Download Microbiology

Food
Microbiology
Sven-Olof Enfors
KTH - Biotechnology
Stockholm 2008
S.-O. Enfors: Food microbiology
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. Foodborne pathogens..................................................................38
4.1 Microbial food intoxications .................................................39
4.2 Foodborne microbial infections .............................................44
Chapt 5. Food preservation.......................................................................51
5.1 Heat sterilisation and pasteurisation ......................................51
5.2 Chemical preservatives..........................................................65
5.3 Classification of preserved food ............................................65
Chapt 6 Fermented foods .........................................................................73
6.1 Beer brewing.........................................................................74
6.2 Fermented milk products......................................................81
6.3 Fermented meat products .....................................................88
6.4 Fermented vegetables ...........................................................89
S.-O. Enfors: Food microbiology
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
foodborne 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
Enterobacteriacae
Genus
Escherichia
Salmonella
Species
Escherichia coli
Salmonella typhimurium
Salmonella enterica
Bacillacae
Bacillus
Bacillus subtilis
Bacillus cereus
Bacillus anthracis
Clostridium
Clostridium botulinum
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 which prevents 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
S.-O. Enfors: Food microbiology
Introduction
2
(with e.g. nitrate as electron acceptor) or fermentation. Oxygen is often toxic
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
nr
2
4
5
Description
Food related organisms
Gram-neg., aerobic, mobile, vibrioformed
Gran-neg., aerobic rods or cocci
Gram-neg., facultatively anaerobic
rods
Campylobacter
17
Gram-pos. cocci
18
Gram-pos endospore formers
aerobic or facultatively anaerobic:
19
obligate anaerobes:
Gram-pos, non-sporulating rods
Pseudomonas, Shewanella, Legionella
Family Enterobacteriacae
(e.g. Escherichia, Enterobacter,
Salmonella, Shigella, Yersinia, Erwinia)
Vibrio
Staphylococcus, Streptococcus,
Lactococcus, Enterococcus, Micrococcus,
Leuconostoc
Bacillus
Clostridium
Lactobacillus
Brochothrix
Listeria
There is a number of often used group names of microorganisms. 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.
S.-O. Enfors: Food microbiology
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 (together with the coliforms) are called indicator bacteria.
3. Hemolytic streptococci. There are two types of hemolytic streptococci, and
these organisms remain in the genus Streptococcus: α-hemolytic and ß-hemolytic.
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 ßhemolytic 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, and others.
S.-O. Enfors: Food microbiology
Introduction
4
However, this fermentation is also involved in food spoilage. Then the type of
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
all
all
some
Streptococcus
Lactococcus
Pediococcus
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
most
Leuconostoc
Lactobacillus
S.-O. Enfors: Food microbiology
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 viewpoint. 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 fate of the
food, namely microbial degradation. However, it means that our interest in a
long shelf-life of food is in conflict with the natural processes.
CO2 + N2
Light
Animals
Plants
Organic
materia
Archae
Bacteria
Fungi
Algae
Protozoa
Fig 2.1. Microorganisms,
especially bacteria and fungi,
account
for
the
main
recirculation of carbon and
nitrogen to the atmosphere
from where it is adsorbed for
generation of plants which
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
becomes 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
S.-O. Enfors: Food microbiology
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
S 2N2
H2O
+
NAD
NADH
Cred
S 2-
Fe 2+
H2
2
Fe 3+
H2O
ATP
NAD+
Ethanol
O2
NO3
SO42Electron acceptors
NADH
Pyruvate
ADP
Fermentation
NH3
CO2
NO3
-
SO4-
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
(e.g. 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
little role in the handling of food.
One alternative type of energy metabolism which 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 coenzymes. There is a number of different fermentative metabolic pathways,
S.-O. Enfors: Food microbiology
2. The ecological basis of food spoilage
7
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 "psychrotrophic, Gramnegative 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 materials 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 which 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
S.-O. Enfors: Food microbiology
2. The ecological basis of food spoilage
8
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
Egg shell
Primary flora ≈ 103 cells/ cm2
Milk
Contamination flora ≈ 103 cells / ml
Meat
Fish fillet
Contamination flora ≈ 103 cells / cm2
Spoilage flora on most food types
≈ 107 - 108 cells / cm2 or gram
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
- Water activity (aw)
- pH and buffer capacity
- Oxygen concentration and transfer
- Mechanical barriers
- Metabolisable energy sources
- Metabolisable nitrogen sources
- 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
S.-O. Enfors: Food microbiology
2. The ecological basis of food spoilage
9
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
(cfu)
9
10°C
8°C
20°C
4°C
0°
C
7
5
3
1
0
150
Time (days)
Fig 2.3. Influence of temperature on the total bacterial count (colony forming units, cfu)
on fresh meat. The dotted line indicate the typical level of spoilage. Note that the growth
initially is exponential.
Relative growth rate
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 .
Mesophiles
Psychrotrophes
Thermophiles
Psychrophiles
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.
S.-O. Enfors: Food microbiology
2. The ecological basis of food spoilage
10
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 2035 °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 , the cell number becomes 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). The water activity is one of the main parameters which
determine 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
aw = pH2 O
pH2 O*
a closed vessel in 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
S.-O. Enfors: Food microbiology
2. The ecological basis of food spoilage
11
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
Starch
Relative rate
Fruit
30
20
Rel
reaktionshastighet
Water concentration (%)
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 2.5 which shows sorption isotherms 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.
Meat
10
Cellulose
0
0
0.3
0.6
0.9
0
Lipid
oxidation
Lipolysis
Proteolysis
0.2
Water activity
Fig 2.5. Sorption isotherms for different
materials show that aw is not the same as
water concentration
Fungi
0.4 0.6 0.8
Water activity
Bacteria
1
Fig 2.6. Schematic view of how the
aw influences the rate of enzyme
reactions 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 water activity and many enzymes retain 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.
S.-O. Enfors: Food microbiology
2. The ecological basis of food spoilage
12
Table 2.2 Examples of typical minimum water activity for growth of some
microorganisms and corresponding aw in some foods.
Organism
Min aw
Food examples
Food aw
Milk, fish, meat
0.99
Pseudomonas
0.97
E. coli
0.96
Sausage, 7% salt
0.96
Clostridium
0.95
Brochothrix
0.94
thermosphacta
Bacillus
0.93
Ham, 12% salt
0.93
Lactobacillus
0.93
Streptococcus
Lactococcus
0.93
Micrococcus
Salmonella
0.91
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% water
0.6
None
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,
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2. The ecological basis of food spoilage
13
temperature, and nutritional conditions. Thus, such data are only approximate
and indicative of relative sensitivities.
pH is another parameter with 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 (Table 2.4), with the
exception lactic acid bacteria wich 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 juice
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
6-9
7±1
Lactic acid bacteria
4-7
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
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2. The ecological basis of food spoilage
14
on the exudate, i.e. the glucose and amino acid rich liquid which 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, which 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 which 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
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
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2. The ecological basis of food spoilage
15
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:
amino acid + O2
deaminase
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.
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2. The ecological basis of food spoilage
16
Proteolysis. One could expect that proteolysis should 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:
Proteins
proteinase
peptides
peptidase
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.
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:
amino acids
Anaerobic
metabolism
Amines
Organic acids
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 fishes of mackerel
type, 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.
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2. The ecological basis of food spoilage
17
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
H3 C - N = O
CH3
TMAO
CH3
TMAOreductase
H3 C - N
CH3
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.
S.-O. Enfors: Food microbiology
2. The ecological basis of food spoilage
18
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 scavenge 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.
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2. The ecological basis of food spoilage
19
ß-oxidation is the common metabolic route for degradation of fatty acids and
each cycle results in generation of one acetyl-CoA 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 is
present in linolic and linolenic acids in plants and in arachidonic acids in
animal tissues. To prevent this type of rancidification during storage some
vegetables, e.g. frozen spinach and peas, are heat treated to inactivate the plant
enzyme. However, these aldehydes and ketones are not always unwanted
products in food. They are also important ingredients in certain types of
cheeses (see Chapter 6).
Degradation of carbohydrates
Microorganisms 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 2.10). The most common fermentative
pathways are listed in Table 2.7.
S.-O. Enfors: Food microbiology
2. The ecological basis of food spoilage
Table 2.7. Common fermentation types
Fermentation type
Alcohol fermentation
Homofermentative lactic acid fermentation
Heterofermentative lactic acid fermentation
Propionic acid fermentation
Butyric acid fermentation
Mixed-acid fermentation
2,3-butanediol fermentation
20
Products
Ethanol, CO2
Lactic acid
Lactic acid, Acetic acid, Ethanol,CO2
Propionic acid, Acetic acid, CO2
Butyric acid, Acetic acid, CO2, H2
Lactic acid, Acetic acid, CO2, H2, Ethanol
CO2, Ethanol, Butanediol, Formic acid
Of these fermentation types, it is the butyric acid, mixed acid and butanediol
fermentations which 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
Ethanol fermentation
+
Lactic acid fermentation
NAD
ATP
NADH
+
+
NAD
Acetaldehyde
Pyruvate
Lactate
Formate
AcetylCoA
Oxaloacetate
AcetylCoA +
+
NAD
Acetate
NAD
Succinate
Ethanol
CO2
+H2
ATP
ATP
ATP
+
NAD
Ethanol
H2
CO 2
Mixed acid fermentation
Acetoin
AcetacetylCoA
Acetate
ATP
+
NAD
+
NAD
Propionate
Butyrate
Butandiol
+
NAD
+
Propionic acid
fermentation
Acetone
Butandiol
fermentation
NAD
Butanol
2-propanol
Butyric acid fermentation
Fig 2.10 Summary of the six main fermentative pathways. The main end products are
emphasised by frames. Sites of co-enzyme generation and ATP formation are indicated.
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2. The ecological basis of food spoilage
21
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 which 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
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. The slime formation on cold-stored fresh meat usually
comes after the meat has become unacceptable due to smelling. Some species
of lactic acid bacteria produce polysaccharides and this is sometimes utilised in
various fermented milk products to give a higher viscosity (yoghurt, Swedish
långmjölk). However, the viscosity of yoghurt is mainly caused by protein
precipitation due to low pH.
S.-O. Enfors: Food microbiology
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 3.1.
Table 3.1 Food categories with different protection against microbial spoilage.
Food properties
Example
Protection
Water-rich
Protein-rich
Relatively neutral pH
Meat
Fish
Milk
Cooked food
None
Water-rich
Protein-poor
Relatively sour
Fruits
Vegetables
Root-fruits
Low pH
Inhibitors
Mechanical structure
Water-poor
Grains
Flour
Bread
Low aw
Fermented food
See Chapter 6
Often low aw + low pH
Microbial competitors
Microbial inhibitors
Preserved food
Salted/dried
Pickled
Smoked
Sterilised
Pasteurised
Low aw
Low pH
Low pH, low aw, inhibitors
No microflora
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 which keeps the
S.-O. Enfors: Food microbiology
3.
Spoilage of different types of food
egg protected from microbial attack for several weeks at room temperature.
This is described in Fig 3.11.
Meat
At the moment of slaughter, the animal's breathing and the 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 3.2). 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 is Pseudomonas (and other Gram negative psychrotrophic rods) and
these organisms are quite sensitive to pH below about 5.5-6, the final pH of
the meat is considered important for the shelf-life.
S.-O. Enfors: Food microbiology
23
3.
Spoilage of different types of food
Table 3.1. Typical pH of meat from different animals and lenth of rigor mortis.
Animal type
Cow
Swine
Chicken
Fish
Rigor mortis
10-20 h
4-8 h
2-4 h
min-h (longer on ice)
final pH
6 - 5.5
6
6.4 - 6
6.8 - 6.4
The meat contains many nutrients for the microorganisms (Table 3.3) which
only grow on the exudate from damaged tissue. Furthermore, it is only on the
surface of meat the microorganisms 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 nutrients 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
Fresh 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 the time of 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 which 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 developing under these conditions.
During storage, the bacteria initially grow exponentially, sometimes after a
lag phase which is caused by a 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
S.-O. Enfors: Food microbiology
24
3.
Spoilage of different types of food
25
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 bacteria, which is strongly related to the hygiene during and after
slaughter.
slime
odour
chicken
2
log N/cm
8
7
slem
odör
kyckling
6
pork
griskött
5
Fig 3.2. Example of microbial
growth measured as "total
aerobic count" during storage of
fresh pork and chicken meat at
refrigerator temperature.
4
3
2
1
0
2
4
6
Tid (d)
Days
8
10
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 of 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=
Glucose (µg/g)
2.7
Fig
3.3. Glucose concentration
gradients
and
microflora
development during cold storing of
fresh meat. At N=32*107 cm-2 the
meat was classified as spoilt and this
coincides with glucose exhaustion at
the surface.
6.3
32
110
0
280
0
20
0
Distance from surface (mm)
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3.
Spoilage of different types of food
26
Carbon dioxide and vacuum packages
Vacuum packaging of meat, both fresh and cured meat, dramatically prolongs
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 which dominates under anaerobic conditions
produces more off-flavour, unless the dominating microflora is composed of
lactic acid bacteria. 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 and subjected to air, the microbial growth rate
immediately increased.
logN / cm2
9
Luft
air
8
N2
Kväve
air
Luft
7
6
CO2
Luft
air
5
4
CO2
3
0
8
16
Tid (dagar)
24
32
Fig 3.4. Influence of
the gas atmosphere on
the growth rate of
microorganisms
on
refrigerated fresh pork
meat. Some of the CO2
stored samples were
opened and further
exposed to air, as
indicated in the CO2plot.
When the composition of the microflora was investigated under these
conditions it became clear that the atmosphere exerts a selecting pressure, see
table 3.4. In air the dominating microflora usually is Pseuomonas. These
organisms are obligate aerobes or use nitrate respiration in absence of oxygen.
In nitrogen atmosphere different species from the Enterobacteriacae family
dominate. These organisms possess a strong fermentation capacity with illtasting 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 which favours growth of
Lactobacillus, which with their lactic acid fermentation have less impact on
the spoilage than the Pseudomonas .
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3.
Spoilage of different types of food
27
Table 3.4. Dominating spoilage flora on cold stored pork in different atmospheres.
O2
%
20
N2
%
80
Pseudomonas
Enterobacteriacae
Aeromonas
Brochothrix Lactobacillus
+
80
100
80
10
CO2
%
+
+
20
20
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 plastic film of the vacuum package has a low gas
permeability and the gas headspace is removed by the vacuum, the partial
pressure of CO2 raises rapidly and exerts a protecting function. Also the shelflife promoting effect of vacuum packing of cured meat products is similar but
in that case it is the metabolic activity of the microflora which produces the
CO2. Table 3.5 lists some properties of bacteria which contribute to the
selection pressure in vacuum packed fresh and cured meat.
S.-O. Enfors: Food microbiology
3.
Spoilage of different types of food
28
Table 3.5 Some characteristics of the organisms that dominate the
spoilage flora on cold-stored fresh and cured meat in different
atmospheres.
Organism
Properties
Pseudomonas
Fast growing
Aerobic
Very CO2-sensitive
Sensitive to low aw
Enterobacteriaceae
Facultative
Intermediate CO2-sensitivity
Aeromonas
Facultative
Intermediate CO2-sensitivity
Brochothrix thermosphacta
Facultative
Relatively CO2 resistant
Resistant to low aw
Lactobacillus
Very CO2-resistant
Indifferent to oxygen
Resistant to low aw
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 which 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.
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3.
Spoilage of different types of food
The antimicrobial effect of CO2 on many spoilage organisms has been utilised
also for direct packaging of food in gaseous atmosphere. These so called
"controlled atmosphere" packages contain mainly carbon dioxide as growth
inhibiting compound but also some oxygen to avoid anaerobic metabolism
and decolourization of the haeme in meat.
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 contributes 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.
ATP
ATPase
ADP
Myokinase
AMP
AMP-deaminase
IMP
Phosphomonoesterase
Fig 3.7. During the post mortem
Inosine
metabolism in the fish tissue inosine
Nucleoside phosphorylase
monophosphate (IMP) is transiently
accumulated.
hypoxhantine + ribose-P
This metabolism has been utilised to develop a "fish-freshness" biosensor in
Japan (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
S.-O. Enfors: Food microbiology
29
3.
Spoilage of different types of food
including also the 5'-nucleotidase the oxygen consumption also includes the
IMP.
IMP
Inosine
Hypoxanthine
OH
N
N
1 5’-nucleotidase
OH
N
O
N
CH2 - P O
3 xanthine oxidase
OH
OH
2 nucleotide
phosphorylase
OH
Enzymer = analys
3 = Hx
2 3 = I + Hx
1 2 3 = IMP + I +Hx
Index =
IMP
IMP + I +Hx
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 relation 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 freshwater
fishes
and
Shewanella
with
marine fishes.
S.-O. Enfors: Food microbiology
30
3.
Spoilage of different types of food
Milk
Milk is a very good substrate for microbial growth. However, it is protected
by several antimicrobial mechanisms which 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 uses the
hydrogen peroxide to oxidise the thiocyanate 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
H2 O2
H2 O
LP
thiocyanate
SCN
OSCN
hypothiocyanate
HO-S-protein
HS-protein
Fig 3.10. The lactoperoxidase system. The lactoperoxidase in milk uses 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 the cow house the
milk is infected with several types of microorganisms as shown in Table 3.6
Table 3.6 The initial milk contamination microflora
Infection
Source
Feces
E. coli
Enterococcus
Micrococcus
Bacillus spores
Air
Mold spores
Yeasts
Lactococcus
Lactobacillus
Milking equipment
Gram-negative rods
S.-O. Enfors: Food microbiology
31
3.
Spoilage of different types of food
If the milk is stored at room temperature the "lactic streptococci", i.e.
Lactococcus spp. will first dominate the microflora and protect it from most
of the other microorganisms by means of lactic acid production. Eventually
Lactobacillus, which can grow at lower pH than the other bacteria (below 5)
will dominate. This fermented milk is similar to yoghurt and it 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
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
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 are not influenced at all by the pasteurisation.
After the pasteurisation the milk becomes 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 the milk 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 of milk by lactic acid
bacteria is not the common fate of refrigerated pasteurised milk.
Egg
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 interior of the
egg due to a number of defence mechanisms, which are illustrated in Fig 3.11.
If this protection fails and the egg becomes invaded by bacteria it is usually
Pseudomonas fluorescens which dominates (80%). These infections can be
detected by illumination of the egg with UV-light.
S.-O. Enfors: Food microbiology
32
3.
Spoilage of different types of food
Albumin: viscous, high pH
(pH9,5), riboflavin + pyridoxin
complexing
No
protect
ion
Con-albumin: Fe2+ complexing
Avidin: Biotin complexing
Lysozyme: kills G+ bacteria
outer mucin layer
1-10µm pores in shell
inner keratin
membrane
Fig 3.11. The egg is protected against bacterial infections an multiple ways: The shell and
the two membranes provide mechanical hinders for the bacteria. The high pH in the egg
white is non-optimal for many bacteria. The egg white contains several protection
mechanisms: Lysozyme ruptures cell walls of many bacteria. Albumin, conalbumin and
avidin make several nutrients 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 is 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 dominate in 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 Nacylated 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 generate antimicrobial chemicals when the plant is attacked.
S.-O. Enfors: Food microbiology
33
3.
Spoilage of different types of food
34
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 damaged 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 many bacterial cells have accumulated 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 has 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 3.7 gives an example of a modified atmosphere for fruits. The exact
composition is optimised for each product.
S.-O. Enfors: Food microbiology
3.
Spoilage of different types of food
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 have a primary microflora 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. Regulations set a maximum water concentration of 13%
for storage of grains for human food 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 microorganisms are transferred to the flour. Typical microbial counts
are 102-103 bacteria plus about 100 mold spores per gram sifted flour and
about ten 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 too 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 produce 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
propionates are often used a preservatives in industrial baking. Dry bread
(knäckebröd) is not subject to any microbial spoilage, provided it is stored
S.-O. Enfors: Food microbiology
35
3.
Spoilage of different types of food
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 spray-drying the food is exposed temperatures that kill
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.
Table 3.8. Summary of common spoilage floras on different types of food
Cured meat products are usually protected by the low water activity created
by salt addition. If the products are fermented 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
S.-O. Enfors: Food microbiology
36
3.
Spoilage of different types of food
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-time limitation is usually rancidification of the fat.
Table 3.9.Properties of common food related organisms
Organsim
Properties
Gramneg. rods:
- Psychrotropic
- Pseudomonas
- Aerobic
- Sensitive to low aw
- Sensitive to low pH
- CO2-sensitive
- Shewanella
- H2S-producer
putrefaciens
Lactic acid bacteria:
-Lactobacillus
-Lactococcus
-Pediococcus
Enterococcus
B. thermpsphacta
Grampositive cocci:
-Micrococcus
-Staphylococcus
Spore formers:
-Bacillus
-Clostridium
Enterobacteriaceae:
-E.coli
-Enterobacter m.fl.
-Erwinia
Molds
Yeasts
- O2-indifferent
- Resistant to low aw
- Resistant to low pH
- CO2-resistant
- Facultative
- Resistent to low aw
- Resistant to low pH
- Heat resistant
- Lipo-/proteolytic
- Extremt värmeresistenta
- Mesofila
- Starkt fermentativa
- Strongly fermentative
- Pektinase-active
- Aerobic
- CO2-sensitive
- Pektinase-active
- Resistant to low aw
- Resistant to low pH
- Lipo-/proteolytic
- Facultative
- CO2-resistant
- Resistant to low aw
- Resistant to low pH
Products
Refrigerated fresh:
-Meat
-Fish
-Milk
Fish
Fish preserves
Vacuum packed
Fermented food
Smoked/salted/dried meat/fish
Pickles
Fish preserves
Vacuum packed
Fermented food
Smoked/salted/dried meat/fish
Heat sterilised food
Reconstituted dried food.
Pre-cooked
Milk: B.cereus
Milk
Pre-cooked food
Vegetables
Vegetables
Fruit
Dried food
Vegetables
Fruit
Low-pH preserves
Sweet products
S.-O. Enfors: Food microbiology
37
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
Membrane damaging toxins (several)
Epidermolytic toxin
Toxic shock syndrom toxin
Pyrogenic exotoxin
Enterotoxin ( 6 serotypes)
Exoenzymes
Coagulase
Staphylokinase
Proteases
Phospholipase
Lipase
Hyaluronidase
4. Foodborne pathogens
40
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
4. Foodborne pathogens
41
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.
4. Foodborne pathogens
42
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.
4. Foodborne pathogens
43
Table 4.2 Examples of mycotoxins, mycotoxigenic molds, and associated food
Toxin
Aflatoxins
Organism
Aspergillus flavus
Asp. parasiticus
Associated food
Nuts, figs, corn
Effect
Liver cancer
Ochratoxin A
Asp. ochraceus
Penicillium viridicatum
Grains, coffee, wine,
beans
Kidney/liver
damage, teratogenic
Patulin
Pc. exapnsum
Pc roqueforti
Fruits, berrys
Diarrhoea
Penicillinic acid
Pc. cyclopium
Pc viridicatum
Peas
Zearalenon
Fusarium graminearum
Grains
Roquefortin
Pc. roqueforti
Bread
zone
1
2
3
4
Infertility
µg aflatoxin/ kg bread
Bread 1
Bread 2
Bread 3
>> 15 000
600
100
n.d
150-300
n.d
n.d
40-80
20
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
4. Foodborne pathogens
44
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
4. Foodborne pathogens
45
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, unpasteurised 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.
4. Foodborne pathogens
46
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
4. Foodborne pathogens
47
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
ETEC
EPEC
EIEC
enterohemorrhagic E. coli
enterotoxigenic E. coli
enteropathogenic E. coli
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.
4. Foodborne pathogens
48
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
4. Foodborne pathogens
49
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
4. Foodborne pathogens
50
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. Food preservation
There are two main principles to preserve food from microbial spoilage (Fig
5.1): Inactivate the microorganisms or create conditions which slow down the
growth rate. The dominating method to inactivate microorganisms in food is by
heat (sterilisation and pasteurisation) but to some extent also inactivation by
irradiation is used and recently also exposure to high pressure is emerging as a
food preservation method. The most important method to reduce the growth
rate is by reducing the temperature (refrigeration or freezing) or by reducing the
pH or water activity (drying or salting). Addition of chemical food
preservatives is common. Sterilisation by filtration is important for production
of sterile liquids in the pharmaceutical industry, but this is hardly applicable in
the food industry.
Inactivation of microorganisms:
Heat
Irradiation
Hydrostatic pressure
Chemical disinfection
Inhibition of microorganisms:
Cooling/Freezing
Low aw, pH
Chemical preservatives
Removal of microorganisms:
Filtration
Fig 5.1 Principles of preservation against microbial spoilage.
5.1 Heat sterilisation and pasteurisation
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.
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, Sporolactobacillus and Thermoactinomyces may form endospores. Endospores are
extremely resistant to heat, UV and ionising radiation, drying and chemical
5. Food preservation
52
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 5.2 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
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 5.3
Core: DNA, Ca-DPA, few ribosomes,
key enzymes, no water
Cortex: Negatively charged peptidoglucanes
Coat: chemically resistant proteins
Fig 5.2 Structure of a bacterial endospore.
Transformation of a spore to a vegetative cell involves a number of reactions
(Fig 5.3). The activation is a reversible reaction, which is poorly understood.
Activation may be needed to make a spore competent for the next stage,
germination.
S.-O. Enfors: Food microbiology
5. Food preservation
activation
dormant spore
53
germination
activated spore
germinated spore
outgrowth
lysis
sporulation
vegetative cell
growth
Fig 5.3 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 which 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
S.-O. Enfors: Food microbiology
5. Food preservation
54
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 5.3). 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.
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
S.-O. Enfors: Food microbiology
5. Food preservation
55
"N%
ln$ ' = (kt
# N0 &
(2)
ln( N ) = ln(N 0 ) " kt
(3)
N = N 0e"kt
(4)
which can be rearranged to
!
or to
!
for calculation of the number of surviving cells after a given time. Eq. 3 is
illustrated in Fig 5.4.!Experimentally determined inactivation curves often show
deviations from this model. Some examples of this are shown and explained in
Fig 5.4.
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 below.
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
-1
(5)
where A (min ) 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.
S.-O. Enfors: Food microbiology
5. Food preservation
56
Fig 5.4. 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:
ln(k) = ln(A) "
#E 1
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 5.5.
1
k (1/min)
spores
!E 280
kJ/mole
Thiamine
!E 92
kJ/mole
0.1
Fig 5.5 Arrhenius plots of inactivation
of B. stearothermophilus spores and
thiamine. Note that a temperature
increase has a larger effect on the spore
inactivation rate than on the vitamin
inactivation rate.
96 °C
112 °C
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
S.-O. Enfors: Food microbiology
5. Food preservation
57
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 5.6.
high T, low aw, high pH
Fig 5.6. 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.
S.-O. Enfors: Food microbiology
5. Food preservation
58
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
Calculation of sterilisation time
According to the model for heat inactivation, eq. 3 and Fig 5.4, 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 5.4. 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
!
S.-O. Enfors: Food microbiology
(10)
5. Food preservation
59
which, after substitution of k according to the Arrhenius model (eq 4), gives the
wanted sterilisation time at a different temperature T °K:
FT = Fref e
"#E $ 1
1'
&&
" ))
R % Tref
T(
(11)
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 5.7). 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
" = Fref Ae
#$E /RTref
(14)
Division of both sides of eq 13 by eq 14 gives the ratio between ∇(t) and ∇:
!
t
% (e
)dt
"(t)
0
=
#$E / RTref
"
Fref e
(
#$E / RT
)
(15)
Fig 5.7 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
! only with some 20 per cent of the total sterilisation dose.
and it contributes
Since cooling from the highest temperature first is very efficient, the
contribution to sterilisation from the cooling phase is very small. Note also that
S.-O. Enfors: Food microbiology
5. Food preservation
60
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 5.7. 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 .
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 5.8.
S.-O. Enfors: Food microbiology
5. Food preservation
30
61
1
F
C / Co
C / Co
F (min)
20
10
0
115
120
125
130
T
135
140
0
145
Fig 5.8 Effect of sterilisation
temperature on a chemical
reaction
in
a
continuous
sterilisation. C/Co is the fraction
of
non-reacted
chemical
compound. F is the sterilisation
time according to eq. 9. C/Co
was obtained from eq. 7 and the
temperature dependence of the
two reaction rate constants, k,
was obtained from the Arrhenius
equation (eq. 5). Parameters as
for the batch sterilisation, Fig 5.7.
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
D
# N0 &
(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 5.9). The decimal reduction time
(D, min), is the time needed to reduce the number of cells to one tenth of the
previous value.
S.-O. Enfors: Food microbiology
5. Food preservation
62
Fig 5.9 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:
D=
ln(10)
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 5.4 or 5.9. 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.
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5. Food preservation
63
Table 5.2 D-values (min) for heat inactivation of microorganisms
D121
D60
D50
Organism
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 5.10
Fig 5.10 The temperature dependence of the D-value and the definition of the Z-value.
The mathematical expression of the temperature dependence shown in Fig 5.10
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.
S.-O. Enfors: Food microbiology
5. Food preservation
64
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).
!
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5. Food preservation
65
5.2 Chemical preservation
A number of chemical additives is used by the food industry to improve
different properties of the food. These additives are usually identified and
referred to by serial E-numbers. Table 5.3 lists different categories. Only the
E200 series containing the chemical food preservatives will be described here.
Table 5.3 E-numbers for different categories of
food additives
Color additives
Preservatives
Antioxidants
Emulgators / thickening agents
Inorganic salts
Flavour improving agents
Sweeteners
Starch derivatives
E 100
E 200
E 300
E 400
E 500
E 600
E 900
E 1400
Table 5.5 on next page lists in detail all currently accepted preservatives in
Sweden. This list is not static and components may be removed or added and
it varies somewhat from country to country. For each component there are
detailed specifications on maximum concentration and in which products the
specific preservative may be used.
Week organic acids. A closer look on the list shows that a large part of the
preservatives are weak organic acids and their corresponding salts. It is the
undissociated acid which is the active component in this category of food
preservatives even if it often is a salt which is used. With this view, the list of
organic acid preservatives is reduced from 24 to 7 components, as shown in
Table 5.4
Table 5.4 Week organic acids used as food preservatives
Code
E26E28E270
E20E21E296
E297
Active substance
Acetic acid
Propionic acid
Lactic acid
Sorbic acid
Benzoic acid
Malic acid
Fumaric acid
Application examples
Antibacterial No conc. limit
Antimold. Only in bread and snuff
Antibacterial. No conc. limit
Antimold/yeast
Antimold/yeast
No concentration limit
Sweets, deserts
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5. Food preservation
66
Table 5.5 Food preservatives and corresponding E-number.
E200 Sorbic acid
E232 Sodium ortophenylphenol
E202 Potassium sorbate
E234 Nisin
E203 Calcium sorbate
E235 Natamycin
E210 Benzoic acid
E239 Hexamethylenetetraamine
E211 Sodium benzoate
E242 Dimethyldicarbonate
E212 Potassium benzoate
E249 Potassium nitrite
E213 Calcium benzoate
E250 Sodium nitrite
E214 Parahydroxybenzoic acid ethylester
E251 Sodim nitrate
E215 Parahydroxybenzoic acid ethylester-Na
E252 Potassium nitrate
E216 Parahydroxybenzoic acid propylester
E260 Acetic acid
E217 Parahydroxybenzoic acid propylester-Na
E261 Potassium acetate
E218 Parahydroxybenzoic acid methylester
E262 Sodium(hydrogen)acetate
E219 Parahydroxybenzoic acid methylester-Na
E263 Calcium acetate
E220 Sulfur dioxide
E270 Lactic acid
E221 Sodium sulfite
E280 Propionic acid
E222 Sodium hydrogensulfite
E281 Sodium propionate
E223 Sodium disulfite
E282 Calcium propionate
E224 Potassium disulfite
E283 Potassium propionate
E226 Calcium sulfite
E284 Boric acid
E227 Calcium hydrogensulfite
E285 Sodium tetraborate
E228 Potassium hydrogensulfite
E290 Carbon dioxide
E230 Diphenyl
E296 Malic acid
E231 Orthophenylphenol
E297 Fumaric acid
Several organic acids, like acetic acid, are often good carbon/energy sources
for microorganisms. However, it is the dissociated ion, e.g. acetate, which
then is taken up by active transport mechanisms, while the undissociated acid
has the inhibitory effect. This means that the food pH has a large influence on
the effect of this class of food preservatives. Table 5.6 shows the pKa values
for some of the most common food preservatives and the relationship between
pH and concentration of acid.
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5. Food preservation
67
Table 5.6. pKa-values for some food preservatives and formula for the acid-base
equilibrium, showing the pH influence on concentration of undissociated acid.
Acid
Acetic acid
Propionic acid
Lactic acid
Sorbic cid
Benzoic acid
pKa
4,8
4,9
4,3
4,8
4,2
pH = pK a + log
[base]
[acid]
!
The food preservative acids are all relatively week acids (Table 5.6), which
means that at the relatively low pH in most foods, a considerable part of the
total acid/base system is present as undissociated acid. This is illustrated as
illustrated in Fig 5.11 which shows a graphic illustration of the dissociation of
the acid HA to the base A- and a proton (in H3O+)
log C
pKa
14
0 0
log Ctot
pH
-
HA
A
OH-
H3O+
Fig 5.11 Diagram illustrating the acid-base equilibrium for the dissociation HA " H + + A #
Ctot is the total concentration HA+A-. Not the logarithmic scale.
The hypothesis that it is mainly the undissociated form of
! the acid which has
the inhibitory effect is illustrated in the experiments the inhibitory effects of
several weak organic acids on E. coli shown in Fig 5.12. In these experiments
the concentration of undissociated acid was varied either by varying the total
concentration or by varying the medium pH (see also Fig 5.11). The left panel
in Fig 5.12 shows that the growth rate depends on the concentration of
undissociated acetic acid irrespectively whether the concentration was varied
by total concentration or pH. The middle panel shows that this resulted in
S.-O. Enfors: Food microbiology
5. Food preservation
68
reduced intracellular pH from 7.4 at max growth rate to about 6.2 when the
growth rate was as lowest. The right panel shows that the growth rate declines
with the intracellular pH irrespectively of which organic acid was used to
reduce the intracellular pH.
100
Growth rate (%)
80
60
40
20
0
7.4
6.2 6.6
7
0 0.1 0.2 0.3
Undissociated acid (mM)
Intracellular pH
= pHo varied / Total conc. 2.5 mM
= Total conc. varied / pHo 5.0
6.2
7.4
6.6
7
Intracellular pH
Propionic acid
Cinnamic acid
Sorbic acid
Bencoic acid
Fig 5.12 The growth rate of E. coli depends on the intracellular pH which is reduced by the
concentration of undissociated acid. See text above for further explanation.
Possible mechanisms behind these effects of undissociated acids are
illustrated in Fig 5.13.
Fig 5.13 Two mechanisms contributing to the inhibitory effects of undissociated organic
acids. Left: The undissociated acid HA diffuses through the cell membrane and dissociates
in the cytoplasm (pH ≈ 7.4), which reduces the pH. Protons are pumped out on expense of
ATP. Right: The reduced pH gradient caused by reduced intracellular pH reduces the
driving potential for ATP regeneration in respiration.
S.-O. Enfors: Food microbiology
5. Food preservation
69
At least two mechanisms may contribute. Only the non-polar undissociated
acid can diffuse through the cytoplasmic membrane. In the relatively high pH
in the cytoplasm the acid dissociates and reduces the pH. The cell tries to
control the intracellular pH by pumping out protons, which costs energy in
form of ATP. Too high diffusion rate of undissociated acid into the cell
therefore uncouples energy from growth. It can not be ruled out that inhibition
of enzymatic reactions in the declining cytoplasmic pH also plays a role. A
second mechanism, relevant for respirating organisms, may be that the
declining intracellular pH also diminishes the proton gradient over the cell
membrane which is needed for the ATP generation in respiration.
Parabens. The different forms of p-hydroxybenzoic acid esters are called
parabens. The inhibitory mechanism of some of the parabens is inhibition of
the phosphotransferas system for uptake of sugars but other mechanisms are
also likely.
Sulfites. A third group of chemical food preservatives is the various forms of
substances which form SO2. When hydrogensulfites, sulfates or disulfites are
dissolved in water dissociation reactions results in small concentrations of
dissolved gaseous sulfur dioxide SO2 which probably is the active
components. Also this compound then diffuses into the cell where it also
reduces the pH but other more specific interactions with enzymes probably
constitute the main inhibiting mechanism.
Nitrites and nitrates is a group of salts which in water solution generate small
amounts of nitrous acid HNO2. It is likely that also these compounds enter the
cell by diffusion. The nitrite and nitrate ions per se have no preservative
effect. Also these ions are common nutrients for microorganisms, e.g. in
denitrification reactions. The hypothesis that it is the undissociated nitrous
acid rather than nitrite which exerts the inhibition is illustrated in Table 5.7,
which shows that irrespectively of the nitrite concentration it is the
concentration of undissociated acid which determines the inhibition.
Table 5.7. Maximum concentration of nitrite for growth of Staphylococcus aureus
at different pH and corresponding concentration of undissociated nitrous acid.
pH
Max concentration of NO2- Concentration of HNO2 (ppm)
for growth (ppm)
6,9
3500
1,00
5,8
300
1,06
5,7
250
1,11
5,2
80
1,13
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5. Food preservation
70
The exact mechanisms of nitrous acid inhibition in the cells is not known, but
it generates the gas nitric oxide, NO, which is strongly toxic through reaction
with sulfhydryl containing enzymes. The use of nitrate and nitrite as food
preservative has been controversial. Firstly, nitrate has probably an effect only
through its partial conversion to nitrite, a reaction which may be catalysed by
many bacteria through nitrate respiration, which generates an unknown
amount of nitrite. Secondly, nitrite can generate the cancerogenic
nitrosamines when heated in sour environment. On the other hand, nitrite is an
efficient inhibitor of germination of Cl. botulinum spores, and for his reason it
is used to increase the safety in products where these spores occur, like
preserved meat and fish products.
The fear for botulism in preserved food has resulted in some standards for
food preservation. In chemical preservation it is generally assumed that either
of the following criteria is sufficient to prevent growth of Cl. botulinum:
pH>4.5; NaCl > 8%; or acetic acid > 2.5%.
In practice a combination of chemical preserving factors is often used. Fig
5.14 shows an example of how much nitrite is needed for the prevention of
the growth of Cl. botulinum at different pH and salt concentrations.
Fig 5.14. 3D diagram showing combined effect of sodium nitrite, salt and low pH on the
inhibition of Clostridium botulinum. Areas with absence of a cube symbol means that
growth is prevented.
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5. Food preservation
71
5.3 Classification of preserved foods
Preserved food products are classified in categories which demand specific
storage conditions. See Table 5.8.
Fully preserved food. To this category belongs heat sterilised canned food.
The product must be hermetically contained, usually in glass or metal cans.
Canned soups, ham, vegetables and fruits belong to this group. The sterility
shall guarantee that these products have a shelf-life of minimum one year at
room temperature. In reality the shelf-life is usually longer. Since it is not
limited by microbial growth, it is usually rancidifiaction or other chemical
reactions which limits the shelf-life. To reduce oxidation reactions, these
products may have been supplemented with antioxidants. The concept
“commercially sterile” is sometimes used in food sterilisation. It means that
the processing has eliminated all endospores which can germinate and grow
out at room temperature, but there may still be a few extremely heat resistant
spores of thermophilic Clostridium spp. These cells will not spoil the product
under the storage condition “room temperature”.
Table 5.8. Classification of food preserves and storage conditions
Cold-stored preserves is another type of preserved food, which may also be
canned or packed in plastics. They are either pasteurised, rather than heat
sterilised and they are usually further preserved by chemical preservatives.
The shelf-life demand is minimum half a year at refrigerator temperature. For
each product the maximum storage temperature should be indicated on the
package. Fish preserves are common in this category and the storage
S.-O. Enfors: Food microbiology
5. Food preservation
72
temperature is usually maximum 4°C. The shelf-life is usually limited by
rancidification and/or by slow growth of lactic acid bacteria, especially
Pediococcus and yeasts.
Frozen products is a large category of preserved food, which is usually
preserved only by the low temperature which shall be below -18°C. The
shelf-life of these products are usually limited by rancidification. To reduce
the activity of certain endogenous enzymes in the product, vegetables and
fruits are sometimes blanched, i.e. subjected to a short heat treatment, before
the freezing.
S.-O. Enfors: Food microbiology
73
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
74
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)
Sauerkraut
Lactic fermentation
Pickles
Lactic fermentation
Bread
Ethanol fermentation
Beer
Enzymatic hydrolysis and ethanol
Vegetables
Cereals
fermentation
Fruits
Soy sauce
Enzymatic hydrolysis by moulds, lactic and
ethanol fermentation
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.
6. Fermented foods
75
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
6. Fermented foods
76
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 polyglucose, 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-
6. Fermented foods
77
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 αamylase 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 nonreducing 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.
6. Fermented foods
78
Table 6.3 Temperature and pH optima
of the main malt enzymes
Enzyme
pH
Temperature
α-amylase
5.7
70
ß-amylase
ß-glucanase
proteinase
5.5
5.1
4-5
60
57
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 ßglucanolysis 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.
6. Fermented foods
79
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
E (%)
60
EtAc (mg/L)
5
50
0
0
N
E
0
0
EtAc
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
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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 postfermentation 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 αacetolactate 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
6. Fermented foods
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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
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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 cremoris
Lactococcus diacetilactis
Leuconostoc cremoris
Lactococcus thermophilus
Lactobacillus bulgaricus
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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.
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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
of curd, whey
separation
Formation of the cheese
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.
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Table 6.9. Main ingredients of cow's milk
Component
Concentration (%)
Water
87
Lactose
5
Fat
3.8
Protein
3.4
Caseins
2.8
1.7
α0.6
β0.1
γ0.4
κWhey proteins
0.6
albumins
globulins
Salts
0.9
CalciumCitrates-
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
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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
Gorgonzola
Stilton
Penicillium roqueforti
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.
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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 cell bound 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
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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
6. Fermented foods
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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.