of cells in a fast,
efficient and reduced labor-intensive manner (Bianchi et al. 2004, Lahtinen
et al. 2006a, Lahtinen et al. 2006b, Maukonen et al. 2006). As mentioned
previously, in dead/live methods we can employ permanent and non-
permanent DNA stains (Propidium Iodide, Propidium Monoazide, TOTO-
1) to assess viability of cells (Bunthof et al. 2001, Bunthof and Abee 2002,
Lahtinen et al. 2006a).
Non Sequence-based Whole Cell Typing

Mass spectrometry detection of microbial nucleic acids was first reported
by Ecker et al. (2005), showing a method for rapid identification and strain-
typing of respiratory pathogens for epidemic surveillance. The use of
electrospray ionization mass spectrometry and base composition analysis
of PCR amplification products gave identification and quantification of
pathogenic bacteria present in the samples. Most recently Massire et al.
(2013) used and evaluated PCR coupled with electrospray ionization mass
spectrometry (PCR/ESI-MS) as a novel means for identification of fungal

pathogens while Kern et al. (2014) were differentiating Lactobacillus brevis
strains using Matrix-Assisted-Laser-Desorption-Ionization-Time-of-Flight
Mass Spectrometry with respect to their beer spoilage potential.
Currently, due to the mass spectrometry revolution for microbial
diagnostics and the low rates of inter-laboratory reproducibility of some of
the DNA based techniques there has been a boost in the efforts developing
methods that are simple, reliable, with high specificity and uniformity
while analyzing multiple groups of microorganisms. At the same time
reducing the cost of analysis is a prerequisite. Robust and mass scale
mass spectrometry methods and technologies exist in our daily life. Mass
spectrometry instruments are the “absolute analyzers” from which we
expect, globally, most if not all the answers for life on earth and beyond.
Matrix assisted laser desorption/ionization time of flight mass
spectrometry (MALDI-TOF-MS) has been introduced in chemo taxonomy
since 1994 (Cain et al. 1994, Böhme et al. 2010, 2011, 2012a, 2012b, Tanigawa
et al. 2010). Doan et al. (2012) have shown the potential of MALDI-TOF
MS-MS to replace molecular techniques based on genomic fingerprinting
while an excellent review is published by Sandrin and coworkers (2012).
Also, as any other existing fingerprinting method, it is of equal importance
to develop reference databases with microbial spectra. Bohme et al. (2012a)
have launched a publicly accessed MALDI TOF MS library of 79 bacterial
spectra.
Conclusion and Further Challenges

Culture independent molecular approaches are increasingly used in most
microbiology laboratories worldwide, whether dealing with clinical,
environmental or dairy-based. Methods and instrumentation continue to
be developed or refined. In tandem with reduction in costs, commercially
based out-sourcing and the abundant use of commercial kits for relatively
complex procedures have made these methods very accessible to most
laboratories. For example at the time of writing, the biology community was
expecting the miniature sequencer of the size of a large USB memory stick
called MinION (Oxford Nanopore Technologies Ltd.) and the production
of a whole genome sequence in real-time and at a cost of under 1,000 US
dollars. Indeed it is pertinent to note that a number of methods described in
this chapter may well become obsolete in a relatively short period of time!
Microbial ecology is moving to the study of microbial function via
metatranscriptomic approach (Cardenas and Tiedje 2008) and structure-
function studies will provide new knowledge in dairy ecosystems (Irlinger
and Mounier 2009). However, as powerful as molecular methods are, it
should be noted that if we lose sight of a polyphasic approach employing a
range of methods including cultivation, physiological and chemotaxonomic

approaches it will be at our peril and to the detriment of microbiology as
a whole.
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CHAPTER 7
Application of Food Safety

Management Systems (FSMS)
in the Dairy Industry
Thomas Bintsis
INTRODUCTION
It is a generally accepted fact that milk from a healthy udder is sterile.
Therefore, pathogens that enter the milk originate either from the milking
parlor (i.e., equipment, environment, personnel), or from means of
transportation (i.e., equipment) to the factory for processing. This chapter
deals with the application of Hazard Analysis and Critical Control Points
(HACCP), in dairy products (including Risk Assessment) and it highlights
the importance of on-farm HACCP.
HACCP in Dairy Products—Principles

The implementation of food hygiene and HACCP system has been reported
to be an effective and cost-effective approach to food safety regulation
(Mortimore 2001, Unnevehr and Jensen 1999). Although the HACCP system
per se does not make food safe, its proper implementation can make a
difference. Despite the fact that the food hygiene basic text from Codex
Alimentarius (Codex Alimentarius Commission 2003, 2009) serves as the
basis for all food safety management systems, a number of countries have
PhD, Dairy Science, 25 Kappadokias St., 55134 Thessaloniki, Greece.

developed national standards for the supply of safe food and individual
companies and groupings in the food sector have developed their own
standards or programmes for auditing their suppliers such as BRC, IFS,
Dutch HACCP, etc. (National Advisory Committee on Microbiological
Criteria for Foods 1997, Bernard 1998, Forsythe and Hayes 1998, BRC
2005, Arvanitoyannis and Traikou 2005, Frost 2006). The plethora of more
than 20 different such schemes worldwide generates risks of uneven
levels of food safety, confusion over requirements, and increased cost
and complication for suppliers that find themselves obliged to conform
to multiple programmes (Papademas and Bintsis 2010). To fill this gap,
the International Organization for Standardization (ISO) published a
new food safety management system, the ISO 22000:2005—Food safety
management systems—Requirements for any organization in the food chain
(International Organization for Standardization 2005). These standards
provide a framework of internationally harmonized requirements for the
global approach to food safety issues. However, ISO 22000:2005 doesn’t
contain the non-exhaustive list of Good Manufacturing Practices present
in the Global Food Safety Initiative (GFSI) guidance document (GFSI
2007). Thus, the Publicly Available Specification—PAS 220:2008 developed
by British Standards Institution (BSI 2008) and published in Oct 2008 in
association with major branded dairies (Unilevel, Kraft, Nestle, Danone)
and is a new complementary standard to ISO 22000:2005. It specifies the
prerequisite programmes requirements in detail to assist in controlling
food safety hazards. In fact, it provides harmonization of prerequisite
programmes and industry best practice for food manufacturing. GFSI
agreed that the combination of ISO 22000:2005 and PAS 220:2008 contained
adequate content for approval, but that an industry-owned scheme
governing the combination of these two standards must exist. Consequently,
the Foundation for Food Safety Certification developed the FSSC 22000, an
auditable standard which incorporates food safety elements already known
from previous standards such as HACCP, ISO 22000:2005, BRC (2005) and
IFS as well as from specifications such as PAS 220:2008 (Sansawat and
Muliyil 2009) which has approved by the GFSI as a global benchmark in
food safety management. In addition, ISO, through the Technical Committee
ISO/TC 34 and based on PAS: 2008, has published ISO 22002-1:2009—
Prerequisite programmes on food safety—Part 1: Food manufacturing
(International Organization for Standardization 2009). Thus, ISO 22002-
1:2009, which has completed with the publication of ISO 22002-2:2013—
Prerequisite programmes on food safety—Part 2: Catering (International
Organization for Standardization 2013) and ISO 22002-3:2011—Prerequisite
programmes on food safety—Part 3: Farming (International Organization
for Standardization 2011), is now used in conjunction with ISO 22000:2005
throughout the whole food chain to manage the food safety aspects.
Food Safety Management Systems 219
According to the European Legislation, the HACCP system has

been mandatorily applied in the food industry since 1993, and has been
incorporated in the new food hygiene package since January 2006 with
the Regulations 852/2004 and 853/2004 (EC 2004a, EC 2004b). However,
an exception was made for producers of primary products (e.g., dairy
farmers), even though they should follow the principles of food safety and
hygiene codes and the best way is to use HACCP-based systems (Maunsell
and Bolton 2004). Well, all food businesses must have a documented food
safety management system appropriate to its size and nature and this must
be based upon the principles of HACCP. Operators have to identify and
regularly review the critical points in their processes and ensure controls are
applied at these points. In addition, management personnel responsible for
HACCP must receive HACCP training. The procedure needs to be reviewed
and any necessary changes made, when any modification is made in the
product, process, or any other step.
According to the Regulation 852/2004 (Article 5), the HACCP principles
consist of the following (EC 2004a):
a) identifying any hazards that must be prevented, eliminated or reduced
to acceptable levels;
b) identifying the critical control points at the step or steps at which
control is essential to prevent or eliminate a hazard or to reduce it to
acceptable levels;
c) establishing critical limits at critical control points which separate
acceptability from unacceptability for the prevention, elimination or
reduction of identified hazards;
d) establishing and implementing effective monitoring procedures at
critical control points;
e) establishing corrective actions when monitoring indicates that a critical
control point is not under control;
f) establishing procedures, which shall be carried out regularly, to verify
that the measures outlined in (a) to (e) are working effectively; and
g) establishing documents and records commensurate with the nature
and size of the food business to demonstrate the effective application
of the measures outlined in (a) to (f).
On Farm HACCP
Although the responsibility lies with the manufacturer for ensuring that
the dairy foods manufactured are safe and suitable, there is a continuum of
effective effort or controls needed by other parties, including milk producers,
to assure the safety and suitability of milk products. It is important to
recognize that distributors, competent authorities and consumers also have
a role in ensuring the safety and suitability of milk and milk products. Due
to the special importance of the primary production to the safety of the
dairy products, major branded dairies have introduced their own on-farm
HACCP programmes. For example, Arla Foods has established a quality
program entitled Arlagεrden (‘the Arla farm’) to be used by their farmers.
The program specifies Arla Foods’ requirements not only for food safety and
milk composition, but also for animal welfare and environmental protection
(Junedahl et al. 2008). The Canadian dairy industry has begun implementing
an on-farm food-safety program called Canadian Quality Milk (Young et al.
2010). These quality assurance programmes starting at dairy farm level deals
with food safety, animal health and animal welfare issues to take account
of the demands of consumers and retailers (Noordhuizen and Metz 2005).
The HACCP concept, focused on risk management and prevention,
appears to be very promising to control on-farm processes. It can be easily
linked to both operational management and food chain quality assurance
and is suitable for certification (Noordhuizen 2003, Heeschen and Bluthgen
2004, Noordhuizen and Jorritsma 2006). In fact, introduction of HACCP on
dairy farms means nothing more than ‘structuring and formalising what the
truly good farmer would be doing anyway’ (Ryan et al. 1997). Until now,
the introduction of HACCP principles in on-farm management has hardly
been tested in practice, due to many objectively immeasurable processes
in an on-farm situation (Noordhuizen 2003).
Livestock species are an important reservoir of C. jejuni, shiga-toxin
producing E. coli, L. monocytogenes, Salmonella spp., and Y. enterocolitica
(Jayarao and Henning 2001, Murinda et al. 2002, Jayarao et al. 2006) and
other pathogenic bacteria that have been implicated in a number of food-
borne outbreaks (Papademas and Bintsis 2010). These pathogens have
been recovered with various frequencies from dairy-cattle faeces, bulk
milk tanks and the dairy-farm environment (Troutt 1995, Jayarao and
Henning 2001, Murinda et al. 2002, Wiedmann 2006, Van Kessel et al.
2004, Srinivasan et al. 2005, Karns et al. 2007, Vissers and Driehuis 2009).
Molecular epidemiological studies of E. coli O157:H7 have demonstrated
that subtypes of the organism can persist on cattle farms for years (Hancock
et al. 2001, Aspán and Eriksson 2010). The presence of food-borne pathogens
in milk is due to direct contact with contaminated sources in the dairy farm
environment and to excretion from the udder of an infected animal (Oliver
et al. 2005, Kousta et al. 2010). Fox et al. (2009) demonstrated the prevalence
of L. monocytogenes in the dairy farm environment and the need for good
hygiene practices to prevent its entry into the food chain and Hussein
and Sakuma (2005) described pre- and postharvest control measures to
ensure safety of dairy cattle products. D’Amico et al. (2008) reported that
the incidence of food-borne pathogens of concern in raw milk utilized for
farmstead cheese production was very low, whereas, Danielsson-Tham et
al. (2004) stated that the conditions on a summer farm can hardly fulfill the
requirements for hygienic and strictly controlled conditions necessary for
safe processing of fresh cheese.
Outbreaks due to the consumption of unpasteurized milk (Peterson
2003, Centers for Disease Control and Prevention 2007, Lind et al. 2008,
Heuvelink et al. 2009, Lejeune and Rajala-Schultz 2009, Oliver et al. 2009),
inadequately pasteurized milk (Fahey et al. 1995) and cheeses made from
unpasteurized milk (Honish et al. 2005, Center for Science in the Public
Interest 2008, 2009) continue to occur. Campylobacteriosis and Salmonellosis
was the most common zoonotic diseases in humans in the European Union
during 2008, but incidences of both have fallen, whereas, the number of
cases of Verotoxigenic Escherichia coli (VTEC) rose by almost 9% and the
number of listeriosis cases in humans decreased by 11.1% (European Food
Safety Authority 2010). In 2008, there were 5,332 food-borne outbreaks in
the EU, sickening over 45,000 people and causing 32 deaths. Some 35 per
cent of these were triggered by Salmonella spp., with viruses and bacterial
toxins detailed as the next most common causes.
When milk is intended to be used for the manufacture of raw milk
products, hygienic conditions used at the primary production are one of the
most important public health control measures, as a high level of hygiene
of the milk is essential in order to obtain milk with a sufficiently low initial
microbial load in order to enable the manufacturing of raw milk products
that are safe and suitable for human consumption (Codex Alimentarius
Commission 2004). In such situations, additional control measures may
be necessary. In addition, increased emphasis in certain aspects of the
production of milk for raw milk products (animal health, animal feeding,
and milk hygiene monitoring) are specified and are critical to the production
of milk that is safe and suitable for the intended purpose. Interestingly,
the FDA/Health Canada risk assessment found that the risk of listeriosis
from soft-ripened cheeses made with raw milk is estimated to be 50 to 160
times higher than that from soft-ripened cheese made with pasteurized
milk (Food and Drug Administration/Health Canada 2012). This finding
is consistent with the fact that consuming raw milk and raw milk products
generally poses a higher risk from pathogens than do pasteurized milk and
its products.
E. coli, S. aureus, Corynebacterium bovis, Klebsiella spp., or Pseudomonas
spp., Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus
uberis may cause, under certain circumstances, clinical mastitis (Hahn 1996,
Barkema et al. 1998). Special care should be taken for the use of antibiotics,
and Sawant et al. (2007) found that enteric bacteria such as E. coli from
healthy lactating cattle can be an important reservoir for tetracycline and
other antimicrobial resistance determinants.
Antimicrobial residues and antimicrobial-resistant bacteria from milk

and milk products can also pose potential health risks to consumers (Katz
and Brady 2000, Straley et al. 2006). Contamination of milk via the exterior of
the cows’ teats occurs when teats, and subsequently milk, are contaminated
with dirt consisting of faeces, bedding material, soil, or a combination of
these. Vissers et al. (2007a) applied quantitative microbial risk analysis of the
microbial contamination of farm tank milk for the amount of dirt transmitted
to milk via the exterior of teats using spores of mesophilic aerobic bacteria
as a marker for transmitted dirt. Silage was the main source of butyric acid
bacteria and clostridia spores in cheese milk (Vissers et al. 2007a, Julien et
al. 2008). When silage fermentation conditions are not prone to rapid pH
decrease and maintenance of uniformly anaerobic conditions, germination
of the spores and subsequent vegetative cell multiplication can occur. Vissers
et al. (2006) applied a modeling approach to identify an effective control
strategy at the farm level and found that contamination level of silage
with the butyric acid bacteria was found to be the most important factor
for the control of contamination of farm tank milk. In addition, Sanaa et
al. (1993) found that poor quality of silage (pH > 4), inadequate frequency
of cleaning the exercise area, poor animal cleanliness, insufficient lighting
of milking barns and parlors, and incorrect disinfection of towels between
milkings were significantly associated with raw milk contamination by
L. monocytogenes.
Cleaning and disinfection of the udder and the teats with appropriate
agents before and after milking can minimize infections during milking
(Burgess et al. 1994). Temperature abuse should be avoided at all stages
in the farm, and temperature should not increase above 6°C during
transportation to the dairy (Dijkers et al. 1995). In recent years, improved
standards of housing and use of separate milking parlors have reduced
the risk of raw milk contamination. Probable control points on the farm for
many of these human pathogens will be: 1) housing and bedding, 2) water
and waste management areas, 3) hospital pens, 4) calving pens, 5) treatment
areas, 6) bulk tank milk, and 7) young stock and cull animals (Cullor 1997).
As part of any on-farm HACCP system, cost-effective, accurate
and reproducible tests should be used to monitor certain control points
(Reybroeck 1996). However, the monitoring is limited by inadequacies and
costs of existing testing methodologies (Gardner 1997).
The dairy industry can adapt and implement Good Dairy Farming
Practice (GDFP) to aid in managing animal health problems and to
begin addressing pathogens of concern for food-borne and waterborne
illness. A joint guidance on GDFP was published from the International
Dairy Federation and the Food and Agriculture Organization of the
United Nations (International Dairy Federation/Food and Agriculture
Organisation 2004). The objective is that milk should be produced on-farm
from healthy animals under generally accepted conditions. To achieve this,

dairy farmers need to apply Good Agricultural Practice in the following
areas: a) animal health, b) milking hygiene, c) animal feeding and water,
d) animal welfare, and e) environment.
In addition, Globalgap has recently published regulations for integrated
farm assurance, which contains all the control points and compliance
criteria that must be followed by the producer and which are audited to
verify compliance (Globalgap 2007). For dairy farms, the protocol includes
specifications for feed, housing and facilities, dairy health, milking,
milking facilities (i.e., milking equipment, milking parlour, milk collection
equipment), hygiene, cleaning agents and other chemicals.
Application of HACCP System in Dairy Products

Several studies have been published concerning the implementation of
HACCP on dairy products such as pasteurized, ultra high temperature
(UHT) and condensed milk (Dijkers et al. 1995, Ali and Fischer 2002, Sandrou
and Arvanitoyannis 2000a), yogurt (Sandrou and Arvanitoyannis 2000a),
a variety of cheeses (Sandrou and Arvanitoyannis 2000b, Mauropoulos
and Arvanitoyannis 1999, Arvanitoyannis and Mavropoulos 2000,
Arvanotoyannis et al. 2009), ice cream (Mortimore and Wallace 1998,
Papademas and Bintsis 2002, Arvanotoyannis et al. 2009) as well as cream
and butter (Sandrou and Arvanitoyannis 2000a, Ali and Fischer 2005).
Risk Analysis
More than 100 countries have signed the “Sanitary and Phytosanitary (SPS)
Agreement” of the World Trade Organization (WTO). This agreement states
that “whilst a country has the sovereign right to decide on the degree of
protection it wishes for its citizens, it must provide, if required, the scientific
evidence on which this level of protection rests.” It follows that if a country
sets a microbiological criterion—or any other limit—for a particular health
hazard in a particular food product, they must be able to explain, based
on scientific data, consideration of risk and societal considerations, the
rationale and justification for the criterion. The “Technical Barriers to Trade
(TBT) Agreement,” also requires that a country must not ask for a higher
degree of safety for imported goods than it does for goods produced in its
own country (International Commission on Microbiological Specifications
for Food 2006).
Risk assessment for food safety is part of the risk analysis framework,
provided by the Codex Alimentarius Commission (Codex Alimentarius
Commission 1999), which also includes risk management and risk
communication as interdependent concepts. Risk analysis is used to

develop an estimate of the risks to human health and safety, to identify and
implement appropriate measures to control the risks, and to communicate
with stakeholders about the risks and measures applied. It can be used to
support and improve the development of standards, as well as to address
food safety issues that result from emerging hazards or breakdowns in food
control systems. It provides food safety regulators with the information and
evidence they need for effective decision-making, contributing to better food
safety outcomes and improvements in public health (Food and Agriculture
Organisation/World Health Organisation 2004).
Risk assessment has been set as a top priority issue on the basic food
legislation document, EC Regulation 178/2002 (EC 2002), and is defined as
a “scientifically based process consisting of four steps: hazard identification,
hazard characterization, exposure assessment and risk characterization”.
Risk is a “function of the probability of an adverse health effect and the
severity of that effect, consequential to a hazard” (EC 2002). Thus, risk
assessment requires the collection of scientific data regarding the nature,
frequency and impact on public health in Europe of food safety hazards.
Indeed, the severity of a food-borne illness caused by a biological hazard
must be combined with its occurrence in humans to accurately define risk
( Food and Agriculture Organisation/World Health Organisation 2004).
The core elements of a risk assessment process are 1) hazard identification
(i.e., the identification of biological, chemical and physical agents capable
of causing adverse health effects which may be present in a particular
food or group of foods), 2) hazard characterization (i.e., the qualitative
and/or quantitative evaluation of the nature of the adverse health effects
associated with biological, chemical and physical agents which may be
present in food—for chemical agents a dose–response assessment should
be performed, for biological or physical agents a dose-response assessment
should be performed if the data are obtainable), 3) exposure assessment
(i.e., the qualitative and/or quantitative evaluation of the likely intake of
biological, chemical and physical agents via food as well as exposures from
other sources if relevant) and 4) risk characterization (i.e., the qualitative
and/or quantitative estimation, including attendant uncertainties, of the
probability of occurrence and severity of known or potential adverse
health effects in a given population based on hazard identification, hazard
characterization and exposure assessment) (ILSI 2012) as presented in Fig.
1. Risk characterization brings together all of the qualitative or quantitative
information of the previous steps to provide a soundly based estimate of
risk for a given population.
Fully quantitative approaches are used nowadays and risks are
expressed as, for example, the number of cases of food-borne disease per
number of people per year, dose–response relationships and exposure
Hazard identification
• What is the problem?
• What is the hazards involved (chemical,
biological or physical)?
• Which foods are associated?
Hazard characterization Exposure assessment

• Which consumers are vulnerable? • What are the levels of the hazard in the
• What are traits of the hazard leading to food eaten?
illness? • How much of the food is eaten?
• What is the dose-response relationship? • How often is the food eaten?
Risk characterization
• What is the risk to consumers and to sub-groups?
• What is the effect of different mitigation actions?
• What is the key assumptions and uncertainties
in the assessments?
Figure 1. The basic steps in risk assessment (After: Codex Alimentarius Commission (1999)).
assessments. Guidelines for chemicals in foods will inevitably have to

address the differences between safety evaluation and a genuine risk
assessment approach. With respect to microbiological hazards, the unique
problems associated with risk assessment of living organisms in food
make it likely that the application of guidelines in the medium term will
more commonly use qualitative approaches. As risk assessment increases
and applied and internationally accepted guidelines become established,
decision criteria for risk management arguably present the greatest
challenge in establishing and maintaining quantitative SPS measures for
food in international trade and in judging their equivalence (Hathaway
1997).
Mathematical/probabilistic modeling is employed to estimate the risk
per serving of a specific food as described in FDA/Health Canada (2012).
In addition, second order Monte-Carlo simulation is used (Frey 1992) and
the variability in the risk (e.g., from serving to serving or from country to
country) is estimated and the uncertainty can be evaluated.
The International Commission on Microbiological Specifications for
Foods (ICMSF) has proposed a scheme for the management of microbial
hazards for food that involves the concept of food safety objectives (FSOs),
i.e., the maximum frequency and/or concentration of a hazard in a food
at the time of consumption that provides or contributes to the appropriate
level of protection (ALOP) (International Commission on Microbiological
Specifications for Foods 2006). To ensure that an FSO is met, it is required
to set performance objectives (POs), which correspond to the levels that
must be met during the earlier steps in the food chain before consumption.
The FSO gives flexibility to the food chain to use different operations and
processing techniques that best suit their situation, as long as the maximum
hazard level specified at consumption is not exceeded (e.g., the replacement
of heat treatment with another equivalent technique, i.e., microfiltration).
The position of these concepts appearing in the food chain can be seen in
Fig. 2. Microbiological standards (Buchanan 1995) have been included in
European Legislation (EC 2005).
Performance Performance Food Safety

Performance
objective, objective, objective,
objective,
PO PO FSO
PO
primary manufacturing transport retail preparation cooking consumption

production
exposure
Control Control Control

Measure, Measure, Measure, Public
e.g., GAPs e.g., GHP, HACCP e.g., cooking health goal
Figure 2. A presentation of a model food chain indicating the position of a Food Safety Objective
and derived Performance Objectives (After: International Commission on Microbiological
Specifications for Foods (2006)).
Risk communication between risk managers and stakeholders is

simplified when the results of a qualitative assessment are available (Clough
et al. 2006, Hauser et al. 2007). However, quantitative assessments are much
more useful tools (Food and Drug Administration/Health Canada 2012,
Peeler and Bunning 1994, Bemrah et al. 1999, Sanaa et al. 2004) for microbial
risk assessments, and the Monte Carlo exposure assessment model for
mycotoxins in dairy milk was developed by Coffie et al. (2009).
Brouillaud-Delattre et al. (1997) used predictive microbiology to
study the influence of biological factors affecting the growth of Listeria
monocytogenes in sterilized milk and raw dairy products and postulated
that it was influenced greatly by bacterial interactions and physiological
state of inoculum cells. Albert et al. (2005) described a Monte Carlo
simulation that forecasts bacterial growth and exposure assessment for
L. monocytogenes in milk. Predictive models are now essential part of risk
assessments (McMeekin and Ross 2002, Notermans et al. 2002).
The development and use of a simple tool for food safety risk
assessment has been described by Ross and Sumner (2002) in spreadsheet
software format that embodies established principles of food safety risk
assessment. Microbial predictive modeling techniques have been developed
by many workers (George et al. 1996, Murphy et al. 1996, McClure et al.
1997, Xanthiakos et al. 2006, Membre and Lambent 2008). The validation of
such models has been investigated (Murphy et al. 1996, Ross 1996, Baranyi
et al. 1999, te Giffel and Zwietering 1999). Notermans et al. (1995) suggested
the use of quantitative risk assessment for setting critical limits at the CCPs
of a HACCP system for realistic levels of control.
Several computer programs have been launched for estimating bacterial
growth and inactivation in different products such as the Pathogen Modeling
Program, PMP (United States Department of Agriculture-Agricultural
Research Service 2006), Combase Predictor (Combase 2010), the online
resource for food safety risk analysis (Joint Institute for Food Safety and
Applied Nutrition 2010), the @RISK spreadsheet software (Palisade 2012),
the Crystal Ball Oracle Risk assessment software (Oracle 2012), Seafood
Spoilage and Safety Predictor (National Institute of Aquatic Resources 2009),
and Safe Foods (2010) for helping in development of HACCP-systems or
in performing quantitative risk assessment (McMeekin et al. 2006, 2008,
Hignette et al. 2008).
International agencies and all levels of government are increasingly
relying on, or at least recognizing the need to rely on, risk assessments
for decision-making in public health protection, international trade,
and to support cost-effective resource allocation including prioritizing
research directions (Council for Agricultural Science and Technology
1994, International Life Science Institute 1996, 2005, Food and Agriculture
Orginisation/World Health Orginisation 1997) and several authors have
highlighted the need for the application of risk assessment methods to food
safety (Jaykus 1996, Kindred 1996, Lammerding 1997, Buchanan et al. 1998,
Voysey and Brown 2000). For these reasons, a web-based quantitative risk
assessment system was developed (Chen et al. 2013), which enables users
to assess, compare and rank the risks posed by multiple food-hazard pairs
at all stages of the food supply system, from primary production, through
manufacturing and processing, to retail distribution and, ultimately, to the
consumer.
Full quantitative risk assessments are provided by the three Joint
Food and Agriculture Organisation/World Health Organisation Expert
Bodies: the Joint Expert Committee on Food Additives (JECFA); the Joint
Meeting on Pesticide Residues (JMPR); and the Joint Expert Meeting on
Microbiological Risk Assessment (JEMRA). Additional risk assessments
may be provided, on occasion, by ad hoc expert consultations, and
by member governments that have conducted their own assessments
(FAO/WHO 2004). Methods for microbial food safety risk assessment

are being developed by various organizations (Food and Agriculture
Organisation/World Health Organisation 1995, PCCRARM 1997, Codex
Alimentarius Commission 1999, FAO/WHO 1997, International Life
Science Institute 1996, 2005, 2012) and, since the mid-1990s, a number of
microbiological risk assessments have been presented (Schlundt 2000).
Conclusions
The integration of HACCP plans with the development of fully quantitative
risk assessments offer very useful tools for controlling the entire farm-
to-table food chain. Moreover, the importance of dairy factory hygiene
needs to be highlighted, as well as the need for efficient controls of the
feed administered to production animals. For example, Salmonella spp. is a
food pathogen that according to the Rapid Alert System for Food and Feed
(RASFF) of the European Union (EU) is isolated in animal’s feed. Finally,
one must be aware and be able to follow changes regarding microbiological
standards as these change both in EU and globally.
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Color Plate Section
Chapter 3
Sample
Primary Enrichment Secondary Enrichment
Enrichment of Listeria in sample In Fraser Broth

with Fraser Broth Incubation: 35 or 37ºC, 48 +/– 2 hours
Incubation: 30ºC, 24+/– 2 hours
Plating
Streaking of enriched cultures on Oxford Agar plate

Incubation: 35ºC, 24 hours
Observe for black colonies at 24 and 48 hours
Streaking of enriched cultures on PALCAM Listeria Selective
Incubation: 30 and 35 or 37ºC 24–48 hours
Observe for green colonies with black halo at 24 and 48 hours
Purification
Select 5 presumptive colonies for confirmation. If well separated colonies

are not available, streak one colony on Tryptone Soya Yeast Extract Agar
Confirmation
Purified colonies are confirmed with standard tests:
• Gram staining
• Motility test
• Carbohydrate fermentation test
• D-hemolysis and CAMP test (lysis tests)
Figure 2. ISO 11290 for detection of L. monocytogenes in food samples (After: Scharlau 2007).
Figure 3. ISO 6579 Detection of Salmonella in Food (After: Scharlau 2007).

Figure 4. Procedure for Isolation and Identification of Campylobacter spp. from Milk (After:
United States Food and Drug Administration).
Figure 5. Detection of Shigella spp. in Food Samples (After: AES Chemunex 2008).
Chapter 5
SCFAs production
Enhancement of
barrier function
Improved lactose
tolerance and lowering Suppression of
of cholesterol levels pathogenic bacteria
infection
Probiotics effects on the

Immune human health Increased
modulation mucin
production
Stabilization of
Improvement of gut microbiota Production of
mineral absorption and composition antimicrobials/
production vitamins/ bacteriocins
micronutrients
Figure 2. Effects of probiotics on the human health. Microbe-microbe interactions are

represented in light blue, microbe-host interactions are represented in orange; interactions
leading to effects directed either to host and microbes are represented in pink.
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Dairy

Editor: Photis Papademas

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© 2015 by Taylor & Francis Group, LLC

No claim to original U.S. Government works

International Standard Book Number-13: 978-1-4822-9868-0 (eBook - PDF)

and the CRC Press Web site at

The book is dedicated to

Dairy products have been consumed in numerous forms over

Basic Concepts of Food

sophisticated functional properties like cheese with bacteriocins, cottage

Current Perspective Through Selected Past Events

for human consumption (Arvanitoyannis et al. 2009). Microbial spoilage

Pasteurization was defined as a heating process of not less than 61.1°C

contaminating microorganisms. The preservation of food by fermentation

of fermented milk products. Tradition and experiences tell us that some

Challenges for Starter Cultures

Milk-borne infections were relatively common before the advent of

Raw milk contains numerous microorganisms that originate from the

Challenges for Milk-borne Pathogens

Milk-borne pathogens are traditionally considered as microorganisms,

Microbiology as Tool for Food Safety and Quality Issues

HACCP systems, verification and validation of HACCP procedures, other

Challenges for Milk Quality and Safety

of microbiological concerns and the maintenance of a cold chain along

Milk Supply Chain Issues

Current Microbial Issues

Challenges for Fast and Reliable Microbiological Analytics

The industry decision-makers, facing a scenario of increasingly more

International Organization for Standardization. ISO 9000:2000. 2000. Quality management

The Microbiology of Raw Milk

Legislative Requirements for Raw Milk in the EU

Assistant Professor, Laboratory of Milk Hygiene and Technology, School of Veterinary

the most recent version of Regulation 853. A way to do this is by performing a

measurement error) on the mean. An example illustrating the “smoothing”

The geometric mean of a range of n values can be easily calculated.

Pre-Harvest Food Safety—Raw Milk Production

production. Many food-borne pathogens (e.g., Salmonella spp., Campylobacter

of protocols in “multiplex” format, enabling the identification of more

Sources of Microbial Contamination of Raw Milk

precursors supplied by the animal’s bloodstream. The (biosynthesized)

investigation of different anatomical sites of nulliparous heifers revealed

the necessity of combining culture-dependent and culture independent

Enumeration of the Raw Milk Microbial Flora

performance sheets. In recent years, automatic flow-cytometry based milk

Microbial Groups Associated with Raw Milk

analyses must be standardized to ensure accuracy of the results (Murphy

Gram-negative bacteria of concern in raw milk include genera, species or

Spore-forming bacteria are a group of microorganisms that exhibit variable

environmental stresses compared to their vegetative counterparts because

genera of Gram-negative and Gram-positive bacteria (both Bacillus spp. and

As mentioned previously, upon withdrawal from the mammary gland,

or even inhibited at low temperatures (e.g., refrigeration). The principle

The results of different studies focusing on psychrotrophs in raw milk

hydrophila. L. monocytogenes and Y. enterocolitica have been identified as the

The term “thermoduric” is used to denote bacteria (Gram-positive

survive pasteurization can affect the shelf-life of pasteurized milk. This is

Lactic acid bacteria

Mastitis (intra-mammary infection, IMI) is one of the costlier diseases of