Microbiology: KTH - Biotechnology

Food
Microbiology
Sven-Olof Enfors
KTH - Biotechnology
Stockholm 2007
Content
Chapt 1 Introduction...................................................................................1
Chapt 2. The ecological basis of food spoilage ...........................................5

2.1 The microflora ........................................................................5
2.2 The physico-chemical properties .............................................8
2.3 Chemical reactions ................................................................15
Chapt 3. Spoilage of different types of food .............................................22
Chapt 4. Food borne pathogens.................................................................38

4.1 Microbial food intoxications .................................................39
4.2 Food borne microbial infection .............................................44
Chapt 5. Food preservation.......................................................................51

5.1 Heat sterilisation and pasteurisation ......................................51
5.2 Irradiation .................................................................................
5.3 High-pressure preservation........................................................
5.4 Freezing ....................................................................................
5.5 Chemical preservatives..............................................................
Section 2-5 are not yet available. Please refer to the lecture notes
Chapt 6 Fermented foods .........................................................................64

1
Chap 1 Introduction
Living organisms are usually classified as animals, plants, algae, protozoa,

bacteria, archae or viruses. All viruses, archae, bacteria and protozoa plus the
unicellular algae and some fungi, so called micro-fungi, are collectively called
microorganisms. The microfungi can be further divided into yeast and molds, a
classification that is based on the cell morphology. Based on DNA analysis, the
group previously called bacteria is further divided into eubacteria and archae
and today the word bacteria is usually used as synonym to eubacteria.
Most microorganisms that we encounter in the normal spoilage of food belong

to the eubacteria, here called “bacteria”, yeasts and molds. When it comes to
food born diseases, also viruses, some protozoa and archae, i.e. the “blue-green
algae”, are involved.
A full species name is composed of two parts: the genus name plus the
specification defining the species within that genus. sometimes these genera are
grouped into families. This is illustrated in Table 1.1. Note that the genus name
is spelt with leading capital letter, while the species name is spelled with lower
case letters: Eschericia coli, Penicillium chrysogenum. The family, genus, and
species names should always be written with italic letters. It is common in food
microbiology literature that the full species name is not used since many species
within the same genus are discussed. Then, Bacillus sp. means one not defined
Bacillus species and Salmonella spp. means several not defined Salmonella
species.
Table 1.1. Examples of family names, genus names and species names
Family Genus Species
Enterobacteriacae Escherichia Escherichia coli
Salmonella Salmonella typhimurium
Salmonella enterica
Bacillacae Bacillus Bacillus subtilis
Bacillus cereus
Bacillus anthracis
Bergey’s Manual of Determinative Bacteriology divides bacteria into 35 groups. Groups,
families, and genera which are most relevant in food microbiology are listed in Table 2.1.
In bacterial classification, the cell morphology, the relation to oxygen, and the
Gram staining reaction are important parameters. Most common
morphological types are rods, cocci (spheric cells), and vibrioforms (short bent
rods). The Gram reaction gives information about the cell envelope. Gram
negative cells have an outer membrane outside the cell wall that prevent the
staining. Obligate aerobes require molecular oxygen for their energy
metabolism (aerobic respiration). Anaerobes have an alternative energy
metabolism that does not need oxygen. It may either be anaerobic respiration
(with e.g. nitrate as electron acceptor) or fermentation. Oxygen is often toxic
Introduction 2
for anaerobic cells. Facultative anaerobic cells use oxygen and aerobic
metabolism if oxygen is available but switch to anaerobic metabolism in
absence of oxygen. Microaerophilic cells require low concentrations of
oxygen, while normal air contact is inhibitory. Lactic acid bacteria (e.g.
Lactobacillus and Lactococcus) have an obligately anaerobic metabolism but
are still resistant to oxygen.
Table 2.1. Some of the bacterial groups (according to Bergey’s Manual of Determinative
Bacteriology) which are commonly encountered in food microbiology.
Group Description Food related organisms
nrp
2 Gram-neg., aerobic, mobile, vibrio- Campylobacter
formed
4 Gran-neg., aerobic rods or cocci Pseudomonas, Shewanella, Legionella
5 Gram-neg., facultatively anaerobic Family Enterobacteriacae

rods (e.g. Escherichia, Enterobacter,
Salmonella, Shigella, Yersinia, Erwinia)
Vibrio
17 Gram-pos. cocci Staphylococcus, Streptococcus,

Lactococcus, Enterococcus, Micrococcus,
Leuconostoc
18 Gram-pos endospore formers:

aerobic or facultatively anaerobic: Bacillus
obligate anaerobes: Clostridium
19 Gram-pos, non-sporulating rods Lactobacillus

Brochothrix
Listeria
There is a number of common groups of microorganisms that is not well

defined but often used. Some food related examples are:
”Gram-negative psychrotrophic rods”: This includes the genera Pseudomonas,

Achromobacter, Alcaligenes, Acinetobacter, and Flavobacterium.
”Lactic acid bacteria” (LAB) includes the food related genera Lactobacillus,
Lactococcus, Pediococcus och Leuconostoc.
”Coliform bacteria” is not synonymous to E. coli but includes Escherichia coli

and Enterobacter.
Introduction 3
A special problem with the microbial taxonomy is that the names often are
“date dependent” due to repeated re-classification of species. One example is
the lactic acid bacteria which previously were called Streptococcus lactis,
Streptococcus cremoris a.o. These so called “lactic streptococci” are now
referred to a new genus and galled Lactococcus lactis, Lactococcus cremoris
etc. Other previous Streptococcus spp. wich are associated with the intestines
are now called Enterococcus, while yet another group of the previous
Streptococcus genus remain as Streptococcus. When it comes to pathogenic
organisms a further classification problem is that only some strains of a certain
species may be pathogenic while other strains are harmless. An example is
Escherichia coli to which species the feared EHEC (enterohaemorrhagic E.
coli) belong. In such cases immunological or DNA analyses are required for
proper classification.
Streptococcus is a genus with species of very different impact for humans. Some of todays
Lactococcus and Enterococcus were previously classified as Streptococcus. They were then
referred to as the lactic group and the enteric group of the streptococci, respectively. A
classification of the old streptococci according to current nomenclature is:
1. Lactococci (Lactococcus lacits, L. cremoris a.o.). These organisms are often used for
fermentation of food.
2. Enterococci (Enterococcus faecalis, E. faecium a.o.) are in most cases not pathogenic,
but certain strains have been reported to cause serious infections. Such contradictions are
due to the limitation in the current nomenclature which is based on phenotypic properties.
These organisms are common in the intestinal flora. The presence of enterococci in food is
not considered to be a health risk per se, but it is used as an indication of bad hygiene and
that constitutes a risk, since other organisms of faecal origin like Salmonella may be present
For this reason enterococci are called as indicator bacteria.
3. Hemolytic streptococci. There are two types of hemolytic streptococci, and these
organisms remain in the genus Streptococcus: α-hemolytic and ß-hemolyic. the α-
hemolytic streptococci are named the viridans group and they are common on mucous
membranes in the mouth and respiratory tract and on the teeth. The ß-hemolytic
streptococci are named the pyogenes group and among them there are serious pathogens
involved in several diseases and wound infections. α-hemolytic organisms produce a
greenish discolorisation zone around the colonies on blood agar while ß-hemolytic cells
produce a clear zone.
Lactic acid fermentation is to a large extent also employed for production of

food, namely some of the fermented foods: cheese, yoghurt, fermented
sausages, and fermented vegetables like sauerkraut, pickles, olives a.o.
However, this fermentation is also involved in food spoilage. Then the type of
Introduction 4
lactic acid fermentation may be important for the taste development. Some
lactic acid bacteria mainly produce lactic acid which while others also produce
other products.
The lactic acid bacteria are grouped according to their type of lactic acid
fermentation. Homofermentative lactic acid bacteria produce mainly lactic
acid from the sugar, and no CO2. To this category belong
all Streptococcus
all Lactococcus
all Pediococcus
some Lactobacillus
Heterofermentative lactic acid bacteria produce, besides lactic acid, also

acetic acid, ethanol, CO2 and formic acid. Some can also convert citric acid
(in milk) to diacetyl. In this group are
all Leuconostoc
most Lactobacillus
5
Chapter 2 The ecological basis of food spoilage
2.1 The microflora

Food consists to a large extent of cells from plants or animals (meat, fish,
fruits, vegetables) and biological material with this origin (milk, juice, fat,
starch etc). When discussing the shelf life of food it must be done from an
ecological view. All biological material in Nature is degraded to simple
molecular components, eventually down to inorganic components. This is
called mineralization and it is a integrated part of the carbon and nitrogen
cycles in Nature (Fig 2.1) which is a prerequisite for life on Earth. If the
process is interrupted all nutrients would eventually be bound in dead
biological material. The circumstance that we select some part of this
biological material for food purpose does not change the natural destination of
the food for degradation. However, it means that our interest in a long shelf-life
of food is in conflict with the natural processes.
CO2 + N2
Light Fig 2.1. Microorganisms,

especially bacteria and fungi,
account for the main
Archae recirculation of carbon and
Animals Plants Organic Bacteria nitrogen to the atmosphere
materia Fungi from where it is adsorbed for
Algae generation of plants which
Protozoa constitute the original source
of food.
Dead organisms
The degradation of biological material is mainly catalysed by microorgansims,

which together carry an enormously diversified metabolic capacity. This is
illustrated in fig 2.2 which summarises the main paths of the biological energy
metabolism.
All energy is generated, with exception of photosynthesis, by oxidation

(combustion) of reduced substances (energy sources). Higher organisms like
animals and also some microorganisms make this by oxidation of reduced
carbon compounds, e.g. sugars. These compounds are oxidised in many steps
in which oxidised co-enzymes (e.g. NAD+) constitute the oxidant, which then
become reduced (e.g. NADH). These co-enzymes must be re-oxidised and
eventually molecular oxygen in the air is used as the ultimate oxidant for this in
the respiration. The reduced compound or energy source is called electron
donor and the ultimate oxidant (oxygen) is called electron acceptor in this
2. The ecological basis of food spoilage 6
energy metabolism. The electron donor in this case ends up as carbon dioxide
while the electron acceptor oxygen is reduced to water. this respiration process
is also coupled to phosphorylation of ADP to ATP.
Electron donors (energy source)
Re-oxidation of co-enzymes
Cred NH3 S 2- Fe 2+ H2
S 2- ATP
N2
H2O
NAD+
Ethanol
+
NAD
O2
Pyruvate NO3
- NADH
NADH
SO42-
Electron acceptors
- 2
ADP CO2 NO3 SO4- Fe 3+ H2O
Fermentation Respiration
Fig 2.2. Summary of different types of energy metabolism. Common principle is that energy
is derived by oxidation in several steps of a reduced compound (C, N, S, Fe, H2 a.o.) by
means of co-enzymes, here represented by NAD+. Re-oxidation of the reduced co-enzyme
can be achieved with respiration, in which molecular oxygen, nitrate or nitrite, and sulphate
are common oxidants (electron acceptors). An alternative to respiration is fermentation, in
which a partially oxidised carbon compound from the metabolic path (e.g. pyruvate) is used
as electron acceptor for re-oxidation of the co-enzyme and then becomes reduced, in this
case to ethanol.
When oxygen is used as electron acceptor the process is called aerobic

respiration, while the use of alternative electron acceptors like nitrate, nitrite,
sulphate etc. is called anaerobic respiration. Many facultatively anaerobic
bacteria use oxygen if it is available but can switch to anaerobic respiration
(mainly nitrate respiration) or fermentative metabolism in absence of molecular
oxygen. Of these respiration types, it is mainly the aerobic respiration and
nitrate respiration that take place in food.
Some microorganisms can use other reduced compounds than carbon

compounds as energy source. Some examples are ammonia and nitrite which
are oxidised by nitrifying bacteria, and sulphide, ferrous iron, and hydrogen
gas. These reactions are very important in the environment but seem to play a
little role in the handling of food.
One alternative energy metabolism that is common in microorganisms growing

in food is fermentation, in which a reduced intermediate is used as electron
acceptor in the re-oxidation of reduced co-enzymes. There is a number of
different fermentative metabolic pathways , named according to the dominating
products, like ethanol fermentation, lactic acid fermentation, mixed-acid

fermentation etc. Some of these reactions are detrimental for the food while
others are utilised in processing of food. The main fermentative pathways and
their role in food microbiology are further discussed in the section on
degradation of carbohydrates.
To increase the shelf-life of food means that the progress of the natural
degradation path must be prevented or delayed. However, food spoilage is not
exclusively a matter of microbial degradation. Other spoilage reactions are
dehydration, oxidation of fat, and endogenous metabolism (over-maturation of
fruits and vegetables), but microbial metabolism is the most important type of
reaction that reduces the quality of food during storage.
The common microbial food spoilage usually does not make the food unsafe or
even reduce its nutritional value, but it makes the product unpalatable. The
negative perception of food which is severely contaminated by microorganisms
is an important defence mechanisms for us, since the risk associated with
eating food increases considerably if it is spoilt by microbial metabolism. This
is due to the risk that some organisms among the spoilage flora may be
pathogens.
It is impossible to give a simple and yet comprehensive description of the

microbial spoilage of food since this is a very diversified process. What is said
in this booklet must be seen as typical and common cases, to avoid the use of
very large lists of microbial names. When, for instance, it is stated below that
the activities of Pseudomonas spp. limits the shelf-life of refrigerated fresh
meat and fish, it means that most investigations - but not all- show that
Pseudomonas species dominate the spoilage flora but there are usually a
number of other species involved, usually in the group "psychrotrophc, Gram-
negative rods". Another problem is that it is not always sure that the
dominating microflora is responsible for the main spoilage reactions. An
example is that it may require 10 times more Achromobacter cells than
Shewanella cells to make fresh fish unacceptable in taste. Another example is
the lactic acid bacteria of the homo-fermentative type which
have a relatively low impact on the spoilage due to the domination of lactic
acid in the metabolic products.
Most food raw material have a primary flora of microorganisms which origins
from the production environment. During the continuing processing of the raw
material and additional contamination (or secondary) flora infects the food. It
may come from the air, especially from dust in the air, from process water,
process equipment, or from humans that handle the food. During the
subsequent storage of the product the different species develop differently
depending on the environment. The primary plus initial contamination flora
usually is in the order of 103 cells/cm2 of solid foodstuff if the quality is very
good (see table 2.1). Depending on the conditions for growth some of these
species will grow exponentially (see Fig 2.3) up to concentrations above 107/
cm2 (or per gram). The finally dominating microflora may origin from the
primary or the contamination microflora. When the number of cells exceed 107
to 108 cells/cm2 (or per gram) the product usually develops bad smell and the
microflora is then called the spoilage flora. It is the nutritional (for
microorganisms) properties of the food and the environment (temperature,
water activity, pH etc.) that determine which species will dominate the
spoilage flora, their metabolic products and how fast this spoilage process will
proceed. In the sections below the environmental parameters will be discussed
and in Chapter 2.2 the most important chemical reactions of food spoilage are
presented.
Table 2.1 Typical size of different food microfloras at good

production hygiene
Product Microbial concentration
Internal tissues of
healthy animals 0
Plant surfaces
Fish skin Primary flora ≈ 103 cells/ cm2
Egg shell
Milk Contamination flora ≈ 103 cells / ml
Meat Contamination flora ≈ 103 cells / cm2

Fish fillet
Spoilage flora on ≈ 107 - 108 cells / cm2 or gram

most food types
2.2 The physico-chemical properties

The possibility of the food to serve as a substrate for microbial growth depends
on a number of physical and chemical properties:
- Temperature - Mechanical barriers

- Water activity (aw) - Metabolisable energy sources
- pH and buffer capacity - Metabolisable nitrogen sources
- Oxygen concentration and transfer - Chemical inhibitors
Temperature. The temperature influences of course the rate of growth, and

thereby the shelf-life of the product. But it has also an impact on selection of
species in the microflora. This is probably the explanation why reduction of
temperature in the refrigeration range (0-8°C) has such a dramatic influence on
the growth rate, as demonstrated by experimental data Fig 2.3. The organisms
growing at 20°C have an initial generation time of about 4.8 h, while the
generation time at 0°C is about 25 h, and represents psycrotrophic organisms.
10
Log
9 10°C 4°C
(cfu) 20°C 8°C
7 0°
C Fig 2.3. Influence of
5 temperature on the total
bacterial count (colony
forming units, cfu) on
3 fresh meat. The dotted
line indicate the typical
1 level of spoilage. Note
that the growth initially
0 150 is exponential.
t(d)
Microorganisms are usually classified in four groups according to their

relationship to temperature. Fig 2.4 illustrates this. In general the mesophiles
have the highest maximum growth rate and an optimum temperature in the
range of 30-40 °C .
Relative growth rate
Mesophiles
Psychrotrophes Thermophiles
Psychro-
philes
0 10 20 30 40 50 60 °C
Fig 2.4. Schematic illustration of the temperature dependence of the growth rate of different
classes of microorganisms. There are no general and exact limits for the temperature ranges.
The psychrophiles have the lowest maximum growth rate, but can grow quite
fast at refrigerator temperature. Thermophiles have an optimum above 40°C
and some can grow even above 100°C. The psychrotrophic organisms
constitute an important group in food microbiology. They grow well in the 20-
35 °C range like the mesophiles but they can also grow relatively fast at
refrigerator temperature.
The growth rate of microorganisms is expressed either with the generation time
(tg, h) or with the specific growth rate constant (µ, h-1). The generation time is the
time needed to double the amount of cells. The specific growth rate expresses the
rate of cell formation per cell. The correlation between these parameters can be
derived from a mass balance of the cell number:
dN
= µN
dt
where N is the number of cells, µ (h-1) is the specific growth rate and t (h) is
time. Integration with N0 cells at t = 0 and Nt cells at time t, gives:
! ln(N t )
= µt
ln(N 0 )
After one generation time, tg is the cell number 2N0. Insertion of this in the
equation above gives::
! ln(2N 0 )
= µt g
ln(N 0 )
from which the correlation between generation time and specific growth rate is
obtained:
! ln(2) 0.69
= tg !
µ µ
Water activity (aw) This is one of the main parameters that determines how fast
and by which type of organisms the food is spoilt. The water activity of food
can be determined as the water vapour pressure (pH2O) in a closed vessel in
aw = pH2 O
pH2 O*
which the product is enclosed in relation to the water vapour pressure of pure
water (pH2O*):
For a water solution with low molecular weight compounds (e.g. salt or sugar)
the water activity is approximately:
aw = nw
nw + ns
where
nw = number of moles water

ns = number of moles of dissolved molecules
Some common food components that reduce the water activity are:
- Ions (e.g. salts)

- Dissolved molecules (e.g. sugars)
- Hydrophilic colloids (e.g. starch)
- Ice
The water activity is a measure of the availability of the water for the
microorganisms. It is not only the water concentration that determines the
water activity but also the capacity of the material to bind water. This is
illustrated in Fig 4.2 which shows sorption isoterms for some materials with
different water binding capacity. Cellulose get a relatively high water activity
and starch a lower water activity at the same water concentration.
Water concentration (%)
Fruit
30
reaktionshastighet
Relative rate
Lipid
20 oxidation
Starch
Meat
Rel
10 Lipolysis
Molds
Cellulose Proteolysis
Yeast Bacter
0 s iar
0 0.3 0.6 0 0. 0. 0. 0. 1
0.9 Water activity 2 Water
4 activity
6 8
Fig 2.5. Sorption isoterms for different Fig 2.6. Schematic view of how the aw
materials show that aw is not the same as influences the rate of enzyme reactions
water concentration and microbial growth.
Most biochemical reaction rates decline with declining water activity.

However, the sensitivity to reduced water activity varies, as illustrated in Fig
2.6. Among microorganisms, molds and yeasts are generally more resistant to
low aw and many enzymes keep their activity at even lower water activity. But
there are many exceptions to this rule. Three types of microorganisms prefer
reduced water activity. These are osmophilic (sugar preferring) yeasts,
xerophilic (drought preferring) fungi, and halophilic (salt preferring) bacteria.
These organisms not only grow faster than most other organisms at lower
water activity, but they also prefer a reduced water activity. See further in
Table 2.2.
Table 2.2 Examples of typical minimum water activity for growth of

some microorganisms and corresponding aw in food.
Organism Min aw Food Food aw
Milk, fish, meat 0.99
Pseudomonas 0.97
E. coli 0.96 Sausage, 7% salt 0.96
Clostridium 0.95
Brochothrix thermosphacta 0.94
Bacillus 0.93 Ham, 12% salt 0.93
Lactobacillus 0.93
Streptococcus
Lactococcus 0.93
Micrococcus
Jam, 50% socker 0.91
Hard cheese, bread
Herring, 20% salt 0.87
Staphylococcus 0.86
Yeasts in general 0.85
Molds in general 0.80
Halophilic bacteria 0.75
Grains w.10% water 0.7
Xerophilic molds 0.65
Osmophilic yeasts 0.60 Dried fruits, 15% 0.6
water
Dry milk, soups etc < 0.5
Dry bread
Halophilic = salt preferring; xerophilic = drought preferring;
osmophilic = preferring high osmotic pressure (of sugar).
The water activity of food has a large impact on the rate of spoilage but also on
the type of spoilage since it exerts a selection pressure on the microflora. Many
of the common food spoiling microorganisms are very sensitive to reduced
water activity and the growth rate of these declines rapidly when the water
activity drops below the optimum, which is close to 1 for Pseudomonas and
Enterobacteriacae. Many conclusions can be drawn from Table 2.2.
Pseudomonas, which dominate the spoilage of refrigerated fresh meat and fish
does not create problems in sausages and salted herrings or if meat and fish is
dried. Such products get a spoilage flora of more low-aw resistant organisms
like lactic acid bacteria, molds and yeasts. The table also explains why molds
are the main problem during storage of cheese and bread, and why dried
products like flour, grains, dry milk are not attacked by microorganisms at all,
provided they are stored in a dry environment so they do not absorb water. It is
also obvious that the toxin producing Staphylococcus, which are commonly
present on human hands, constitute a threat at "smörgåsbord" and other buffets.
Note that the figures in Table 2.2 are collected from different sources. The
actual minimum aw for and organism depends on other parameters like pH,
temperature, and nutritional conditions. Thus, such data are only approximate
and indicative of relative sensitivities.
pH is another parameter of large impact for the shelf-life of food. The pH

influences both the growth rate and the type of organisms that will dominate
during storage. Most food products have pH below 7 (Table 2.3) and most
food spoiling bacteria require a relatively neutral pH, with the large exception
lactic acid bacteria (Table 2.3). The lactic acid bacteria grow well down to a
pH in the range 4-5. In Nature there are many examples of bacteria that can
grow at very low and very high pH values, but these organisms are not relevant
in food microbiology. Comparing these tables give one reason why fruits and
many vegetables mainly are degraded by molds and sometimes yeasts.
Table 2.3. Typical pH-values of common food products

Shrimps 7 Cabbage 5.5
fish 6.7 Potatoes 5.5
Corn 6-7 Tomatoes 4.2
Milk 6.5 Orange juicee 4
Melon 6.5 Yoghurt 3.5
Butter 6.2 Apples ≈3
meat 5.1-6.4 Lemon ≈2
Cheese 5.9
Oysters 5-6
Table 2.4. Generalised picture of pH ranges for microbial growth

pH range pH optimum
Most food spoilage bacteria 4-9 7±1
Molds 2 - 11 5±1
Yeasts 2.5 - 7 4-5
Oxygen availability and the diffusion rate of oxygen are important parameters
that influence the type of metabolism. The rate of growth may be slower in
anaerobic than in aerobic environments but on the other hand is the anaerobic
metabolism associated with much more detrimental products for the shelf-life.
An exception to this is the lactic acid bacteria which have anaerobic
metabolism but usually produce less ill-smelling compounds than most other
anaerobic organisms. Anaerobic conditions are a prerequisite for growth of the
dangerous pathogen Clostridium botulinum, and therefore special precautions
must be taken when storing some types of food under anaerobic conditions.
The mechanical structure may be important for the shelf-life of food. On

whole meat bacteria grow only on the contaminated surface, where they dwell
on the exudate, i.e. the glucose and amino acid rich liquid that leaks from
damaged cells and blood vessels. If the meat is minced this surface and
exudate increase enormously which leads to much higher microbial activity
and growth in the inner anaerobic parts of the minced meat. Fruits and
vegetables are protected from microorganisms by the outer shell or skin and by
the gelatine-like pectins which cements adjoining plant cells together. Outside
the skin/shell the water activity is low and there is a lack of nutrients for
growth of the contaminating microflora. But if the product is mechanically
damaged or if the organism can produce pectinases the nutrients become
available and the spoilage rate increases. It is mainly molds that produce
pectinases, and this, together with the often low pH of these products, explains
why this type of food often is spoilt by molds. Yeasts that also grow well at
low pH often come as a second infection after the initial mold attack. Erwinia
is one of few bacterial genera with pectinase producing species that attack
plant material.
Antimicrobial substances. Many food raw materials, especially vegetables and

other food with plant origin, contain antimicrobial compounds which hamper
the microbial growth. Some examples are listed in Table 2.5.
Many microorganisms produce antimicrobial substances (antibiotics) and in

food there is often growth of lactic acid bacteria some of which produce known
antibiotics (Table 2.6). Nisin is a polypeptide antibiotic naturally produced in
fresh (unpasteurised) milk by Lactococcus lactis which belong to the normal
flora transmitted during milking. Other antibiotics, like acidocin B and
reuterin are mainly produced in processed milk if it is inoculated with the
producing organism.
Table 2.5. Some examples of naturally occurring antimicrobial substances.

Food Inibitor
Horseradish Allyl isothiocyanate
Onion and garlic Allicin and diallylthiosulphinic acid
Tomato Tomatin
Radish Raphanin
Lingonberry Bensoic acid
Oregano Eteric oils
Table 2.6. Antibiotic substances produced by lactic acid bacteria

Antibiotic Organism
Nisin (in milk) Lactococcus lactis
Salvaricin Lactococcus. salvaricus
Acidocin B (fermented milk) Lactobacillus acidophilus
Reuterin (fermented milk) Lactobacillus reuterii
Some definitions of antimicrobial compounds
Antibiotics Microbial product with an antimicrobial (bactericide/

fungicide or bacteristatic/fungistatic) activity and which
have low toxicity to humans. If the latter is not added to
the definition most mycotoxins would also be classified as
antibiotics.
Probiotics Microbial cultures, mainly lactic acid bacteria, which are

consumed for stabilisation of the intestinal microflora of
humans or animals. They are believed to act by
establishing on the intestinal mucouse membrane and
prevent, possibly by production of antibiotics, the growth
of other disturbing organisms.
Prebiotics Components (oligosaccharides) in the food that are not

digested in the intestines but are assumed to promote the
beneficial microflora.
Bacteriocines Bacterial proteins or peptides with bactericidal effect

mainly on related species and strains.
bactericide = bacteria killing; fungicide = fungi killing;

bacteri/fungi-static = inhibiting growth of bacteria/fungi.
2. 3 The chemical reactions

The most important chemical reactions involving food components during
microbial spoilage of food are:
- Degradation of N- compounds
- Degradation of fat
- Degradation of carbohydrates
- Pectin hydrolysis
Degradation of nitrogen compounds

The dominating and usually the first reaction is oxidative deamination of
amino acids:
deaminase
amino acid + O2 NH3 + organic acid
This reaction is assumed to be the dominating spoilage reaction in refrigerated

fresh meat and fish. The amino acid is then used as energy source by splitting
off the amino group with an oxidative deaminase, which leaves the organic
acid that enters the energy metabolism.
Proteolysis. One would think that proteolysis would be a common spoilage

reaction. However, most microorganisms do not secrete proteases and those
who do usually do not produce them until there is a lack of nitrogen source. In
later stages of spoilage, however, proteases and peptidases may degrade the
protein:
proteinase peptidase
Proteins peptides amino acids
Many peptides have strong taste, bitter or sweet, and this sometimes
contributes to the spoilage. These reactions are also important for the
development of characteristic tastes of many fermented products (Chapter 6).
Putrification is a set of anaerobic reactions with amino acids which results in a

mixture of amines (e.g. cadaverine, putrescine, histamine), organic acids, and
strong-smelling sulfur compounds like mercaptans and hydrogen sulphide:
Amines
Anaerobic Organic acids
amino acids
metabolism S-compounds
Indol
Many of these compounds have terrible odour. Cadaverine, putrescine, and

histamine are formed by decarboxylation of lysine, ornithine, and histidine,
respectively (Fig 2.6) While cadaverine and putrescine in food probably have
no health impacts, only spoil the food due to the odour, histidine causes
intoxication problem since it may induce a serious anaphylactic shock. This is
often associated with microbial activity in histidine rich fish products (e.g. tuna
fish).
Putrification is typical for microbial degradation of meat and other protein rich
foods at higher temperature (> 15°C). Bacillus and Clostridium species may
then grow fast and rapidly make the food toxic, but under refrigeration
conditions these organisms are usually not active and under these conditions
the oxidative deamination spoils the food before the putrification becomes
dominating.
Fig 2.6. Histamine, cadaverine and other amines are formed by decarboxylation of amino
acids.
Reduction of trimethylamine oxide (TMAO). Marine animals may contain high

concentrations of trimethylamine oxide, which is believed to have a function in
protecting proteins from denaturation at low temperatures, high pressure and
high osmolarity. Certain microorganisms, like Pseudomonas and Shewanella,
can utilise TMAO as electron acceptor in anaerobic respiration:
CH3 CH3
TMAO-
H3 C - N = O H3 C - N
reductase
CH3 CH3
TMAO TMA
This results in formation of trimetylamin (TMA) which gives a typical "fishy"
smelling. TMA can also be formed by enzymatic hydrolysis of lecithin.
Degradation of fat
When fat is degraded it becomes rancid and this rancidification depends on
many different reactions which are not all well known in detail. One attempt of
classification is shown in Fig 2.7. The hydrolytic rancidification results in free
fatty acids (FFA) and glycerol. Our organoleptic tolerance of free fatty acids
depend on the type of the fatty acids, especially the carbon chain length. Up to
15% FFA is said to be acceptable in beef, which has long fatty acids, while
only up to 2% is acceptable in olive oil. If very short FFA are formed, e.g.
butyric acid from butter, only traces of the acids can be accepted. The
hydrolysis can be spontaneous but then at a very low rate, while it may proceed
fast if lipolytic enzymes from the foodstuff or from the contaminating
microflora are present.
Fig 2.7. Different types of rancidification reactions.
The oxidative rancidification requires presence of oxygen. Autooxidative

rancidification is catalysed by metal ions and is accelerated by light. In this
process peroxide radicals (ROO*) are produced and they react with other fatty
acids to form instable hydroperoxides (R-OOH) which later on decompose to
aldehydes and ketones which give the rancid taste (Fig 2).
Fig 2.8. Autooxidation of a fatty acid (RH) results in aldehydes and ketones.
The chain reaction is initiated by a radical (R*) which is produced from the
fatty acid under catalysis of Fe2+ and other metal ions and light. The radical
reacts with molecular oxygen to form a peroxide radical (ROO*).
Antioxidants in food are used to scavange the peroxide radical that otherwise
continuous the chain reaction by reacting with another fatty acid to produce a
new radical (R*) and a hydroperoxide (R-OOH). The hydroperoxide is
instable and decomposes to ketones or aldehydes.
ß-oxidation is the common metabolic route for degradation of fatty acids and
each cycle results in generation of one acety-çoA and a new fatty acid with 2 C
shorter C-chain (Fig 2.9). Some microorganisms have a side reaction in the last
step of the ß-oxidation cycle, by which very aromatic methyl ketones are
formed and may contribute to bad taste (rancidity) of the food.
Fig 2.9. Methyl ketones may be formed as by-products in the ß-oxidation of fatty acids.
Lipoxydaser are common enzymes in plant and animal tissues and they are also
produced by some molds. The enzyme oxidises unsaturated fatty acids with
cis-cis 1-4 pentadien configuration to hydroperoxides which decompose
spontaneously to ill-tasting aldehydes and ketones. This configuration occurs
in linolic and linolenic acids in plants and in arachidonic acids in animal
tissues. To prevent this type of rancidification during some vegetables , e.g.
frozen spinach and peas, are heat treated to inactivate the plant enzyme. These
aldehydes and ketones are not always unwanted products in food. They are
also important ingredients in certain types of cheeses (see Chapter 6,
Fermented foods).
Degradation of carbohydrates
Microorgansims growing on food mainly use various sugars as carbon- and
energy source. Under aerobic conditions the energy source is combusted to
carbon dioxide and water but under oxygen limiting or anaerobic conditions
many species switch to fermentative metabolism which results in various

fermentation products (see Fig 2.2). The most common fermentative pathways
are listed in Table 2.7.
Table 2.7. Common fermentation types

Fermentation type Products
Alcohol fermentation Ethanol, CO2
Homofermentative lactic acid fermentation Lactic acid
Heterofermentative lactic acid fermentation Lactic acid, Acetic acid, Ethanol,CO2
Propionic acid fermentation Propionic acid, Acetic acid, CO2
Butyric acid fermentation Butyric acid, Acetic acid, CO2, H2
Mixed-acid fermentation Lactic acid, Acetic acid, CO2, H2, Ethanol
2,3-butanediol fermentation CO2, Ethanol, Butanediol, Formic acid
Of these fermentation types, it is the butyric acid, mixed acid and butanediol
fermentations that are most detrimental for the food taste. The mixed-acid and
butanediol fermentations are typical for organisms in the Enterobacteriacae
family. Butyric acid fermentation is common among saccharolytic
Clostridium. Lactic acids is mainly produced by lactic acid bacteria but it
proceeds also under aerobic conditions since these bacteria are relatively
indifferent towards oxygen although they always use the fermentative
metabolism. A more detailed picture of the different fermentation pathways
from glucose via the common intermediate pyruvate is shown in Fig 2.10.
Glucose
Lactic acid fermentation + Ethanol fermentation
ATP NAD
NADH
+ +
NAD NAD
Lactate Pyruvate Acetaldehyde Ethanol
Oxaloacetate AcetylCoA Formate AcetylCoA + CO2 +H2
ATP ATP
ATP +
NAD
Acet- Acetate
+ acetylCoA
NAD Acetate Ethanol H2 CO 2
Succinate Acetoin Mixed acid fermentation ATP
+
NAD
+ Butyrate Acetone
NAD
Propionate Butandiol
+
+ NAD
NAD
Propionic acid Butandiol
fermentation fermentation Butanol 2-propanol
Butyric acid fermentation
Fig 2.10 Summary of the six main fermentative pathways. The main end product are
emphasised by frames. Sites of co-enzyme generation and ATP formation are indicated.
Pectin hydrolysis
Pectins are carbohydrate polymers mainly composed of partially methylated
poly-α-(1,4)-D-galacturonic acid. They are present in all fruits and vegetables
where they function as a glue between the plant cells and gives mechanical
rigidity. During ripening of fruits and berries indigenous pectinases are
synthesised or activated and start hydrolysing the pectins which makes the
structure soft. Also mechanical damages on fruits and vegetables activate the
pectinases and this opens for microbial attack. However, also some
microorganisms produce and secrete pectinases. Many molds have this
capacity and among bacteria plant pathogens in the genus Erwinia also
produce pectinases which serve as tools for the microbial invasion resulting in
soft rot.
Slime production
Microbial spoilage of meat and fish sometimes results in a slimy surface layer,
composed of microbial polysaccharides. Such polysaccharide slime can also
appear as a result of microbial growth on vegetables, wine and vinager. A
special case of slime formation is the so called ropiness of bread which is
caused by B. subtilis which may survive the baking as spores and then
germinate and grow if the water activity is high and the temperature kept too
high after the baking. Usually the slime formation on cold-stored fresh meat
comes after the meat has become unacceptable due to smelling. Some species
of lactic acid bacteria produce polysaccharides and this sometimes utilised in
various fermented milk products to give a higher viscosity (yohurt, Swedish
långmjölk). However, the viscosity of yoghurt is mainly caused by protein
precipitation due to low pH.
22
Chapter 3. Spoilage of different types of food
From a microbiological viewpoint it is convenient to classify different types

of food according to the conditions they provide for microbial growth which
gives an indication of the food shelf-life. One such classification is shown in
Table 1.3.
Food properties Exampel Protection

Water-rich Meat None
Protein-rich Fish
Relatively neutral pH Milk
Cooked food
Water-rich Fruits Low pH

Protein-poor Vegetables Inhibitors
Relatively sour Root-fruits Mechanical structure
Water-poor Grains
Flour Low aw
Bread
See Chapter 6 Often low aw + low pH

Fermented food
Microbial competitors
Microbial inhibitors
Salted Low aw
Preserved food
Pickled Low pH
Smoked Low pH, low aw, inhibitors
Sterilised No microflora
Pasteurised Small initial microflora
Often in combination with
chemical preservatives
3.1 Water and protein rich foods

Fresh meat, fish and milk belong to this category. They have a water activity
close to 1, contain lots of energy sources and other nutrients for microbial
growth, are relatively pH neutral and contain no or little microbial inhibitors.
If not treated by preservation methods these food stuffs are spoilt by microbial
activity in a couple of days or shorter at room temperature. Therefore these
products are always stored at refrigerator temperatures to reduce the rate of
microbial growth.
At a first look one would expect that eggs should belong to this category, but
for obvious reasons Nature has build a sophisticated system that keeps the egg
3. Spoilage of different types of food 23
protected from microbial attach for several weeks at room temperature. This
is described in Fig 3.11.
Meat
At the moment of slaughter, the breathing and aerobic respiration cease
abruptly but the cells in the body tissues continue their metabolism for several
hours and these reactions are important for the later microbial development.
During the post mortem metabolism glucose is metabolised through the
glycolysis, but due to lack of oxygen, lactic acid is produced from the
pyruvate. Glycolysis generates two ATP molecules per glucose molecule,
which is much less than in the aerobic respiration but still enough to prevent
the formation actomyosin complex in the muscle (See Fig 3.1). However, the
formation of lactic acid reduces the tissue pH from neutral towards pH 5.5-6.
Eventually the low pH inhibits the glycolysis and the ATP generation ceases
which results in formation of actomyosin from the components actin and
myosin which are kept dissociated by ATP. Formation of actomyosin results
in muscle contraction and it is observed as rigor mortis.
Fig 3.1. The post mortem glycolysis generated protons and ATP. The ATP forces the
equilibrium between actin + myosin and the actomyosin towards the dissociated state.
When pH has dropped too much the ATP generation through glycolysis ceases and the
equilibrium shifts towards formation of the actomyosin complex, which results in muscle
contraction, i.e. rigor mortis. After some time (Table 3.2) the actomyosin complex is
hydrolysed by proteases (cathepsins and calpains).
The time course of this most mortem metabolism and the final pH depends on
the animal species (Table 3.1). The final pH is considered important for the
shelf-life. This pH is not only dependant on the animal species but also on the
condition of the animal before slaughtering. An animal that has been stressed
has a lower blood glucose level and the post mortem metabolism can then
cease due to glucose limitation rather than pH inhibition and the result is a
meat with higher pH. Since the dominating spoilage flora on refrigerated fresh
meat and fish is Pseudomonas (and other Gram negative psychrotrophic rods)
and these organisms quite sensitive to pH below about 5.5-6, the final pH of
the meat is considered important for the shelf-life.
Table 3.1. Typical pH of meat from different animals and lenth of rigor mortis.
Animal type Rigor mortis final pH
Cow 10-20 h 6 - 5.5
Swine 4-8 h 6
Chicken 2-4 h 6.4 - 6
Fish min-h (longer on ice) 6.8 - 6.4
The meat contains many nutrients for the microorganisms (Table 3.2) which
only grow on the exudated from damaged tissue. Furthermore, it is only on
the surface of meat the cells grow, unless the meat has been mechanically
perforated or minced. Therefore, the microbial count is expressed as cells/
cm2 or cfu/ cm2, where cfu means colony forming units on agar plates.
Table 3.2 .Example of microbial nutriens in meat exudate

Component Concentration g/Kg
Lactic acid 9
Creatine 5
Inosine 3
Carnosine 3
Amino acids 3
Glucose-6P 1
Nucleotides 1
Glucose 0.5
Meat is usually stored at refrigerator temperature which gives a shelf life

around one week, however longer for beef, but this shelf life depends strongly
on other factors like the hygiene during slaughter and handling of the meat. It
is often assumed that also a low pH after rigor mortis is important. Under
these conditions the microflora at spoilage is dominated by Gram negative
psychrotrophic rods of the genera Pseudomonas, Achromobacter, Alcaligenes,
Acinetobacter och Flavobacterium. These organisms are often obligate
aerobes. Many investigations report Pseudomonas, and especially P. fragi as
common spoilage flora on fresh cold-stored meat. there are also reports that
state that this type of microflora on meat is universal and not dependent on
which animal the meat comes from. The flora is always dominated by
bacteria, only small amounts of yeasts and molds are developoing under these
conditions.
During storage, the bacteria initially grow exponentially, sometimes after a

lag phase that can be due to shift of domination microflora. The cell
concentration increases from about 103 cells/cm2 on a meat of highest
hygienic quality towards 107 - 108 cells /cm2. Then the spoilage becomes
apparent through bad odour, and sometimes discolorisation and slime
formation. Typical growth curves on refrigerated pork and chicken are shown
in Fig 3.2 It is apparent that the shelf life of such products depends on the
growth rate, which is mainly determined by the temperature, and the initial
amount of cells that can grow, which is strongly related to the hygiene during
and after slaughter.
2
log N/cm slime
slem
8 odour odör
7 chicken
kyckling
6
pork
griskött Fig 3.2. Example of microbial
5
4 growth "total aerobic count"
3 during storage of fresh pork and
2 chicken meat at refrigerator
1 temperature.
0 2 4 6 8 10
Tid (d)
Days
According to one hypothesis, the shelf-life of fresh meet depends on the

availability of glucose at the surface. As long as glucose is available, this is
the main energy source for the bacteria, but when it is exhausted, other
organic compounds, e.g. amino acids provide the energy. When aminoacids
are used as energy source, ammonia is split off by oxidative deamination and
produces bad odour. This is supported by the data shown in Fig 3.3 which
shows how the glucose gradually is exhausted at the surface when the
microflora approaches the spoilage stage. It can also be an explanation to why
meat from stressed animals has a lower shelf-life, since short intensive stress
before the slaughter may reduce the blood glucose concentration.
.
400 400
N*10-7=
2.7
Glucose (µg/g)
Fig 3.3. Glucose concentration

gradients and microflora
6.3 development during cold storing of
fresh meat. At N=32*107 cm-2 the
32 meat was classified as spoilt and this
coincides with glucose exhaustion at
110 280 the surface.
0 0
0 20
Distance from surface (mm)
Carbon dioxide and vacuum packages

Vacuum packaging of meat, both fresh and cured meat, increases dramatically
the shelf-life. It was originally believed that the main mechanisms of vacuum
packaging is that oxygen is removed and that this hampered the main spoilage
flora. However, storing meat under nitrogen atmosphere does not improve the
shelf-life. Fig 3.4 shows that the microflora develops slower, but the
fermentative metabolism that dominates under anaerobic conditions produces
more off-flavour, unless it is a lactic acid microflora that dominates. The
figure also shows that storing the meat under CO2 atmosphere significantly
reduces the rate of microbial growth. When the CO2 packed meat was opened
subjected to air, the microbial growth rate immediately increased.
logN / cm2
9
air
Luft
8 N2
Kväve
7 air
Luft
Fig 3.4. Influence of
the gas atmosphere on
6
CO2 the growth rate of
air
Luft microorganisms on
5 refrigerated fresh pork
meat. Some of the CO2
4 stored samples were
opened and further
CO exposed to air, as
3 indicated in the CO2-
0 82 16 24 32 plot.
Tid (dagar)
When the composition of the microflora was investigated under these

conditions it became clear that the atmosphere exerts a selecting pressure, see
table 3.3. In air the dominating microflora usually is Pseuomonas. These
organisms are obligate aerobes or use anaerobic respiration in absence of
oxygen. In nitrogen atmosphere different species from the Enterobacteriacae
family dominate. These organisms possess a strong fermentation capacity
with ill-tasting products from the mixed-acid fermentation or 1,3 butandiol
fermentation pathways. The CO2 not only reduces the rate of growth on the
meat, but it also exerts a selective pressure that favours growth of
Lactobacillus, which with their lactic acid fermentation have less impact on
the spoilage than the Pseudomonas .
Table 3.3. Dominating spoilage flora on cold stored pork in different atmospheres.
O2 N2 CO2 Pseudomonas Entero- Aeromonas Brochothrix Lactobacillus
% % % bacteriacae
20 80 +
100 +
80 20 +
80 20 + +
10 90 + +
100 +
The selective pressure of CO2 is explained by the different inhibitory effect

this gas has on various microorganisms. Pseudomonas belongs to the most
CO2 sensitive bacteria while lactic acid bacteria are very resistant to this gas.
Most molds are very sensitive while yeasts are very resistant to CO2.
Fig 3.5 Relative sensitivity of

microorganisms to inhibition of growth
by carbon dioxide.
When fresh meat is vacuum packed after slaughter, which is often the case for
meat that is to be stored for tendering, CO2 is released from the tissues during
the first day and since the vacuum package plastic film has a low gas
permeability and the gas headspace is removed by the vacuum, the partial
pressure of CO2 raises rapidly and exert a CO2 protecting function. Also the
shelf-life promoting effect of vacuum packing of cured meat products is
similar but in that case it is the metabolic activity of the microflora that
produces the CO2. The strong protecting effect of CO2 on meat and meat
products have
The selective pressure of the gas atmosphere, can be explained by the

relationship of the dominating types of microorganisms to oxygen and CO2,
as summarised in Table 3.4
Table 3.4 Some characteristics of the organisms that dominate the

spoilage flora on cold-stored meat in different atmospheres.
Organism Properties
Pseudomonas Aerobic
Very CO2-sensitive
Enterobacteriaceae Facultative
Intermediate CO2-sensitivity
Aeromonas Facultative
Intermediate CO2-sensitivity
Brochothrix thermosphacta relatively CO2 resistant
Facultative
Resistant to low aw
Lactobacillus Very CO2-resistant
Indifferent to oxygen
The inhibitory effect of CO2 seems to be synergistic with low temperature in

storage of meat as shown in Fig 3.6. This may partly be due to the increasing
solubility of CO2 at declining temperature. Even if CO2 dissolves in water and
partly is hydratized and dissociates to bicarbonate, it is the gaseous CO2
molecule that has the inhibitory effect. This also means that the effect is
strongly pH dependent and declines with increasing pH.
°C
6 -2
Fig 3.6. Time needed to reach 10 cells cm on pork meat stored at different temperatures
in air or in CO2.
The antimicrobial effect of CO2 on many spoilage organisms has been utilised
also for direct applications in which food is stored under so called controlled
atmosphere in which carbon dioxide is the growth inhibiting compound and
oxygen often is present to avoid anaerobic metabolism or discolorisation.
Vacuum packing of food is applied also for other reasons than to provide
microbial inhibition via CO2. One common reason for vacuum packing is to
prevent oxidative rancidification or other oxidising reaction with molecular
oxygen (e.g. peanuts), or to prevent evaporation of flavour compounds (e.g.
coffe). When cheese is packed in vacuum tight plastic films it is likely that a
mold inhibiting CO2 atmosphere develops, but on the other hand, molds are
obligately aerobic so the lack of oxygen is also a mold-protecting mechanism.
Fish. The post mortem metabolism is important also in the fish. An important
reaction is the degradation of ATP which results in a transient accumulation
of inosine monophosphate (IMP). this compound is considered to contribute
to the sensoric appreciation of "fresh fish" taste. IMP is also utilised as a
flavour improving additive in the food industry, in analogy with the meat
flavour enhancing effect of glutamine.
Fig 3.7. During the post mortem metabolism in the fish tissue inosine monophosphate
(IMP) is transiently accumulated.
This metabolism has been utilised to develop a "fish-freshness" biosensor (Fig

3.8). Since the absolute level of the IMP varies much between fish sorts and
even between individuals, it is not sufficient to analyse only the concentration
of IMP . Instead the ratio IMP/(IMP + inosin + hypoxanthine) is used as a
fish-freshness index. The enzymatic biosensor measures the oxygen
consumption catalysed by xanthine oxidase. If only xanthine oxidase is
present in the analysis, the oxygen consumption represents the concentration
of hypoxanthine. If also the nucleotide phosphorylase is present, the oxygen
consumption represents the concentration of hypoxanthine + inosine. By
including also the 5'-nucleotidase the oxygen consumption also includes the
IMP.
Fig 3.8. Principle of a "fish-freshness" biosensor based on analysis of the degradation of

IMP degradation. The oxygen consumption catalysed by xanthine oxidase is analyses with
or without the enzymes nucleotide phosphorylase and 5'-nucleotidase and a index that
represents the concentration of IMP In reölation to the sum of the metabolites is calculated.
The microbial spoilage of refrigerated fresh fish has large similarities with
that of fresh meat. Pseudomonas is often dominating in the spoilage flora (Fig
3.9). A similar organism, Shewanella putrifaciens (previously called
Pseudomonas putrifaciens or Alteromonas putrifaciens) is another spoilage
organism specifically associated with marine fishes. It has the capacity to
produce both hydrogen sulfide from cysteine and trimetylamine (TMA) by
anaerobic respiration with TMAO as electron acceptor. Due to this capacity to
produce bad odour the fish may be spoilt at 10 times lower total microflora if
Shewanella putrifaciens dominates.
Fig 3.9. Distribution of
spoilage organisms on
refrigerated fresh fish.
Aeromonas is mainly
associated with fresh-
water fishes and
Shewanella with
marine fishes.
Milk is a very good substrate for microbial growth. However, it is protected

by several antimicrobial mechanisms that favour the development of lactic
acid bacteria if the temperature is not too low. The lactoperoxidase system is
one of these antimicrobial systems in milk (Fig 3.10). Milk contains the
enzyme lactoperoxidase and small concentrations of its substrate thiocyanate.
The milk is contaminated with lactic acid bacteria during the milking. These
bacteria are catalase negative and therefore the hydrogen peroxide, which
always is produced as a by-product in the metabolism, is not removed by
catalase as in other microbial systems. Instead The lactoperoxidase oxidises
the thiocyanate with the hydrogen peroxide to hypothiocyanate. This
compound is strongly oxidising and reacts with sulfhydryl groups in transport
proteins in the bacterial membrane, especially in Gram negative bacteria,
while the lactic acid bacteria are relatively resistant. The lactoperoxidase
system has been reported to have an antimicrobial function also in tears and
other body-fluids.
O2
oxidase catalase
thiocyanate
- HO-S-protein
H2 O2 SCN
LP -
H2 O OSCN HS-protein
hypothiocyanate
Fig 3.10. The lactoperoxidase system. The milk contains thiocyanate. H2O2 is produced in
small quantities by all aerobic cells and usually degraded by catalase. However, lactic acid
bacteria, which are first contaminating milk during the milking process, do not have
catalase. The lactoperoxidase use the hydrogen peroxide to oxidise thiocyanate to the
strongly oxidising hypothiocyanate which oxidises transport proteins in bacterial
membranes. Especially Gram negative bacteria are sensitive to the hypothiocyanate.
When the milk leaves the udder it becomes infected by about 100 so callled
udder cocci per milliliter. During the further handling in tha cow house the
milk is infected with several types of microorganisms as shown in Table 3.5
Table 3.5 The initial milk contamination microflora

Infection Source
E. coli Feces
Enterococcus
Micrococcus
Bacillus spores Air
Mold spores
Yeasts
Lactococcus
Lactobacillus Milking equipment
Gram-negative rods
If the milk is stored at room temperature the "lactic streptococci", i.e. the
Lactococcus will first dominate the microflora and protect it from most of the
other microorganisms with lactic acid. Eventually Lactobacillus, which can
grow at lower pH will dominate. This fermented milk similar to yoghurt was
previously produced on the farms (Swedish filbunke). If the milk is stored
further proteolytic molds will finally raise the pH and it will be further
destroyed by putrification by Clostridium and Bacillus. These reactions do not
take place in a refrigerated milk.
When the milk is cooled after milking and stored refrigerated on the farm,
psychrotrophic gram negative rods (Pseudomonas and similar) will dominate.
These bacteria will not make it sour as does the lactic acid bacteria. If stored
too long the milk is spoilt by ammonia, peptides and free fatty acids. This
psychrotrophic microflora, which itself is very heat sensitive, is known to
produce comparatively heat resistant proteases and lipases which may create
problems in the later storage. When the milk reaches the dairy it is pasteurised
(see chapter 5) which efficiently eliminates the psychrotrophic Pseudomonas
flora and most other bacteria. However, some of the more heat resistant
organisms, mainly Lactobacillus and Micrococcus will survive, and the
bacterial endospores from Bacillus will not be influenced at all by the
pasteurisation.
After the pasteurisation the milk is re-infected with the dairy equipment
microflora. This may restore the psychrotrophic Pseudomonas flora or at bad
hygiene even the Enterobacteriacae flora. The final spoilage of the
refrigerated milk therefore differs depending on the contamination flora.
Members of the Enterobacteriacae family may spoil the milk with
fermentation, Bacillus spores my germinate and spoil by proteolysis. This is
especially common in fatty products like cream. Also proteolysis and
lipolysis by enzymes from the early Pseudomonas flora may contribute to the
final spoilage of milk. However, the old days souring by lactic acid bacteria is
not the common fate of refrigerated pasteurised milk.
The egg is infected on the surface when the hen lays the egg. this flora is
dominated by Pseudomonas, Staphylococcus, Micrococcus and fecal
bacteria. It is not uncommon that the hen is infected with Salmonella and
during the 1990ths many reports on Salmonella infected egg yolks appeared
in England. The surface microflora is usually not infecting the egg due to a
number of defence mechanisms, which are illustrated in Fig 3.11. When this
protection fails and the egg becomes invaded by bacteria it is Pseudomonas
fluorescens that dominates (80%). These infections can be detected by
illumination of the egg with UV-light.
Albumin: viscous, high pH

(pH9,5), riboflavin + pyridoxin
complexing
No
protect
ion outer mucin layer
Con-albumin: Fe2+ complexing 1-10µm pores in shell
Avidin: Biotin complexing
Lysozyme: kills G+ bacteria inner keratin
membrane
Fig 3.11. The egg is protected against bacterial infections an multiple ways: The shell and
the two membranes gives is the first and mechanical hinder. The high pH in the egg white
is non-optimal for many bacteria. Lysozyme ruptures cell walls of many bacteria.
Albumin, conalbumin and avidin make several nutrient unavailable by strong complex
formations.
3.2 Fruits and vegetables

Fruits and vegetables do have a high water activity but they develop another
spoilage scenario than meat, fish and milk. Many of these products are
protected mechanically by the pectins which constitute a "glue" between the
cells and gives rigidity. When fruits and berries ripen, endogeneous pectinases
start to hydrolyse the pectin and this also makes the products more susceptible
to microbial attacks. Another common protection the low pH of some of these
products. This group of foods also has a much lower concentration of free
amino acids and other nutrients than meat, fish, and milk. For these reasons it
is usually not the Pseudomonas and other spoilage bacteria mentioned above
which dominates the spoilage. Instead it is often pectinase producing
organisms, which mostly means molds, that initiate the spoilage of fruits and
vegetables. In the later phase, when the pectinolytic organisms have opened
up the defence structure, also yeasts participate in the spoilage.
One of few bacteria involved in spoilage of vegetables is the plant pathogen

Erwinia carotovora. This organism has been subject to studies of the corum
sensing phenomenon which plays a central role in the ecology of many
organisms. In this case the corum sensing is based on accumulation of N-
acylated homoserine lactones (AHL) which accumulates around the cells (Fig
3.12). When the concentration of AHL is high enough this compound induces
the pectinase synthesis. The strategic advantage of not producing the
pectinase constitutively is obvious, since the plants have their defence
systems which generated antimicrobial chemicals when the plant is attacked.
Only by delaying the pectinase synthesis until the number of bacteria is large
enough, can the hydrolysis of the pectins be fast and efficient enough. Once
the pectinases have damage the structure of the fruit/vegetable, other
organisms follow and contribute to the soft rot. Due to the often low pH,
molds and yeasts, rather than bacteria are common in the spoilage of these
products.
plant cell
pectinolytic
bacteria
AHL
AHL
AHL
AHL
AHL
Fig 3.12. Erwinia carotovora utilises corum sensing to invade plants. They start by
hydrolysing the protecting pectin layer with extracellular pectinases. When the plant
recognises a microbial attack it defends itself by producing antimicrobial ( )
compounds. Instead of initiating this defence response at low concentration of Erwinia
cells, they first accumulate acylated homoserine lactones (AHL) and when the
concentration is high enough this is a signal for induction of the pectinase ( )
production. By delaying the attack until there are many cells that can produce much
pectinase, Erwinia gains increased virulence.
It is estimated that only about 20% of the fruits and vegetables are spoilt by
microorganisms. The endogenous metabolism of the products, which leads to
over-maturation plays a major role for the spoilage. Furthermore, drying also
contributes to the spoilage. To reduce and better control the endogenous
metabolism, fruits and to some extent also vegetables are stored in modified
atmospheres (Controlled Atmosphere, CA-storage). Common principles are to
increase the CO2-concentration, which also ha a microbial inhibition effect,
and to reduce the oxygen concentration by adding nitrogen gas. Many fruits
produce ethylene gas, which acts as a maturation hormone, and for some
products absorption of the ethylene is included in the CA storage. Addition of
ethylene or cessation of the absorption is then used to initiate the ripening.
Table 3.6 gives an example of a modified atmosphere for fruits. the exact
composition is optimised for each product.
Table 3.6. Example of modified atmosphere for storage of fruits

O2 : 0-5%
CO2 : 2 - 10 %
N2 : 90-95 %
Relative humidity: 90-95%
3.3 Cereals
Grains on the field usually has a primary flora of 103 - 106 bacteria g-1. Lactic
acid bacteria, coliform bacteria and Bacillus spores dominate. A weather
dependent flora of fungal spores is also present. At humid conditions the mold
spore count can be 105 g-1. Different species of Aspergillus and Penicillium
usually dominate. If the grains are soaked in water the bacterial flora will
dominate, the regulations set a maximum water concentration of 13% for
storage of grains and then no significant microbial activity is expected due to
the low water activity. If the water content exceed 15% mold growth begins.
Even if the grains are kept dry enough according to the regulations, local
humid zones may appear in the silos, e.g. due to water condensation on walls.
Under these conditions mold growth and mycotoxin formation may appear.
During the milling of the grain most of the microflora follows the hull but
some is of course transferred to the flour. Typical microbial counts are 102-
103 bacteria plus about 100 mold spores per gram sifted flour and about 10
times more in course flour. At correct dry storage of the flour there is no
microbial activity, but as soon as water is added a vigorous growth starts.
The surface of the bread becomes sterilised in the oven and a dry hard bread
surface protects the bread against mold growth. If the bread is cut before
packing the surfaces are usually infected and if the bread is kept to moist in a
plastic bag mold growth will spoil it. The inner part of a bread is usually
heated to 95-99°C which means it is essentially sterile with respect to
vegetative cells and mold spores. There is however a rare bakery problem
called ropiness, which is caused by polysaccharide formation by Bacillus
subtilis. The organism has then survived the baking in spore form and if the
temperature is kept at 30-45 °C too long and the bread has not become dry
enough during the baking the B. subtilis spores germinate and grow very fast
and produces the polysaccharides.
During storage of the bread, spoilage is entirely caused by molds which have
contaminated the bread after the baking. To reduce the rate of mold growth
propionic acid or propionates are often used a preservatives in industrial
baking. Dry bread (knäckebröd) is not subject to any microbial spoilage,
provided it is stored dry. Under such conditions the very slow spoilage is
eventually caused by rancidification.
3.4 Preserved foods

The spoilage of preserved food depends on the type of preservation. In
general, if the preservation prevents microbial spoilage, the ultimate fate is
usually spoilage by rancidification, which usually is a very slow process.
Dried products. In the drying process the water activity is reduced to so low
levels that no microorganisms are active. If the storage conditions are not dry
enough, mold formation may occur, but otherwise the shelf-life is limited by
rancidification processes, which depend very much on the fat composition of
the product. During a spray-drying process the food is exposed temperatures
that kile the most sensitive bacteria, but endospores, mold spores and more
heat resistant vegetative bacteria as Enterococcus, Lactococcus, Micrococcus,
and Lactobacillus may survive. When such products (e.g. dry milk, soups,
sauces, etc.) are reconstituted with water they are usually very susceptible to
fast microbial spoilage and considerable risks for food poisoning.
Cured meat products are usually protected by the low water activity created
by salt additions. If the products are fermented (salami and other fermented
sausages) they are also protected by the lactic acid and the competitive effects
of the lactic acid bacteria. These products are often further protected with
nitrite. A common bacterium in vacuum packed cured meat products, is
Brochothrix thermosphacta. This organism is similar to Lactobacillus (CO2
resistant and tolerant against low aw) which usually dominates vacuum packed
meat products, but it is a severe spoilage organism since it produces stinking
metabolites. Also the low-aw tolerant Micrococcus and Lactobacillus are
common in these products.
Salted fish and fish preserves are also protected mainly by the low water
activity and chemical preservatives. In salted fish products mainly halophilic
strains of Pediococcus, Micrococcus, and yeasts grow and they do this at a
very low rate with slow spoiling. Usually these products are to be cold stored.
and the main shelf-lime limitation is usually rancidification of the fat.
Table 3.7. Summary of common spoilage floras on different types of food

Fresh meat Fresh fish Vac/CO2-pack Salted/Smoked Fish preserves
Pseudomonas Pseudomonas Lactobacillus Lactic acid bacteria Lactic acid bact
Shewanella Aeromonas Brochotrix Micrococcus
putrefaciens Enterobacteriacae thermosphacta Yeasts
Aeromonas Brochotrix Micrococcus
(freshwater) thermosphacta Enterococcus
Molds (not in
vacuum)
Table 3.8.Propertie of common food related organisms

Organsim Properties Products
Gramneg. rods: - Psychrotropic Refrigerated fresh:
- Pseudomonas - Aerobic -Meat
- Sensitive to low aw -Fish
- Sensitive to low pH -Milk
- CO2-sensitive
- Shewanella - H2S-producer Fish
putrefaciens
Lactic acid bacteria: Fish preserves
-Lactobacillus - O2-indifferent Vacuum packed
-Lactococcus - Resistant to low aw Fermented food
-Pediococcus - Resistant to low pH Smoked/salted/dried meat/fish
Enterococcus - CO2-resistant Pickles
B. thermpsphacta
Grampositive cocci: - Facultative Fish preserves
-Micrococcus - Resistent to low aw Vacuum packed
-Staphylococcus - Resistant to low pH Fermented food
- Heat resistant Smoked/salted/dried meat/fish
- Lipo-/proteolytic Pickles ade livsmedel
Spore formers: - Extremt värmeresistenta Heat sterilised food
-Bacillus - Mesofila Reconstituted dried food.
-Clostridium - Starkt fermentativa Pre-cooked
Milk: B.cereus
Enterobacteriaceae: - Strongly fermentative Milk
-E.coli Pre-cooked food
-Enterobacter m.fl. Vegetables
-Erwinia - Pektinase-active
Molds - Aerobic Vegetables
- CO2-sensitive Fruit
- Pektinase-active Dried food
- Resistant to low aw
- Resistant to low pH
- Lipo-/proteolytic
Yeasts - Facultative Vegetables
- CO2-resistant Fruit
- Resistant to low aw Low-pH preserves
- Resistant to low pH Sweett products
38
Chapter 4. Foodborne pathogens
Most cases of so called food poisoning are caused by microorganisms. Only a

few per cent of the food poisoning cases are reported to be caused by toxic raw
materials like toxic mushrooms or plants or contamination by toxic impurities
like heavy metals. The remaining cases of food poisoning can be divided into
microbial food intoxication, when microorganisms have produced toxins in the
food and microbial food borne infections, when pathogenic microorganisms in
the food are ingested and infect the human body.
Intoxications and infections caused by microorganisms in food and water

account for a large number of fatal cases and large economic loss in the
society. Food borne pathogens causes millions of death cases every year,
especially in poor countries and it is especially children that are the victims. It
is difficult to estimate the true statistics behind the food borne diseases, since
most cases are never confirmed by clinical analysis. This is especially true for
"mild" but common diseases like Bacillus cereus intoxication and Clostridium
perfringens infections, since they are usually confirmed by analyses only in
large outbreaks. On the other hand, statistics on the severe Clostridium
botulinum intoxication is probably reflecting the true cases, at least in the
industrial world.
Fig 4.1 Number of cases with food borne diseases reported to the Swedish Institute for
Infectious Disease Control according to the law for report on certain diseases (Average
number per year during 1997-2005).
4. Foodborne pathogens 39
Only some of the microbial food poisoning diseases are reported to authorities
according to law. See Fig 4.1. Other sources of statistics that also include
organisms that are not covered by obligatory reporting gives a similar picture,
namely that Campylobacter, Salmonella and Norovirus (earlier called
calicivirus) are among the most frequent causes of food borne illness, but it
also shows that Clostridium perfringens and Staphylococcus often occur in the
outbreaks (Fig 4.2).
Cases Outbreaks
Fig 4.2 Statistics of food borne diseases in Sweden for a 5 year period. Calicivirus =
Norovirus .Source: Vår Föda, nr 5, 1999.
4.1 Microbial food intoxications

Staphylococcus aureus. The probably most common microbial food
intoxication is caused by certain strains of Staphylococcus aureus. This
organism is also known as a common pathogen causing infections in wounds
and blood, but these infections are not considered to be transferred via food. S.
aureus produces a series of toxins and other virulence factors (Table 4.1) but it
is mainly the enterotoxins that cause food poisoning after ingestion of food on
which S. aureus has grown and produced the enterotoxins.
Table 4.1 Some virulence factors of S. aureus

Toxins Exoenzymes
Membrane damaging toxins (several) Coagulase
Epidermolytic toxin Staphylokinase
Toxic shock syndrom toxin Proteases
Pyrogenic exotoxin Phospholipase
Enterotoxin ( 6 serotypes) Lipase
Hyaluronidase
S. aureus is a common inhabitant on animals and humans where it grows on

mucose membranes, for instance in the nose, even on healthy individuals, and
it is frequently found in pus and wounds. The common source of food
contamination is therefore human hands. This organism is very resistant to low
water activity (Table 2.2) which means that they can grow on salted and
relatively dry products. It does not grow under refrigerator conditions, and
without growth no toxin is produced. It has low competitive power compared
to many other bacteria, like lactic acid bacteria and Pseudomonas. For this
reason, a small amount of S. aureus is usually accepted in food ( e.g. 102 - 103
g-1) before it is classified as not acceptable (Swedish: otjänligt) which means
the product must be withdrawn from the market.
The intoxications are associated with a large number of foods, often food that
has been cooked which eliminates competing microorganisms and food that is
handled by human hands: Chicken, ham, salads, pizza, kebab, sauses, paseries
etc. The enterotoxins are very heat stable and contaminated food may therefore
still be poisonous after re-heating when all vegetative cells have been killed.
The disease caused when eating S. aureus enterotoxis is characterised by a

violent nausea with vomiting, diarrhoea and convulsions. It is one of the few
cases when the eating of infected food results in an almost immediate illness,
within one or a couple of hours. The patient usually recovers in 1-2 days and
the disease is not associated with further complications.
Bacillus cereus. This organism is a facultatively anaerobic endospore former

that is ubiquitously present in Nature. Therefore vegetables are usually
contaminated with this organism. It is also frequently present in milk, probably
since the dusty air in the barn contaminates the milk and the subsequent
pasteurisation has no effect on the endospores, while most competing organism
are killed. B. cereus produces three enterotoxins which cause relatively mild
diarrhoeal illness with an incubation time of 6-24 hours, and an emetic toxin,
cereluid. The haemolysins are inactivated in the stomach and this type of
disease is actually an infection where the toxin is produced locally by B. cereus
growing in the intestine. The cereluid is a heat stable protein and this disease is
considered to be a true intoxication. It has a shorter incubation time, 0.5-6
hours, and is especially associated with rice dishes. B. cereus is assumed to be
a very common agent of food poisoning, but both diseases are usually
proceeding fast with little complications and therefore isolated outbreaks are
normally not identified and the statistics becomes unsure. B. cereus is not so
competitive but after heating of a product the spores may become the
dominating organisms and if the food after that is kept too long in the
temperature range 15-45°C the spores may germinate and grow and produce
the toxin. Like most Bacillus this organism is typical mesofilic with respect to
temperature, but certain strains are reported to be psychrotrophic and may
grow down to about 4 °C.
Clostridium botulinum. The most well-known and feared microbial

intoxication is botulism, which is caused by one of several toxins of Cl.
botulinum. This organism is an obligately anaerobic spore forming bacterium
that is very common in soil and water. The toxin is classified according to
serotype A-F, where type A, B, E, and F are toxic to humans. Cl. botulinum
type E is commonly found on fishes and this toxin is relatively heat labile,
destroyed by boiling, while type A is more heat resistant.
The endospores make also heat treated food potentially dangerous since
surviving spores may grow out. The botulin toxin is a very toxic protein that is
produced during growth of the vegetative cells in food. Cl. botulinum does not
grow at temperatures below 4°C, at pH below 4.5, or in presence of oxygen.
The toxin acts as a neurotoxin paralysing the central nervous system. It is one
of the most potent toxins known with a lethal dose of about 10-6 g. After an
incubation time of 18-36 hours, the illness sometimes starts with nausea and is
followed by the effects on the CNS caused by blocking of the acetyl choline
release at the nerve synapses: double-seeing, difficulties to swallow and finally
paralysing of the breathing. At this stage the mortality is high. In US statistics
during 1950 - 1970 the number of fatal cases was almost as high as the number
of reported cases. After that an anti-toxin became available but mortality is still
considerable. Fortunately, the number of cases is low, in Sweden the average is
less than one/year.
The few cases of botulism in Sweden are associated with home preserved
(marinaded or smoked) fish and home preserved meat. The precautions that
must be taken to avoid botulism in association with food preservation are low
pH (often vinegar), high salt concentration and storage below 4°C. In
commercial preservation nitrate also plays an important role. This is further
described in Chapter 6.
The so called infant botulism has another mechanism. It is caused by a Cl.

botulinum infection of the intestines where the spores germinate, grow and
produce the toxin. This disease is only associated with babies under one year
age who have not obtained the normal competitive intestinal microflora, and
the infection origin has exclusively been honey which often (10%) contains
spores of Cl. botulinum. For this reason authorities recommend not to give
honey to babies.
Mycotoxins. Intoxications by fungal toxins, mycotoxins, are not found in the

statistics on food borne diseases. The reason is that these diseases, contrary to
the other diseases discussed here, do not cause acute symptoms. Most reports
on mycotoxins describe their effects as cancerogenic or liver or kidney
damaging, with symptoms emerging long time after consumption of the food.
An exception is patulin, which is associated with intestinal illness but it is also
a suspected carcinogen.
There are hundreds of mycotoxins described in the literature. Biochemically

they are typical secondary metabolites produced by moulds. It means they are
mainly produced late in or after the growth phase. Most mycotoxins are
resistant to temperatures used in cooking. Fig 4.3 shows the chemical structure
of some mycotoxins. For some of the mycotoxins (e.g. aflatoxins, ochratoxins
and patulin, there are regulatory concentration limits for food, based on TDI
values (TDI=tolerable daily intake). TDI-values are usually in the range below
1 mg/Kg body weight.
Aflatoxin B1 Ochratoxin A
Fig 4.3 Examples of mycotoxins
Table 4.2 lists some well-known mycotoxins, producing organisms and food
they are typically associated with. The table demonstrates two characteristics
of mycotoxins: several species, even from different genus, may produce the
same mycotoxin and one mycotoxigenic organism may produce several
mycotoxins.
There are several variants of chemically related aflatoxins. Aflatoxin B1 is the

most commonly observed and most toxic of the aflatoxins and it is a strongly
potent carcinogen. In animal experiments daily intake of less that 100 ng/kg
body weight causes liver tumours. Aflatoxin M1 is found in milk and it is a
degradation product of aflatoxin E.
Table 4.2 Examples of mycotoxins, mycotoxigenic molds, and associated food

Toxin Organism Associated food Effect
Aflatoxins Aspergillus flavus Nuts, figs, corn Liver cancer
Asp. parasiticus
Ochratoxin A Asp. ochraceus Grains, coffee, wine, Kidney/liver
Penicillium viridicatum beans damage, teratogenic
Patulin Pc. exapnsum Fruits, berrys Diarrhoea
Pc roqueforti
Penicillinic acid Pc. cyclopium Peas
Pc viridicatum
Zearalenon Fusarium graminearum Grains Infertility
Roquefortin Pc. roqueforti Bread
µg aflatoxin/ kg bread
zone Bread 1 Bread 2 Bread 3
1 >> 15 000 150-300 40-80
2 600 n.d 20
3 100 n.d n.d
4 n.d
Fig 4.4 Analysis of aflatoxin distribution in three breads that were inoculated with A. flavus
and incubated until a colony was formed (Vår Föda, 31, 390-399, 1979).
The extremely high toxicity of aflatoxin and the fact that mould colonies often
grow on bread raises the question about how far the toxin reaches from the
fungal colony. In an investigation 3 breads were inoculated at the surface with
an aflatoxin producing strain of Aspergillus flavus, as indicated in Fig 4.4.
Samples were taken from 4 zones at different distances from the colony and
analysed for aflatoxin B1, B2, G1, and G2. The table in Fig 4.4 shows the sum
of the aflatoxin concentrations in the zones after one week. The permitted level
in bread was 5 µg/Kg.
While aflatoxins are mainly associated with nuts and figs, ochratoxins are
generally found in food and especially in food that is consumed in large
amounts, like cereals. Ochratoxin is also spread via meat from animals fed on
grains. It has been shown to cause damages on liver and kidney and it is also
teratogenic. The TDI is 14 ng/Kg body weight but due to expected but not
proved cancerogenic effects the TDI value used by some authorities is

considerably lower.
Patulin was first studied as a potential antibioticum but is now classified as a

mycotoxin. The source in nature is fruits and berries, and it is frequently found
at very low concentrations in commercial fruit juices and jam.
Several strains of P. roqueforti isolated from commercial blue cheeses have

been shown in the laboratory to produce mycotoxins, among them PR toxin
and roquefortine. This organism is also a common contaminant in many foods
and it is a predominant organism in silage where it is said to have a positive
effect on the acceptance by cattle. Roquefortine C has been reported to have a
neurotoxic effect and it is an inhibitor of Gram positive bacteria.
The uncertainty of the real effects of consumption of mycotoxins with food has
resulted in the general recommendation to avoid mold infected food.
Algal toxins. Planktonic algae called dinoflagellates are responsible for

different types of shellfish poisoning: Paralytic shellfish poisoning (PSP),
diarrheic shellfish poisoning (DSP) and others. The PSP is observed as
respiratory paralysis within 0.5 - 2 hours after consumption of the toxic food
and it may get severe consequences if not treated. The DSP causes diarrhea
within 0.5 - 3 hours and lasts for 2-3 days with no after effects. These types of
poisoning are associated with filter-feeding molluscs, like mussels, clams,
scallops and oysters.
Cyanobacteria, previously called blue-green algae, are involved in so called

algal blooms, some of which may make the water toxic. Nodularia spumigena
is one of the most common toxic cyanobacteria in algal blooms in the Baltic
sea. These intoxications are normally not associated with food or drinking
water, however.
4.2 Food borne infections

Food borne diseases caused by microbial infection of the consumer is much
more frequent than the intoxications caused by ingestion of microbial toxins
produced in the food. Two of the most frequent diseases in the statistics (Fig
4.1) namely Campylobacter and Salmonella are infections caused by eating
contaminated food. The pathogens may then grow in the intestines and cause
the disease. In general, this type of disease has an incubation time of one to
several days, which often results in difficulties to identify the food that was
causing the problem. The incubation times reported for infections varies much
with the status of the individual and with the infecting dose. Also reported
minimal infectious doses are very unsure figures and depend on the condition
of the person. Mostly elderly people and children are much more sensitive that
grown-up and healthy individual.
In Table 4.3 common infections are grouped according to the probable source
of contamination. Bacteria with fecal origin may enter the food from water or
raw material that has been in contact with feces, which is the natural
environment for these organisms. Alternatively, the food has got this infection
directly from feces contaminated hands of someone handling the food. Most of
the food pathogens of fecal origin belong to the family Enterobacteriaceae,
which includes among others the genera Salmonella, Shigella, Yersinia and
Escherichia belong to the
Table 4.3 Classification of common food pathogens based on their probable source
Fecal origin Water origin Soil origin
Campylobacter Listeria monocytogenes Clostridium perfringens
Salmonella Aeromonas hydrophila Bacillus cereus (diarrhoeal)
Shigella Vibrio parahaemolyticus
Yersinia enterocolitica
Pathogenic E. coli
Campylobacter is a common inhabitant of intestines of many types of warm

blooded animals without causing any symptoms in the animal. It is mainly the
species C. jejuni that causes the food borne infections. It is a Gram negative
bacterium and it is environmentally sensitive: it grows only in the range
between 25 and 42 °C, is microaerophilic, and very sensitive to drying,
freezing and disinfectants. The food contamination source is often chicken, un-
pasteurised milk or water. Flies are also suspected to transmit the bacteria from
feces to food. Several large outbreaks have been caused from municipal water,
but mostly it is chickens that are associated with Campylobacter infections.
The chicken (and also other types of meat) becomes contaminated from its
feces during the slaughter and since the infectious dose is very small ( 500
cells) infected meat can cause disease even if it has been stored so that no
further growth has been possible. The only way to avoid this disease is to
apply good hygiene in the food preparation and to heat the food enough to kill
the cells, which requires 65 °C through all parts of the meat. The infection
gives diarrhoea and other typical gastroenteritis symptoms for about 2-5 days
but sometimes reactive arthritis prolongs and complicates the disease.
Salmonella. There are more than 2000 different serotypes of Salmonella and
some cause relatively mild diseases while other strains cause severe illness. S.
typhi and S. paratyphi cause the most dangerous infections. Salmonella is

frequently found in poultry and swine. It is environmentally very resistant
which explains why they are widely spread in Nature even if they grow mainly
in animals. The organisms are usually distributed via meat that is contaminated
with feces during slaughter. Infected animal feed is another carrier of
Salmonella. It is also common in spices and vegetables, probably through
contamination with infected water or soil.. The disease breaks out after 12-48
hours and lasts for a couple of days, with some exceptions when there are
complications with reactive arthritis or septicemia with subsequent infection of
organ systems. Humans may also become carriers of Salmonella without
showing any symptoms. The infective dose varies much but as little as 15-20
cells has been reported. According to FDA the number of cases of
salmonellosis is 2-4 millions/year in the US and the frequency is rapidly
increasing. Especially S. enteritides is rapidly spreading in US and Europe.
Shigella. Contrary to Salmonella these organisms are very host specific and
grow only in the intestines of humans and apes. The food borne infections are
mostly caused by bad personal hygiene but also by vegetables that have been
contaminated with water containing human feces. Infected humans may
recover and still be "healthy carrier" of the organisms. This, together with the
very low infectious dose (10 cells), also makes shigellosis (bacillus dysenteri)
directly transferable between individuals. Shigella multiply intracellulary in the
epitheleal cells which results in tissue destruction. Some strains produce shiga
toxin which is similar to the toxin produced by EHEC. This protein, when
produced by the bacteria in the infected human host cell, inhibits the protein
synthesis and results in cell death with severe hemorrhage in the patient.
Yersinia enterocolitica. There are three pathogenic Yersinia species. Y. pestis,

Y. pseudotuberculosis and Y. enterocolitica. The latter is associated with food
borne infections, mainly from pork, since swine is a common reservoir of this
organism. Also dogs and cats are frequent carriers of Y. enterocolitica which
grows not only in the intestinal tract but also in mucous membranes in the
mouth and throat. This is one of few psychrotrophic pathogens which can grow
at high rate in the refrigerator, even down to 0°C. Most cases are associated
with pork and vacuum packed meat products but also water and un-pasteurised
milk have been involved in out-breaks. Vacuum packed meat products have
often been heat treated, which removes most vegetative cells inclusive the
quite heat sensitive Y. enterocolitica, and if the product then is re-infected and
stored for long time in the refrigerator the product may cause infection. The
disease breaks out 3-7 days after the infection and it lasts for 1-3 weeks. The
disease is relatively rare in the statistics which partly may be due to the
difficulties to isolate the bacteria. It is also assumed that only certain strains of
Y. enterocolitica are pathogenic.
Pathogenic E. coli. There are four enteropathogenic groups of E. coli. They are
classified according to serotype. The nomenclature is not strict, but a common
classification is:
EHEC enterohemorrhagic E. coli

ETEC enterotoxigenic E. coli
EPEC enteropathogenic E. coli
EIEC enteroinvasive E. coli
EHEC (enterohemorrhagic E. coli ) produces shiga-like toxins, also called

verotoxins. The most well-known EHEC are characterised and analysed as the
serotype O157:H7, but these shiga-like toxins are produced also by other E.
coli serotypes. Alternative names of EHEC are STEC ( shiga-toxin producing
E. coli ) or VTEC (verotoxin producing E. coli). The natural reservoir of
EHEC is probably the intestines of cows, who are not themselves showing any
symptoms, and then the distribution occurs via fecal contamination. EHEC
infections have been associated with hamburgers, un-pasteurised milk, water,
and alfalfa sprouts. In some cases it has been assumed that humans keeping
indoors in a cow-house can be infected directly from this environment. The
very low infectious dose,10 cells, means that the bacteria do not need to grow
on food to make it infective. EHEC has unusually high resistance to low pH,
and the cells can survive extended periods in sour products like juice, yoghurt,
and fermented sausages, products that usually have been considered as safe in
this respect. The two shiga-like toxins are coded by genes (stx1 and stx2)
which are located on lambda phages and integrated as inactive prophage genes
in the bacterial genome. Only after induction, which can be by agents resulting
in the SOS response or by iron limitation, does the prophage enter the lytic
phase which induces the toxin production. The toxin kills the cells in the
intestines and causes bloody diarrhoea. In severe cases, especially in children,
the infection is spread to the kidney which may be permanently destroyed.
ETEC, or enterotoxigenic E. coli causes a relatively mild gastroenteritis with

watery diarrhea, often called travelers' diarrhea. These infections are also
common among children in poor countries. Large infective doses (> 108 cells)
are required and the incubation time is about 1 day. ETEC is not common in
countries with good sanitary standards but when the water is contaminated
with human feces there is a risk for ETEC infections in food.
EPEC are strains of E. coli that cause the infantile diarrhea in newborn babies.
It is not assumed to be food associated.
EIEC invade the epithelial cells of the intestine, resulting in a mild form of
dysentery. It is not known if this is a food associated infection.
Water and soil are reservoirs for several pathogenic bacteria that may
contaminate food: Listeria monocytogenes, Clostridium perfringens, Bacillus
cereus, Aeromonas hydrophila, and Vibrio parahaemolyticus.
Listeria monocytogenes is widely spread in nature both in water, soil, plants,

and in animal intestines. It grows often in biofilms which are common in food
manufacturing facilities. L. monocytogenes is therefore very common in food.
Also humans are often carrying this organism in its intestinal flora without any
symptoms. Many strains are pathogenic to some extent. Listeriosis is not
primarily a gastroenterit but it is rather manifested as septicemia, meningitis,
and cervicial infections in pregnant women which may result in spontaneous
abortion. Sometimes the symptoms are preceded by gastrointestinal symptoms
like nausea, vomiting, and diarrhea. The bacteria invade the human phagocytic
cells and propagate intracellulary. In this way the infection is spread to organs
with the blood. The organism is one of the psychrotrophic pathogens which
can grow to dangerous concentrations also in a refrigerator. It is frequently
found in vacuum packed smoked or marinaded fish and in soft cheeses made
on un-pasteurized milk. Cooking at 70°C kills the bacteria. Food associated
with Listeria outbreaks are often such food where the organism gets the chance
to grow during production and then is consumed without further heating, e.g.
in soft cheeses, marinaded meat, and smoked fish. The infective dose is
unknown. The reason why the number of identified disease cases in not larger
while Listeria is often present in food is probably that only some strains are
pathogenic and the pathogenecity factors are not known enough to be the goal
for analysis.
Clostridium perfringens is a common spore forming soil bacterium which

means that vegetables often are contaminated. It can also grow in the intestines
of humans and animals without causing any symptoms. Many strains of Cl.
perfringens produce enterotoxins. There are a number of facts that together
makes this one of the most frequent diseases associated with large-scale
cooking, especially with soups and casserols: 1) Being a common soil
bacterium it is often added to food as spores in contaminating vegetables,
2) The spores not only survives cooking at 100°C but even become activated to
germinate, while most competing bacteria are killed. 3) The boiling also
removes the oxygen which otherwise prevent growth of this obligately
anaerobic bacterium. 4) In rich media and optimal temperature it can grow

extremely fast (8 min doubling time at 45°C). If the cooling from this
temperature down to below 15°C is not fast enough, or if the food is kept warm
at too low temperature (should be ≥ 60°C), conditions for growth of Cl.
perfringens are excellent. The common form of perfringens poisoning is
characterized by intense abdominal cramps and diarrhea that come within 8-24
hours after consumption of foods with large numbers of cells and lasts for
about 24 hours. The infective dose is very large , over 108 cells.
Bacillus cereus. This organism produces the toxin cereluid that act as food
intoxication causing vomiting. But many strains of B. cereus also produce one
or several of three enterotoxins: haemolysin BL, non-haemolytic enterotoxin,
and cytokine K. These proteins do not survive the passage through the
stomach, and therefore its is considered that the bacteria also can establish
themselves in the intestines and produce the enterotoxins there. This diarrhoeal
disease is often associated with meat and vegetable dishes and sauces. Also the
spores may germinate and grow in the intestines, so contaminated food may
cause disease even after heating. This is assumed to be a very common source
of mild illness, that is seldom investigated clinically, and therefore the
statistics is uncertain.
Vibrio parahaemolyticus is widespread in marine environments and brackish

waters all over the world, especially in areas with warm climate. It is
associated with infections from fish, shellfish, shrimps and other seafood,
especially raw food that has not been heat treated. The organism attaches itself
to the small intestine and secretes a toxin. The illness comes after about 24
hours and lasts for a couple of days. All the common food poisoning symptoms
may be involved: diarrhea, abdominal cramps, nausea, vomiting, headache,
and fever.
Aeromonas hydrophila. This organism has only recently been recognised as a

food pathogen and there is not much information available on this. However,
in several cases it has been isolated from stools of patients with gastroenteritis
without any other sign of infection. A. hydrophila is a common bacterium in
soil and water, even in drinking water pipes. It grows well down to 5°C and it
grows in vacuum packed food. Shrimps, ham, sausages are examples of food
that has been associated with these infections.
Virus. There are several virus infections spread with food and water. Viral
gastroenteritis is usually a mild illness characterized by nausea, vomiting,
diarrhea, and fever. The infectious dose is not known but is presumed to be
low. These infections are either spread via contaminated water or food or
through direct contacts between people. Norovirus is one of these viruses that
cause short but intensive gastroenteritis especially in children. It has previously
been named Calicivirus. The virus is present in the feces of infected persons. It
is assumed that only 10 virus particles is enough for an infection and this may
explain why this disease also is very contagious and not only distributed via
food and water.
51
Chapter 5. Heat sterilisation and pasteurisation
Sterilisation is based on either of two principles: All living cells, including

spores, are physically removed from the medium or they are killed. Each
principle involves a number of methods. Heating is the most coming method for
killing microorganisms in food. If this is made with the goal to kill even
endospores temperature in the range of 120 °C or higher must be used and this
can result in real sterilisation, i.e. also the endospores are killed. If the goal is to
eliminate the majority of vegetative cells, temperatures in the range of 70-90°C
are used and this is called pasteurisation, and it has no inactivating effect on
endospores. Also ionising irradiation (Co60 or electron beams) can be used for
sterilisation, and if the doses used only kills the more sensitive cells and leaves
the endospores, also this is called pasteurisation. Killing of microorganisms can
also be made with chemical agents, and this is usually referred to as
disinfection, and applied mainly to surfaces
5.1 Mechanisms of heat inactivation of microorganisms.
Microorganisms may be classified in two groups with respect to heat

sensitivity: 1) Bacterial endospores and 2) Vegetative cells and spores of other
types, e.g. fungal spores. Endospore formation is mainly found in the genera
Bacillus and Clostridium, but also Sporosarcina, Desulfotomaculum, Sporo-
lactobacillus and Thermoactinomyces may form endospores. Endospores are
extremely resistant to heat, UV and ionising radiation, drying and chemical
agents. It takes heat treatment in the range of 100°C and higher to inactivate
these spores. Note that other spores, e.g. fungal spores, may be quite resistant to
drying but they are only slightly more resistant to heating than are the
corresponding vegetative cell. To inactivate these spores and vegetative cells in
general, heat treatment in the range of 50 to 90 °C (pasteurisation) may be
efficient and it does not provide complete sterility since it has no effect on the
endospores.
Fig 5.1 shows the main structures of a bacterial endospore. The exact
mechanisms behind the extraordinary resistance of bacterial endospores are not
known, though some information is available from mutants lacking different
components in the spore: The spore has three distinctive structures: The core,
containing the DNA, a few key enzymes and 2-10% dipicolinic acid (DPA) in
complex with Ca2+ and the DNA. The core also contains some basic proteins
that are quickly hydrolysed and serve as amino acid source during the
germination. The water content of the core is low, which together with DPA is
5. Sterilization and pasteurization 52
assumed to contribute to the large thermal stability of the spore. The basic
proteins contribute to the high UV radiation resistance. The surrounding cortex
contains negatively charged peptidoglucans and the water in the cortex is freely
exchangeable with surrounding water. The difference in water concentration
between the core and the cortex makes the spore refractile and gives it a light
appearance in a phase contrast microscope, while vegetative cells appear dark.
The cortex is surrounded by a spore coat of proteins that confer the chemical
resistance to the spore. The size of a spore is somewhat smaller than the
vegetative cell, as indicated in Fig 5.2
Core: DNA, Ca-DPA, few ribosomes,

key enzymes, no water
Cortex: Negatively charged peptidoglucanes
Coat: chemically resistant proteins
Fig 5.1 Structure of a bacterial endospore.
Transformation of a spore to a vegetative cell involves a number of reactions

(Fig 5.2). The activation is a reversible reaction, which is poorly understood.
Activation may be needed to make a spore competent for the next stage,
germination.
activation germination
dormant spore activated spore germinated spore

outgrowth
lysis
sporulation vegetative cell
growth
Fig 5.2 The endospore germination-sporulation cycle. The dormant spore may need
activation before the initiation of germination can take place. Activation is a reversibel
raction and does not change the resistance or appearance of the spore. At the initiation of
germination all resistance properties disappear and the spore then grows out to a vegatative
cell which divides a number of times until harsh environmental conditions induce
sporulation. The spore is eventually liberated by cell lysis.
Agents that cause activation are, for instance, sub-lethal heat treatment, high
pressure and extreme pH. Spores that are difficult to activate are called super
dormant spores. It is difficult to differentiate between super dormant spores and
dead spores, since it is only when the spore has been provoked to germinate that
it has been proven that it was not a dead spore. The activation reaction does not
result in any visible change of the spore structure or composition nor any
observable metabolic reaction.
An activated spore may be initiated to germinate by several chemicals like

amino acids, nucleotides etc. The initiation of germination is seen as a swelling
of the spore and it is associated with migration of the Ca2+ ions from the DPA
complex in the core to the cortex where they neutralise the electronegatively
expanded cortex, which shrinks and in this process water enters the core which
swells. The germination of one spore takes only a couple of minutes. In a phase
contrast microscope the appearance of the spore is changed from a bright
reflecting structure of the ungerminated spore to a dark colour, like that of the
vegetative cell, of the germinated spore. Germination of a whole spore
population can also be observed as a reduction of the absorbance in a
spectrophotometer. The germination process of a whole population of spores
may be completed as fast as within 15 minutes, but it may also take much
longer time. The initiation of the germination is characterised by a complete
loss of all the resistance factors of the spore. Electron microscopy reveals that
the germination is associated with an expansion of the core and a thinning of
the surrounding cortex (Fig 5.2). During this phase a sequence of metabolic
reactions and synthesis of enzymes is initiated.
The last phase of the germination is called outgrowth. During this phase, which
takes about one generation time, all the normal metabolic reactions are restored
and the spore is gradually converted to a vegetative cell.
Heat inactivation of the endospore is believed to be a matter DNA damage, but

also heat denaturation of essential proteins in the core may be involved. Heat
inactivation of vegetative cells involves quite different reactions, and it is
mainly a matter of disorganisation of the cell membrane. This is indicated by
several phenomena observed in the heat surviving fraction of a population, as
for instance the increased osmosensitivity and increased leakage of cell
components. Also DNA damages and denaturation of proteins may be observed
during heat killing of vegetative cells. The thermal resistance of vegetative cells
is also influenced by the level of its heat shock proteins, which participate in the
protection against thermal denaturation of proteins. Since the heat shock
proteins may be induced by e.g. thermal (sub-lethal) chock and other stress
agents, the thermal stability of a vegetative cell depends not only on the
environment but also on its history. Also endospore stability depends on

environmental factors like the composition of the medium during the
sporulation. Metal ions like Ca2+ and Mn2+ are often required for the endospore
to aquire full heat resistance.
5.2 Kinetics of heat inactivation of cells
Heat inactivation of spores as well as vegetative cells can be described with the
same mathematical model. Therefore the same methods of calculation may be
employed for sterilisation and pasteurisation.
The rate of heat inactivation of a population is proportional to the number of

cells, N. If N represents the number of organisms in the total volume of
medium to be sterilised, the rate of inactivation becomes:
dN
= "kN
dt (1)
-1
where k (min ) is the specific heat inactivation constant, also called the death
rate constant and t (min) is the time. Integration from time zero with the initial
number of cells (No)!gives
"N%
ln$ ' = (kt
# N0 & (2)
which can be rearranged to
! ln( N ) = ln(N 0 ) " kt (3)

or to
N = N 0e"kt (4)
!
for calculation of the number of surviving cells after a given time. Eq. 3 is
illustrated in Fig 5.3.!Experimentally determined inactivation curves often show
deviations from this model. Some examples of this are shown and explained in
Fig 5.3.
Note that this first order kinetic model does not permit calculation of the time
when the number of cells reaches zero, which is the time it takes to sterilise a
sample! However, when N is below one cell (N < 1) the sample is in practice
sterile. An interpretation of this is that when N < 1 (i.e. ln(N) <0) there is a
certain statistic probability that the sample is sterile. This will be used for
calculation of the sterilisation time in Section 5.3
The inactivation constant, k, is a characteristic of the cell but it depends also on

many environmental parameters. The higher the temperature is the larger is the
k-value. The heat inactivation constant depends on temperature like most rate
constants of chemical reactions. This is usually described with the Arrhenius
equation:
k = Ae"#E /RT (5)
where A (min-1) is a constant which gives the order of magnitude of the
inactivation reaction, ∆E ( J mole-1 ) is the activation energy which describes
!
the temperature dependence of the inactivation reaction, R ( ≈ 8.31 J mole-1 °K-1
) is the universal gas constant, and T (°K) is the temperature.
Fig 5.3. Heat inactivation curves. The left hand figure shows two inactivation curves with
different death rate constants. The right hand figure shows some deviations from the
model:
1. This form may be caused by super-dormant spores, which are activated by the first heat
treatment and do not germinate unless they get this treatment;
2. This may be observed in samples that contain aggregates of cells, since analysis is
usually made by viable count that gives number of colony forming units rather than
number of cells. The viable count does then not decline until the last cell in an aggregate is
killed. This curve form can also be caused by an experimental artefact, if heat transfer is
not fast enough.
3. Non-uniform heat resistance in the population, e.g. when the sample contains species
with different thermal sensitivity. This is the expected curve for a mixed microflora.
Eq. 5 can be only used to calculate the effect of a temperature change on the
rate of heat inactivation within a limited temperature range, where the
inactivation is caused by the same reaction. The constants A and the activation
energy, ΔE, can be obtained from the logarithmic form of the Arrhenius
equation:
#E 1
ln(k) = ln(A) "
R T (6)
!
which shows that a plot of the logarithm for the thermal death rate, k, against
the reciprocal absolute temperature will have the slope ΔE/R and an intercept
with the ln(k) axis corresponding to ln(A). See Fig 5.4.
spores
!E 280
kJ/mole
Thiamine Fig 5.4 Arrhenius plots of inactivation
k (1/min)
0.1 !E 92
kJ/mole of B. stearothermophilus spores and
thiamine. Note that a temperature
increase has a larger effect on the spore
inactivation rate than on the vitamin
112 °C 96 °C inactivation rate.
0.01
2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9
1/T
The heat treatment does not only cause cell death but also increased rates of
other chemical reactions, which may be beneficial or detrimental to the product.
Examples are inactivation of vitamins and other nutrients, lipid oxidation, and
so called Maillard reactions. The latter is a group of reactions involving
reducing sugars and amino groups and it is an important class of reaction in
food processing, including both wanted and unwanted reactions, depending on
the situation. The principle reaction is shown in Fig 5.5.
high T, low aw, high pH
Fig 5.5. A Maillard reaction is a reaction between reducing sugar and an amino acid
favoured by high temperature, low aw and high pH.
Depending on which sugars and amino acids that are involved, the products
may be have different taste, be toxic, and cause colourisation of the product. A
large group of such mellanoidines are important for the organoleptic properties
of food.
Also chemical reactions like vitamin inactivation and Maillard reactions can be
modelled with first order kinetics. In analogy with eq. 2 :
"C%
ln$ ' = ( kC t
# C0 & (7)
where C and Co are the time dependent and the initial concentration of the
compound, respectively,
! and kc is the inactivation rate constant. The
temperature dependence of these reactions also follows the Arrhenius equation
(eq. 5) and can be characterised with the activation energy. Table 5.1 lists some
examples of activation energies for inactivation of endospores and for some
other chemical reactions. There is a tendency that the activation energy for cell
killing is higher than the activation energy of most of the chemical reactions.
This can be utilised to minimise the chemical reactions during sterilisation by
applying continuous sterilisation.
Table 5.1 Examples of ∆E values for heat

inactivation of spores and some chemical
reactions.
Inactivation of ∆E (kJ mol-1 )
B. subtilis spores 318
B. stearothermophilus spores 283
Cl. botulinum spores 343
Folic acid 70
d-panthotenyl alcohol 88
Cyanocobalamin 97
Thiamine HCl 92
Maillard reactions ≈125
5.3 Calculation of sterilisation time
According to the model for heat inactivation, eq. 3 and Fig 5.3, it is not possible
to calculate the time needed to reach zero concentration of viable cells. Yet,
when the cell number, N, in eq. 3 is below 1, the medium is sterile. If eq. 3 is
used to calculate the time needed to reach e.g. 10-3 cells, it means that there is a
probability that one batch of 1000 sterilised batches will be infected. The time
needed to reach this probability of sterility does not only depend on the death
rate constant, k, but also on the initial number of organisms, No, as is obvious
from Fig 5.3. Thus, a sterility criterion, ∇ (nabla) has to be defined:
#N &
" = ln%% 0 ((
$Nf ' (8)
where Nf is the final number of organisms. Eq. 2 can now be used to estimate
the sterilisation time,
! F (min), needed to satisfy the sterility criterion " :
"
F=
k ! (9)
This sterilisation time depends also on the temperature applied since k is a
function of temperature. The sterilisation time (FT ) required to satisfy the same
!
sterility criterion at another temperature (T °K) than the reference temperature
(Tref °K) at which the sterilisation time Fref once has been assessed, can be
obtained from eq. 9 written for the two sterilisation temperatures:
" = Fref k ref = FT kT

(10)
which, after substitution of k according to the Arrhenius model (eq 4), gives the
wanted sterilisation time at a different temperature T °K:
!
"#E $ 1 1'
& " ))
R &% Tref T(
FT = Fref e (11)
5.3.1 Batch sterilisation

If the death rate constant,
! k, is known and constant during the sterilisation, eq. 9
gives the answer for the sterilisation time required to satisfy the sterility
criterion. However, in batch sterilisations the temperature is first increasing,
then constant, and finally declining (See Fig 5.6). Since the thermal death rate
constant, k, depends on the temperature, this effect must be included in the
calculation. In the beginning of the sterilisation, when the temperature is low,
the rate of sterilisation is low. We must then introduce a time dependent relative
sterilisation dose ∇ (t)= ln(No/N(t)), during the sterilisation. The sterility
criterion is then satisfied when ∇(t) = ∇. Combining eq. 9 with eq. 5 gives an
expression that shows how the sterilisation dose depends on time:
#$E
"(t) = t k(T) = t Ae RT (12)
!
Since the temperature varies with time during batch sterilisation, the total
sterilisation dose is obtained as the integral of eq. 12
t
"(t) = A % (e#$E / RT )dt
0
(13)
The batch sterilisation has reached the criterion on sterility when ∇t = ∇. This
can be calculated
! without knowledge of the constant A in equation 11.
According to eq 4 and eq 8, the sterility criterion can be written
#$E /RTref
" = Fref Ae (14)
Division of both sides of eq 13 by eq 14 gives the ratio between ∇(t) and ∇:
t
!
"(t)
% (e )dt
#$E / RT
= 0 (15)
" Fref e (
#$E / RTref
)
Fig 5.6 shows an example of a batch sterilisation temperature profile and the
sterility according to eq.15. In this example the heating phase is relatively short
and it contributes
! only with some 20 per cent of the total sterilisation dose.
Since cooling from the highest temperature first is very efficient, the
contribution to sterilisation from the cooling phase is very small. Note also that
the exponential dependence of the sterilisation rate on the temperature means
that good temperature control at the holding phase is important for the precision
of the sterilisation.
Fig 5.5. Simulation of the progress of the sterilisation (∇t /∇) and inactivation of a
temperature sensible compound (C/Co) during a batch sterilisation. The sterility was
calculated according to eq.15 and the concentration of compound according to eq. 7 and eq.
5. Parameters: ∇ =20, A= 1035.8 sec-1 and 109 sec-1 for sterilisation and chemical reaction,
respectively. ΔE= 282 kJ mole-1 and 92 kJ mole-1, respectively. R= 8.31 J mol-1 °K-1 .
5.3.2 Continuous HTST sterilisation

During the low temperature part of the batch sterilisation, the rate of
sterilisation is relatively low, but other chemical reactions, like vitamin
inactivation, lipid oxidation and Maillard reactions may take place at a
considerable rate, which is sometimes detrimental for the food quality. An
interesting property of the sterilisation reaction is that it has a relatively high
activation energy, as pointed out in Table 5.1. The HTST sterilisation (High
Temperature Short Time) utilises the different sensitivity to temperature of the
two reactions "sterilisation" and "chemical reaction", which was expressed as
different values of the activation energy. This is exemplified in Fig 5.7, which
shows how the rate constant for spore inactivation increases much faster with a
temperature increase than does the rate constant of thiamine inactivation,
because the former has a higher activation energy (slope of the curve).
Therefore, the vitamin inactivation will be reduced if an increased sterilisation
temperature is combined with a reduced sterilisation time to give identical
sterility criterion, ∇. This effect of increased sterilisation temperature is
demonstrated in Fig 5.6.
30 1 Fig 5.7 Effect of sterilisation

temperature on a chemical
reaction in a continuous
F C / Co sterilisation. C/Co is the fraction
20 of non-reacted chemical
compound. F is the sterilisation
F (min)
C / Co
time according to eq. 9. C/Co

was obtained from eq. 7 and the
10 temperature dependence of the
two reaction rate constants, k,
was obtained from the Arrhenius
equation (eq. 5). Parameters as
0
115 120 125 130 135 140
0
145
for the batch sterilisation, Fig 5.6.
T
5.4 The D-value and the Z-value
The theory of heat sterilisation was developed in the food industry during the
first part of the 20th century. It was then common to use logarithms with the
base of 10 and much literature on sterilisation, and especially constants on heat
sensitivity and temperature dependency, are still based on this nomenclature,
which uses a D-value and a Z-value to describe the inactivation rate and the
temperature sensitivity of the inactivation rate, respectively.
The rate of heat inactivation according to eq. 2 can be written on a 10log basis
as
10
"N% 1
log$ ' = ( t
# N0 & D (16)
where 1/D is the slope of the curve when the number of surviving cells (N) is
plotted against time
! during heat exposure (Fig 5.8). The decimal reduction time
(D, min), is the time needed to reduce the number of cells to one tenth of the
previous value.
Fig 5.8 Inactivation curve plotted on 10log basis showing the definition of the D-value.
Solving eq. 2 for t = D and N = No/10 gives the correlation between the
inactivation constant k and the D-value:
ln(10)
D=
k (17)
D-values for inactivation of spores as well as for inactivation of vegetative cells

are available in! the literature and, if not found, can be determined
experimentally by plotting the data as in Fig 5.3 or 5.8. Table 6.2 shows some
examples of D-values. For endospores D-values are often standardised to
minutes at 121 °C but for vegetative cells lower temperatures, e.g. 60°C, are
often used.
These D-values (and k) depend much on the environment in which the heating
is performed. As a general rule one may say that the heat resistance increases
with reduced water activity but it decreases when the organism is subjected to
other extreme conditions like extreme pH, toxic compounds etc. The effect of
the water activity means that it may be very difficult to heat sterilise media with
suspended solids like starch, in which spores may stay relatively dry.
Sterilisation of dry materials like glass and other equipment requires much
higher temperature and/or prolonged heating. While water solutions mostly are
sterilised by some 15 minutes at 120°C (steam sterilisation) the corresponding
sterilisation of dry materials (dry heat sterilisation) may require about 4-6 hours
at 160°C or 1.5 h at 170°C to give similar effect. Data given in this chapter
refers to sterilisation in water solutions.
Table 5.2 D-values (min) for heat inactivation of microorganisms

Organism D121 D60 D50
Endospores (general) 0.1-4
Cl .botulinum 0.2
Cl. thermosaccharolyticum 10-20
Micrococcus spp. 5-20
Streptococcus spp. 5-20
Fungal spores 5-20
Virus 1-10
Mesophilic bacteria 1
Psychrotrophic bacteria 1-5
Psychrophilic bacteria <1
The temperature influence on the D-value is expressed by the Z-value, (°C)

which is the temperature increase that is needed to reduce the D-value by a
factor of 10. The definition is exemplified in Fig 5.9
Fig 5.9 The temperature dependence of the D-value and the definition of the Z-value.
The mathematical expression of the temperature dependence shown in Fig 5.9

is
10 1
log(D) = " T+10 log(DT = 0 )
Z (18)
where log (DT=0) is just a constant that represents the extrapolation to

temperature zero.! This constant has no physical meaning, since the model is
relevant only for a limited temperature range where the nature of the heat
inactivation reactions are identical.
In analogy with the calculation of sterilisation time at another temperature than

the reference temperature (eq. 11), it is possible to show that this calculation
can be done based on the Z-value:
(Tref "T )/Z
FT = Fref 10
(19)
The Z-value for endospores is usually in about 10°C while it is lower, about
5°C for inactivation of vegetative cells (pasteurisation).
!
64
Chapter 6. Fermented foods
All food raw materials are contaminated by microorganisms, which take part in
the mineralisation of organic materials in Nature. Therefore, Man had early to
learn to live with microbially infected food. The microbial reactions mostly
resulted in spoilage of the food. However, Man learnt to handle some foods in
ways that extended their shelf-life. These preservation methods were mainly
based on drying or fermentation. Food fermentations are still used to produce so
called fermented food, but today preservation is not the main objective of the
fermentation, but it is rather the specific taste and texture that is the goal of the
fermentation. Food fermentation is applied to a all main types of food, as meat
(sausage), milk (cheese and yoghurt), grains (beer and bread), fruit juice (wine)
and vegetables (sauerkraut and pickles). In Africa and Eastern Asia many other
types of food fermentation are applied. For a European, the most well-known of
these products is soy sauce, which is produced by fermentation of soy, sometimes
supplemented with rice. Table 6.1 lists the main types of fermented food in the
Western world together with the main biochemical reactions employed in these
fermentations.
In most food fermentation the basis of fermentation control is inoculation and
adjustment of the oxygen concentration and the water activity:
1) Inoculation with a microflora. In traditional fermentations the inoculum was a
contamination from earlier production via the equipment or addition of some
product that had already been fermented. Some processes still rely on the
spontaneous natural microflora. This method is now gradually replaced by the use
of pure starter cultures, as the production becomes more industrial, since
inoculation increases the control and reproducibility of the process.
2) Adjustment of the oxygen concentration. Ethanol fermentations are inhibited by
oxygen and therefore require un-aerated conditions. Lactic acid bacteria are
independent on the oxygen concentration, but since anaerobic metabolism of
competing organisms is slower than aerobic metabolism, also lactic acid
fermentation is favoured by anaerobic conditions. Acetic acid fermentations
require oxygen. Also moulds, which are important producers of hydrolytic
enzymes in some food fermentation, are obligately aerobic organisms.
3) Reduction of water activity. Several food fermentation processes are controlled
by reduction of the water activity by addition of salt. This is the case in
fermentation of meat, fish, vegetables and soy sauce (the lactic acid stage). The
background to this is that lactic acid bacteria, which are active in these
fermentations, are relatively resistant to reduced water activity and therefore are
favoured in this environment. In sausage fermentation the salt is mixed with the
minced meat and in the other cases the raw material is placed in a salt brine.
6. Fermented foods 65
Table 6.1 Fermented foods, their raw materials and main biochemical reactions
Raw material Products Main type of reaction
Meat Sausages Lactic fermentation
Fish Sour herring Enzymatic hydrolysis and lactic
fermentation
Milk Cheese Enzymatic hydrolysis and lactic
fermentation and (sometimes mold)
fermentation
Yoghurt Lactic (thermophilic) fermentation
Fermented milk Lactic (mesophilic) fermentation
Butter Lactic fermentation1)
Vegetables Sauerkraut Lactic fermentation
Pickles Lactic fermentation
Cereals Bread Ethanol fermentation
Beer Enzymatic hydrolysis and ethanol
fermentation
Soy sauce Enzymatic hydrolysis by moulds, lactic and
ethanol fermentation
Fruits Wine Ethanol (and malo-lactic) fermentation
Cider Ethanol fermentation
Vinegar Ethanol and acetic acid fermentation
Cocoa Ethanol and acetic acid fermentation
Coffee Microbial pectin hydrolysis
Olives Lactic fermentation
1) In some countries the cream is fermented before the churning of butter to provide
diacetyl as aroma compound.
6.1 Beer brewing

Production of beer by ethanol fermentation of grains dates back to at least 4000
BC, when it was applied in Egypt. In the ancient beer production lactic acid
fermentation probably played a role, and certain beer types are still produced with
mixed cultures of yeast and lactic acid bacteria. Hops was introduced as an aroma
compound and preservative during the 15th century. Around 1840 the lager type
of beer was introduced in Bavaria, characterised by slow fermentation at low
temperature (below 10 °C) and maturation before bottleing.
The beer brewing process is outlined in Fig 6.1. It contains a large number of
biochemical reactions. The raw materials of beer are malt, sometimes supplied
with other grains called adjunct, hops and water. Yeast, either Saccharomyces
cerevisiae or Saccharomyces uvarum, is added as a biocatalyst and sometimes
also additional enzymes of microbial origin are added to improve the enzymatic
reactions.
Fig 6.1 Summary of the beer production process.
Malting. The first stage of beer production is the malting of barley. The barley
should be of low nitrogen type, as opposite to the fodder barley. The grains are
first soaked in water in a steeping process during about two days to raise the
water content to 45%, which initiates sprouting of the grains. The grain content of
giberellic acid is important for the resulting germination. This germination
involves respiration, and the grains must be aerated to provide oxygen and remove
the carbon dioxide. Since the reaction is exothermic cooling must also be provided
and the grains are mechanically turned to provide homogeneous conditions.
During the malting process many of the barley enzymes are activated and start to
hydrolyse the grain: Hemicellulases, proteinases, α- and ß- amylases. Roots also

develop from the grain during the germination, which may take about 4-6 days to
be completed.
The germination and the emerging enzymatic reactions are interrupted by the
kilning, in which the temperature is gradually raised to 65-85 °C. During the
kilning, the high temperature results in Maillard reactions between reducing
sugars and amino-groups, that colour the malt, darker the higher the temperature is
used. This is the main way of controlling the beer colour. Maillard reaction
products also contribute to the taste of the malt and the beer. During the kilning
the water content is reduced so the malt can be stored for later use. Thus, malt can
be considered as a package of hydrolytic enzymes, notably α- and ß-amylases,
packed with the enzyme substrates, mainly starch. The last stage of the malting is
the removal of the rootlets which, like most other by-products from the beer
production, are used as fodder. Malt is not always produced by the brewer, but
often obtained from specialised malting companies.
Table 6.2 Composition of barley grains before

and after malting
Compound % in barley % in
malt
Starch 64 59
Sugar 2.5 9
ß-glucans 9 7
Cellulose 5 5
Amino acids and
peptides 0.5 1.5
Mashing. The malt is milled, coarsely to facilitate the later separation of the husk.
The milled malt is mixed with hot water to extract starch and enzymes from the
grains in the mashing process at about 65 °C. Some brewers supply additional
starchy materials, adjuncts, that are cheaper than malt, like maize, wheat or rice.
Even sugar may be used. This also reduces the protein concentration of the wort,
which may be an advantage if the malt is too protein rich, since proteins may
cause problems with precipitations in the beer. On the other hand, the use of
starchy adjuncts requires higher enzyme activity in the malt.
Starch is composed of amylose, that is a straight chain of α-1,4-linked poly-

glucose, and amylopectin which besides α-1,4 bonds also contains branching
points with α-1,6 bindings (Fig 6.2). During starch hydrolysis α-amylase
randomly hydrolyses α-1,4 bonds between the glucose units in the starch, which
results in smaller poly-glucose molecules called dextrins. Thus, hydrolysis by α-
amylase gradually reduces the mean molecular weight and the viscosity of the
starch solution but little fermentable sugar is produced in this reaction.
Fig 6.2. Hydrolysis of amylopectin to dextrins, maltotriose and maltose by α-

amylase and ß-amylase. Both enzymes hydrolyse at the α-1,4 site leaving the
branching α-1,6 sites in low molecular weight dextrins. Oligosaccharides larger than
maltotriose are not fermented by the yeast.
The ß-amylase hydrolyses α-1,4 bindings two glucose units from the non-
reducing terminal of amylopectin, amylose or dextrin to produce the disaccharide
maltose, which is the main fermentable sugar in the wort (Table 6.4). Thus, the
longer the mashing continues the higher becomes the concentration of fermentable
sugar. However, these enzymes can not hydrolyse the branching points (α-1,6
bonds) of the amylopectin and therefore small branched dextrins are left. These
dextrins are not fermentable and they remain in the beer and contribute to
sweetness and viscosity of the product.
Additional enzymes like proteases or ß-glucanases, may also be added to improve
the proteolysis or the ß-glucan hydrolysis. Pullulanase, a debranching enzyme that
hydrolyses α-1,6 bonds in the amylopectin, may also be used to increase the
concentration of fermentable sugar from the starch.
Table 6.3 Temperature and pH optima

of the main malt enzymes
Enzyme pH Temperature
α-amylase 5.7 70
ß-amylase 5.5 60
ß-glucanase 5.1 57
proteinase 4-5 40-50
These enzymes have different temperature optima (Table 6.3). During the
mashing different temperature programmes can therefore be used to control the
hydrolysis of the macromolecules. The proteolysis should furnish the wort with
amino acids for the growth of the yeast during the fermentation but it should also
degrade proteins that would otherwise precipitate in the beer. Likewise, the ß-
glucanolysis is important to reduce later precipitations and it yields
oligosaccharides. The main reaction during mashing is the degradation of starch to
fermentable sugars and non-fermentable dextrins. A typical composition of the
wort is shown in Table 6.4
Table 6. 4. Components of starch hydrolysis in wort.

Product % of total starch
Maltose 51
Maltotriose 12
Glucose 9 Fermentable
Fructose 2
Sucrose 2
Maltotetrose 3 Non-fermentable
Dextrins 21
The enzymatic hydrolysis is interrupted by boiling of the wort for 1-2 hours. pH
has then dropped from 5.8 to 5.4. Before this, the husks and precipitated proteins
are removed from the wort and hops are added. It is the dried non-fertilized female
flower of Humulus lupulus that is used. Today also pelleted hops and even hops
extract is used by the brewer. During the subsequent wort boiling, aromatic
compounds are extracted from the hops, some unwanted aroma compounds are
evaporated, all enzymatic activity ceases and the wort becomes essentially sterile.
Hops contain two main types of flavour compounds: humulones (the so called
alpha acids) and lupulones (called beta acids).
Fig 6.3 Basic structure

of the humulones of hops.
The molecules isomerise during the wort boiling which makes them more water
soluble and more bitter. Negatively charged tannins are also extracted from the
hops and they form precipitate with proteins. After the wort boiling the hops
residuals are separated off together with the precipitated proteins and used as
fodder. The so clarified wort is cooled and inoculated with yeast.
Fermentation. The fermentation process is performed in a batch according to

either of two principles. In top fermentation Saccharomyces cerevisiae is used.
This yeast flotates to the top when the fermentation has ceased due to lack of
fermentable sugar. The bottom fermentation processes utilise Saccharomyces
uvarum (carlsbergensis) which sediments to the bottom after the fermentation.
Bottom fermentation is typical for lager beer and pilsner and it is performed at
low temperature: 5-10 °C for about one to two weeks, until all visible
fermentation has ceased. Top fermentations is applied to produce the beers of ale,
stout and porter type and this fermentation is made at higher temperature, around
20°C, which results in more ester production.
N*10-6/mL EtAc (mg/L)

E (%)
60 5 50
E
EtAc
0 0 0
0 50 100 150
Time (hrs)
Fig 6.4 Progress of a lager beer fermentation at 10°C. N = yeast cell
number; E = ethanol concentration; EtAc = concentration of ethyl acetate.
During the fermentation, the yeast biomass concentration increases about four
times (Fig 4.4). Cells separated from the beer after fermentation are partly used to
inoculate next batch and partly used as fodder. To permit growth of the yeast
during the conditions in the wort, oxygen must be available for synthesis of cell
membrane constituents. Therefore the wort is saturated with oxygen from air
before inoculation. This oxygen is quickly consumed by the cells and then the
process is strictly anaerobic. From this time in the process much effort is focused
on keeping the beer free from oxygen since the shelf-life is strongly reduced by
oxidations in the beer. All fermentable carbohydrates (Table 6.4) are converted
during the fermentation to biomass carbon dioxide, ethanol and other organic
compounds that contribute to the taste. Since the yield coefficient for ethanol from
maltose is about 0.5 g/g, the final alcohol concentration can be predicted from the
concentration of wort used to start the fermentation. However, it depends also on
the extent of the starch hydrolysis to fermentable sugars. To make a low-caloric
beer there is only one way: reduce the wort concentration, since most of the
energy of the sugar is preserved in the ethanol. Depending on the extent of starch
hydrolysis, the low caloric beer can either be a low alcohol beer with a normal
alcohol to dextrin ratio or a low dextrin beer with normal alcohol content.
Ethanol is a major contributer to the taste of beer, but minor quantities of organic
acids, higher alcohols, esters and other aroma compounds are also produced and
make important contributions to the taste of the beer. However, also less pleasant
compounds are produced and for this reason a post-fermentation process is
included. One of these unwanted compounds is diacetyl. It is not produced
directly by the yeast cells, but α-acetolactate is secreted by the cells during the
later phase of the primary fermentation (see the ethyl acetate curve in Fig 6.4) and
then spontaneously decarboxylated to diacetyl.
The main fermentation results in a "green" beer which must be matured in a post-
fermentation process at 0 - 10 °C before use. Lager beer is generally matured for
a longer period, 2 weeks to 2 months at a temperature close to 0 °C, while ale is
stored at higher temperature for a much shorter period of time. Many less
characterised reactions takes place during the post- fermentation. One of the
products from the main fermentation, α-acetolactic acid, spontaneously
decarboxylates to diacetyl, which is considered unpleasant in beer. However,
during the late stage of the fermentation, and further during the post fermentation,
this diacetyl is resorbed by the remaining yeast cells, and the concentration of
remaining diacetyl is sometimes used as a measure of the post-fermentation
progress. A problem in this process is that it is the decarboxylation of the α-
acetolactate that is the rate limiting step. New technology has been developed to
achieve the postfermentation by means of an accelerated decarboxylation induced
by continuous heat treatment in a heat exchanger followed by diacetyl removal by
immobilised yeast in a packed bed column. In this way, the post fermentation
reactions can be accomplished with about 2 hours residence time during which
almost all diacetyl is resorbed by the cells.
After the post-fermentation the beer is clarified by centrifugation or filtration. To
reduce effects of microbial infections, the beer is often pasteurised or sometimes
sterile filtered. It is mainly other yeasts and lactic acid bacteria that can interfere
with beer during storage, due to the low pH, the alcohol content and the high
partial pressure of carbon dioxide. As long as these infections can be avoided the
shelf life of some 3-6 months is mainly limited by oxidation reactions. To reduce
these reactions ascorbic acid is commonly added as an anti-oxidant in beer.
6.2 Fermented milk products

Lactic acid bacteria is a group of species that are characterised by fermentation of
sugar to lactic acid. The group is divided into two categories, homofermentative
and heterofermentative lactic acid bacteria, depending on whether the metabolism
yields mainly lactic acid (homofermentative) or also considerable amounts of
acetic acid, ethanol and carbon dioxide is formed (heterofermentative). This
classification is not strict, since cultivation conditions may influence the product
pattern. Table 6.5 lists typical representatives of lactic acid bacteria in these
groups.
Table 6.5 Lactic acid bacteria classification according to
the product pattern
Homofermentative Heterofermentative
Lactococcus spp . (all) Leuconostoc spp .(all)
Pediococcus spp. (all) Lactobacillus spp. (some)
Lactobacillus spp . (some)
The lactic acid bacteria play an important role in fermentation of food. Table 6.1
shows that they are involved in fermentation of milk, meat, fish and vegetables. In
these cases the lactic acid fermentation plays an important role to stabilise the
product against microbial spoilage. The mechanism of this food preservation
effect is not at all generally known. It is well known, however, that many lactic
acid bacteria, when grown in mixed culture in the laboratory, are very
competitive. This competitiveness has been ascribed a number of factors like
production of inhibitors and resistance against low pH and low water activity (aw)
as depicted in Table 6.6.
Table 6.6 Competition advantages associated with lactic acid bacteria
Antagonistic products
Lactic acid
Acetic acid
Hydrogen peroxide
Antibiotics, e.g. nisin and reuterin
Properties of the bacteria

Ubiquitous on food raw materials (inoculum)
Oxygen indifferent
Relatively fast growing
Tolerant to carbon dioxide
Tolerant to low pH
Tolerant to low aw
6.2.1 Fermented milk and yoghurt.

Fermentation of milk with lactic acid bacteria is probably the oldest method to
preserve milk. It is widely used all over the world, probably because it has been
the safest way to consume milk. Milk that is not quickly fermented with lactic
acid bacteria soon becomes infected with a number of potentially pathogenic
bacteria. Only lately has it become possible to store non-fermented milk safely for
several days in refrigerators. Milk is fermented with lactic acid bacteria in many
different ways in different countries. Here only two types of fermentation will be
considered: a mesophilic fermentation employing a mixture of Lactococcus spp.
and yogurt, that is a thermophilic fermentation employing Lactobacillus spp. as
well. These two types are summarised in Table 6.7 and Fig 6.4.
Note that the Lactococcus genus in older literature is called Streptococcus. Only
the so called lactic streptococci are re-named Lactococcus. Streptococcus of the
enteric, viridans and pyogenes types are still classified in the Streptococcus genus.
The mesophilic fermentation employs two types of Lactococcus spp.; the
acidifiers Lactococcus lactis and Lactococcus cremoris, which are
homofermentative and have the task to quickly reduce pH and produce lactic acid,
and the heterofermentative aroma bacteria Lactococcus diacetilactis and
Leuconostoc cremoris, which are slow fermenters but produce diacetyl, which is a
desired aroma contributor in dairy products. The species mentioned in Table 6.7
are used by Swedish dairies, but many variants of this concept may be utilised.
The American fermented buttermilk, Swedish filmjölk, Danish ymer and Finnish
villi belong to this category . Villi is, however, also inoculated with a surface
growing mould, Geotrichium candidum, that contributes to the flavour and the
surface crust.
Lactobacillus spp. are generally slower to initiate the lactic acid fermentation, but
they are more resistant to low pH. Reduction of pH inhibits the glycolysis in all
starter organisms but Lactococcus spp stop the fermentation at about pH 4.5,
while the Lactobacillus fermentation continues to pH 3.9. Thus, pH in the
Lactococcus fermented milk is higher than in yogurt.
Table 6.7 Examples of two starter cultures for

fermentation of milk
Mesophilic (20°C) Thermophilic (44°C)
"Filmjölk" Yogurt
Lactococcus lactis Lactococcus thermophilus

Lactococcus cremoris Lactobacillus bulgaricus
Lactococcus diacetilactis
Leuconostoc cremoris
Another difference between the two types of fermented milk is the consumption of
lactose. The starter culture is inoculated to a concentration of about 106-107
cells/ml which grow to about 108-109 cells/ml. For this purpose lactose is used as
the energy source. The organisms of the yogurt starter culture do hydrolyse
lactose to glucose and galactose, but only glucose is consumed leaving the
galactose. Since the total biomass produced is similar or even higher in yoghurt,
the result is that yoghurt has lower concentration of lactose than the common
mesophilically fermented milk (Fig 6.5).This may be of significance in many parts
of the world, since adults generally do not accept too much lactose. The so called
lactose intolerance among adults, expressed as abdominal pains and diarrhoea
because of inability to hydrolyse the lactose in the intestines, is unevenly
distributed over the world. Generally, North Europeans and the white population
in America have a large tolerance to lactose while Asians and Africans generally
have very low lactose tolerance.
Many alternative species of lactic acid bacteria are used for fermentation of milk,
sometimes with the claim to give a more healthy product. The basis of these
properties would be that the cells colonise the intestine. Examples of such starter
organisms are Lactobacillus acidophilus, which grow very slowly compared to
other starter bacteria, and Bifidobacterium spp., which is frequently isolated from
the gastrointestinal tract. Other fermented milk types, like kefir and koumiss
contain yeast species, e.g. Candida spp and Saccharomyces spp , which contribute
to the flavour by production of alcohols and esters in very small quantities.
Fig 6.5 Schematic presentation of the lactose consumption in a fast thermophilic yoghurt
fermentation and mesophilic 'filmjölk' fermentation with a Lactococcus based starter culture.
6.2.2 Cheese. Like beer production, manufacturing of cheese is a combination of

enzymatic and microbial processes and the origin of the product dates back to
prehistoric times. The main steps of hard cheese production is outlined in Table
6.8. However, the variety of cheeses available on the market is reflected by a large
number of process variations. Only some common features and examples from
two main types of hard cheeses and the mould fermented cheeses will be treated.
The milk selected for cheese production is pasteurised (with some exceptions) at
for instance 72°C for 15 seconds. It is extremely important that it is antibiotic free,
since the starter cultures used are very sensitive to antibiotics. Especially
penicillin, which is often used to treat mastitis, may accidentally be present in the
milk. Lactic acid bacteria are extremely sensitive to penicillin. Antibiotics in the
milk may delay the lactic fermentation and it gives the opportunity for
Clostridium spores to germinate. Especially Cl. tyrobutyricum is a problem and a
spore concentration below 10 spores per 100 ml milk is required. Clostridial
growth in cheese may cause excessive gas production, butyric acid off-flavour and
even health hazards. Thus, special quick-test kits have been developed to analyse
the presence of antibiotics in the milk before cheese production.
Table 6.8 Main stages of cheese production

Action Main purpose
Pasteurisation Inactivate pathogenic and
competing organisms
Fermentation Reduce pH.

Produce lactic acid
Produce cells for later function
Addition of rennet Hydrolyse and precipitate

casein to a curd
Cutting and pressing Formation of the cheese

of curd, whey
separation
Storing Maturation of the cheese
Cow's milk contains about 87% water. The main ingredients of the dry matter are
shown in Table 6.9. Cheese is composed mainly of the caseins, except for part of
the κ-casein that is removed by enzymatic hydrolysis, the fat and part of the salts.
Table 6.9. Main ingredients of cow's milk

Component Concentration (%)
Water 87
Lactose 5
Fat 3.8
Protein 3.4
Caseins 2.8
α- 1.7
β- 0.6
γ- 0.1
κ- 0.4
Whey proteins 0.6
albumins
globulins
Salts 0.9
Calcium-
Citrates-
The milk is inoculated with starter cultures that have much concordance with
those used to produce fermented milk. Two main types may be distinguished for
hard cheese production: The Emmentaler and Gruyère type of cheese is based on
thermophilic Lactobacillus and Propionibacterium mixture while the Cheddar
and Gouda type is based on a mesophilic Lactococcus mixture (Table 6.10). the
purpose of the fermentation is to initiate the casein precipitation by reduction of
pH and to provide cells which are entrapped in the precipitated curd to take part of
the later maturation process.
Also the soft cheeses like Camembert, Brie, Roquefort, Stilton and Gorgonzola
are started with Lactococcus mixtures but they are also inoculated with a mould
species before the maturation and the action of these organisms takes place during
the maturation(Table 6.11). Since moulds are obligate aerobes, they grow only on
the surface, unless the cheese is perforated by holes.
Table 6.10 Examples of starter cultures for cheese production

Cheddar / Gouda Emmentaler / Gruyère
Lactococcus cremoris Lactococcus. thermophilus
Lactococcus lactis Lactobacillus helveticus
Lactococcus diacetylactis Lactobacillus lactis
Leuconostoc spp. Lactobacillus bulgaricus
Propionibacterium shermanii
The declining pH during the fermentation contributes to precipitation of casein at

its isoelectric point 4.6, as in the case of milk fermentation. However, proteases
are also added to the milk during cheese manufacturing, and these enzymes
contribute to an efficient precipitation of the main part of the casein. The major
protease preparation is calf-rennet, which is an enzyme extract from young
calves. The proteases of rennet are mainly chymosin (rennin) and pepsin. When
the calf grows older the proportion of pepsin increases, which makes the extract
less useful for cheese production, since pepsin hydrolysis is too extensive which
reduces the curd yield. A relative lack of calf rennet has provoked the
development of microbial proteases for cheese production. Such microbial rennet
is in extensive use in some countries. Calf chymosin has been cloned to a yeast,
Kluyveromyces sp., to produce chymosin in bioreactors. The process has been
scaled up and introduced on the market.
Table 6.11 Mould species used for maturation cheeses

Cheese type Example Mold specie (example)
White moulded cheeses Camembert Penicillium camemberti
Brie
Blue-vein cheeses Roquefort Penicillium roqueforti

Gorgonzola
Stilton
The casein is present in milk as colloidal micelles of very complex structure as

illustrated in Fig 6.6. The micelles with a diameter around 100 nm, are composed
of submicelles. The inner part of the submicelle is composed of α- and ß- caseins
which interact by their hydrophobic parts and via Ca2+ ions also between their
hydrophilic parts. The stabilisation of the submicelle is achieved by a surface
layer of κ-casein which is divided into a very hydrophilic part, turned outside, and
a hydrophobic part turned inwards the submicelle.
The main action of the rennet enzymes is a selective cleavage in the region
between the hydrophobic and hydrophilic parts of κ-casein. This removes the
hydrophilic surface layer from the micelle which, by hydrophobic interactions,
start to aggregate and precipitate as the cheese curd. Thus, the hydrophilic parts of
the κ-casein make up the whey proteins together with the globulins and the
albumins. The whey also contains the lactic acid and most of the remaining
lactose. It is important that the cells and some of the rennin enzymes and a little
lactose are entrapped in the curd, since they form the basis for the maturation
process.
Fig 6.6 Schematic illustration of the composition of a casein submicelle in milk. The casein
molecules have characteristic hydrophobic (dotted) and hydrophilic (white) regions. ß- casein
forms chains which are interlinked by hydrophobic interaction. α-casein binds to the
hydrophobic areas of this chain and Ca2+ ions stabilises the complex by ionic bindings between
the hydrophilic parts. Finally, the submicelle increases its hydrophilicity of the surface by
attracting κ-casein units which bind their hydrophobic ends inwards against the hydrophobic
sites. Chymosin and pepsin act by specific hydrolysis in the region between hydrophilic and
hydrophobic parts of κ-casein, thus exposing a hydrophobic surface. The degraded micelles
start to interact by hydrophobic binding and precipitate as a cheese curd.
The precipitated curd is cut in pieces, separated from the whey, washed and
pressed etc., according to different procedures for the different types of cheeses.
During the subsequent storing for some months, a large number of biochemical
reactions takes place to give the product its special texture and taste. First the
starter culture cells resume a slow growth, since the pH, that had declined to stop
the glycolysis during the initial fermentation, is increased after removal of most of
the lactic acid with the whey. During this stage heterofermentative lactic acid
bacteria or propionic bacteria produce gas that is entrapped in the cheese to give
the characteristic holes. Heterofermentative lactic acid metabolism also results in
diacetyl formation which is important for the flavour, as is the lactic acid and in
some cases propionic acid and other microbial products of the primary
metabolism.
Eventually the microorganisms die and lyse, thus releasing proteases and lipases.
These enzymes, together with the traces of the rennet proteases and the low
activity extracellulary cellbound proteases of the lactic acid bacteria induce a very
slow proteolysis and lipolysis that produce peptides and fatty acids to contribute
to the flavour. Furthermore, in mould inoculated cheeses, extracellular proteases
and lipases gradually diffuse from the mycelium to slowly soften and mature the
cheese. Moulds also produce lipoxydases, enzymes that catalyse oxidative
degradation of fatty acids which results in methylketones. Among these
degradation product are 2-heptanone and 2-nonanone considered to be especially

important for the cheese flavour.
6.3 Fermented meat products

In Europe, fermented meat is mainly found in some types of sausage, like the
salami and many other of the hard, often smoked sausages. Meat is normally
contaminated by an aerobic psychrotrophic flora dominated by Pseudomonas spp.
that normally spoil the meat by growth on the surface. When meat is minced this
flora is mixed into the product and it is furnished by a surplus of nutrients from
the damaged meat cells. Thus, minced meat is extremely sensitive to microbial
spoilage. However, since long time ago, Man learnt that if the minced meat was
salted and stuffed in a gut it did not develop the unpleasant odour but stabilised
and could be used as food for very long time. This is still the basic procedure in
production of fermented sausages.
There are several mechanisms that stabilise the meat in a fermented sausage:
Addition of salt reduces the water activity to prevent the Pseudomonas spp. to
develop. These organisms are especially sensitive to reduced water activity, while
lactic acid bacteria are especially tolerant in this respect. It is also essential that
lactic acid bacteria are present in the mixture so that lactic acid is quickly
produced and pH declines. This prevents organisms of the Enterobacteriaceae
family, Clostridium spp. and Bacillus spp., which are normally present at low
concentrations, to develop. Traditional formulations often included garlic or
spices with antimicrobial compounds that further increased the stability. To
increase the safety with respect to the dangerous Clostridium botulinum , also
nitrite is added nowadays, since the undissociated acid, HNO2, is known to be
very efficient in preventing endospore germination. Actually, the name botulinum
comes from Latin botulus = sausage, since botulism was formerly often
associated with infected sausages. The package of the sausage, originally guts
from animals but nowadays often synthetic materials, also protects the meat from
infection during the storage. It is quite common that the surface becomes covered
with certain species of mould during the storage. This growth of moulds is even
utilised for the processing in certain case, like the production of Salami.
In the traditional procedures the inoculum was obtained either automatically from
the not too clean vessels used to mince and mix the meat. Some formulations also
contain milk or other sources of lactic acid bacteria. It is also common to mix old
product into the fresh unfermented mixture to inoculate the meat. Today it has
been common practice to use starter cultures to make the process more safe and
reproducible. Lactobacillus plantarum, Pediococcus spp , Lactococcus spp. and
in some cases even Micrococcus spp are used as starter cultures for fermentation
of sausages. After the fermentation the sausages are often smoked, which further
contributes to the preservation of the product.
6.4 Fermented vegetables

Vegetables are not fermented to a large extent in Europe. Some common products
are sauerkraut, that is fermented cabbage, pickles, that is a mixture of fermented
vegetables and fermenter cucumber. The history of fermented vegetables is,
however, probably as old as that of the other fermented foods. It is documented
that large scale fermentation was applied to furnish the workers with food during
construction of the Great Chinese Wall during the third century BC.
The microbiology of fermented vegetables is not so well documented as that of
beer, wine or cheese production. It is also just recently that starter cultures has
been adopted. The common method is still to place the vegetables in a 3-6% salt
brine and wait till the natural flora starts the fermentation. This takes some time
since the lactic acid bacteria are present only at very low concentrations.
Meanwhile, the main flora that may be Enterobacteriaceae members and Bacillus
spp. are retarded by the salt and they gradually die. Typically, this fermentation
starts with Leuconostoc mesenteroides and is followed by Lactobacillus brevis,
that can ferment pentoses and Lactobacillus plantarum that is the main acid
producer. Also Pediococcus cerevisiae is commonly found in fermented
vegetables. After the main fermentation a slow post-fermentation by yeasts is
common
During the fermentation, that may take a couple of weeks, the low concentration
of fermentable sugars is further reduced, which is part of the stabilisation of the
product against other microorganisms. Lactic acid is also produced at
concentrations determined by the available sugar concentration. 1-2% lactic acid
is achievable. The product obtains special characteristics not only by the taste of
the acid produced, but also by the effect of the low pH that makes the vegetable
crispy. Another important function of the fermentation is that it may inactivate
some of the plant enzymes, like pectinases, that otherwise would hydrolyse
pectins to soften the product.

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Food

Chapt 2. The ecological basis of food spoilage ...........................................5

Chapt 3. Spoilage of different types of food .............................................22

Chapt 4. Food borne pathogens.................................................................38

Chapt 5. Food preservation.......................................................................51

Chapt 6 Fermented foods .........................................................................64

Living organisms are usually classified as animals, plants, algae, protozoa,

Most microorganisms that we encounter in the normal spoilage of food belong

4 Gran-neg., aerobic rods or cocci Pseudomonas, Shewanella, Legionella

5 Gram-neg., facultatively anaerobic Family Enterobacteriacae

17 Gram-pos. cocci Staphylococcus, Streptococcus,

18 Gram-pos endospore formers:

obligate anaerobes: Clostridium

19 Gram-pos, non-sporulating rods Lactobacillus

There is a number of common groups of microorganisms that is not well

”Gram-negative psychrotrophic rods”: This includes the genera Pseudomonas,

”Coliform bacteria” is not synonymous to E. coli but includes Escherichia coli

Lactic acid fermentation is to a large extent also employed for production of

Heterofermentative lactic acid bacteria produce, besides lactic acid, also

Chapter 2 The ecological basis of food spoilage

2.1 The microflora

Light Fig 2.1. Microorganisms,

The degradation of biological material is mainly catalysed by microorgansims,

All energy is generated, with exception of photosynthesis, by oxidation

When oxygen is used as electron acceptor the process is called aerobic

Some microorganisms can use other reduced compounds than carbon

One alternative energy metabolism that is common in microorganisms growing

products, like ethanol fermentation, lactic acid fermentation, mixed-acid

It is impossible to give a simple and yet comprehensive description of the

Table 2.1 Typical size of different food microfloras at good

Product Microbial concentration

Milk Contamination flora ≈ 103 cells / ml

Meat Contamination flora ≈ 103 cells / cm2

Spoilage flora on ≈ 107 - 108 cells / cm2 or gram

2.2 The physico-chemical properties

- Temperature - Mechanical barriers

Temperature. The temperature influences of course the rate of growth, and

Microorganisms are usually classified in four groups according to their

nw = number of moles water

- Ions (e.g. salts)

Most biochemical reaction rates decline with declining water activity.

Table 2.2 Examples of typical minimum water activity for growth of

pH is another parameter of large impact for the shelf-life of food. The pH

Table 2.3. Typical pH-values of common food products

Table 2.4. Generalised picture of pH ranges for microbial growth

The mechanical structure may be important for the shelf-life of food. On

Antimicrobial substances. Many food raw materials, especially vegetables and

Many microorganisms produce antimicrobial substances (antibiotics) and in

Table 2.5. Some examples of naturally occurring antimicrobial substances.

Table 2.6. Antibiotic substances produced by lactic acid bacteria

Some definitions of antimicrobial compounds

Antibiotics Microbial product with an antimicrobial (bactericide/

Probiotics Microbial cultures, mainly lactic acid bacteria, which are

Prebiotics Components (oligosaccharides) in the food that are not

Bacteriocines Bacterial proteins or peptides with bactericidal effect

bactericide = bacteria killing; fungicide = fungi killing;

2. 3 The chemical reactions

Degradation of nitrogen compounds

This reaction is assumed to be the dominating spoilage reaction in refrigerated