Food microbiology

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Feb 12, 2013 (4 years and 7 months ago)

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S.
-
O. Enfors: Food 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 preser
ved 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





1


S.
-
O. Enfors: Food microbiology


Chap 1
Introduction


Living organisms are usually classified as animals, plants, algae, pro
tozoa,
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
classifi
cation 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 encou
nter 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 specie
s 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 sinc
e many species
within the same genus are discussed. Then,
Bacillus sp.
means one not defined
Bacillus
species and
Salmonella spp
. means several not defined
Salmonella

species.


Table 1.1. Examples of family names, genus names and species names

Family

Genus

Species

Enterobacteriacae

Escherichia

Escherichia coli


Salmonella

Salmonella typhimurium



Salmonella enterica

Bacillacae

Bacillus

Bacillus subtilis



Bacillus cereus



Bacillus anthracis


Clostridium

Clostridium botulinum

Bergey’s Manual of Dete
rminative 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 rea
ction 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 wal
l 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

Introduction

2





S.
-
O. Enfors: Food microbiology

(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 l
ow 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

Description

Food related organisms

2

Gram
-
neg., aerobic, mobile, vibrio
-
formed

Campylobacter

4

Gran
-
neg., aerobic rods or cocci

Pseudomonas, Sh
ewanella, Legionella

5

Gram
-
neg., facultatively anaerobic
rods

Family
Enterobacteriacae


(e.g.
Escherichia, Enterobacter,
Salmonella, Shigella, Yersinia, Erwinia)

Vibrio

17

Gram
-
pos. cocci

Staphylococcus, Streptococcus,
Lactococcus, Enterococcus, Microco
ccus,
Leuconostoc

18

Gram
-
pos endospore formers

aerobic or facultatively anaerobic:


obligate anaerobes:


Bacillus


Clostridium

19

Gram
-
pos, non
-
sporulating rods

Lactobacillus

Brochothrix

Listeria


There is a number of often used group names of microorg
anisms. Some food
related examples are:


”Gram
-
negative psychrotrophic rods”: This includes the genera
Pseudomonas,
Achromobacter, Alcaligenes, Acinetobacter,
and
Flavobacterium.


”Lactic acid bacteria” (LAB) includes the food related genera
Lactobacillus,

Lactococcus, Pediococcus
och
Leuconostoc.



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



Introduction

3





S.
-
O. Enfors: Food microbiology

A special problem with the microbial taxonomy is that the names often are “date
dependent” due to repeated re
-
cla
ssification 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 classi
fication 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 anal
yses 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 grou
p
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.
En
terococci
(
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 or
igin 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 ß
-
hemolytic streptococci are named the
pyogenes group
and among them the
re 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 la
rge 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.

Introduction

4





S.
-
O. Enfors: Food microbiology

However, this fermentation is also involved in food spoilage. T
hen 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 lact
ic acid
fermentation.
Homofermentative
lactic acid bacteria
produce mainly lactic
acid from the sugar, and no CO
2
. To this category belong



all

Streptococcus




all

Lactococcus




all

Pediococcus




some

Lactobacillus


Heterofermentative
lactic acid
bacteria
produce, besides lactic acid, also
acetic acid, ethanol, CO
2
and formic acid. Some can also convert citric acid
(in milk) to diacetyl. In this group are


all

Leuconostoc



most

Lactobacillus






5




S.
-
O. Enfors: Food microbiology


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 discussin
g 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 c
arbon 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 materia
l 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.



Fig 2.1. Microorganisms,
especially bacteria and f
ungi,
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.


The degradation of biological material is mainly catalysed by microorgansim
s,
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
+
) constitu
te 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
do
nor and the ultimate oxidant (oxygen) is called electron acceptor in this
Anima
ls

Dead organisms

Plants

Organic
materia

CO
2
+ N
2

Light

Archae

Bacteria

Fungi

Algae

Protozoa


2. The ecological basis of food spoilage

6




S.
-
O. Enfors: Food microbiology

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 phosp
horylation of ADP to ATP.


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, H
2
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 th
e 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 fermenta
tive 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 co
-
enzymes. There is a number of different fermentative metabolic pathways,

C

red

NH

3

Fe

2+

S

2
-

CO

2

NO

3

-

SO

4

2

-

Fe

3+



NADH



NAD

+

ATP

ADP

S

2
-

N

2

H

2

O

O

2

NO

3

SO

4

2
-

-

H

2

H

2

O

Ethanol

Pyruvate

NAD

+

NADH

Fermentation

Respiratio
n

Electron donors (energy source)

Re
-
oxidation of co
-
enzymes

Electron
acceptors



2. The ecological basis of food spoilage

7




S.
-
O. Enfors: Food microbiology

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 pathw
ays 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 i
s 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 redu
ces 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 micr
obial 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 flor
a but there are usually a
number of other species involved, usually in the group "psychrotrophic, Gram
-
negative rods". Another problem is that it is not always sure that the
dominating microflora is responsible for the main spoilage reactions. An
example i
s 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 secon
dary) 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
dep
ending on the environment. The primary plus initial contamination flora

2. The ecological basis of food spoilage

8




S.
-
O. Enfors: Food microbiology

usually is in the order of 10
3
cells/cm
2
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 exponent
ially (see Fig 2.3) up to concentrations above 10
7
/
cm
2
(or per gram)
. The finally dominating microflora may origin from the
primary or the contamination microflora. When the number of cells exceed 10
7

to 10
8
cells/cm
2

(or per gram)
the product usually dev
elops 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 si
ze 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
≈ 10
3
cells/ cm
2

Milk

Contamination flora ≈ 10
3
cells / ml

Meat

Fish fillet

Contamination flora ≈ 10
3
cells / cm
2

Spoilage flora on most food types

≈ 10
7

-
10
8
cells / cm
2
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 (a
w
)

-
pH and buffer capacity

-
Oxygen concentration and transfer

-
Mechanical barriers

-
Metabolisable energy sources

-
Met
abolisable 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 e
xplanation 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

2. The ecological basis of food spoilage

9




S.
-
O. Enfors: Food microbiology

growing at 20°C have an initial generation time of about 4.8 h, while t
he
generation time at 0°C is about 25 h, and represents psycrotrophic organisms.













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.


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 te
mperature in the
range of 30
-
40 °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.



C

4°C

8°C

10°C

20°C



0

0

Time (days)

15

10
Log

(cfu)

3

5

7

9

1

0 10 20 30 40
50 60 °C



Relative growth rate

Psychro
-

philes

Psychrotrophes

Mesophiles

Thermophiles


2. The ecological basis of food spoilage

10




S.
-
O. Enfors: Food microbiology

The psychrophil
es 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 g
row well in the 20
-
35 °C range like the mesophiles but they can also grow relatively fast at
refrigerator temperature.



The growth rate of microorganisms is expressed either with the
generation time

(
t
g
, 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
dt


N

where
N
is the number of cells,
µ
(h
-
1
) is the specific growth rate and
t
(h) is time.
Integration with
N
0
cells at
t
= 0 and
N
t
cells at time
t
, gives:



ln(
N
t
)
ln(
N
0
)


t

After one generation time,
t
g
, the cell number becomes 2
N
0
. Insertion of this in
the equation above give
s:



ln(
2
N
0
)
ln(
N
0
)


t
g


from which the correlation between generation time and specific growth rate is
obtained:


l
n(
2
)


t
g

0.69





Water activity

(a
w
). The water activity is one of the main parameters which
determine how fast and by which type of organisms t
he food is spoilt. The
water activity of food can be determined as the water vapour pressure (p
H2O
) in
a closed vessel in which the product is enclosed in relation to the water vapour
pressure of pure

water (p
H2O
*):


For a water solution with low molecula
r weight compounds (e.g. salt or sugar)
the water activity is approximately:




a
w

=

p
H
2
O
p
H
2
O
*


a
w

n
w
n
w

n
S

2. The ecological basis of food spoilage

11




S.
-
O. Enfors: Food microbiology

where

n
w

= number of moles water

n
s

= number of moles of dissolved molecules


Some common food components that reduce the water activity are:


-
Ions (e.g. salts)


-
Dissol
ved molecules (e.g. sugars)


-
Hydrophilic colloids (e.g. starch)


-
Ice

The water activity is a measure of the availability of the water for the
microorganisms. It is not only the water concentration that determines the
water activity but also the ca
pacity 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.















Fig 2.5. Sorption isotherms for different
materials show that a
w
is not the same as
water concentration


Fig 2.6. Schematic view of how the
a
w
influences the rate of enzyme
reactions and microbial growth.


Most biochemical r
eaction 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 the
ir 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
p
referring) 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.


0
0.3 0.6 0.9

Water activity

Water concentration
(%)

30

20

10

0

Fruit

Starch

Cellulose

Meat

Water activity

Bacteria

Fungi

Lipolysi
s

Proteolysis

Lipid

oxidation

Rel
reaktionshastighet

0

0.2

0.4

0.6

0.8

1

Relative rate


2. The ecological basis of food spoilage

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S.
-
O. Enfors: Food microbiology

Table 2.2 Examples of typical minimum water activity for growth of some
m
icroorganisms and corresponding a
w
in some foods.

Organism

Min a
w

Food examples

Food a
w



Milk, fish, meat

0.99

Pseudomonas

0.97



E. coli

0.96

Sausage, 7% salt

0.96

Clostridium

0.95



Brochothrix
thermosphacta

0.94



Bacillus

0.93

Ham, 12
% salt

0.93

Lactobacillus

0.93



Streptococcus

Lactococcus

Micrococcus


0.93



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 b
acteria

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.

Dry bread

< 0.5

Halophilic
= salt preferring;
xerophilic
= drought preferring;

osmophilic
= preferring hig
h 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 sensiti
ve 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
-
a
w
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 a
w

for and organism depends on other parameters like pH,

2. The ecological basis of food spoilage

13




S.
-
O. Enfors: Food microbiology

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
infl
uences 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 wi
ch 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 ma
ny
vegetables mainly are degraded by molds and sometimes yeasts.


Table 2.3. Typical pH
-
values of common food products

Shrimps

7

Fish


6.7

Corn


6
-
7

Milk


6.5

Melon


6.5

Butter

6.2

Meat

5.1
-
6.4

Cheese


5.9

Oysters

5
-
6

Cabbage


5.5

Potatoes


5.5

Tomatoes


4.2

Orange juice

4

Yoghurt


3.5

Apples

≈3

Lemon

≈2




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 dif
fusion 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 product
s 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 conta
minated surface, where they dwell

2. The ecological basis of food spoilage

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O. Enfors: Food microbiology

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 m
olds 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.
Erwin
ia

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 mic
robial 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 antib
iotic 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 pro
ducing
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





2. The ecological basis of food spoilage

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O. Enfors: Food microbiology


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 a
dded 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 o
f 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 hyd
rolysis



Degradation of nitrogen compounds

The dominating and usually the first reaction is
oxidative deamination
of
amino acids:



amino acid + O
2
NH
3
+ organic acid


This reaction is assumed to be th
e 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.

deaminase


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O. Enfors: Food microbiology

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, protea
ses and peptidases may degrade the
protein:



Proteins

peptides

amino acids



Many peptides have strong taste, bitter or sweet, and this sometimes
contributes to the spoilage. These reactions are also important for the
development of characteristi
c 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 hy
drogen sulphide:


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.


Putrificati
on 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.

proteinase

peptidase

amino acids

Anaerobic


metabolism

Amines

Organic acids

S
-
compounds

Indol


2. The ecological basis of food spoilage

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S.
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O. Enfors: Food microbiology



Fig 2.6. Histamine, cadaverine and other amines are formed by decarboxylation of amino
acids.


Reduction of trimethylamine oxid
e (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:









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 fa
t

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.
H
3
C
-
N = O

CH
3

CH
3

H
3
C
-
N

CH
3

CH
3

TMAO
-


reductase

TMAO

TMA


2. The ecological basis of food spoilage

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O. Enfors: Food microbiology

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 contam
inating
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 perox
ide 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 a
ldehydes and ketones. The
chain reaction is initiated by a radical (R*) which is produced from the fatty acid
under catalysis of Fe
2+
and other metal ions and light. The radical reacts with
molecular oxygen to form a peroxide radical (ROO*). Antioxidants i
n 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 aldehyde
s.


2. The ecological basis of food spoilage

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O. Enfors: Food microbiology

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 an
d 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
pres
ent 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 a
ldehydes 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 sou
rce. 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 mos
t common fermentative
pathways are listed in Table 2.7.



2. The ecological basis of food spoilage

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S.
-
O. Enfors: Food microbiology

Table 2.7. Common fermentation types


Fermentation type

Products

Alcohol fermentation

Ethanol, CO
2

Homofermentative lactic acid fermentation

Lactic acid

Heterofermentative lactic acid fermentati
on

Lactic acid, Acetic acid, Ethanol,CO
2

Propionic acid fermentation

Propionic acid, Acetic acid,

CO
2

Butyric acid fermentation

Butyric acid, Acetic acid, CO
2
, H
2

Mixed
-
acid fermentation

Lactic acid, Acetic acid, CO
2
, H
2
, Ethanol

2,3
-
butanediol ferme
ntation

CO
2
, 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 organis
ms 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 t
owards 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.























Fig 2.10 Summary of the six ma
in fermentative pathways. The main end products are
emphasised by frames. Sites of co
-
enzyme generation and ATP formation are indicated.


Glucose

Pyruvate

Acetaldehyde

Ethanol

Lactate

Formate

AcetylCoA

+H
2

CO
2

Oxaloacetate

Succinate

Acetate

Acet
-
acetylCoA

Propionate

Acetoin

Butandiol

Butyrate

Acetone

Butanol

2
-
propanol

AcetylCoA +

Propionic acid
fermentation

Ethanol fermentation

Lactic acid fermentation

Mixed acid fermentation

Butyric acid fermentation

Butandiol
fermentation

NAD
+

NAD
+

NAD
+

NAD
+

NAD
+

NAD
+

NAD
+

NAD
+

NAD
+

NADH

ATP

ATP

ATP

ATP

ATP

Acetate Ethanol H
2
CO
2


2. The ecological basis of food spoilage

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O. Enfors: Food microbiology

Pectin hydrolysis

Pectins are carbohydrate polymers mainly composed of
partially methylated
poly
-
α
-
(1,4)
-
D
-
galacturoni
c 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 bact
eria 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.



22


S.
-
O. Enfors: Food microbiology


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 a
w

Fermented food

See Chapter 6

Often low a
w
+ low pH

Microbial competitors

Microbial inhibitors

Preserv
ed food


Salted/dried

Pickled

Smoked

Sterilised

Pasteurised

Low a
w


Low pH

Low pH, low a
w
, inhibitors

No microflora

Small initial microflora

Often in combination with

chemical preservatives


3.1 Water and protein rich foods

Fresh meat, fish and milk belon
g 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 s
tuffs 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 be
long to this category, but
for obvious reasons Nature has build a sophisticated system which keeps the

3. Spoil
age of different types of food

23




S.
-
O. Enfors: Food microbiology

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 metabolis
ed
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 actom
yosin 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 be
tween 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 considere
d 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 sensitiv
e to pH below about 5.5
-
6, the final pH of
the meat is considered important for the shelf
-
life.


3. Spoil
age of different types of food

24




S.
-
O. Enfors: Food microbiology


Table 3.1. Typical pH of meat from different animals and lenth of rigor mortis.

Animal type

Rigor mortis

final pH

Cow

10
-
20 h

6
-
5.5

Swine

4
-
8 h

6

Chicken

2
-
4 h

6.4
-
6

Fish

min
-
h (longer on ice)

6.8
-
6.4


The meat contains many nutrients for the microorganisms (Table 3.3) which
only grow on the exudate from damaged tissue. Furthermore, it is only on the
surface of meat the microorganisms grow, unless th
e meat has been
mechanically perforated or minced. Therefore, the microbial count is
expressed as cells/ cm
2
or cfu/
cm
2
, where cfu means colony forming units on
agar plates.


Table 3.2 .Example of microbial nutrients in meat exudate

Component

Concentratio
n 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 n
egative 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 fre
sh
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 developin
g 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 10
3
cells/cm
2
on a meat of highest
hygienic q
uality towards 10
7

-
10
8
cells /cm
2
. Then the spoilage becomes

3. Spoil
age of different types of food

25




S.
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O. Enfors: Food microbiology

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.






Fig 3.2. Example of microbial
growth measured as "total
aerobic count" during storage of
fresh pork and chicken meat at
refrigerator temperature.




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 th
e 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 stres
s
before the slaughter may reduce the blood glucose concentration.







Fig 3.3. Glucose concentration
gradients and microflora
development during cold storing of
fresh meat. At N=32*10
7
cm
-
2
the
meat was classified as spoilt and this
coincides with gl
ucose exhaustion at
the surface.





0
400
Glucose
(
µ
g/g
)
0
400
0
20
Distance
from
surface
(mm)

N*10
-7
=
2.7
6.3
32
110
280
odör
slem
griskött
kyckling
Tid (d)
0 2 4 6 8 10
1
2
3
4
5
6
7
8
log N/cm
2
chicken

slime

Days

odour

pork


3. Spoil
age of different types of food

26




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O. Enfors: Food microbiology

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 rem
oved 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 conditi
ons
produces more off
-
flavour, unless the dominating microflora is composed of
lactic acid bacteria.

The figure also shows that storing the meat under CO
2

atmosphere significantly reduces the rate of microbial growth. When the CO
2

packed meat was opened an
d subjected to air, the microbial growth rate
immediately increased.








Fig 3.4. Influence of
the gas atmosphere on
the growth rate of
microorganisms on
refrigerated fresh pork
meat. Some of the CO
2

stored samples were
opened and further
exposed to ai
r, as
indicated in the CO
2
-
plot.





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
. Thes
e
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 ill
-
tasting products from the
mixed
-
acid fermentation or 1,3 butandiol
fermentation pathways. The CO
2
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 imp
act on
the spoilage than the
Pseudomonas
.


0 8 16 24 32
3
4
5
6
7
8
9
CO2
Luft
Luft
Kväve
Luft
Tid (dagar)

?

logN / cm2
air

N
2

air

air

CO
2


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S.
-
O. Enfors: Food microbiology

Table 3.4. Dominating spoilage flora on cold stored pork in different atmospheres.

O2

%

N2

%

CO2

%

Pseudomonas

Entero
-

bacteriacae

Aeromonas

Brochothrix

Lactobacillus

20

80


+






100



+




80


20




+



80

20


+

+



10


90




+

+



100





+


The selective pressure of CO
2
is explained by the different inhibitory effect
this gas has on various microorganisms.
Pseudomonas
belongs to the most
CO
2
sensitive bacteria while lactic acid bacteria are very resist
ant to this gas.
Most molds are very sensitive while yeasts are very resistant to CO
2
.













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, CO
2
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
CO
2
raises rapidly and exerts a protecting function. Also the shelf
-
life promoting effect of vacuum packing of cured meat products is similar but
in that case it is the metabolic activity of the microflora which produces the
CO
2
. Table 3.5 lists some pro
perties of bacteria which contribute to the
selection pressure in vacuum packed fresh and cured meat.








3. Spoil
age of different types of food

28




S.
-
O. Enfors: Food microbiology

Table 3.5 Some characteristics of the organisms that dominate the
spoilage flora on cold
-
stored fresh and cured meat in different
atmospheres.

Org
anism

Properties

Pseudomonas

Fast growing

Aerobic

Very CO
2
-
sensitive

Sensitive to low a
w

Enterobacteriaceae

Facultative

Intermediate CO
2
-
sensitivity

Aeromonas

Facultative

Intermediate CO
2
-
sensitivity

Brochothrix thermosphacta

Facultative

Relatively C
O
2
resistant

Resistant to low a
w

Lactobacillus

Very CO
2
-
resistant

Indifferent to oxygen

Resistant to low a
w



The inhibitory effect of CO
2
seems to be synergistic with low temperature in
storage of meat as shown in Fig 3.6. This may partly be due to th
e increasing
solubility of CO
2
at declining temperature. Even if CO
2
dissolves in water and
partly is hydratized and dissociates to bicarbonate, it is the gaseous CO
2

molecule which has the inhibitory effect. This also means that the effect is
strongly pH
dependent and declines with increasing pH.

















Fig 3.6. Time needed to reach 10
6
cells cm
-
2
on pork meat stored at different temperatures
in air or in CO
2
.




°C


3. Spoil
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-
O. Enfors: Food microbiology

The antimicrobial effect of CO
2
on many spoilage organisms has been utilised
also f
or 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.


Va
cuum packing of food is applied also for other reasons than to provide
microbial inhibition via CO
2
. One common reason for vacuum packing is to
prevent oxidative rancidification or other oxidising reaction with molecular
oxygen (e.g. peanuts), or to preven
t evaporation of flavour compounds (e.g.
coffe). When cheese is packed in vacuum tight plastic films it is likely that a
mold inhibiting CO
2
atmosphere develops, but on the other hand, molds are
obligately aerobic so the lack of oxygen is also a mold
-
prote
cting 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
appreciat
ion of "fresh fish" taste. IMP is also utilised as a flavour improving
additive in the food industry, in analogy with the meat flavour enhancing
effect of glutamine.









Fig 3.7. During the
post mortem
metabolism in the fish tissue inosine
monophospha
te (IMP) is transiently
accumulated.




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 th
e 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
ATP

ATPase

ADP

Myokinase

AMP

AMP
-
deaminase

IMP



Phosphomonoesterase

Inosin
e

Nucleoside phosphorylase

hypoxhantin
e +
ribose
-
P



3. Spoil
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30




S.
-
O. Enfors: Food microbiology

including also the 5'
-
nucleotidase the oxyge
n consumption also includes the
IMP.




















Fig 3.8. Principle of a "fish
-
freshness" biosensor based on analysis of the degradation of
IMP degradation. The oxygen consumption catalysed by xanthine oxidase is analyses with
or without the enzym
es 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
She
wanella putrifaciens
dominates.


Fig 3.9. Distribution of
spoilage organisms on
refrigerated fresh fish.
Aeromonas
is mainly
associated with fresh
-
water fishes and
Shewanella
with
marine fishes.






CH



2





-

P O



N



N



OH



N



N



OH



OH



OH



OH



O



IMP



Inosine



Hypoxanth
ine

1 5







-



nu

c

l

e

o

ti

d

a

se



2



nuc

l

e

ot

ide



ph

o

s

p

h

o

r

y

l

a

se



3



xan

t

h

i

ne



ox

id

a

se



1



= IMP + I +



Hx



2



3



= I +



Hx



2



3



3



=



Hx



Enzym

e

r



=









analys



Index =



IMP



IMP + I +



Hx




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-
O. Enfors: Food microbiology

Milk


Milk is a very good substrate for microbial growt
h. 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
e
nzyme 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 group
s 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
-
fl
uids.











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 negativ
e 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

E. coli

Enterococcus

Feces

Micrococcus

Bacillus
spores

Mold spores

Yeasts


Air

Lactococcus

Lactobacillus

Gram
-
negative rods


Milking equipment

oxidase

catalase

LP

SCN

-

HO
-
S
-
p
rotein

OSCN

-

thiocyanate

hypothiocyanate

HS
-
protein

H
2
O
2

H
2
O

O
2


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O. Enfors: Food microbiology

If the milk i
s 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 t
han 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 destr
oyed 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 he
at 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 microflor
a. 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 spo
il 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 s
poilage 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, Staphylococcu
s, 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
e
gg 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.



3. Spoil
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33




S.
-
O. Enfors: Food microbiology














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 hav
e 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, end
ogeneous 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 ami
no 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 mo
lds, 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 pla
nt pathogen

Erwinia carotovora
. This organism has been subject to studies of the
corum
sensing
phenomenon which plays a central role in the ecology of many
organisms. In this case the
corum sensing
is based on accumulation of N
-
acylated homoserine lactones
(AHL) which accumulates around the cells (Fig
3.12). When the concentration of AHL is high enough this compound induces
the pectinase synthesis. The strategic advantage of not producing the
pectinase constitutively is obvious, since the plants have their
defence
systems which generate antimicrobial chemicals when the plant is attacked.
No
protect
i
on

inner keratin

membrane

1
-
10µm pores in shell

outer mucin layer

Con
-
albumin
: Fe2+ complexing

Avidin:
Biotin complexing

Lysozyme:
kills G+ bacteria

Albumin
: viscous, high pH
(pH9,5), riboflavin + pyridoxin
complexin
g


3. Spoil
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O. Enfors: Food microbiology

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 ha
ve 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.




















Fig 3.12.
Erwinia c
arotovora
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 ( )