Microbiology Book PDF

Classification of
UNIT 1 CLASSIFICATION OF Microorganisms

Important in the Food
MICROORGANISMS IMPORTANT Industry: Bacteria,
Yeasts and Mold
IN THE FOOD INDUSTRY:
BACTERIA, YEASTS AND MOLD
Structure
1.0 Objectives
1.1 Introduction
1.2 Various Types of Microorganisms
1.3 Characteristics (Morphological, Cultural and Physiological) of Various
Microorganisms
Bacteria
Molds
Yeasts
1.4 Let Us Sum Up
1.5 Key Words
1.6 Answers to Check Your Progress Exercises
1.7 Some Useful Books
1.0 OBJECTIVES
The objectives of this unit are to enable you to understand the important genera
of microorganisms associated with food. This unit gives a brief account of the
morphological, physiological and cultural characteristics of various
microorganisms. After going through this unit, you should be able to:
• know the various types of microorganisms;
• explain the requirements for their growth;
• learn the classification of these organisms based on their characteristics;
and
• distinguish between the useful and harmful microorganisms.
1.1 INTRODUCTION
We already know that the microorganisms use our food as a source of nutrients
for their own growth. This, of course can result in deterioration of the food. By
increasing their numbers, utilizing nutrients, producing enzymatic changes and
contributing off flavors by means of breakdown of a product or synthesis of
new compounds they can “spoil” a food. When the microorganisms involved
are pathogenic, their association with our food supply is critical from a public
health point of view. Therefore a classification of different organisms and their
growth requirements is required to prevent spoilage of foods.
1.2 VARIOUS TYPES OF MICROORGANISMS

Microbes are single-cell organisms so tiny that millions can fit into the eye of a
needle. They are the oldest form of life on earth. Microbe fossils date back
more than 3.5 billion years to a time when the Earth was covered with oceans
that regularly reached the boiling point, hundreds of millions of years before
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Introduction dinosaurs roamed the earth. Without microbes, we couldn’t eat or breathe.
Without us, they’d probably be just fine.
Bacteria
Many of us know bacteria only as “germs,” invisible to naked eyes that can
invade our bodies and make us sick. Few know that many bacteria not only
coexist with us all the time, but help us do an amazing array of useful things
like make vitamins, break down garbage, and even maintain our atmosphere.
These are unicellular microorganisms that are classed as plants. A bacterial cell
is about 1µm in length and somewhat smaller in diameter. Bacteria are
classified according to their shape. Cocci are spherical, bacilli are cylindrical
and spirilla and vibrios are spiral. Bacterial spores are more heat resistant than
yeast or mold spores to most processing conditions. Bacteria, with a few
exceptions cannot grow in acid media in which yeasts and molds thrive. They
multiply by ‘binary fission’. When a bacterium becomes mature it divides into
two, these two become four and so on. Bacteria can be found virtually
everywhere. They are in the air, the soil, and water, and in and on plants and
animals, including us. A single teaspoon of topsoil contains about a billion
bacterial cells (and about 120,000 fungal cells and some 25,000 algal cells).
The human mouth is home to more than 500 species of bacteria. Some bacteria
(along with archaea) thrive in the most forbidding, uninviting places on Earth,
from nearly-boiling hot springs to super-chilled Antarctic lakes buried under
sheets of ice. Microbes that dwell in these extreme habitats are aptly called
extremophiles. The growth of bacteria is very rapid and depends upon the
nature of the food material, moisture, temperature and air. Some bacteria do
not grow in air but temperature plays a major role in their growth, the optimum
being generally 37°C for bacteria pathogenic to humans.
Bacteria are very sensitive to acids and are destroyed in their presence even at
temperature of boiling water. Hence, most fruits being acidic can be easily
sterilized at 100°C whereas vegetables being non-acidic require a higher
temperature of 116°C.
A bacterium’s genetic information is contained in a single DNA molecule
suspended in a jelly-like substance called cytoplasm. In most cases, this and
other cell parts are surrounded by a flexible cytoplasmic membrane that is
itself surrounded by a tough, rigid cell wall. A few species, such as the
mycoplasmas, don’t have cell walls.
Even though bacteria have only one cell each, they come in a wide range of
shapes, sizes, and colours.
The important groups of bacteria are:
a) Bacillus: rod-shaped.
b) Coccus: spherical.
c) Coccobacillus: oval-shaped.
d) Aerobes: require atmospheric oxygen for growth, e.g., Acetobacter aceti.
e) Facultative anaerobes: can grow with or without atmospheric oxygen.
f) Obligate anaerobes: do not grow in atmospheric oxygen.
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Classification of
g) Mesophiles: require a temperature below 38°C for growth.
Microorganisms
h) Obligate thermophiles: grow between 38°C and 82°C. Industry: Bacteria,
Yeasts and Mold
i) Facultative thermophiles: grow over a wide range of temperatures covered
by mesophiles and obligate thermophiles and below.
j) Psychrotrophs: grow fairly well at refrigeration temperatures and some can
even grow slowly at temperatures below freezing.
Some bacteria have natural colours. Certain species contain pigments, such as
various chlorophylls, that make them naturally green, yellow, orange, or
brown. Colonies of millions of bacteria may appear pink, yellowish, or white.
Important Food Spoilage Bacteria
Group Genus
Acetics Acetobacter and Gluconobacter
Lactics Lactobacillus, Leuconostoc,
Pediococuus, Sreptococcus
Butyrics Clostridium
Propionics Propionibacterium
Proteolytics Bacillus, Pseudomonas, Closridium,
Proteus etc.
Some useful bacteria

The following bacteria are of great importance in the food processing industry.
Acetobacter sp.
These bacteria, also known as “vinegar bacteria”, cause significant spoilage in
the wine industry but are necessary for vinegar production. The important
species are Acetobacter aceti, A. orleansis and A.schutzenbachi. They are very
small, usually non-motile and generally do not form spores. These bacteria are
aerobes and in the presence of oxygen convert ethyl alcohol to acetic acid.
These bacteria can be easily destroyed by heating to 65°C.
Lactobacillus sp.
Different organisms of this group, also known as “lactic acid bacteria”, have
different properties but all of them produce lactic acid from carbohydrates. The
important species include Lactobacillus plantarum, Pediococcus cerevisiae,
Leuconostoc mesenteroides, Streptococcus faecalis and Lactobacillus brevis.
These bacteria cause “lactic souring” and spoil wines, which can be easily
prevented by maintaining a sulphur dioxide concentration of 0.007 per cent in
wine.
Yeasts
Yeasts are unicellular fungi which are widely distributed in nature. They are
somewhat larger than bacteria. The cell length is about 10µm and the diameter
is about a third of this. Most yeasts are spherical or ellipsoidal. Yeasts that
multiply by means of ‘budding’ are known as ‘true yeasts’. Yeasts grow
luxuriously at a moderate temperature in a solution of sugar in plenty of water.
9
Introduction Under suitable conditions the sugar is converted into alcohol and carbon
dioxide is evolved. This is the reason that carbon dioxide is evolved from food
materials spoiled by yeasts and pushes out corks from bottles with great force.
Most of them do not develop in media containing more than 66% sugar or
0.5% acetic acid. Boiling destroys the yeast cells and spores completely. Some
of the yeasts which grow on fruits are Saccharomyces, Candida and
Brettanomyces.
Pseudo-yeasts
These are like true yeasts but do not form spores. All the members of this
group are particularly unsuitable for fermentation purposes as they produce
off-flavours and cloudiness.
Yeasts causing food spoilage
Yeast Product Spoilage

Saccharomyces Low sugar products
Candida High-acid foods, salty foods, butter
Brettanomyces Beers, wines
Zygosaccharomyces (osmophillic) Honey, syrups, molasses, wines, soy
sauce
Pichia Wines
Hansenula Beers
Torulopsis Milk products, fruit juices, acid
foods
Rhodotorula Meat, sauerkraut
Fungi (Molds)
Fungi are eukaryotic organisms. This means that their DNA-containing
chromosomes are enclosed within a nucleus inside their cells. (The
chromosomes of bacteria and archaea are not walled off inside nuclei, making
them prokaryotic organisms). Molds are multicellular, filamentous fungi which
are devoid of chlorophyll. They are larger than yeasts. They are strict aerobes
and require oxygen for growth and multiplication and tend to grow more
slowly than bacteria.
Fungi are lower thallophytic plants but do not make their own food via
photosynthesis like green plants. They feed on organic matter like rotting
leaves, wood, and other debris, or upon the tissues of living plants and animals.
Fungi, along with bacteria, are the planet’s major composters and recyclers.
Although fungi may seem like a nuisance when they grow in your fruit bowl or
refrigerator, their ability to degrade some of the toughest organic materials,
including tree wood and insect exoskeletons, means that our planet is not
cluttered with a mass of debris. Fungi secrete digestive enzymes in order to
break down complex food sources, such as animal corpses and tree stumps,
into smaller components they can absorb.
The principle parts of a mold are a web-like structure known as mycelium and
the spore. The mycelium is often white and cottony and penetrates into the
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attacked foodstuff. After fixing itself the mold produces viable spores which Classification of
Microorganisms
resist the favourable conditions after the dispersal and germinate when they get Important in the Food
favourable conditions. They thrive best in closed, damp and dark situations Industry: Bacteria,
with an adequate supply or warm, moist air but require less free moisture than Yeasts and Mold
yeasts and bacteria. They prefer sugar containing substances and may spoil
jams, jellies and other sugar-based products. Acid medium favours their
growth and, therefore, they grow well in pickles, juices etc. this is the main
reason that fruit and fruit products are attacked by molds which not only
consume nutrients present in the food thereby lowering its food value but also
spoil the flavour, texture and appearance of the product. Molds are sensitive to
heat; boiling quickly destroys molds and their spores. The most important
molds are:
a) Penicillium sp. (Blue moulds)
b) Aspergillus sp. (Black moulds)
c) Mucor sp. (Gray moulds)
d) Bysslchlamyces fulva
Classification of Microorganisms
A) On basis of temperature for growth
Microorganisms can be classified into:
− Thermophillic: Microbes who require high temperature for their growth
and survival (optimum temperature=45-65ºC).
− Thermoduric: Microbes which do not grow at high temperatures but
can survive in it.
− Mesophillic: Microorganisms which require optimum temperature of
20-50ºC for growth and multiplication.
− Psychrophillic: Microorganisms requiring less than 20ºC as optimal
temperature for growth.
− Psychroduric: Microorganisms which do not grow at low temperature
but can survive.
B) On basis of oxygen requirement for growth:
− Obligate Aerobes: Require oxygen for growth and multiplication e.g.
molds.
− Obligate Anaerobes: Strictly grow only in absence of oxygen.
− Facultative: Microorganisms than can grow in both presence and
absence of oxygen e.g. yeasts.
− Microaerophillic: Organisms which are able to grow at very low
oxidation-reduction potential.
11
Introduction
C) On basis of requirement of water activity.
In general, bacteria require more moisture than yeasts and yeasts more than
molds.
The classification according to requirement of aw is as follows:
Group of microorganism Minimal aw value
Bacteria 0.91
Yeast 0.88
Molds 0.80
Halophillic bacteria 0.75
Xerophillic fungi 0.65
Osmophillic Yeasts 0.60
− Halophillic bacteria: Bacteria which grow in high salt solutions

− Osmophillic Yeasts: Yeasts which can grow best in high concentrations
of sugar
− Xerophillic Fungi: Fungi which can grow in low water activity
D) On basis of nutrient degradation capacity:
− Proteolytic: Microorganisms which are capable of protein degradation
because of extracellular proteinases produced.
− Lipolytic: Microbes which catalyxe the hydrolysis of fats to fatty acids
and glycerol.
− Sacchrolytic: These microorganisms hydrolyse disaccharides or
polysaccharides to simpler sugars.
− Pectinolytic: These microorganisms hydrolyse pectin.
E) On basis of staining:
On basis of staining the bacteria can be classified as:
− Gram positive: Those bacteria that stain violet after Gram stain test. In
these the cell wall is mostly comprised of peptidoglycan layer.
− Gram negative: Those bacteria that do not stain violet after Gram stain
test. Cell wall mainly comprised of lipopolysaccharides.
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Check Your Progress Exercise 1 " Classification of
Microorganisms
Note: a) Use the space below for your answer. Industry: Bacteria,
b) Compare your answers with those given at the end of the unit. Yeasts and Mold
1. Classify bacteria according to their morphology.

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2. Differentiate between yeasts and molds.
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3. Classify microorganisms on basis of the temperature requirements, oxygen
requirements, water activity requirement, staining procedure and nutrient
degrading capability.
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1.3 CHARACTERISTICS (MORPHOLOGICAL,

CULTURAL AND PHYSIOLOGICAL) OF
MICROORGANISMS
1.3.1 Bacteria
Morphological Characteristics
One of the first step in the identification of bacteria in food is microscopic
examination to ascertain the shape, size, aggregation, structure and staining
13
Introduction reactions of the bacteria present. The following characteristics may be of
special significance:
Encapsulation: The presence of capsules or slime may account for sliminess or
ropiness of a food. Most capsules are polysaccharides of dextrin, dextran or
levan and they serve as a source of reserve nutrients and increase the resistance
of bacteria under adverse conditions.
Formation of Endospores: Bacteria of genera Bacillus, Clostridium,
Sporosarcina etc have the ability to form endospores. Endospores are formed
at an intracellular site and are resistant to heat, ultraviolet light and dessication.
Lysis of the vegetative cell releases the free endospore, which may remain
dormant with no detectable metabolism for years. Sporulation usually appears
in the late logarithmic phase of growth, possibly because of nutrient depletion
or product accumulation. The acquisition of heat resistance is closely related to
the formation of dipicolinic acid and the Ca2+ uptake. Germination is favoured
by conditions that are favourable for growth.
Formation of Cell Aggregates: It is characteristic of some bacteria to form
long chains or of others to clump under certain conditions. It is more difficult
to kill all bacteria in intertwined chains or sizable clumps than to destroy
separate cells.
Cultural Characteristics
Bacterial growth in and on foods often is extensive enough to make the food
unattractive in appearance or otherwise objectionable. Pigmented bacteria
cause discolouration on the surfaces of foods; films which may cover the
surfaces of liquids; growth may make surfaces slimy; or growth throughout the
liquids may result in undesirable cloudiness or sediment.
Physiological Characteristics
Most bacteria may be placed into one of three groups based on their response
to gaseous oxygen. Aerobic bacteria thrive in the presence of oxygen and
require it for their continued growth and existence. Other bacteria are
anaerobic, and cannot tolerate gaseous oxygen, such as those bacteria which
live in deep underwater sediments, or those which cause bacterial food
poisoning. The third group are the facultative anaerobes, which prefer growing
in the presence of oxygen, but can continue to grow without it.
Bacteria may also be classified both by the mode by which they obtain their
energy. Classified by the source of their energy, bacteria fall into two
categories: heterotrophs and autotrophs. Heterotrophs derive energy from
breaking down complex organic compounds that they must take in from the
environment − this includes saprobic bacteria found in decaying material, as
well as those that rely on fermentation or respiration.
The other group, the autotrophs, fix carbon dioxide to make their own food
source; this may be fueled by light energy (photoautotrophic), or by oxidation
of nitrogen, sulfur, or other elements (chemoautotrophic). While
chemoautotrophs are uncommon, photoautotrophs are common and quite
diverse. They include the cyanobacteria, green sulfur bacteria, purple sulfur
bacteria, and purple nonsulfur bacteria. The sulfur bacteria are particularly
interesting, since they use hydrogen sulfide as hydrogen donor, instead of
water like most other photosynthetic organisms, including cyanobacteria.
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Microbe is a term for tiny creatures that individually are too small to be seen Classification of
Microorganisms
with the unaided eye. Microbes include bacteria (back-tear-ee-uh), archaea Important in the Food
(are-key-uh), fungi (fun-jeye) and protists (pro-tists). You've probably heard of Industry: Bacteria,
bacteria and fungi before. Archaea are bacteria-like creatures that have some Yeasts and Mold
traits not found in any true bacteria. Protists include primitive algae (al-gee),
amoebas (ah-me-buhs), slime molds and protozoa (pro-toe-zoh-uh). We can
also include viruses (vye-rus-is) as a major type of microbe, though there is a
debate as to whether viruses can be considered living creatures or not.
1.3.2 Molds
General Characteristics
The term “mold” is a common one applied to certain multicellular, filamentous
fungi whose growth on foods usually is readily recognized by its fuzzy or
cottony appearance. The main part of the growth commonly appears white but
may be coloured or dark or smoky. Coloured spores are typical of mature mold
of some kinds and give colour to part or all of the growth. The thallus, or
vegetative body, is characteristic of thallophytes, which lack true roots, stems
and leaves.
Morpohological Chactacteristics
The morphology, i.e. the form and structure, of molds, as judged by their
macroscopic and microscopic appearance, is used in their identification and
classification.
Hyphae and Mycelium: The mold thallus consists of a mass of branched,
intertwined filaments called hyphae (singular hypha), and the whole mass of
these hyphae are known as the mycelium.
Reproductive Parts or Structures: Molds can grow from a transplanted piece
of mycelium. Reproduction of molds is chiefly by means of asexual spores.
Some molds also form sexual spores.
Culture Characteristics
The gross appearance of a mold growing on a food often is sufficient to
indicate its class or order. Some molds are loose and fluffy; others are
compact. Some look velvety on the upper surface, some dry and powdery, and
others wet or gelatinous. Some molds are restricted in size, while others seem
limited only by the food or container. Pigments in the mycelium – red, purple,
yellow, brown, gray black, etc. – are characteristic, as are the pigments of mass
of asexual spores; green, blue-green, yellow, orange, pink, lavender, brown,
gray, black, etc.
The physiological characteristics of molds will be reviewed only briefly here
and will be discussed in more detail subsequently.
Moisture Requirements: In general most molds require less available moisture
than do most yeasts and bacteria. It has been claimed that below 14 to 15
percent total moisture in flour or some dried fruits will prevent or greatly delay
mold growth.
Temperature Requirements: Most molds would be considered mesophilic i.e.
able to grow well at ordinary temperature. The optimal temperature for most
15
Introduction molds is around 25 to 30°C, but some grow well at 35 to 37°C or above, e.g.
Aspergillus spp. And some at still higher temperatures. A number of molds are
psychrotophic or psychroduric i.e. they grow fairly well at temperatures of
refrigeration, and some can grow slowly at temperatures below freezing.
Growth has been reported at as low as – 5 to 10°C. A few are thermophilic; i.e.
they have a high optimal temperature.
Oxygen and pH Requirements Molds are aerobic; i.e. they require oxygen for
growth; this is true at least for the molds growing on foods. Most molds can
grow over a wide range of hydrogen-ion concentration (pH 2 to 8.5), but the
majority are favoured by an acid pH.
Food Requirements: Molds in general can utilize many kinds of foods, ranging
from simple to complex. Most of the common molds possess a variety of
hydrolytic enzymes, and some are grown for their amylases, pectinases,
proteinases, and lipases.
Inhibitors: Compounds inhibitory to other organisms are produced by some
molds, such as penicillin from Penicillium chrysogenum and clavacin from
Aspergillus clavatus. Certain chemical compounds are mycostatic, inhibiting
the growth of molds (sorbic acid, propionates, and acetates are examples), or
are specifically fungicidal, killing molds.
Initiation of growth of molds is slow compared to that of bacteria or yeasts, so
that when conditions are favourable for all these organisms, molds usually lose
out in the competition. After mold growth is under way, however, it may be
very rapid.
1.3.3 Yeasts
Like mold, the term “yeast” is commonly used but hard to define. As used here
it refers to those fungi which are generally not filamentous but unicellular and
ovoid or spheroid and which reproduce by budding or fission.
Yeasts may be useful or harmful in foods. Yeast fermentations are involved in
the manufacture of foods such as bread, beer, wines, vinegar, and surface-
ripened cheese, and yeasts are grown for enzymes and for food. Yeasts are
undesirable when they cause spoilage of sauerkraut, fruit juices, syrups,
molasses, honey, jellies, meats, wine, beer, and other foods.
Morphological Characteristics
Form and structure: The form of yeasts may be spherical to ovoid, lemon-
shaped, pear-shaped, cylindrical, triangular, or even elongated into a false or
true mycelium. They also differ in size.
Reproduction: Most yeasts reproduce asexually by multilateral or polar
budding, a process in which some of the protoplasm bulges out the cell wall;
the bulge grows in size and finally walls off as a new yeast cell. A new species
or yeasts reproduce by fission, and one reproduces by combination of fission
and budding.
Sexual reproduction of “true” yeasts (Ascomycotina) results in the production
of ascospores, the yeast cell serving as the ascus. The ascospores may differ in
colour, in smoothness or roughness of their walls, and in their shape (round,
oval, reniform, bean or sickle-shaped, hemispherical, angular, fusiform, or
needle-shaped).
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“False” yeasts, which produce no ascospores or other sexual spores, belong to Classification of
Microorganisms
the Fungi Imperfecti. Cells of some yeasts become chlamydospores by Important in the Food
formation of a thick wall about the cell, for example, Candida, Rhodotorula, Industry: Bacteria,
and Cryptococcus. Yeasts and Mold
Cultural Characteristics
For the most part, the appearance of massed yeast growth is not useful in the
identification of yeasts, although growth as a film on the surface of liquid
media suggests an oxidative or film yeasts, and production of a carotenoids
pigment indicates the genus Rhodotorula. However, the appearance of the
growth is important when it causes coloured spots on foods.
Yeasts are oxidative, fermentative, or both. The oxidative yeasts may grow as a
film, pellicle, or scum on the surface of liquid and then are termed film yeasts.
Fermentative yeasts usually grow throughout the liquid and produce carbon
dioxide.
Most common yeasts grow best with a plentiful supply of available moisture.
But since many yeasts grow in the presence of greater concentration of solutes
(such as sugar or salt) than most bacteria it can be concluded that these yeasts
require less moisture than the majority of bacteria. Most yeast require more
moisture than molds, however, on the basis of water activity or aw yeasts may
be classified as ordinary if they do not grow in high concentrations of solutes,
i.e. in a low aw, and as osmophilic if they do. However limits of aw for
ordinary yeasts tested thus far ranges from 0.88 to 0.94.
The range of temperature for growth of most yeasts is, in general, similar to
that for molds, with the optimum around 25°C to 30°C and the maximum
about 35°C to 47°C. Some kinds can grow at 0°C or less. The growth of most
yeasts if favoured by an acid reaction in the vicinity of pH 4 to 4.5, and they
will not grow well in an alkaline medium unless adapted to it. Yeasts grow best
under aerobic conditions, but the fermentative types can grow anaerobically,
although slowly.
In general, sugars are the best source of energy for yeasts, although oxidative
yeasts, e.g., the film yeasts, oxidize organic acids and alcohol. Carbon dioxide
produced by bread yeasts accomplishes the leavening of bread, and alcohol
made by the fermentative yeasts is the main product in the manufacture of
wines, beer, industrial alcohol, and other products. The yeasts also aid in the
production of flavors or “bouquet” in wines.
Nitrogenous foods utilized vary from simple compounds such as ammonia and
urea to amino acids and polypeptides. In addition, yeasts require accessory
growth factors.
Microorganisms, namely, bacteria, yeasts and molds can be found in any
environment. The eight environmental sources of organisms to foods are: soil
and water, plants and plant products, food utensils, intestinal tracts of humans
and animals, food handlers, animal feeds, animal hides, air and dust. Although
we see that the microorganisms are beneficial to the humans in many ways,
there are many microorganisms that are the causative agents for food borne
diseases. e.g. Staphlococcus aureus and Clostridium botulinum cause food
borne intoxication whereas Salmonella, E.coli, Campylobacter, Listeria,
17
Introduction Yersinia, Bacillus etc cause food borne infections. Molds are responsible for
causing food intoxication by production of mycotoxins, which are lethal for the
human body e.g. Aflatoxin produced by Aspergillus flavus, patulin produced
by Penicillium expansum, ochratoxins produced by Aspergillus ochraceus etc.
All these will be discussed in Unit 3.
# Check Your Progress Exercise 2
Note: a) Use the space below for your answer.
b) Compare your answers with those given at the end of the unit.
1. Explain the formation of special structures by bacteria.
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2. What are the physiological requirements of molds?
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3. Yeasts maybe useful or harmful. Explain.
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1.4 LET US SUM UP

This unit briefly outlines the identification and classification of food
microorganisms. After reading this unit, you will be able to classify
microorganisms broadly into three categories: bacteria, yeasts and molds.
Further you will be able to categorize these microorganisms on the basis of
their requirements of temperature, oxygen, water activity for growth and also
on the basis of their ability to degrade certain nutrients. To prevent the spoilage
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of food products, appropriate measures have to be taken for preventing their Classification of
Microorganisms
growth and multiplication. Hence food microbiologists have to be well versed Important in the Food
with the various morphological, cultural and physiological characteristics of Industry: Bacteria,
the different microorganisms so as to prevent their growth and proliferation. Yeasts and Mold
After reading this unit, you will get a knowledge of the factors that favour or
inhibit the growth of microorganisms which are essential to give an
understanding of the principles of food spoilage and preservation.
1.5 KEY WORDS

Bacteria : Unicellular microorganisms 1µm in length,
lacking chlorophyll and multiply by binary
fission.
Yeasts : Unicellular fungi, larger than bacteria, which

multiply by budding.
Molds : Multicellular, filamentous fungi devoid of

chlorophyll.
Endospore : Heat resistant structures formed by bacteria under

unfavourable conditions (nutrient depletion or
product accumulation) which remain dormant till
exposed to favourable environment.
Capsule : Slimy material composed of polysaccharides that

serve as a source of reserve nutrients and make
cell resistant to adverse conditions.
Mycostatic : Substances which are inhibitory to mold growth.
Fungicidal : Substances capable of killing the fungi.
Thermophillic
organisms : Microorganisms which require high optimum
temperature for their growth and multiplication
(45-60ºC).
Mesophillic
organisms : Microorganisms whose optimum temperature for
growth is 25-40ºC.
Proteolytic
microorganisms : Microorganisms that have the capacity to break
down complex proteins to amino acids due to
production of extracellular protein degrading
enzymes.
Gram positive
bacteria : Bacteria that stain violet after Gram staining.
Gram negative
bacteria : Bacteria that stain red after Gram staining.
19
Introduction Psychrophillic
microorganisms : Microorganisms requiring less than 20ºC as
optimal temperature for growth.
Obligate aerobes : Microorganisms that grow and multiply only in
presence of oxygen.
Obligate anaerobes : Microorganisms that grow and multiply only in
absence of oxygen.
Facultative
microorganisms : Those microorganisms that can grow and survive
under both aerobic and anaerobic conditions.
Microaerophillic
microorganisms : Those microorganisms that can grow and survive
under low concentrations of oxygen.
Thermoduric
microorganisms : Those microorganisms that can survive at high
temperatures but grows in mesophillic range.
# 1.6 ANSWERS TO CHECK YOUR PROGRESS

EXERCISES
Check Your Progress Exercise 1
1. On basis of morphology bacteria can be classified as:
a) Bacilli: rod-shaped
b) Coccus: spherical
c) Coccobacillus: oval-shaped
d) Spirilla: spiral shaped
2. Yeasts are unicellular fungi and are smaller in size whereas molds are
multicellular and filamentous and are larger than yeasts. Yeasts multiply
asexually by budding whereas molds reproduce sexually by spores. Yeasts
can grow in anaerobic conditions but molds are strictly aerobic.
3. Classification of microorganisms on basis of temperature requirement:
a) Thermophillic(45-60ºC)
b) Thermoduric
c) Mesophillic (25-40ºC)
d) Psychroduric
e) Psychrophillic
Classification of microorganisms on basis of oxygen requirement:
a) Obligate aerobes: Strictly grow in presence of oxygen
b) Obligate anaerobes: Strictly grow in absence of oxygen
c) Facultative: Can grow both in presence and absence of oxygen
d) Microaerophillic: Grow at low oxygen concentrations
20
Classification of microorganisms on basis of aw requirement: Classification of
Microorganisms
a) Bacteria (aw =0.9) Important in the Food
Industry: Bacteria,
b) Yeast (0.88) Yeasts and Mold
c) Mold (0.80)
d) Halophillic bacteria (0.75)
e) Xerphillic molds (0.65)
f) Osmophillic yeasts (0.60)
Classification of microorganisms on basis of nutrient degradation:
a) Lipolytic: Fat degrading
b) Saccharolytic: Sugar degrading
c) Pectinolytic: Pectin degrading
d) Proteolytic: Protein degrading
Classification of microorganisms on basis of staining:
a) Gram positive bacteria: Stain violet after staining
b) Gram negative bacteria: Stain red after Gram staining

1. Bacteria form special structures such as endospores, capsules and cell
aggregates to combat the adverse environmental conditions. Bacteria of
genera Bacillus, Clostridium, Sporosarcina etc have the ability to form
heat resistant endospores. Sporulation usually appears in the late
logarithmic phase of growth, possibly because of nutrient depletion or
accumulation of toxic products. Endospores may remain dormant with no
detectable metabolism for years and germinate when exposed to favourable
conditions for growth. Capsules serve as a source of reserve nutrients and
increase the resistance of bacteria under adverse conditions. They are
composed of dextran and levan and account for sliminess or ropiness of a
food. Bacteria may also form aggregates which helps them to combat
unfavourable environments.
2. Physiological requirements of molds: Molds are mesophillic and grow well
at 25-30°C. Some are psychrotrophic and can grow at –5 to 10°C. They are
aerobic in nature and can grow at wide range of pH 2-8.5. They require a
minimum moisture range of 14-15 per cent to grow. Owing to a number of
hydrolytic enzymes, molds can proliferate on complex media also.
3. Yeasts may be useful or harmful in foods. Yeast fermentations are involved
in the manufacture of foods such as bread, beer, wines, vinegar, and
surface-ripened cheese, and yeasts are grown for enzymes and for food.
Yeasts are undesirable when they cause spoilage of sauerkraut, fruit juices,
syrups, molasses, honey, jellies, meats, wine, beer, and other foods.
21
Introduction
1.7 SOME USEFUL BOOKS
1. Banwart, G.J. (1979) Basic Food Microbiology, AVI Publishing Co. Inc.,
Westport, Connecticut.
2. Frazier, W.C. and Westoff, D.C. (1996) Food Microbiology. Tata McGraw
Hill Publishing Co. Ltd., New Delhi, pp 539.
3. Pelczar, M. Jr., Chan, E.C.S. and Krieg, N.R. (1993) Microbiology, Tata
McGraw Hill Inc., New York, pp 918.
4. Stanier, R.Y., Adelberg, E.A. and Ingraham, .J (1976) The microbial
world. Prentice-Hall, Inc., Englewood Cliffs, N.J
22
Factors affecting
Growth and Inhibition
of Microorganisms in
UNIT 2 FACTORS AFFECTING GROWTH Food
AND INHIBITION OF
MICROORGANISMS IN FOOD
Structure
2.0 Objectives
2.1 Introduction
2.2 Hydrogen-Ion Concentration (pH)
Effect on Microbial Growth
Effect on Microbial Ecology and Food Spoilage
Inhibition of Microbes by Weak Acids
Buffers in Food
2.3 Moisture Requirement/Water Activity
Effect on Microbial Growth and Activity
Ways of Reducing Water Activity
Factors Affecting Water Requirement
2.4 Oxidation Reduction Potential
Redox Couples in Food
Effect of Microbial Growth on Redox Potential of Food
Effect on Microbial Growth and Ecology
Poising Capacity of Food
2.5 Nutrient Content
Foods for Energy
Foods for Growth
Accessory Food Substances or Vitamins
2.6 Biological Structure
Antimicrobial Barriers
Effect of Destruction of Microbial Barriers
2.7 Inhibitory Substances
Biological Inhibitory Substances Originally Present in Food
Inhibitory Substances Developed/ Destroyed in Food Due to the Activity of
Microorganisms
Inhibitory Substances Developed During Processing of Food
2.8 Let Us Sum Up
2.9 Key Words
2.0 OBJECTIVES
After reading this unit you should be able to:
• list out the various factors that favour/inhibit the growth of
microorganisms;
• explain the role played by pH in inhibition of microbial growth;
• explain the effect of water activity on microbial growth and activities;
• explain the influence of redox potential on the natural microflora of food
and the type of spoilage occurring in food;
• understand the role played by nutrient composition on type of
microorganisms growing in food;
• understand the role played by antimicrobial barriers in retarding microbial
spoilage of food; and
• understand the role played by inhibitory substances in retarding microbial
spoilage of food.
2.1 INTRODUCTION
23
Introduction
Microorganisms use our food supply as a source of nutrients and energy. They
increase their numbers by utilizing nutrients. This can result in a deterioration
of the food. They produce enzymatic changes and off-flavours in food by
breaking down a nutrient or synthesizing new compounds. Thus, they "spoil"
our food and make it unfit for consumption. To prevent this we reduce the
contact between microorganisms and our foods (prevent contamination) and
also eliminate microorganisms from our foods, or adjust conditions of storage
in such a way that their growth is prevented (preservation) and thus, there is
no spoilage of food.
If the microorganisms involved are pathogenic, then their presence in our food
will lead to outbreak of food borne diseases also. Many of our foods support
the growth of pathogenic microorganisms or serve as a source of them. Here
again, we attempt to prevent their entrance and growth in our foods or
eliminate them by processing.
Interactions between microorganisms and our foods are also beneficial. Many
of the cultured products consumed and enjoyed for example cultured
buttermilk, yoghurt, sauerkraut, pickles and tofu are produced as a result of
beneficial activities of microorganisms.
Food is the substrate for growth of microorganisms, so the characteristics of a
food are important. Food or substrate will determine which microorganisms
can or cannot grow on it so there is a need to understand the characteristics of
the food or substrate. Then only one can make predictions about the microbial
flora that may develop and flourish in it. This microflora will bring about the
biochemical changes in food due to their activities. The types of biochemical
changes will determine whether those changes are beneficial or harmful.
Knowledge of the factors that favour or inhibit the growth of microorganisms
is very important. It will help us in understanding the principles of food
spoilage and preservation. The chief compositional factors of food that
influence microbial activity are hydrogen-ion concentration, moisture,
oxidation-reduction (O-R) potential, nutrients, biological structure and
presence of inhibitory substances.
2.2 HYDROGEN-ION CONCENTRATION (pH)

The acidity and alkalinity (pH) of an environment has a strong influence on
the activity and stability of macromolecules such as enzymes. These enzymes
play an important role during growth of microorganisms and in their
metabolism. Thus, growth and metabolism of microorganisms are influenced
by pH.
2.2.1 Effect on Microbial Growth

Every microorganism has a minimal, a maximal, and an optimal pH for
growth. In general, bacteria grow in the pH range of 6.0–8.0, yeasts 4.5-6.0
and filamentous fungi 3.5-4.0. Molds can grow over a wider range of pH than
most yeasts and bacteria, and many molds grow at acidities too high for yeasts
and bacteria. Most fermentative yeasts grow well in pH range of about 4.0 to
4.5, eg. fruit juices, and film yeasts grow well on acid foods, such as
sauerkraut and pickles. On the other hand, most yeast do not grow well in
alkaline foods and thus do not have a significant role to play in the spoilage of
food products with high pH. However, large number of yeasts grow well in
near neutral pH. There are some exceptions for example some bacteria can
grow in moderate acidity particularly those bacteria that produce large acids as
24
Factors affecting
a result of their activities like lactobacilli and acetic acid bacteria. These have of Microorganisms in
Food
pH optima between 5.0 and 6.0 and others like the proteolytic bacteria can
grow in foods with a high (alkaline) pH, as found in the stored egg white.
Bacteria are more sensitive to pH than molds and yeasts, with the pathogenic
bacteria being the most sensitive amongst them. The pH values of some of the
common foods along with the pH range for growth of some groups of
microorganisms and a few of food associated pathogenic bacteria are given in
Table 2.1.
Table 2.1: The pH ranges of some common food items and pH range of
some common food microflora
Food pH range Organism pH range

Citrus fruits 2-5 Molds 0-11
Soft drinks 2.5-4 Yeasts 1.5-8.5
Beer 3.5-4.5 Lactic acid bacteria 3.2-10.5
Meat 5.5-6.2 Staphylococcus aureus 4-9.8
Fish 6.5-7.3 Salmonella spp. 4.1-9
Egg white 8.6-9.6 Escherichia coli 4.3-9
Milk 6.5-7 Yersinia enterocolitica 4.5-9
Flour 6.2-7.2 Clostridium botulinum 4.8-8.2
Vegetables 4.8-7 Clostridium perfringens 5.4-8.7
Fermented shark 10-12 Bacillus cereus 4.7-9.3
pH minima and maxima of microorganisms also varies due to other important

factors like temperature, moisture content, salt concentration, redox potential
etc. For example, in the presence of 0.2 M NaCl, Alcaligenes faecalis can
grow over a wider pH range than in the absence of NaCI or in the presence of
0.2 M sodium citrate. The pH minima of certain lactobacilli also depends upon
the type of acid used, for example with citric, hydrochloric, phosphoric and
tartaric acids growth can occur at lower pH than in presence of acetic or lactic
acids. In general, yeast and molds are more acid-tolerant than bacteria.
When microorganisms are grown at pH either higher or lower than their
optimum pH there is an increase in lag phase of the microbe. The increased
lag would be of longer duration if the food has a good buffering capacity in
contrast to one that has poor buffering capacity. Good buffering capacity of
food would result in slower change in pH of food due to microbial activity. A
respiring microbial cell is adversely affected by pH since it affects the
functioning of enzymes and the transport of nutrients into the cell. In addition
to the effect of pH on rate of growth of microorganisms, pH also affects rate
of survival of microorganisms during storage, heating, drying and other forms
of processing. Many times the initial pH may be suitable, but growth of the
organism itself may alter the pH, thereby making it unfavourable. Conversely,
the initial pH may be restrictive, but the growth of a limited number of
microorganisms may alter the pH to a more favourable range for the growth of
many other microorganisms.
The inherent pH of foods varies, although most are neutral or acidic. Materials
with an alkaline pH generally have a rather unpleasant taste with some
exceptions like egg white where the pH increases to around 9.2, as CO2 is lost
from the egg after laying. The pH of a product can be easily determined with a
25
Introduction
pH meter. However, this value alone is not sufficient for predicting microbial
spoilages. It is also desirable, for example, to know the acid responsible for a
given pH, because some acids, particularly the organic acids, are more
inhibitory than others.
2.2.2 Effect on Microbial Ecology and Food Spoilage

The acidity of a product plays an important role in deciding the type
microflora present in food and the rate and type of its spoilage. For example,
most of the meats and seafoods have a final ultimate pH of about 5.6 and
above. Thus, these products are susceptible to bacterial as well as to mold and
yeast spoilage. Similarly, most vegetables have higher pH values than fruits,
and thus vegetables would be more prone to bacterial than fungal spoilage
since such pH values favour bacterial growth. Soft-rot producing bacteria such
as Erwinia carotovora and pseudomonads play a significant role in their
spoilage. In fruits, however, a lower pH (below 4.5) prevents bacterial growth
and yeasts and molds dominate spoilage.
Fish is spoiled more rapidly than meat under chilled conditions. This is due to
the fact that the pH of post-rigor mammalian muscle is around 5.6 and this
contributes to the longer storage life of meat. On the other hand, fish have a
pH between 6.2-6.5. Shewanella (formerly Alteromonas) mainly causes
spoilage under chilled conditions. It is a pH-sensitive microbe and hence,
plays a significant role in fish spoilage but not in normal meat (pH<6.0).
Those fishes that have a naturally low pH such as halibut (pH~5.6) as a result
have better keeping qualities than other fish. Thus, a food with inherently low
pH would tend to be more stable microbiologically than a neutral food.
Upon the death of a well-rested meat animal, the usual 1% glycogen is
converted into lactic acid, which directly causes a depression in pH values
from about 7.4 to about 5.6. Most of the bacteria cannot tolerate lower pH,
hence meat has a longer storage life. Meat from fatigued animals spoils faster
than that from rested animals. This is because most of the glycogen present
had already been used during its lifetime and hence, final pH attained upon
completion of rigor mortis is not as low as that of a well-rested animal. Thus,
bacteria are able to grow and spoil it.
The excellent keeping quality of certain foods is related to their restrictive pH,
for example fruits, soft drinks, fermented milks, sauerkraut and pickles which
have an acidic pH. Fruits, soft drinks, vinegar, and wines have an excellent
keeping quality mainly due to pH, which falls far below the point at which
bacteria normally grow. Fruits generally undergo mold and yeast spoilage, and
this is due to the capacity of these organisms to grow at pH values < 3.5,
which is considerably below the minima for most food spoilage and all food
poisoning bacteria.
Some foods have a low pH because of inherent acidity; others, for example,
the fermented products like sauerkraut, pickles and fermented milks have a
low pH because of acidity produced due to the activity of microorganisms.
This acidity is also known as biological acidity and is generally due to the
accumulation of lactic acid during fermentation. Regardless of the source of
acidity, the effect upon keeping quality appears to be the same. This ability of
low pH to restrict microbial growth has been employed since the earliest times
for preservation of foods using acetic acid and lactic acids.
2.2.3 Inhibition of Microbes by Weak Acids

With the exception of those soft drinks that contain phosphoric acid, in most
26
Factors affecting
other acidic foods acidity is due to the presence of weak organic acids. These of Microorganisms in
Food
do not dissociate completely into protons and conjugate base in solution but
establish equilibrium:
HA H + + A−
The partial dissociation of weak acids, such as acetic acid, plays an important
role in their ability to inhibit microbial growth. Although addition of strong
acids has a more profound effect on pH but at the same pH, they are less
inhibitory than weak lipophilic acids. This is because microbial inhibition by
weak acids is directly related to the concentration of undissociated acid
(Figure 2.1). These undissociated lipophilic acid molecules can pass freely
through the membrane, in doing so they pass from an external environment of
low pH where the equilibrium favours the undissociated molecule to the high
pH of the cytoplasm. At this higher pH, the equilibrium shifts in favour of the
dissociated molecule, so the acid ionizes producing protons. These protons
tend to acidify the cytoplasm. The cell tends to maintain its internal pH by
expelling protons leaking in. This process requires energy and the microbe
diverts energy from growth related functions to removing protons from the
cell thereby slowing its growth. The burden on the cell becomes too great. The
cytoplasmic pH drops to a level where growth is no longer possible and the
cell eventually dies. Strong acids on the other hand dissociate completely into
protons and conjugate base in solution. These dissociated acid molecules
cannot pass freely through the cell membrane. Hence there is not much change
in the pH of the cytoplasm. As a result these are less inhibitory than weak
acids at the same pH.
Bacterial cell
Neutral pH Plasma membrane
A−+H
+
HA
More Less ATP
ADP
Use of energy to
Free passage of − + remove protons
HA A +H
undissociated molecule More Less from the bacterial cell
Low pH external environment

Figure 2.1: Inhibition of bacterial growth by weak acids
2.2.4 Buffers in Foods

Some foods are better able to resist changes in pH than others. These tend to
resist changes in pH since these are buffered and the ability to resist changes
in pH is known as buffering capacity. The buffers are the compounds present
in food that resist changes in pH and thus are important. These are especially
effective within a certain pH range. Buffers permit an acid (or alkaline)
fermentation to go on longer with a greater yield of products and organisms
27
Introduction
than would otherwise be possible. In general, meats are more buffered than
vegetables. Contributing to the buffering capacity of meats are their various
proteins. Vegetables are generally low in proteins and consequently lack the
buffering capacity to resist changes in their pH by the growth of
microorganisms. Hence these permit an appreciable decrease in pH with the
production of small amounts of acid by the lactic acid bacteria during the early
part of sauerkraut and pickle fermentations. This is desirable since it enables
the lactic acid bacteria to suppress the undesirable pectin-hydrolyzing and
proteolytic organisms which cause spoilage. Low buffering power makes for a
more rapidly appearing succession of microorganisms during fermentation
than high buffering power. Milk is fairly high in protein (a good buffer) and
therefore permits considerable growth and acid production by lactic acid
bacteria during the manufacture of fermented milks before growth is
suppressed.
1. Why do fishes spoil more rapidly than meat?
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2. Why does the meat from fatigued animal spoil faster than that from a well-
rested animal?
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3. What is biological acidity?
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28
Factors affecting
……………………………………………………………………………… of Microorganisms in
Food
………………………………………………………………………………
4. Why are the weak organic acids more inhibitory to growth of
microorganisms than the strong acids?
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5. How does adverse pH affect the microorganism?
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2.3 MOISTURE REQUIREMENT/WATER ACTIVITY

One of man's oldest methods of preserving foods is drying or desiccation. The
preservation of foods by drying is a direct result of removal of moisture.
Microorganisms need water for growth. Without water no growth can occur.
The exact amount of water needed for growth of microorganisms varies. This
water requirement of microorganisms is best expressed in terms of available
water or water activity aw, the vapour pressure of the solution (of solutes in
water in most foods) divided by the vapour pressure of the solvent (usually
water). Thus aw, for pure water would be 1.00. The water activity depends on
the number of molecules and ions present in solution, rather than their size.
Thus a compound like sodium chloride, which dissociates into two ions in
solution, is more effective at reducing the water activity than a compound like
sucrose on mole-to-mole basis.
2.3.1 Effect on Microbial Growth and Activity

Bacteria require higher values of aw for growth than fungi. Gram-negative
bacteria have higher requirements than gram positives. Most spoilage bacteria
do not grow below aw 0.91, while spoilage molds can grow as low as 0.80.
However, food-poisoning bacteria like Staphylococcus aureus can grow at aw
as low as 0.86, while Clostridium botulinum does not grow below 0.94. Yeasts
and molds can grow over a wider aw range than bacteria. The lowest aw values
for bacteria is 0.75 for halophilic (meaning salt-loving) bacteria, while
29
Introduction
xerophilic (dry-loving) molds and osmophilic (preferring high osmotic

pressures) yeasts can grow at aw values of 0.65 and 0.60, respectively. The
limiting value of water activity for the growth of microorganisms is about 0.6
and below this value the spoilage of foods is not due to microorganisms but
may be due to insect damage or chemical reaction such as oxidation. At a
water activity of 0.6, corresponding to a water potential of -68MPa (Mega
Pascals), the cytoplasm would need to contain very high concentrations of an
appropriate compatible solute and it is probable that the macromolecules such
as DNA would no longer function properly and active growth may stop.
Most bacteria grow well in a medium with a water activity aw approaching
1.00 (at 0.995 to 0.998), i.e., they grow best in low concentrations of sugar or
salt. Culture media for most bacteria contain not more than 1 per cent of sugar
and 0.85 per cent of sodium chloride (physiological salt solution). As little as
3 to 4 percent sugar and 1 to 2 percent salt may inhibit some bacteria. The
optimal aw and the lower limit of aw for growth vary with the bacterium, as
well as with food, temperature, pH, and the presence of oxygen, carbon
dioxide, and inhibitors. The optimal aw and the lower limit of aw for growth is
lower for bacteria which are able to grow in high concentrations of sugar or
salt. Some examples of lower limits of aw for growth of some food bacteria are
given in Table 2.2. These figures would vary depending on conditions used for
growth of the microorganisms as mentioned above.
Table 2.2: Minimum aw values for growth of microorganisms of
importance in food
Organisms Water Organisms Water

activity (aw) activity
Groups Specific organisms
(aw)
Most spoilage 0.90 Pseudomonas spp. 0.97
bacteria
Most spoilage yeasts 0.88 Escherichia coli 0.96
Most spoilage molds 0.80 Bacillus subtilis 0.95
Halophilic bacteria 0.75 Enterobacter 0.945
aerogenes
Xerophilic molds 0.61 Clostridium botulinum 0.93
Osmophilic yeasts 0.60 Staphylococcus 0.86
aureus
Molds differ considerably in optimal aw and range of aw for the germination of

asexual spores. The minimal aw for spore germination is as low as 0.62 for
some molds and as high as 0.93 for others (e.g., Mucor, Rhizopus, and
Botrytis). Each mold also has an optimal aw and range of aw for growth.
Examples of optimal aw are 0.98 for Aspergillus sp., 0.995 to 0.98 for
Rhizopus sp., and 0.9935 for Penicillium sp. The aw would have to be below
0.62 to stop all chances for mold growth, although aw below 0.70 inhibits most
molds that cause food spoilage. The reduction of the aw below the optimum
for a mold delays spore germination and reduces the rate of growth and
therefore is an important factor in food preservation. Many of the molds can
grow in foods with aw approaching 1.00 (pure water).
With a reduction of water activity of food, the number of microorganisms
30
Factors affecting
capable of maintaining active growth in it decreases. On the other hand, there of Microorganisms in
Food
are microorganisms that grow better at reduced aw. These microorganisms are
generally associated with foods having low water activity. Since low water
activities are associated with three distinct types of food, the following three
terms are used to describe the microorganisms especially associated with these
foods:
i) Halotolerant – able to grow in the presence of high concentrations of salt
ii) Osmotolerant – able to grow in the presence of high concentrations of
nonionized organic compounds such as sugars.
iii) Xerotolerant – able to grow on dry foods.
The halobacteria are obligately halophilic and cannot grow in the absence
of high concentration of salt.
2.3.2 Ways of Reducing Water Activity

Water is made unavailable in various ways:
1. Solutes and ions tie up water in solution. Solutes lower aw and this
reduction in aw depends on the total concentration of dissolved molecules
and ions. Since these bind to water molecules, there is reduction in aw.
Therefore, an increase in the concentration of dissolved substances such as
sugars and salts is in effect a drying of the material (Graph 2.1). Not only
is water tied up by solutes, but also water tends to leave the microbial cells
by reverse osmosis to maintain equilibrium between the concentration of
solute outside and inside the cells.
1.05
Water activity
0.95
0.9
0.85
0.9 1.7 3.5 7 10 13 16 19 22
Sodium chloride concentrations (% w/v)
Graph 2.1: Effect of sodium chloride concentration on water activity
2. Hydrophilic colloids (gels) make water unavailable. As little as 3 to 4

percent agar in a medium may prevent bacterial growth by leaving too
little available moisture.
3. Water of crystallization or hydration is usually unavailable to
microorganisms. Water itself, when crystallized as ice, no longer can be
used by microbial cells. The aw of water-ice mixtures (vapour pressure of
ice divided by vapour pressure of water) decreases with a decrease in
temperature below 0oC (Graph 2.2). In a food, as more and more ice is
formed, the concentration of solutes in the unfrozen water increases, thus
lowering available water and thereby it’s aw is reduced.
1.2
1
activity
0.8 31
0.6
Introduction
Graph 2.2: Effect of temperature on water activity
The water activity aw varies with temperature; these variations are only slight
within the range of temperatures that permit microbial growth. Variations in
temperature increase in importance with increasing concentrations of solutes
and increasing effects on ionization of solutes.
Each microorganism has a maximal, optimal, and minimal aw for growth. As
the aw is reduced below the optimal level, there is a lengthening of the lag
period of growth, a decrease in the rate of growth and a decrease in the
amount of cell substance synthesized, changes that vary with the organism and
with the solute employed to reduce aw. This range depends on a number of
factors which are mentioned below.
2.3.3 Factors Affecting Water Requirement
Factors that may affect aw requirements of microorganisms include:
1. Kind of solute employed to reduce the aw: For some organisms, like molds,
the lowest aw for growth is independent of the kind of solute used. For
other organisms, however, lower limiting aw values differ from solute to
solute. For example potassium chloride usually is less toxic than sodium
chloride, and it in turn is less inhibitory than sodium sulphate. Thus,
sodium sulphate at a lower concentration may be as effective in reducing
aw as potassium chloride at a higher concentration.
2. Nutritive value of the culture medium: In general, the better the medium
for growth, the lower the limiting aw permitting growth of microorganism.
3. Temperature: Most organisms have the greatest tolerance to low aw at
about optimal temperatures.
4. Oxygen supply: Growth of aerobes takes place at a lower aw in the

presence of air than in its absence, and the reverse is true of anaerobes.
5. pH: Most organisms are more tolerant of low aw at pH values near
neutrality than in acid or alkaline media.
6. Inhibitors: The presence of inhibitors narrows the range of aw for growth
of microorganism.
Each organism has its own characteristic optimal aw and its own range of aw
for growth in a given set of environmental conditions. This range of aw
permitting growth is narrowed if any of the above mentioned environmental
factors are not optimal and is narrowed still more if two or more conditions
are not favourable. An unfavourable aw will result not only in a reduction in
the rate of growth but will also reduce the yield of cells. The delay (lag) in
initiation of growth or germination of spores will be more under more
unfavourable aw of the substrate. It is known that growth of at least some cells
32
Factors affecting
may occur in high numbers at reduced aw values, but the production of certain of Microorganisms in
Food
extracellular products may be limited or these may not be produced at all. For
example, reduced aw results in the cessation of enterotoxin B production by
Staphylococcus aureus even though high numbers of cells are produced at the
same time This often is as important in food preservation as reduction in the
rate of growth of the organism. Microorganisms that can grow in high
concentrations of solutes, e.g., sugar and salt, obviously have a low minimal
aw. Halophilic bacteria require a certain minimal concentration of dissolved
sodium chloride for growth. Osmophilic yeasts grow best in high concen-
trations of sugar.
"
1. List out the types of microorganisms associated with foods having low
water activity.
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2. Define water activity.
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3. How water is made unavailable to microorganisms?
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4. Describe the factors that may affect aw requirements of microorganisms.
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33
Introduction
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2.4 OXIDATION REDUCTION POTENTIAL

The tendency of a substrate to accept or donate electrons, is termed its redox
potential (Eh). The O/R potential of a substrate may be defined generally as
the ease with which the substrate loses or gains electrons. When a substrate
loses electrons, the substrate is oxidized while a substrate that gains electrons
becomes reduced. Therefore, a substance that readily gives up electrons is a
good reducing agent, while one that readily takes up electrons is a good
oxidizing agent. In the equation below, this is represented in its most general
form to include the many redox reactions, which also involve protons and
have the overall effect of transferring hydrogen atoms.
[Oxidant]+H++ ne [Reductant]
Where n is the number of electrons, e, transferred.
The tendency of an atom or molecule to accept or donate electrons is
expressed as its standard redox potential, Eo'. When electrons are transferred
from one compound to another, a potential difference is created between the
two compounds. This difference may be measured by use of an appropriate
instrument and expressed as millivolts (mv). It can be measured against an
external reference by an inert metal electrode, usually platinum. The more
highly oxidized a substance, the more positive will be its electrical potential,
and the more highly reduced a substance, the more negative will be its
electrical potential.
2.4.1 Redox Couples in Food

Pair of oxidizing and reducing agents present in food are known as redox
couples. A large positive Eo' of food indicates that the oxidized species of the
couple is a strong oxidizing agent and the reduced form only weakly reducing.
A large negative Eo' of food indicates the reverse. When the concentration of
oxidant and reductant is equal, a zero electrical potential exists. The relative
proportions of oxidized and reduced species present will also influence the
measured Eh. If the balances of the various redox couples present favours the
oxidized state then there will be a tendency to accept electrons from the
electrode creating a positive potential, which signifies an oxidizing-
environment. If the balance is reversed, the sample will tend to donate
electrons to the electrode, which will then register a negative potential – a
reducing environment.
With the notable exception of oxygen, most of the couples present in foods,
e.g. glutathione and cysteine in meats and ascorbic acid and reducing sugars in
plant products, would on their own tend to establish reducing conditions.
Oxygen, which is present in the air at a level of around 21 %, is usually the
most influential redox couple in food systems. It has a high Eo' and is a
powerful oxidizing agent. If sufficient air is present in food, a high positive
potential will result and most other redox couples present will, if allowed to
equilibrate, be largely in the oxidized state. Hence, increasing the access of air
to food material by chopping, grinding or mincing will increase its Eh.
Similarly, exclusion of air as in modified vacuum packing or canning will
reduce the Eh.
34
Factors affecting
2.4.2 Effect of Microbial Growth on Redox Potential of Food of Microorganisms in
Food
Microbial growth in food reduces its Eh. This is usually because during their
growth, microorganisms consume oxygen and produce reducing compounds
such as hydrogen. Oxygen is the most important terminal electron accepter in
the electron transport chain, especially in case of aerobes. During passage of
electrons through the electron transport chain, microorganisms generate
energy and thereby oxygen is depleted. As the oxygen content of the medium
decreases, so the redox potential declines from a positive potential to a
negative potential.
The decrease in Eh as a result of microbial activity is the basis of some rapid
tests for determination of microbial load of food, particularly dairy products.
Redox dyes such as methylene blue or resazurin are used to indicate changes
in Eh, which are correlated with microbial levels. These dyes become
colourless when these are reduced. The time taken for reduction of the dyes
will be inversely proportional to the microbial load of food i.e. more the
microorganisms in food, less is the time taken for dye to be reduced and vice
versa. The factors influencing redox potential of foods are summarized in
Table 2.3 given below:
Table: 2.3 Factors affecting redox potential of foods
1. Redox couples present

2. Ratio of oxidizing species to reducing species
3. pH
4. Poising capacity
4. Availability of oxygen
5. Microbial activity
2.4.3 Effect on Microbial Growth and Ecology

Redox potential exerts an important selective effect on the microflora of a
food since it will decide the type of microorganism which can grow in that
food. Microbial growth can occur over a wide spectrum of redox potential.
However, individual microorganisms have their own redox ranges over which
they can grow. They are classified into one of several physiological groups on
the basis of the redox range over which they can grow and their response to
oxygen. Based on their ability to use free oxygen, microorganisms have been
classified as
1. Aerobic when they require free oxygen.
2. Anaerobic when they grow best in the absence of free oxygen.
3. Facultative when they grow well either aerobically or anaerobically.
Molds are aerobic, most yeast grow best aerobically and bacteria may be
aerobic, anaerobic, or facultative. A high (oxidizing) potential favours aerobes
but will permit the growth of facultative organisms also, and a low (reducing)
potential favours anaerobic or facultative organisms. Growth of an organism
may alter the O-R potential of a food enough to inhibit other organisms.
Anaerobes, for example, may lower the O-R potential to a level which is
inhibitory to aerobes.
Obligate aerobes are those organisms that generate their energy from oxidative
phosphorylation using oxygen as the terminal electron acceptor.
Consequently, they have a requirement for oxygen and a high Eh and will
predominate at food surfaces exposed to air or where air is readily available,
for example, pseudomonads, such as Pseudomonas fluorescens, which grows
35
Introduction
at an Eh of +100 to +500 mv, and other oxidative Gram-negative rods. These

grow on meat surfaces and produce slime and off-odours. Bacillus subtilis (Eh
-100 to +135 mv) produces ropiness in the open texture of bread and
Acetobacter species growing on the surface of alcoholic beverages oxidize
ethanol to acetic acid to produce vinegar or spoil the alcoholic beverage.
Plant juices, tend to have Eh values of +300 to +400 mv. It is not surprising to
find that aerobic bacteria and molds are the common cause of spoilage of
products of this type. Minced meats have Eh values of around +200 mv while
in solid meats the Eh is generally around -200 mv. Cheeses have Eh values on
the negative side from −20 to around −200 mv.
Obligate anaerobes grow only at low or negative redox potentials and require
absence of oxygen. Anaerobic metabolism gives the organism a lower yield of
utilizable energy than aerobic respiration. A reducing environment minimizes
the loss of reducing power from the microbial cell and thus, is favoured.
Hence, presence of oxygen, which provides an oxidizing environment to the
microbes is not favoured. However, for many anaerobes, oxygen itself exerts a
specific toxic effect. For example, Clostridium acetobutylicum can grow at an
Eh as high as +370 mv maintained by ferricyanide, but would not grow at
+110mv in an aerated culture. This effect is due to the inability of obligate
anaerobes to scavenge and destroy toxic products of molecular oxygen such as
hydrogen peroxide and superoxide anion radical (02−) produced by one
electron reduction of molecular oxygen. They lack the enzymes catalase and
superoxide dismutase, which catalyse the breakdown of these radicals.
Thus, in a highly oxidized food, there will be a predominance of aerobic
organisms especially at food surfaces exposed to air. Whereas, in food with
negative Eh, the anaerobic microflora requiring reduced conditions will be
favoured. For example, anaerobic bacteria do not multiply until the onset of
rigor mortis (stiffening of body after death) of muscles of horse because of the
high Eh (+250 mv) in prerigor meat. At 30 h postmortem (after death), the Eh
falls to about -130 mv in the absence of bacterial growth and this low Eh
values favour the growth of obligate anaerobes like Clostridium. Obligate
anaerobes, such as clostridia, have the potential to grow wherever conditions
are anaerobic such as deep in meat tissues and stews, in vacuum packs and
canned foods causing spoilage and C. botulinum is of major public health
concern, since it causes botulism.
Aerotolerant anaerobes are incapable of aerobic respiration, but can
nevertheless grow in the presence of air. Many lactic acid bacteria fall into this
category. They can only generate energy by fermentation and lack both
catalase and superoxide dismutase, but are able to grow in the presence of
oxygen because they have a mechanism for destroying superoxide.
Microorganisms affect the Eh of their environment during growth. This is true
especially of aerobes, which can lower the Eh of their environment while
anaerobes cannot. As aerobes grow, oxygen in the medium is depleted,
resulting in the lowering of Eh. Growth is not slowed, however, due to the
ability of cells to make use of oxygen donating or hydrogen-accepting
substances in the medium. The result of this is that the medium becomes
poorer in oxidizing and richer in reducing substances. Microorganisms can
reduce the Eh of a medium by their production of certain metabolic by-
products such as hydrogen sulphide, which has the capacity to lower Eh to
−300 mv. Since hydrogen sulphide reacts readily with oxygen, it will
accumulate only in anaerobic environments.
36
Factors affecting
2.4.4 Poising Capacity of Food of Microorganisms in
Food
As redox conditions change, there will be some resistance to change in a
food's redox potential. This is known as poising capacity of food. This
capacity is dependent on the concentration of the redox couple. Poising is
greatest when the two components of a redox couple are present in equal
amounts.
Most fresh plant or animal foods have a low and well-poised O-R potential in
their interior: the plants because of reducing substances such as ascorbic acid
and reducing sugars and the animal tissues because of SH (sulfhydryl) and
other reducing groups. As long as the plant or animal cells respire and remain
active, they tend to poise the O-R system at a low level, resisting the effect of
oxygen diffusing from the outside. Therefore, a piece of fresh meat or a fresh
whole fruit would have aerobic conditions only at and near the surface. The
meat could support aerobic growth of slime-forming or souring bacteria at the
surface at the same time as anaerobic putrefaction could be proceeding in the
interior.
Processing procedures may alter this situation. For example, heating may
reduce the poising power of the food by destroying or altering the reducing
and oxidizing substances present and also allow more rapid diffusion of
oxygen inward, either because of the destruction of poising substances or
because of changes in the physical structure of the food. Processing also may
remove oxidizing or reducing substances. For example, clear fruit juices loose
reducing substances by their removal during extraction and filtration and
therefore become more favourable to the growth of yeasts than the original
juice containing the pulp.
In the presence of limited amounts of oxygen the same aerobic or facultative

organisms may produce incompletely oxidized products, such as organic
acids, from carbohydrates, while with plenty of oxygen available, complete
oxidation to carbon dioxide and water might result. Protein decomposition
under anaerobic conditions may result in putrefaction, whereas under aerobic
conditions, the products are likely to be less obnoxious. Thus, the redox
potential of the food would decide the course of spoilage and the type of end
products being produced due to microbial activities.
1. Define redox potential.
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37
Introduction
2. Enlist the physiological groups of microorganisms based on their oxygen

requirement.
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3. Define the poising capacity of food.
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4. How is the poising capacity of food destroyed?

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5. Enlist the factors which affect the redox potential of food.
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38
Factors affecting
of Microorganisms in
2.5 NUTRIENT CONTENT Food
Microorganisms use foods as a source of nutrients and energy. Each kind of

microorganism has a definite range of food requirements. For some species
that range is wide and growth takes place in a variety of substrates e.g.
coliform bacteria; but others, e.g., many of the pathogens, being fastidious in
their nutrient requirements can grow in limited kinds of substrates. The better
the medium for an organism, the wider the ranges of temperature, pH, and aw
over which growth can take place.
The food based on their nutrient composition can be classified as (1) foods for
energy, (2) foods for growth, and (3) accessory food substances, or vitamins,
which may be necessary for energy or growth.
2.5.1 Foods for Energy
The carbohydrates, especially the sugars, are most commonly used as an

energy source, but other carbon compounds may also serve the purpose, e.g.,
esters, alcohols, peptides, amino acids, organic acids and their salts.
Comparatively few organisms can utilize complex carbohydrates, e.g.,
cellulose and starch. Microorganisms differ even in their ability to use some of
the simpler soluble sugars. Many organisms cannot use the disaccharide
lactose (milk sugar) and therefore do not grow well in milk. Some yeast do not
attack maltose. Most organisms, if they utilize sugars at all, can use glucose.
The ability of microorganisms to hydrolyze pectin, which is characteristic of
some kinds of bacteria and many molds, is important in the softening or
rotting of fruits and vegetables or fermented products got from them. The
ability to synthesize amylolytic (starch degrading) enzymes will favour the
growth of an organism on cereals and other starchy products. The addition of
fruits containing sucrose and other sugars to yoghurt increases the range of
carbohydrates available and allows the development of a more diverse
spoilage microflora of yeasts.
Bacteria differ in their ability to utilize different foods as a source of energy.

Some can use a variety of carbohydrates, e.g., the coliform bacteria and
Clostridium spp., and others only one or two. Some can use other carbon
compounds like organic acids and their salts, alcohols, and esters
(Pseudomonas spp.). Some can hydrolyze complex carbohydrates, although
others cannot.
A limited number of microorganisms can obtain their energy from fats but do
so only if a more readily usable energy food, such as sugar, is absent. First, the
fat must be hydrolyzed with the aid of lipase to glycerol and fatty acids, which
then can serve as energy source for the hydrolyzing organism or others
microbes that grow on products of lipid hydrolysis. Aerobic microorganisms
are more commonly involved in the decomposition of fats than anaerobic
ones, and the lipolytic organisms usually are also proteolytic.
Split products of proteins, for example, peptides and amino acids, serve as an
energy source for many proteolytic organisms when a better energy source is
lacking. These also serve as source of energy for some non-proteolytic
organisms. Meats for example, may be low in carbohydrate and therefore will
be decomposed by proteolytic species, e.g., Pseudomonas spp.
Molds in general can utilize many kinds of foods as energy source, ranging
from simple to complex. Most of the common molds possess a variety of
39
Introduction
hydrolytic enzymes and some are grown for their amylases, pectinases,
proteinases, and lipases.
Not only is the kind of energy food important but also its concentration in
solution and hence it’s osmotic effect and the amount of available moisture,
which will determine its growth rate. For a given percentage of sugar in
solution, the osmotic pressure will vary with the weight of the sugar molecule,
Therefore, a 10% solution of glucose has about twice the osmotic pressure of a
10% solution of sucrose or maltose; i.e., it ties up twice as much moisture.
Molds can grow in the highest concentrations of sugars and yeasts in fairly
high concentrations but most bacteria grow best in fairly low concentrations.
There are, of course, some exceptions to this generalization: osmophilic yeasts
grow in as high concentrations of sugar as molds and some bacteria can grow
in fairly high concentrations of sugar.
An adequate supply of foods for growth will favour utilization of the foods for
energy. More carbohydrate will be used if a good nitrogen food is present in
sufficient quantity than if the nitrogen is in poor supply. Organisms requiring
special accessory growth substances might be prevented from growing if one
or more of these vitamins were lacking, and thus the whole course of
decomposition might be altered due to a change in the microflora.
2.5.2 Foods for Growth
Microorganisms differ in their ability to use various nitrogenous compounds

as a source of nitrogen for growth. The primary nitrogen sources utilized by
heterotrophic microorganisms are amino acids. A large number of other
nitrogenous compounds may serve this function for example, nucleotides, free
amino acids, peptides and proteins. Simple compounds such as amino acids
will be utilized by most of the organisms before they utilize complex
compounds such as high molecular weight proteins. The nitrogen require-
ments of some bacteria such as Pseudomonas spp. may be satisfied by simple
compounds like ammonia or nitrates whereas for others like lactics, more
complex compounds like amino acids, peptides, or proteins may be utilized or
even required.
Many molds are proteolytic, but comparatively few bacteria and very few
yeast are actively proteolytic. Proteolytic bacteria grow best at pH values near
neutrality and are inhibited by acidity. Only exceptions are the acid-
proteolytic bacteria that hydrolyze protein while producing acid. Carbon for
growth for most of the microorganisms is derived from organic compounds
but some can use carbon dioxide also.
The minerals required by microorganisms are nearly always present at the low
levels required.
2.5.3 Accessory Food Substances or Vitamins
Bacteria also vary in their need for vitamins or accessory growth factors.
Some microorganisms are unable to synthesize some or all of the vitamins
needed for their growth. For example, Staphylococcus aureus synthesizes part
while Pseudomonas or Escherichia coli all of the factors needed. The lactics
and many pathogens must have all of the vitamins furnished. Most natural
plant and animal foodstuffs contain an array of these vitamins, but some may
be low in amount or lacking. For example, meats are high in B vitamins and
fruits are low, but fruits are high in ascorbic acid.
40
Factors affecting
Microorganisms may require B vitamins in low quantities and most of the of Microorganisms in
Food
natural foods have an abundant quantity of these. Gram positive bacteria are
the least synthetic and must, therefore, be supplied with one or more of these
compounds before they will grow. Gram negative bacteria and molds are able
to synthesize most of their requirements. Consequently, these two groups of
organisms may be found growing on foods low in B vitamins. Fruits tend to
be lower in B vitamins than meats. Thus, the usual spoilage of fruits is by
molds rather than bacteria since fruits also have a low pH and positive Eh,
which favour mold growth.
Egg white contains biotin but also contains avidin, which ties it up, making it
unavailable to microorganisms and thus eliminate spoilage of eggs through
biotin requiring organisms. The processing of foods often reduces the vitamin
content. For example, thiamine, pantothenic acid, folic acid and ascorbic acid
(in air) are heat-labile. Drying causes a loss in vitamins such as thiamine and
ascorbic acid. Even storage of foods for long periods, especially if the storage
temperature is elevated, may result in a decrease in the level of some of these
growth factors.

1. What is the effect of sugar concentration on microbial growth?
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2.6 BIOLOGICAL STRUCTURE

The plants and animals that serve as food sources have all evolved
mechanisms of defense against the invasion and proliferation of
microorganisms. By taking these natural phenomena into account, one can
make effective use of these in preventing the microbial spoilage of the
products.
2.6.1 Antimicrobial Barriers

The inner parts of whole, healthy tissues of living plants and animals are either
sterile or low in microbial content. Therefore, unless opportunity has been
given for their penetration, spoilage organisms within raw food may be few or
lacking.
41
Introduction
The first barrier is the integument: a physical barrier to protect the food, e.g.,
the shell on eggs, the skin on poultry, the shell on nuts and the rind or skin on
fruits and vegetables, or these may be surrounded by natural wax. It is usually
composed of macromolecules relatively resistant to degradation and provides
an inhospitable environment for microorganisms either with a low water
activity or nutrients deficiency or antimicrobial compounds e.g. short chain
fatty acids on animal skin, essential oils on plant surfaces etc. This physical
protection to the food may not only help in its preservation but may also
determine the kind, rate and course of spoilage. Layers of fat over meat may
protect that part of the flesh, or scales may protect the outer part of the fish.
2.6.2 Effect of Destruction of Microbial Barriers

Physical damage to the integument allows microbial invasion of the
underlying nutrient-rich tissues and it is a common observation that damaged
fruits and vegetables deteriorate more rapidly than entire products and that this
process is initiated at the site of injury. Consequently, it is important that
during harvesting and transport these barriers are maintained intact as far as
possible.
An increase in exposed surface, brought about by peeling, skinning and chop-
ping may serve not only to distribute spoilage organisms but also to release
juices containing food materials for the microorganisms. The disintegration of
tissues by freezing may accomplish a similar result. In meat the growth of
spoilage bacteria takes place mostly in the fluid between the small meat fibers
and it is only after rigor mortis that much of this food material is released from
the fibers to become available to spoilage organisms.
1. What is the role of antimicrobial barriers in preventing food spoilage by
microorganisms?
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2. What happens when integument is physically damaged?
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42
Factors affecting
Food
2.7 INHIBITORY SUBSTANCES

These may be originally present in the food, added purposely or accidentally,
or developed there by growth of microorganisms or by processing methods.
These may prevent growth of all microorganisms or, more often, may deter
certain microorganisms. The mechanism of action for nearly all antimicrobials
can be classified into one or more of the following groups: (1) reaction with
the cell membrane, (2) inactivation of essential enzymes, or (3) destruction or
functional inactivation of genetic material.
2.7.1 Biological Inhibitory Substances Originally Present in Food

The stability of some foods against attack by microorganisms is due to the
presence of certain naturally occurring substances e.g. plants such as mustard,
horseradish, watercress, cabbage and other brassicas produce antimicrobial
isothiocyanates (mustard oils) (Fig. 2.2) and in Allium species (garlic, onions
and leeks) thiosulfinates such as allicin. Antimicrobials collectively known as
phytoalexins are produced by many plants in response to microbial invasion,
for example phaseollin an antifungal compound is produced in green beans.
Many natural constituents of plant tissues such as pigments, alkaloids and
resins also have antimicrobial properties. Benzoic and sorbic acids found in
cranberries and mountain ash berries respectively are commonly used in their
pure forms as food preservatives. The anthocyanins are a group of water-
soluble pigments, which occur naturally in fruits. The aglycone portion of
these compounds, the anthocyanidins, has antimicrobial powers against
several bacterial spp.
R-C=
R
Physical damage
S C
N-OSO3
Gluc.
+ Myrosinase
R-C=N-OSO3
SH
R-N=C=S
Isothiocyanate
−
CH2
HO −
CH2
N
White mustard Cabbage
Figure 2.2: Production of plant antimicrobials as a result of physical damage
Some spices are known to contain essential oils that possess antimicrobial
activity e.g. eugenol in cloves, allicin in garlic, cinnamic aldehyde and
eugenol in cinnamon, allspice (pimento) and cloves, allyl isothiocyanate in
mustard, eugenol and thymol in sage and carvacrol (isothymol) and thymol in
oregano etc. As a consequence, herbs and spices may contribute to the
microbiological stability of foods in which they are used. It has, for example,
43
Introduction
been claimed that inclusion of cinnamon in raisin bread retards mould

spoilage. However, in some cases, they can be a source of microbial
contamination leading to spoilage or public health problems. Outbreaks of
botulism associated with crushed garlic in oil and home canned peppers
demonstrate that even in relatively high concentrations plant antimicrobials
are not a complete guarantee of safety.
These antimicrobial components differ in their spectrum of activity and
potency. They are present at varying concentrations in the natural product and
are frequently at levels too low to have any effect. Humulones contained in the
hop resin impart the characteristic bitterness of the product but have also been
shown to possess activity against the common beer spoilage organisms, lactic
acid bacteria.
Antimicrobial oleuropein from green olives and its aglycone are also
inhibitory to lactic acid bacteria and if not removed at this early stage, they
would prevent the necessary fermentation occurring subsequently. The
hydroxycinnamic acid derivatives (coumaric, ferulic, caffeic and chlorogenic
acids) found in fruits, vegetables, tea, molasses and other plant sources all
show antibacterial and some antifungal activity.
Animal products too, have a range of non-specific antimicrobial constituents,
for example egg white or albumen possesses a variety of inhibitory
components (Table 2.4). Similar factors can also be found in milk, however, in
lower concentrations e.g. enzyme lysozyme which catalyses the hydrolysis of
glycosidic linkages in peptidoglycan. Destruction or weakening of this layer
causes the cell to rupture (lyse) under osmotic pressure. Lysozyme is most
active against gram- positive bacteria, where the peptidoglycan is more readily
accessible, but it can also kill gram- negatives if their protective outer
membrane is damaged in some way.
Table 2.4: Antimicrobial substances in egg and milk
Egg Milk
Ovotransferrin (conalbumin) Lactoferrin
Lysozyme Lysozyme
Avidin -
Ovoflavoprotein -
Ovomucoid and ovoinhibitor -
- Lactoperoxidase
- Immunoglobulin
Other components limit microbial growth by restricting the availability of key

nutrients. Ovotransferrin and conalbumin in egg white and lactoferrin in milk
are proteins that scavenge iron from the medium. Iron is an essential nutrient
for all bacteria. Infact, lysozyme with conalbumin provides fresh eggs with a
fairly efficient antimicrobial system. In addition, egg white has powerful
cofactor-binding proteins such as avidin and ovoflavoprotein, which remove
biotin and riboflavin restricting the growth of those bacteria for which they are
essential nutrients.
Cows’ milk contains several other antimicrobial substances including
conglutinin, lactenins, anticoliform factor and the lactoperoxidase system.
44
Factors affecting
Casein as well as some free fatty acids that occur in milk have also been of Microorganisms in
Food
shown to be antimicrobial.
2.7.2 Inhibitory Substances Developed/ Destroyed in Food due to the

Activity of Microorganisms
Microorganism growing in food may produce one or more substances
inhibitory to other organisms, products such as acids, alcohols, peroxides, or
even antibiotics. Propionic acid produced by the propionibacteria in Swiss
cheese is inhibitory to molds; alcohol formed in quantity by wine yeasts
inhibits competitors; and nisin a polypeptide produced by Streptococcus lactis
may be useful in inhibiting lactate fermenting, gas-forming clostridia during
curing of cheese. These may however, be undesirable during the
manufacturing process since these would slow down some of the essential
lactic acid streptococci. Streptococcus cremoris produces an inhibitor named
diplococcin. The most pathogenic member of genus - S. pyogenes forms an
inhibitor, streptococcin A-FF22. Streptococcin A-PF22 had many properties in
common with nisin.
Gram-negative organisms and molds are insensitive to nisin. However, its
effectiveness against sensitive gram-positive organisms depends on the
bacterial load. As the number of organisms increases, the inhibitory
effectiveness of nisin decreases. Nisin can be used along with heat processing
since heat treated spores become more nisin sensitive. Thus, sterility might be
attained with less heat treatment than presently used thereby decreasing the
fuel consumption.
In addition to inhibitory polypeptides and bacteriocins, lactic streptococci
produce acids and peroxides. These add up to a formidable array of substances
designed to hinder and suppress other microbes. Hence, lactic acid bacteria are
excellent competitors in foods. S. diacetylactis produces inhibitor inhibiting a
broad-spectrum of gram-positive and gram-negative organisms. The
undissociated molecule is the toxic component.
There is also the possibility of the destruction of inhibitory compounds in
foods by microorganisms. Certain molds and bacteria are able to destroy some
of the phenolic compounds that are added to meat or fish by smoking or
benzoic acid added to foods; yeasts resistant to it destroy sulfur dioxide; and
lactobacilli can inactivate nisin.
2.7.3 Inhibitory Substances Developed During Processing of Food

Heating foods may result in the formation of inhibitory substances e.g. heating
lipids may hasten autoxidation and make them inhibitory and browning
concentrated sugar syrups may result in production of furfural and
hydroxymethylfurfural, which are inhibitory to fermenting organisms. Milk
also has the capacity to generate antimicrobials in the presence of hydrogen
peroxide. The milk enzyme lactoperoxidase will catalyse the oxidation of
thiocyanate by hydrogen peroxide to produce inter alia hypothiocyanate. This
can kill gram-negative bacteria and inhibit gram-positives, possibly by
damaging the bacterial cytoplasmic membrane.
1. Enlist the various mechanisms of action of inhibitory substances.
45
Introduction
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2. Elaborate on the inhibitory substances naturally present in plants with
suitable examples.
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3. Enlist all the antimicrobial constituents present in egg and milk.
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4. What is the mode of action of lysozyme?
………………………………………………………………………………
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46
Factors affecting
Food
5. Give examples of inhibitory substances developed in food due to the
activity of microorganisms.
………………………………………………………………………………
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2.8 LET US SUM UP

Food is the substrate for growth of microorganisms. Since our foods are of
plant and animal origin, all the characters of plant and animal tissues that
affect the growth of microorganisms are important. In other words, food will
dictate what grows or does not grow on it. Thus, knowledge about the factors
that favour or inhibit growth of microorganisms is a must to make predictions
about the microflora that may develop. The main factors, which influence
microbial activity are pH, water activity, redox potential, nutrient
composition, biological structure and presence of inhibitory substances in
foods.
Each microorganism has a minimal, maximal and optimal range for pH, water
activity and redox potential at which they can grow. These in turn would
determine the microflora of food. Buffers in food and poising capacity of food
also play an important role in deciding the succession of microorganisms and
the extent of spoilage occurring in food.
Different microorganisms have different nutritional requirements. Some have
a wide range while others are very fastidious in their nutritional requirements.
The inability of an organism to utilize a component of food limits its growth
and others with not so stringent requirements gain a competitive edge over it
and predominate, thereby typifying the natural microflora of that food.
Plants and animals have also evolved mechanism of defense against invasion
and proliferation of microorganisms. Antimicrobial barriers allow food to
remain relatively free from microorganisms and the other inhibitory
substances present in food/ produced during invasion by microorganisms tend
to maintain low microbial counts in food.
These six parameters represent nature’s way of preserving plant and animal
tissue from microorganisms. All these parameters have an important role to
play in determining microbial ecology of food. These in turn decide the type
of microbial activities likely to occur and the type of spoilage occurring. All
these factors are interlinked and the changes in one factor may affect
microbial requirements. By taking these natural phenomena into account, one
can make effective use of each or all in preventing or retarding microbial
spoilage of the products that are derived from them.
2.9 KEY WORDS

Aerobic : microorganisms are those, which require free
oxygen for growth.
47
Introduction
Anaerobic : microorganisms are those, which grow best in

the absence of free oxygen.
Antibiotics : are substances produced by microorganisms
which inhibit the growth of other
microorganisms.
Antimicrobials : are substances which inhibit the growth of
microorganisms.
Bacteriocins : are substances produced by a strain of bacterial
spp. which inhibit growth of other strains of that
bacterial spp.
Buffers : are the compounds that resist changes in pH.
Facultative : microorganisms are those, which grow well
either aerobically or anaerobically.
Halotolerant : are those microorganisms, which are able to
grow in the presence of high concentrations of
salt.
Osmotolerant : are those microorganisms, which are able to
grow in the presence of high concentrations of
unionized organic compounds such as sugars.
Oxidising agent : is a substance that readily takes up electrons.
Phytoalexins : are a class of antimicrobials which are produced
by many plants in response to microbial
invasion.
Poising capacity : is the ability of food to resist change in a food's
redox potential.
Proteolytic
microorganisms : are those, which are able to hydrolyze proteins.
Redox potential : of a substrate may be defined generally as the
ease with which the substrate loses or gains
electrons.
Reducing agent : is a substance that readily gives up electrons.
Standard redox
potential, Eo' : is the tendency of an atom or molecule to accept
or donate electrons.
Water requirement
or water activity aw : is the vapour pressure of the solution (of solutes
in water in most foods) divided by the vapour
pressure of the solvent (usually water).
Xerotolerant : are those microorganisms which are able to grow
on dry foods.
2.10 ANSWERS TO CHECK YOUR PROGRESS

EXCERCISES
"
48
Factors affecting
1. Fish is spoilt more rapidly than meat under chilled conditions. The pH of of Microorganisms in
Food
post-rigor mammalian muscle, around 5.6, is lower than that of fish (6.2-
6.5) and this contributes to the longer storage life of meat. Those fishes
that have a naturally low pH such as halibut (pH~5.6) have better keeping
qualities than other fishes.
2. Your answer should include the following points:
• pH attained upon rigor mortis of well rested animals
• not much change in pH change in case of fatigued animal
3. Biological acidity is the acidity developed in food due to the activity of
microorganisms growing in it. This generally occurs due to accumulation
of lactic acid during fermentation.
• partial dissociation of weak acids
• free passage of undissociated lipophilic acids into the cytoplasm
• dissociation of lipophilic acids in the microbial cell
• maintenance of internal pH by expulsion of protons
5. When microorganisms are grown on either side of their optimum pH
range, an increased lag phase results. Adverse pH affects at least two
aspects of a respiring microbial cell: the functioning of its enzymes and the
transport of nutrients into the cell.

1. Your answer should include the Halotolerant, Xerotolerant and
Osmotolerant microorganisms.
2. Water activity aw, is the vapour pressure of the solution (of solutes in water
in most foods) divided by the vapour pressure of the solvent (usually
water).
• Solutes and ions tie up water in solution
• Hydrophilic colloids (gels) make water unavailable
• Water of crystallization or hydration is usually unavailable to
microorganisms
• Kind of solute employed to reduce the aw
• Nutritive value of the culture medium
• Temperature
• Oxygen supply
• pH
• Inhibitors
49
Introduction
1. The tendency of a substrate to accept or donate electrons, to oxidize or

reduce, is termed its redox potential (Eh).
2. Your answer should include aerobic, anaerobic and facultative
microorganisms.
3. Poising capacity is the resistance to change in a food's redox potential with
change in the redox conditions.

• Heating destroys/ alters reducing and oxidizing substances
• Processing removes reducing and oxidizing substances
5. Factors affecting redox potential of foods are:
• Redox couples present
• Ratio of oxidant to reductant
• pH
• Poising capacity
• Availability of oxygen
• Microbial activity
• Concentration of sugars in food and their osmotic effect
• Molds grow at high concentration of sugars
• Osmophilic bacteria and yeasts grow at high concentration of sugars
• Type of physical barriers about food
• These barriers provide inhospitable environment for the microbe
• They determine type and rate of spoilage
• Physical damage allows microbial invasion into tissues
• It distributes spoilage microbes
• It releases juices from plant and animal tissues for microbial growth
1. The mechanism of action for nearly all antimicrobials can be classified
into one or more of the following groups:
a) Reaction with the cell membrane,
b) Inactivation of essential enzymes, or
50
Factors affecting
c) Destruction or functional inactivation of genetic material. of Microorganisms in
Food
• Isothiocyanates – mustard oil
• Thiosulfinates – in garlic, onions and leeks e.g. allium

• Antimicrobial substances present in egg
• Antimicrobial substances present in milk
• Lysozyme catalyses the hydrolysis of glycosidic linkages in
peptidoglycan
• Destruction leads to cell lysis
• Very active against gram positive bacteria
• Various types of inhibitory substances produced by microbes
• Inhibitory substances produced by propionibacteria
• Inhibitory substances produced by lactic streptococci

1. Adams, M.R. and Moss, M.O. (1996) Food Microbiology. New Age
International (P) Ltd., Publishers, New Delhi.
2. ICMSF (1980) ‘Microbial Ecology of Foods. Volume I. Factors affecting
life and death of microorganisms’, academic Press, New York, 332pp.
3. McMeekin, J.N., Olley, T. Ross and Ratkowsky, D.A. (1993) ‘Predictive
Microbiology: Theory and Application’, Research Studies Press Ltd.,
Tauton, England, 340pp.
4. Stanier, R.Y., Adelberg, E.A. and Ingraham, J. (1976) The Microbial
World. Prentice-HaIl, Inc., Englewood Cliffs, N.J.
51
Industrially Important
UNIT 3 INDUSTRIALLY IMPORTANT Yeast, Mold and
Bacteria
YEAST, MOLD AND BACTERIA
Structure
3.0 Objectives
3.1 Introduction
3.2 Culturing of Important Microorganism
3.3 Enzymes and Kinetics
3.4 Types of Fermentation
3.5 Types of Fomenters: Concept of Batch and Continuous Fermentation
3.6 Microbial Production and Recovery of Wine, Vinegar, Sauerkraut,
Ethyl Alcohol, Beer, Organic Acids
Wines
Beer
Vinegar
Lactic Acid Bacteria (LAB) and Fermented Foods
Ethanol Production
Enzyme Production
Citric Acid
3.7 Single Cell Proteins
3.8 Waste Water Treatment
3.9 Let Us Sum Up
3.10 Key Words
3.11 Answer to Check Your Progress Exercises
3.0 OBJECTIVES
• state the fermenter, types of microorganisms involved in fermentation and
their processes;
• explains the different products made by fermentation; and
• describe the waste from food processing industry and their utilization.
3.1 INTRODUCTION
Microorganism on one hand are responsible for causing a number of diseases,
on the other hand they are employed to produce a number of useful products.
These useful microorganisms include an array of yeasts, molds and bacteria.
Traditionally, the man prepared wine, curd, vinegar and pickles using
fermentation. Earlier, the term ‘Fermentation’ was used for the production of
wine but at present it encompasses the foods made by the application of
microorganisms including lactic acid bacteria (LAB). Lactic acid fermentation
is one of the oldest method of preserving fruits and vegetables. Apart from
contributing certain desirable physical and flavour characteristics, it also
prolongs the availability and processing period of the products at
relatively low cost. Many of the fermented products are made at industrial
scale making use of microorganisms. Some of the fermented products and
industrially important microorganisms are listed in Table 3.1.
53
Introduction Table 3.1: Production/synthesis of various compounds by micro-organisms
Product Micro-organism(s) involved
Alcoholic beverages & related products
Beer Saccharomyces cerevisiae; S. carlsbergensis

Bourbon whiskey S. cerevisiae
Cider S. cidri
Palm wine Acetobacter spp.; yeasts
Sake Aspergillus oryzae; Lactobacillus spp.;
Leuconostoc spp.; S. cerevisiae
Scotch whiskey S. cerevisiae
Thumba Endomycopsis fibuliges
Tibi Betabacterium vermiforme; S. intermidium.
Vinegar Acetobacter spp.
Wines S. cerevisiae var. ellispoideus
Breads
Idli Leuconostoc mesenteroids
Rolls, cakes etc. S. cerevisiae
Colour
β-carotene Blakeslea trispora; Rhodotorula spp.
Astaxanthine Phiffia rhodozyma
Dairy products
Acidophillus milk Lactobacillus acidophilus
Bulgarian milk L. bulgaricus
Cheeses (brie, Streptococcus lactis; S. cremoris: camembert,
cheddar, S. durans; Penicillium camembertii:
edam, P. candidum; P. roquefortii; Lactobacillus
caseiroqueforte)
Kefir Streptococcus lactis; L. bulgaricus; torula spp.
Kumiss L. bulgaricus; L. leicuhmannii; Torula spp.
Yoghurt L. bulgaricus
Enzymes
Amylases Bacillus spp.; Aspergillus niger; A. oryzae.
Cellulases Trichoderma reesei.
Glucose oxidases Corynebacterium spp.
& catalase
Invertase S. cerevisiae
Lipase Saccharomycopsis lipolytica.
Pectinases Aspergillus spp.
Proteases B. licheniformis; B. subtilis; Aspergillus spp.; S.
cerevisiae
54
Meat and fishery products
Yeast, Mold and
Country cured hams Aspergillus; Penicillium spp. Bacteria
Dry sausages Pediococcus cerevisiae
Microbial cells as fermented products

Bakers’ yeast Saccharomyces cerevisiae
Single cell Candida utilis; C. arborea; protein (SCP)
Methylophilus methylotrophus; Saccharomycopsis
lipolytica; Spirulina
Mushrooms Agaricus bisporus; Morchella hortensis
Non-beverage plant products

Miso Aspergillus oryzae
Saurkraut Neurospora sitophila
Sufu L. delbrueckii
Tempeh A. oryzae; Rhizopus oligosporus; R. oryzae
Organic acids
Acetic acid Acetobacter aceti; C. aceticum
Citric acid Aspergillus niger; Saccharomycopsis lipolytica
Lactic Acid Lactobacillus delbrueckii
Polysaccharides
Alginate Azotobacter vinelandii; Pseudomonas aeruginosa
Dextrans Leuconostoc mesenteroids; Klebsiella;
Acetobacter
Pullulan Aureobasidium; Pullularia spp.
Vitamins and Amino acids

Riboflavin Eremothecium ashbyi
Vit. B-12 Bacillus megaterium; Streptomyces olivaceus,
Propionibacterium
Pro-Vit. A. Rhodotorula gravillis.
3.2 CULTURING OF IMPORTANT MICROORGANISM

The human food supply consists basically of plants and animals or products
derived from them, so our food supply can contain microorganisms in
interaction with the food. The interactions between microorganisms and our
food is beneficial as exemplified by many cultured products developed by
fermentation and are consumed and enjoyed by many people (Bread, bear,
wine etc). To produce such products microorganisms are added as pure culture
or mixed cultures. However, in some cases no cultures may be added if the
desired microorganisms are known to be present in sufficient numbers in the
original raw material.
Starter culture, pure as well as mixed are usually employed in the manufacture
of certain fermented food and dairy products. Cultures for food fermentations
are selected primarily on the basis of their stability and their ability to produce
desired products. Mother culture is usually prepared daily from a previous
55
Introduction mother culture and originally from the stock culture. The mother cultures can
be used to inoculate a large quality of culture medium to produce the mass or
bulk culture to be used in the fermentation process.
Bacterial cultures: Most of the bacterial cultures employed as starters are for
dairy products. Sausage and bread also use pure or mixed cultures of lactic
acid bacteria (LAB) e.g. Streptococcus lactis sub sp lactis, S. mesenteroides
sub-sp. cremoris etc.
Yeast cultures: Most yeasts of industrial importance are of the genus
Saccharomyces. It is used to manufacture wine, beer and other alcoholic
products.
Bakers’ yeast: Yeast for baker’s yeast production: Strains of S.cerevisiae,
S.uvarum are used.
Wine yeast: S. cerevisiae var. ellipsoidus (Plate 1.1)
Plate 1.1: Photo micrograph of yeast cells showing budding (inset yeast cells are mating)
Distillers yeast: High alcohol yielding strains of S.cerevisiae var ellipsoidus.

Mold cultures: Stock of cultures of molds usually are carried in slants of a
suitable agar medium and may be preserved as spore stab for a long period by
freeze drying (Penicillium roquefortii).
3.3 ENZYMES AND KINETICS

Enzymes: Enzymes are biological catalysts possessing extraordinary
efficiency, specificity and are mostly protein in nature. Enzyme commission
has classified various enzymes on the basis of the type of the reactions
catalysed. All the enzymes have been classified into 6 classes.
Class 1 : Oxido-reductase
Class 2 : Transferase
Class 3 : Hydrolase
Class 4 : Lyase
Class 5 : Isomerase
56 Class 6 : Ligase
Properties of Enzymes
Yeast, Mold and
• All enzymes are protein in nature except nucleases. Bacteria
• All enzymes are specific in their functions.

• Enzymes are sensitive to temperature i.e. they are functional at optimal
temperature.
• These are destroyed at higher temperature.
• Enzymes are not destroyed during their use.
Kinetics of Enzyme Reactions
The studies on the kinetics of enzyme reactions must be based on quantitative
measurements of the rate of the catalyzed reactions. Main factors which
influence the kinetics of enzymatic reactions are as follow:
Enzyme concentration: The velocity of enzymatic reactions is directly
proportional to the concentration of the enzyme proteins.
Substrate concentration: When the velocity of the reaction is plotted vs.
substrate concentration, classical enzymes give a rectangular curve.
pH: Almost every enzyme exhibits maximum activity at a particular pH which
is called optimum pH.
Temperature: Almost every enzyme exhibits an optional temperature at
which the enzyme exhibits maximal activity. A graph of enzyme velocity
versus temperature, is a bell shaped curve.
Role of enzymes in food processing: The enzymes play a significant role in
food processing. Pectinase enzymes are used in juice clarification (apple juice,
guava juice), in softening of fruit (apple, tomatoes, peaches, avocadoes), and
thereby resulting in increase in yield of juices and pulps extraction of juice
from fruits. Proteases (papain) results in clarification and removal of
cloudiness in beer and wine. Glucose oxidase enzyme is used in removal of
glucose from egg white and thereby, improve, the colour of dehydrated egg
powder. Pectinase with cellulase has been employed for extraction of oil from
oil containing fruits (olive). Enzyme diastase converts starch to sugar during
beer preparation.
3.4 TYPES OF FERMENTATION

Fermentations have been classified on the basis of relationship of the formation
of product, substrate utilization or the free amount of water as given below:
Solid state fermentation: Fermentation processes which take place in the
absence or near absence of free water in the substrate are termed as solid state
fermentation (SSF). It is imperative, however, that the substrate contain
enough moisture absorbed in the substrate particles within the substrate. SSF
have been used mostly for food fermentation and production of a few enzymes.
Extractive fermentation: There are several industrially important products
being catalysed by enzymes which are susceptible to end product or feedback
inhibition. Hence, the increased concentration of the product inhibits the
enzymes involved in its own synthesis so that the overall rate of conversion of
substrate to the desired product is lowered. When the end product or anyone of
57
Introduction the by-product of fermentation interacts with the enzyme, the synthesis of the
final product proceeds sub-optimally and in extreme case may stop altogether.
This problem has largely been overcome by using a technique called extractive
fermentation. In it there is fast removal of product, or by-product of a
metabolic pathway, so that their subsequent interference with the cellular or
medium component is not possible. Hence, it involves all the actions taken for
the separation of a product from its producing cell. Separation of the product
can be achieved either inside the reactor (internal) or outside the reactor
(external).
Submerged fermentation: Fermentation processes which take place in the
presence of free water in the substrate are termed as sub-merged fermentation.
Such fermentations have been used mostly to the produce fermented food and
beverages.
3.5 TYPE OF FERMENTER, CONCEPT OF BATCH AND

CONTINUOUS FERMENTATION
Fermenter: The industrial usage of micro-organisms often requires that they
be grown in large vessels containing considerable quantities of nutritive media.
These vessels are commonly called fermenters. Therefore, fermenter is the
basic equipment of fermentation.
Types of fermenters: Some of the types of fermenter are listed below:
1. Shake flasks and bottles
2. Stirred tanks
3. Air-lift fermenters
4. Tower fermenter
5. Rotating disc fermenter
6. Fixed bed fermenter
7. Fluidized bed fermenter
Batch fermentation: In this fermentation, starter culture is added to the
medium and the product is withdrawn only after completion of fermentation.
Continuous fermentation: In this fermentation, the substrate is continuously
fed to the fermenter and the product is also withdrawn continuously.
3.6 MICROBIAL PRODUCTION OF WINE, VINEGAR,

SAUERKRAUT, ETHYL ALCOHOL, BEER,
ORGANIC ACID
3.6.1 Wines
The term ‘wine’ is applied to a beverage made by alcoholic fermentation of
grape or grape juice and final production is obtained without distillation. But
now-a-days, any fleshy fruit or flower in the new world may be employed for
this purpose. Wine was suggested to have been made during the Neolithic
period in the near East. These are the part of food of man ever since his
settlement in Tigoris Euphorates basins and have also been used as a
therapeutic agent.
58
Wines are produced by the fermentation of juices/extracts of many fruits such Industrially Important
Yeast, Mold and
as apple, pear, cherries, most of berries, rhubarb, dandelion, honey, besides Bacteria
bananas, pineapple, cashew nut, pomegranate, lemons, tangerines, oranges,
dates and figs. Wines from grapes are classified basically into red and white
wines.
Types of Wines
Still wines: These wines retain none of the carbon dioxide produced during the
fermentation.
Sparkling wines: These are the wines which have considerable amount of
carbon dioxide. Champagne in France is the sparkling wine made in
Champagne region.
Dry wines: These wines contain little or no unfermented sugar.
Sweet wines: Wines having either unfermented sugar or with added sugar later
on are called sweet wines. Both types of wines generally contain 11 to 14% of
alcohol.
Fortified wines: Wines to which distillate of wine called “Brandy” is added
and may contain 15 to 21% of alcohol.
Table wines: It is a wine having comparatively low alcohol content (7 to
11%) and little or no sugar.
Sherry: It is produced by special processing technique from wine, containing
18 to 21% alcohol and could be sweet or dry.
Cider: Cider is a low alcoholic beverage obtained from apple by fermentation.
Perry: It is a wine made from pear juice.
Mead: This type of wine was prepared by the Indians from honey.
Vermouth: Wine flavoured with a characteristic mixture of herbs and spices,
some of which impart an aromatic flavour and odour while others a bitter
flavour. It can be sweet or dry with alcohol content of 15 to 21%.
Toddy: Sweet alcoholic drink, having alcohol content of 4-6%, is made by the
fermentation of sap from coconut palm.
Pulque: National drink of Mexico, contains 6-7% alcohol and B-vitamins.
Method of Table Wine Preparation
Grape is the most widely used fruit to make wine but it can be prepared from
any fruit having fermentable sugars, optimum acidity, nitrogenous compounds
or other growth factors to make wine of acceptable quality. The major
difference is in the extraction of sugar from the pulp of some fruits. From
grape, red and white wines are produced the world over using black/red
coloured and white varieties, respectively. The generalized flow sheets for
wine making from grapes is shown in Figure 3.1.
59
Introduction White Wine production Red Wine production
White -grapes Red -grapes
SO2 De-stemming/crushing De-stemming/crushing SO2
Settling Maceration of juice + grape skins
Addition of sugar,
nitrogen source and pectinase
where needed
Pomace Pressing Must
Inoculation with
yeast selected
Addition of sugar , Fermentation of

nitrogen source macerated contents
and pectinase
where needed
Inoculation Must
with selected
yeast Fermentation Pressing
Pomace
Racking
Racking
Maturation Maturation
Fining & Clarification Fining & Clarification
Filtration Filtration
Bottling Bottling
White Wine Red Wine
Figure 3.1: Flow–sheet of process to make red and white wine
60
Preparation of must: For wine preparation, the first step is the preparation of Industrially Important
Yeast, Mold and
must which is prepared depending upon the type of fruits used and the type of Bacteria
wine to be made. Must is a juice or pulp corrected for sugar, acid/pH, nitrogen
source or other requirements for the alcoholic fermentation. To prepare the
must, the fruits are trimmed and washed and the must Juice is extracted or fruit
is made into pulp. In the preparation of white wine only the free run juice is
used while in the red wine, the skin and seeds along with pulp/juice are
fermented together for some time to get attractive coloured wine. Proper
dilution of fruit pulp is required as fruits like plum and apricot are highly
acidic and effect the fermentability besides making the wine unpalatable. The
sugar content of the juice or pulp is checked with an instrument called
refractometer and is expressed as degree Brix. Sulphur dioxide (S02) is added
to the must to control the wild microflora and to allow the yeast to act
efficiently to conduct the alcoholic fermentation. Amelioration (or correction)
of must for better fermentability with ammonium salt and vitamins like
thiamine, biotin is necessary in some fruits.
Preparation of active yeast culture: An active culture of wine yeast
(Saccharomyces cerevisiae var ellipsoideus) is prepared from the stock culture
in the juice to be used for wine making.
Fermentation: After must preparation, activated yeast starter culture is added
to the must and fermentation is carried out at a temperature of 20-25oC, till the
sugar content or the oBrix stabilizes.
Siphoning/racking
Siphoning or racking is a simple but important process wherein the wine is
transferred through a clean pipe into another container, kept at a lower height
than the vessel with wine. It is done after completion of fermentation. Two or
three rackings are usually done at an interval of 15-20 days to separate the
yeast and other settled materials at the bottoms of container.
Maturation
As the newly made wine is harsh and has yeasty flavour maturation (from
6 months upto a year) is allowed to make the wine mellow (It is the term used
to signify the sensory quality of wine having smoothness i.e. is devoid of any
harsh taste)in taste and fruity in flavour.
Clarification: Clarification of wine is done by using filter aids such as
bentonite, celite and tannin/gelatin using a machine called filter press.
Blending: Blending is also practiced in some cases to make wine sweet or
better flavoured before pasteurization.
Pasteurization: Wine is generally pasteurized at a temperature of 620C for 15-
20 min, after bottling.
Storage: Low temperature storage is preferred for good quality wine.
3.6.2 Beer
Beer is an alcoholic beverage primarily prepared from barley besides other
cereals in limited quantities and is consumed in large quantities throughout the
world. Beer and ale the principal malt beverages made with hops, yeast, water
and malt adjuncts. Adjuncts are the malted cereals other than barley, used in
61
Introduction minor quantities. Brewing was one of the earliest processes undertaken on a
commercial scale and became one of the first process that has developed from
an art into a technology. Beer can be differentiated from ale as in beer bottom
fermenting yeast is employed while in ale the top fermenting yeast is
employed. In the preparation of ale, more hops is used. It is usually pale yellow
in colour, tart in taste and have more alcohol content. On the basis of alcohol
content beers can be classified as light beer having 3-5% v/v and hard beer
having 5-8% alcohol content. Beer production is divided into four distinct
process as described here.
Malting: It is obtained by soaking followed by germination of barley or other
cereals and drying of the germinated cereal. Then, most of sprouts or germs are
removed and the malt remains. The malt is crushed before its use in beer
making.
Mashing: It is the process in which extraction of the ground malted barley
with water is made. The mashing is done so as to make soluble as much as
possible of the valuable constituents of the malt and malt adjuncts. It causes
hydrolysis of starches, other polysaccharides and proteins. The insoluble
material is then filtered. The liquid so obtained is called wort.
Wort boiling: Boiling of wort with hops (Hops is the female flowers of hops
plant used in beer production to give flavour and bitter taste) is carried out to
concentate the wort, inactivate the enzymes, extract soluble substances from
the hops,coagulate and precipitate the proteins and other substances,
caramelize sugar slightly and to contribute antiseptic substances (Chiefly the
alpha resins humulone, co-humulone and adhumulone) to the wort and beer.
Fermentation: A special beer, bottom fermenting yeast strain Saccharomyces
cerevisiae var carlbergensis, is used for the inoculation or pitching of the
cooled wort. The wort temperature during the fermentation varies in different
breweries but is usually in the range from 3.3 to 14oC. The fermentation is
usually completed within 8 to 14 days. During fermentation as the carbon
dioxide is evolved in increasing amounts, the foaming increases; later it
decreases to none when the fermentation is finished. At the later stage, the
bottom yeast flocculates and settles down.
Aging or Maturation: The young, green or draft beer is stored or lagered in
vats at about OoC for several weeks to several months, during which period
precipitation of proteins, settling of yeast, resin and other undesirable
substances takes place and the beer becomes clear and mellowed or matured.
Finishing: After aging, the lager beer is carbonated to a CO2 content of about
0.45 to 0.52 per cent, mostly by means of gas collected during the fermentation
or by addition of CO2 from cylinders. Then, beer is cooled, clarified or filtered
and packaged in the bottles, cans or barrels.
3.6.3 Vinegar
The word vinegar is derived from two French words, vin and aigre meaning
sour wine but the term is used to denote a condiment prepared from various
sugar and starch containing materials by alcoholic and subsequent, acetic acid
fermentation. It is one of the several fermented foods prepared and consumed
by early man, even today. Earlier, it was used as a beverage, a condiment, a
preservative, a household cleansing and medicinal agent. Vinegar mainly
consists of a dilute solution of acetic acid in water, also contains colour,
62
flavour and extracted substances besides fruit acids, esters and inorganic salts Industrially Important
Yeast, Mold and
which vary according to its origin. The minimum legal strength for vinegar is Bacteria
4% acetic acid (w/v).
Types and Composition of Vinegar
1. Synthetic vinegar: This type of vinegar is directly prepared from synthetic
acetic acid with the addition of water and finally, it is coloured by caramel.
2. Brewed vinegar: Virtually, anything having enough sugar to produce
alcohol can be used to make brewed vinegar. The vinegar usually derives
its descriptive name from the material from which it is made such as: cider
vinegar is made from apple juice, alegar from ale, malt vinegar from
malted grains spirit vinegar from alcohol etc.
Vinegar Preparation
It involves two step fermentations as detailed below:
Alcoholic fermentation: The first is alcoholic fermentation, mainly carried
out by yeast Saccharomyces cerevisiae either by pure culture inoculation or
by the natural process of fermentation. The process can be represented by a
simplified equation:
C6H12O6 → 2CO2 + 2C2H5 – OH + 55 Kcal
Glucose Carbon dioxide Ethyl alcohol
In the process, ethyl alcohol is not the only product but small amounts of
other compounds like glycerol, succinic acid, amyl alcohol, propyl alcohol
etc. are also produced in this fermentation. The fermentation is anaerobic.
Acetous fermentation: The second fermentation is acetic acid fermentation. It
is an oxidative fermentation carried out by acetic acid bacteria like Acetobacter
aceti. In the vinegar production, pure culture of acetic acid bacteria is not
used, due to more efficiency of mixed cultures. The oxidation reaction can
be shown as:
C2H5OH + O2 → CH3COOH + H2O + 116 Kcal
Ethyl alcohol Acetic acid
The optimum temperature of fermentation is 26oC which is achieved by the
heat generated in the process.
Process of Vinegar Preparation
Slow process: This process takes a long period and is generally followed in
countries like India. The juice kept in the barrels is allowed to undergo
alcoholic and acetic fermentations slowly with the passage of time. The bung
hole of the barrel is covered with a piece of cloth to screen-off the dust and
flies, and the barrel is placed in a damp but warm place. It takes about 5-6
months to complete the whole alcoholic and acetous fermentation to produce
the vinegar from the juice. The main drawbacks of this process are: alcoholic
fermentation is often incomplete, the acetic fermentation is very slow and the
yield is low coupled with an inferior quality vinegar.
63
Introduction Quick process: In the quick process like generator process alcoholic liquid is
in motion and this process is applied mostly to the production of vinegar from
spirit (alcohol). Fruit or malt liquors are well supplemented with food for the
vinegar bacteria, but to maintain active vinegar bacteria in generator methods
using alcohol denatured with ethyl acetate or vinegar, it must be supplemented
with a combination of organic and inorganic compounds known as vinegar
food. Combinations of substances such as dibasic ammonium phosphate, urea,
peptones, yeast extract, glucose, malt, starch, dextrin, salts etc have been made.
Materials such as pumice, branches of vines and grape stems for packing the
generators are used. Schiizenbach introduced the use of a vat instead of cask
for the acetification process and provided mechanical means for the repeated
distribution of the acidic liquid over the packing.
Generator: The equipment used is known as “Upright Generator” which in its

simplest form is a cylindrical tank that comes in different sizes and is usually
made of wood. Its interior is divided into 3 parts:
i) Upper section: Here, alcoholic liquid is introduced.
ii) Large middle section: In this section, liquid is allowed to trickle down
over beech wood shavings, corn cobs, charcoal, coke, or some other
material that will provide a large total surface area yet not settled into a
compact mass.
iii) Bottom section: This section is for the vinegar collection.
The alcoholic liquid is put at the top through an automatic feed trough or a
sprinkling device (sparger) and trickled down over the shavings or other
material on which a slimy growth of acetic acid bacteria has been developed
and the bacteria oxidize the alcohol to acetic acid and the process is called
acetification. Air enters through the false bottom of the middle section and
after becoming warm, it is exhausted out through a ventilation above. As
considerable heat is released by oxidation process, it is necessary to control
the temperature below 30oC. It is usually done by using cooling coils, by
adjusting the rate of alcoholic liquid, feeding air and by cooling the
alcoholic liquid before it enters the generator or by cooling the partially
acetified liquid that is returned to the top from the bottom section of the tank
for further acetification.
3.6.4 Lactic Acid Bacteria (LAB) and Fermented Foods
Lactic acid bacteria (LAB) are obligate microorganisms producing lactate

from sugars as the main end product, besides producing inhibitory
substances like organic acids, bacteriocin, hydrogen peroxide which are
antagonistic towards other microorganisms. Fermented dairy products are
known to be inhibitory to both pathogenic and spoilage causing
microorganisms and Yoghurt is the best known fermented milk product (with
fruit pulp).Cultured milk and milk products contain lactic acid bacteria that
prevent the occurrence of stomach, colon and other cancers.
Traditional fermentations of vegetables were depended upon growth of

naturally occurring lactic acid bacteria to metabolise sugars in the vegetables to
mainly lactic acid and improve their taste and keeping quality. However,
starter cultures are being used now to develop controlled fermentation
64
It is established that more than one species of lactic acid bacteria are Industrially Important
Yeast, Mold and
responsible for vegetable fermentation. Lactic acid bacteria responsible for Bacteria
natural fermentation of vegetables are within the genera of Streptococcus,
Leuconostoc, Pediococcus and Lactobacillus. Acidity, pH, salt concentration,
temperature, naturally occurring inhibitors, chemical additives, exposed
brined surface to air and sunlight, amount of fermentable carbohydrates in
the vegetables and availability of nutrients in the brine are important
factors affecting the lactic fermentation.
Sauerkraut
It is the clean, sound product of characteristic flavour, obtained by full

fermentation, chiefly lactic of properly prepared and shredded cabbage in the
presence of not less than 2% nor more than 3% of salt. It contains, upon
completion of the fermentation not less than 1.5 per cent of acid expressed as
lactic acid. To prepare sauerkraut rough outer leaves of fully mature solid
cabbage heads are removed. Head are quartered, the cores are removed and
then, shredded the quarters into thin strips which are mixed with salt. About
2.25 to 2.5% of salt by weight should be added to the shredded cabbage to
obtain kraut of the best quality. Pack the cabbage loosely in a jar, place a
wooden board on the top. In order to press out juice from the cabbage, a heavy
stone is placed on the wooden board. The jar is kept at a warm place (24 to
31oC) for 8 to 12 days to allow fermentation to complete. The brine is
separated from the cabbage, boiled and poured hot over the cabbage shreds in
the jars. Sauerkraut can be packed in cans also. The cans are filled with the hot
juice, exhausted and processed till the temperature at the centre of can reaches
82oC.
Prominent bacteria that attain appreciable number early in fermentation are

Enterobacter cloacea and Erwinia herbicola and contribute some flavour.
However, Leuconostoc mesenteroides bacteria begins to outgrow all
organisms and continue acid production upto 0.7 to 1% (as lactic acid). Next,
Lactobacillus plantarum, a non-gas forming lactobacilli continues the
production of acid and can raise the acidity to 1.5 to 2.0%. These bacteria
produce chiefly lactic acid in their fermentation of sugars. A final acidity of
1.7% as lactic acid is most desirable and fermentation can be stopped at this
stage by canning or refrigerating the sauerkraut.
Kanji
Carrots of deep purple variety are fermented in Northern India and Pakistan to
make a ready-to-serve beverage /drink called as Kanji. It is a popular beverage
and is considered to have cooling and smoothing properties besides nutritional
content. To prepare it, the carrots are washed, grated finally. For every Kg of
grated carrot, 7Kg of water, 200g of salt, 40 g of crushed mustard seeds and
8g of hot chillies are added followed by placing the mixture in a glazed
earthenware, leaving a tiny whole for the release of gases produced during
fermentation. The mixture is fermented for 7-10 days. It is strained through a
muslin cloth. The final product is acidic in taste with an attractive purple red
colour and is usually consumed within 3-4 days.
65
Introduction Pickles from Vegetables
Vegetables like cucumber are pickled whole or in slices after washing in

potable water. For every one Kg of cucumber, 15g salt is added which results
in the formation of brine. It is followed by lactic acid fermentation. Depending
upon the ambient temperature it takes one to four weeks. The fermented
cucumbers are stored in clean capped jars after pasteurization.
Radish can also be pickled in a manner similar to sauerkraut as discussed

earlier.
Kimchi
It is a fermented food of Korea with cabbage or radish as the main ingredient.

Cucumbers can also be added. Cabbages are cut and brined in 5 to 7% salt
solution for 12 hr or in 15% brine for 3 to 7 hr. Then, brined cabbage is rinsed
and mixed with 10% seasoning ingredients i.e. garlic, green onions, peppers,
ginger, mustard, parseley, sesame grains and fermented shrimp. This mixture is
allowed to ferment in jars which takes a few days at temperature of more than
20oC for a month below 10oC. ‘Kimchi’ has a pH value of 4-4.5 and lactic acid
content of 0.4 to 0.8%. The main organisms responsible for fermentation of
‘kimchi’ are Leuconostoc mesenteroides and acidifying microorganism is
Lactobacillus plantarum.
3.6.5 Ethanol Production
The material rich in sugar can be converted into ethanol. The fermentation is
carried out using yeast like Saccharomyces cerevisiae. The sugars like glucose
is converted into ethyl alcohol and carbon dioxide, anaerobically. Ethanol is a
liquid fuel or liquid fuel supplement and is used as a solvent in many
industries.
The waste from fruits and vegetable processing industries being rich in
polysaccharides (cellulose, hemicellulose and lignin) has been subjected to
SSF for the production of ethanol. The cellulose and hemicellulose present
in the processing waste like apple pomace are readily fermented by anaerobic
bacteria. For ethanol production, the waste from processing industries has to be
pre-treated due to presence of lignin. A SSF process has been used for
production of ethanol from apple pomace by using Saccharomyces cerevisiae.
Apple, pear, orange peel and cherry wastes have also been utilized for
production of ethanol by fermentation with Saccharomyces cerevisiae.
3.6.6 Enzyme Production
Both submerged fermentation (SF) and solid state fermentation (SSF) are
employed for production of enzymes. But SSF is a better method than SF for
production of enzymes. Various enzymes have been produced by fermenting
food processing waste. Invertase enzyme by fermenting sauerkraut waste
with the help of Canidida utilis has been produced. This enzyme is widely
used in the food processing industry. Subsequently, fungal amylase by using
baked bean waste has been produced. Enzymes like cellulase and xylanase are
produced by fermenting apple pomace, using Trichoderma viridae and
Aspergillus sp. Pectinase is another enzyme which is produced from wastes
like apple pomace.
66
Table 3.3: Food processing waste used as SCP/animal feed after microbial
Yeast, Mold and
fermentation Bacteria
Waste Microorganisms utilised
Apple pomace Saccharomyces cerevisiae

Candida utilis
Torula utilis
Aspergillus niger
Corn cob Aspergillus niger
Dried citrus peel Aspergillus niger
Fodder beets Saccharomyces cerevisiae
Orange peel and grape Pleurotus ostreatus

stalks Agrocybe aegirata
Armillarialla mellea
Sugarcane bagasse Polyporus sp.

Pleurotus
Trichoderma
Sugar beet pulp Trichoderma reesei

Tricoderma viridae
Fusarium oxysporum
3.6.7 Citric Acid

Citric acid is being produced by fermenting brewery waste with Aspergillus
niger. Apple pomace is a potential source of citric acid when fermented with
Aspergillus niger by SSF on various substrates like pineapple juice, molasses,
sweet potatoes residue, sugar cane bagasse impregnated with pineapple juice,
mandarin orange waste, apple pomace, grape pomace. While production of
citric acid by fermenting apple pomace, addition of methanol to the medium
increases the yield of citric acid.
3.7 SINGLE CELL PROTEINS

In developing countries like India, deficiency of proteins leads to mal-
nutrition. It has necessitated to explore new non-conventional resources of
protein production. Amongst the various processes used to supply protein are
those based on the microbial growth and microbial biomass especially using
the waste material (Table 3.3). Microbial cells used as proteins as single cell
protein (SCP) and can be used as protein supplement for feed or food. A
number of micro-organisms like yeast, fungi, algae and bacteria can be
employed production of SCP and each of them has its advantage and
disadvantages. The micro-organism in turn use these substances as starting
materials for fermentation and SCP production by assimilation.
The SCP however, is not without limitations also such as high nucleic acids
which are metabolized to uric acid and can give rise to articular gout in
human beings. Secondly, human being can eat a maximum of 2.0g SCP/kg
67
Introduction body weight/day in their diet. To overcome the nucleic acid levels in SCP
various methods have been tried but with a variable success. The success of
SCP depends upon economics of SCP production.
3.8 MICROBIAL FERMENTATION FOR UTILIZATION

OF WASTE
With the advent of post-harvest technology, the fruits and vegetables are
processed for the production of various products. The processing of fruits
and vegetables in this way generate a large quantity of bio-degradable waste.
The waste from processing of fruits and vegetables include water and various
organic substances e.g. simple and complex polysaccharides (Sugars, starch,
pectin, etc.), vitamins and minerals. The large quantities of waste generated
in this way leads to environmental pollution. In today’s environment conscious
society, there are regulatory laws for the discharge of industrial effluent under
the water conservation and control of Pollution Act 1974 and Environmental
Protection Act, 1986 and these are mandatory for the processing industries.
The waste from the processing industries can either be disposed-off after
necessary treatment as per the directions of the pollution control agencies or it
can be utilized by applying suitable technologies as illustrated in Table 3.4.
Table 3.4: Microbial utilization of food processing industry waste
Sl. Products Waste
1. Ethanol Citrus industry waste, apple pomace, peach waste,

cashew apple pomace, pineapple waste, pear cutting.
2. Biogas Waste from fruit and vegetable industry as a whole,
fermentation (wine and beer) waste.
3. Single Cell Apple pomace, peach waste, cashew apple proteins
pomace, citrus waste extract, molasses, potato peels,
cabbage waste.
4. Cider, beer Apple pomace.
and vinegar
5. Pectin Citrus waste, apple pomace.
6. Citric acid Apple pomace.
7. Baker’s yeasts/ Waste from wine, beer and distillary.
industrial yeast
8. Colour Apple pomace.
9. Flavours/Xanthan Fruits and vegetable waste, citrus waste
gum
10. Animal feed Apple pomace, peach waste, potato industry waste,
olive processing waste.
68
Check Your Progress Exercise 1 Yeast, Mold and
"
Bacteria
1. Define wine, toddy, vermouth, cider, beer and perry.
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2. What is lagering, pitching and draft beer?
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3. Name the yeast used in beer fermentation.
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4. What is role of boiling wort?
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69
Introduction 5. Give various steps for wine and beer production.
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6. What is vinegar and SCP?
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7. Name the microorganisms and their sequence in lactic acid fermentation of
cabbage.
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8. Name the processes used in vinegar preparation.
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9. Which is commercially available single cell protein source?
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70
………………………………………………………………………………. Industrially Important
Yeast, Mold and
………………………………………………………………………………. Bacteria
……………………………………………………………………………….
10. Classify enzymes and their role in industry?
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11. Differentiate SF and SSF fermentation.
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12. Name the microorganisms associated with production of following
products: Sauerkraut, Beer, Wine, Organic acid (acetic acid) and SCP.
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13. Define batch and continuous fermentation.
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71
Introduction 14. Why yeast is preferred as a single cell protein compared to bacteria and
algae?
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15. What are different types of fermenters? Enlist the same.
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16. Write 2-3 lines about the following:
Sauerkraut, Kimchi, vinegar
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3.9 LET US SUM UP

Fermentation is an ancient practice, carried out by natural or inoculated
microflora. Several microorganisms are important as these are employed to
produce the fermented food, additives and products of industrial significance.
These include yeast, bacteria and fungi. Fermentation could be classified as
solid state fermentation, submerged fermentation or extractive fermentation.
Depending upon the mode of operation fermentation could be batch, fed batch
or continuous type. At the industrial scale, the fermentationis carried outin the
vessel called fermentor or bio reactors. Depending upon the type of
fermentation or product, the type of fermentoris employed. Food fermentations
include those to produce wine, beer, brandy, whisky, pickles, sauerkraut,
kimchi, vinegar, yoghurt additives like citric acid, lactic acid, enzymes,
ethanol, single cell proteins being produced commercially using microbial
process.
72
3.10 KEY WORDS Yeast, Mold and
Bacteria
Fermentation : was used for the production of wine but at
present it encompasses the foods made by the
application of microorganisms including lactic
acid bacteria (LAB).
Bakers’ yeast : The strains of Saccharomyces uvarum used to
make bread.
Wine yeast : S. cerevisiae var. ellipsoidus.
Distillers yeast : High alcohol yielding strains of S. cerevisiae var
ellipsoidu used to higher alcoholic beverages.
Controlled “starter”
Culture : pure as well as mixed cultures of microorganisms
are responsible for conducting fermentations,
employed in the manufacture of certain food and
dairy products such as fermented milk, butter,
cheese, bread, malt beverages.
Enzymes : Enzymes are biological catalysts possessing
efficiency and specificity and are mostly protein
in nature.
Solid state
fermentation : Fermentation processes which take place in the
absence or near absence of free water in the
substrate are termed as solid state fermentation
(SSF).
Submerged
fermentation : Fermentation processes which take place in the
presence of free water in the substrate are termed
as sub-merged fermentation.
Fermenter : The industrial usage of micro-organisms often
requires that they be grown in large vessels
containing considerable quantities of nutritive
media. These vessels are commonly called
fermenteors.
Wine : The term ‘wine’ is applied to the product made
by the alcoholic fermentation of grape or grape
juice. But any fleshy fruit or flower in the new
world may be employed.
Beer : Beer is an alcoholic beverage prepared from
barely or other cereals.
Vinegar : The word vinegar is derived from two French
words, vin and aigre meaning sour wine but the
term vinegar is used to denote a condiment
prepared from various sugar and starch
containing materials by alcoholic and
subsequent, acetic fermentation.
73
Introduction Acetous fermentation : or acetic acid fermentation which is oxidative
fermentation carried out by acetic acid bacteria
viz. Acetobacter aceti.
Lactic acid bacteria
(LAB) : These are the microorganisms that are obligate
producing lactate from sugars as the main end
product, besides producing inhibitory substances
to other microorganisms.
Sauerkraut : It is the product of characteristic flavour,
obtained by lactic fermentation of cabbage in the
presence of 2-3% of salt.
Kimchi : It is a group of fermented vegetable foods of
Korea with cabbage or radish as the main
ingredient.
Single cell protein : The microbial biomass used as protein
supplement for feed or food is called as single
cell protein (SCP).

EXERCISES
Wine: It is the product made by the alcoholic fermentation of grape or
other fruit or flower.
Cider: Cider is a low alcoholic beverage from apple juice.
Vermouth: Wine flavoured with a characteristic mixture of herbs and
spices, with an aromatic flavour and odour while others a bitter flavour,
sweet or dry with alcohol content of 15 to 21%.
Toddy: Sweet alcoholic drink, having alcohol content of 4-6%, made by
the fermentation of sap from coconut palm.
Lagering: It is the process of storage of young, green or draft beer in vats
at about OoC for several weeks to several months, to make it clear and
mellow.
Pitching: It is the process of inoculation of yeast culture in the wort.
Draft beer: The freshly prepared beer (not matured) is called draft beer.
The answer is Saccharomyces cerevisiae var carlbergenesis.
The boiling concentrates, sterilizes the wort and inactivates the enzymes.
74
5. Your answer should include the following points: Industrially Important
Yeast, Mold and
Various steps are involved in beer production: Bacteria
Malting
Mashing
Wort boiling
Fermentation
Aging or Maturing
Finishing
Table wine
Juice Extraction
Must preparation
Fermentation
Siphoning/racking
Maturation
Clarification
Blending
Pasteurization
Vinegar: Vinegar is the product obtained by acetic acid fermentation of
ethanolic liquid of any fruit and contains 3.75 %w/w acetic acid. It is
prepared by alcoholic fermentation of fruit juice.
SCP: Single cell proteins (SCP) are the microbial cells used as a source of
proteins. To produce single cell proteins, suitable microorganism and
medium is selected. The optimum conditions of growth are also determined
and the same are given during the fermentation. The grown biomass is then
harvested and the cells are used as a source of proteins.
First of all Pediococus comes, then Streptococcus followed by
Lactobacillus in cabbage fermentation.
Slow process: This process takes long period wherein the juice is allowed
to undergo alcoholic acetic fermentations. It takes about 5-6 months to
complete the fermentation to form the vinegar from the juice.
Quick process: In quick processes like generator process alcoholic liquid
is kept in motion and this process is applied mostly to the production of
vinegar from spirit (alcohol). The alcoholic fermented liquors is well
supplemented with food for the vinegar bacteria, such as a combination of
organic and inorganic compounds. The process needs additional supply of
oxygen.
Spirullina which is used as a SCP commercially.
75
Introduction 10. Your answer should include the following points:
All the enzymes have been classified into six classes.
Class 1 – Oxido-reductase e.g. dehydrogenases, peroxidases.
Class 2 – Transferase
Class 3 – Hydrolase
Class 4 – Lyase
Class 5 – Isomerase
Class 6 – Ligase
Uses of enzymes in food processing: Pectinase enzyme are used in
clarification of juices (apple juice, guava juice), lemon juice etc. Pectinase
enzyme result in softening of apple fruit, tomatoes, peaches, avocadoes and
thereby resulted in increase in yield of juice and pulp during processing.
Pectinase enzyme result in easy extraction of juice from fruits. Proteases
resulted in clarification and removal of cloudiness in beer and wine.
Glucose oxidase enzyme is used in removal of glucose from egg white and
thereby, improve, the colour of dehydrated egg powder. Pectinase with
cellulase enzymes are also used for extraction of oil from oil containing
fruits. Beer cloudiness can be removed by use of proteases e.g. papain.
Enzyme diastase resulted in conversion of starch to sugars during beer
preparation.
The submerged fermentation (Smf) makes use of free liquid while in that
of solid state fermentation (SSF) no free water is available.
The microorganisms used for the products are listed as below:
Sauerkraut: Lactic acid bacteria
Beer: Saccharomyces cerevisiae var carbergenesis
Wine: Saccharomyces cerevisiae var ellipsoideus
Organic acid (Acetic Acid): Acetobacter aceti
SCP: Spirullina, Saccharomyces cerevisiae, Candida utilis
Batch fermentation: Here the starter culture is added to the medium and
the product is withdrawn after completion of fermentation.
Continuous fermentation: Here the substrate is continuously fed to the
fermenter and the product is also withdrawn continuously.
1. Yeasts have nutritive value especially proteins and vitamins.
2. Able to grow on a variety of carbon and nitrogen source.
76
3. Has faster growth and high yield, ability to grow at low pH. Industrially Important
Yeast, Mold and
4. Can grow on a large number of waste including that from processing Bacteria
industries.
Types of Fermentors:
1. Shake flasks and bottles
2. Stirred tanks
3. Airlift fermenters
4. Tower fermenter
5. Rotating disc fermenter
6. Fixed bed fermenter
7. Fluidized bed fermenter
Sauerkraut: It is the clean, sound product of characteristic flavour,
obtained by full fermentation, chiefly lactic of properly prepared and
shredded cabbage in the presence of not less than 2% nor more than 3%
of salt.
Kimchi: It is a group of fermented vegetable foods of Korea with cabbage
or radish as the main ingredient with or without cucumbers.
Vinegar: The word vinegar is derived from two French words,vin and
aigre meaning sour wine but the term vinegar is used to denote a
condiment prepared from various sugar and starch containing materials by
alcoholic and subsequent, acetic fermentation.

1. Green, J.H. and Kramer, A. (1979) Food Processing Waste Management
AVI Publishing Company Westport CT. p.663.
2. Joshi, V.K., Pandey, A. and Sandhu, D.K. (1998) Food Factory Waste
Management Technology. In: Biotechnology Food Fermentation Vol. II
(eds.) V.K. Joshi and Ashok Pandey. Educational Publishers and
Distributors, New Delhi.
3. Joshi, V.K., Sharma, Somesh, Bhushan, Shashi and Attri, Devender (2004)
Fruits based alcoholic beverages. In: Concise Encyclopedia of Bioresource
Technology, Ashok Pandey (eds.) p. 335-345. The Howorth Press, Inc.,
New York.
4. Kharatyan, G.S. (1978) Microbes as food for humans. Ann. Rev.
Microbiol. 32: 301.
5. Verma, L.R. and Joshi, V.K. (Ed.) (2000) Post-harvest Technology of
Fruits and Vegetable – Handling, Processing, Fermentation and Waste
Management. Vol. I & II. Indust Publishing Co., New Delhi.
77
Introduction
UNIT 4 SPOILAGE AND ASSOCIATED
CHEMICAL/PHYSICAL CHANGES
IN FOOD
Structure
4.0 Objectives
4.1 Introduction
4.2 Principles of Food Preservation
4.3 Classification of Foods Based on Perishability
4.4 Factors Governing Spoilage
Number and Kind of Microorganisms
Suitability of Temperature
Suitability of food
pH of Food
Presence of Air
4.5 Chemical and Physical Changes Associated with Food Spoilage
4.6 Microbiology of Fresh Fruits, Vegetables and their Products
Spoilage of Fruits and Vegetables
4.7 Spoilage of Processed Fruit and Vegetable Products
4.8 Preventive Measures
4.9 Let Us Sum Up
4.10 Key Words
4.0 OBJECTIVES
• state the meaning of spoilage;
• principle of preservation of food;
• discuss various causes of spoilage;
• describe different types of spoilages; and
• preventive measures which should be taken.
4.1 INTRODUCTION
We all know that food is the basic necessity of all the living entities. Needless
to say that such a commodity has to be absolutely safe and of highest possible
quality especially free from toxins and spoilage. A food is said to be spoiled if
it has been damaged or injured making it unsuitable for human use. “A product
is fit as a food if a discriminating consumer, knowing the story of its
production and seeing the material itself, will eat it, and conversely, the
same product is spoiled when such an examiner refuses it as a food”. All of
us would agree that a food is spoiled if it is not harvested at proper maturity, is
contaminated with dirt, handled by dirty or diseased person, is fertilized with
sewage and has objectionable changes due to the activity of microorganisms or
action of enzymes of the food. The major causes of spoilage are: the
microorganisms or their enzymes, the native enzymes of food, rodents,
environmental factors and purely chemical reactions.
78
It must be admitted that despite of the improvement in the methods of Spoilage and
Associated Chemical/
production, handling and processing, the microbiological quality still remains Physical Changes in
the most important factor. This aspect assumes significance from toxin Food
production, spoilage of fresh and processed products and quality control and as
sanitation indicators in a processing unit. Microbial quality is also on the top of
the different hazards which are associated with the safety of food for
consumption by human beings. Various fruits, vegetables and their products
may be spoiled by one or more factors like unsuitable packaging, chemical
changes or action of microorganisms, tissue enzymes, insects, rodents or
improper methods of processing, under processing, etc. Different spoilage
causing agents for various fruit and vegetable products, their prevention and
health hazards associated with spoilage are also discussed here.
4.2 PRINCIPLES OF FOOD PRESERVATION

In accomplishing the preservation of food by various methods, three main
principles are involved:
I. Delay or prevention of microbial decomposition of food
II. Delay or prevention of self-decomposition of food
III. Prevention of damage caused by insects, rodents, birds, mechanical causes
etc.
Various principles and sub-principles of preservation of food are summarized
in Table 4.1. First principle of food preservation is based mainly on the
following considerations:
• By delaying the microbial decomposition of food
• By preventing the microbial decomposition of food
Most of the methods of food preservation depend not only on the
destruction or removal of microorganisms but also on the delay in the
initiation of their growth, and hindrance to growth once it has begun.
Knowledge of growth curve of microorganisms is very helpful for developing
the appropriate technique to delay the microbial decomposition of the food.
Table 4.1: Detailed principles of preservation
I. Delay or prevention of microbial decomposition of food

i) by keeping out microorganisms (asepsis).
ii) by removal of microorganism.
iii) by hindering the growth and activity of microorganisms.
iv) by killing the microorganisms.
II. Delay or prevention of self decomposition of food
i) by inactivation of food enzymes.
ii) by delay or prevention of purely chemical reactions.
III. Prevention of damage to food caused by insects, rodents, birds and
mechanical causes
79
Introduction
4.3 CLASSIFICATION OF FOODS BASED ON
PERISHABILITY
1. Perishable foods: The foods which spoil readily unless special preservation
methods are used such as meat, fish, most of fruits and vegetables, egg,
poultry and milk etc.
2. Semi-perishable foods: Semi-perishable foods like waxed potatoes and
some varieties of apple, if handled and stored properly, shall remain
unspoiled for a fairly long period.
3. Non-perishable foods: These foods do not spoil until and unless they are
handled carelessly. Such foods are also called as stable foods such as
cereals, sugar.
4.4 FACTORS GOVERING SPOILAGE

Since microorganisms are the main spoilage causing factors, major
emphasis remains on the factors related to microbial spoilage of the
foods. Here bacteria cause most of the problems since these are not killed at
ordinary temperatures. The yeasts and molds have low resistance to heat
(processing temperatures). The main factors responsible for such spoilage are
described here.
4.4.1 Number and Kind of Microorganisms
The initial number of microorganisms present on the food has direct
relationship with its spoilage. More the number of microorganisms present,
rapid is the spoilage. The effect of initial number of spores on time required to
kill them is shown in Table 4.2. The heat resistance of bacteria involved in
food poisoning is of major concern from public health view point. Usually,
only one type of microorganisms will be there because of the particular
environmental conditions involved. However, contamination may increase the
number as well as new kinds of microorganims.
Table 4.2: Effect of initial number of spores on time required to kill them
Initial concentration of Thermal death time, or time

cells, number/ml required to kill all spores
min. at 121oC
50,000 14
5,000 10
500 9
50 8
In addition to destruction or removal of microorganisms, the delay in

the initiation of growth also prevents microbial spoilage. This is done by
keeping the microorganisms in lag phase as long as possible. Once the
microorganisms enter the log phase, it is very difficult to control them. A
typical growth curve of microorganism is shown in Figure 4.1.
80
Spoilage and
Physical Changes in
Food
Figure 4.1: Growth curve of microorganism. A to B lag phase, B to C positive

acceleration phase, C to D logarithmic phase, D to E negative acceleration
phase, E to F stationary phase, F to G accelerated death phase, G to H death
phase, H to I survival phase
4.4.2 Suitability of Temperature

Different microorganisms grow at different temperatures. Psychrophiles have
affinity for low temperatures (8-10oC), mesophiles grow best at medium
temperatures (25-40oC), while thermophiles appear at higher temperatures
(50-55oC). A large number of microorganisms grows at mesophilic temperature
therefore processed foods are immediately cooled to arrest microbial activity /
spoilage of foods. So, storage temperature is very important in relation to
microbial growth and hence, the spoilage behaviour of foods.
Different types of microorganisms require different times to kill their cells or
spores. The time required to kill all the spores of flat sour bacteria (Bacillus
stearothermophilus) in relation to temperature is shown in Table 4.3.
Table 4.3: Effect of temperature of heating on the time needed to kill
spores of flat sour bacteria
Temperature Thermal death time or time to destroy all spores, min.

(oC)
100 1200
105 600
110 190
115 70
120 19
125 7
130 3
135 1
Source: Adapted from Frazier and Westhoff (1996).
4.4.3 Suitability of food

Different microorganisms prefer different kinds of foods. Some grow best on
proteinacious foods, others on starchy or fatty foods. The physical state of the
food whether heated, frozen, moistened or dried, also has an important
81
Introduction influence on the spoilage it will undergo. The moisture content also influence
the type of microorganisms in the foods since the requirements of moisture for
their growth are different. Bacteria, yeast and molds have different moisture
requirements in terms of water activity (Figure 4.2).
Figure 4.2: Water activity of different microorganisms
4.4.4 pH of Food
The pH of the food influences the kind and growth of microorganisms. The
composition of the vegetable, its pH and moisture contents affect their type of
spoilage. As a general rule, foods having pH<4.5 (acid foods) do not require
heat processing (particularly cooking under pressure), but those with pH > 4.5
(low acid foods) always require processing under pressure.It is because of the
reason that thermophilic bacteria may not be killed at normal temperatures as
most bacteria thrive best at pH of 4-7.5, while the yeasts and molds require a
pH of 2.5-8.0 and 1.5-8.5 for their growth.
4.4.5 Presence of Air

Aerobic or anaerobic microorganisms can be found in the spoiled fruit and
vegetable products, depending upon the presence or absence of air (oxygen) in
the container or package.
4.5 CHEMICAL AND PHYSICAL CHANGES

ASSOCIATED WITH FOOD SPOILAGE
Changes in nitrogeneous organic compounds: Most of the nitrogen in the food
present in the form of proteins which are hydrolysed by enzymes to produce
82
amino acids. The anaerobic decomposition of the protein, peptide or amino
acid results in the production of obnoxious odour which is called as Spoilage and
putrefaction. When the microorganisms act on amino acid they deaminate or Physical Changes in
decarboxylate. Food
E. coli produces glyoxylic acid, acetic acid and ammonia from glycine and
from serine it produces pyruvic acid and ammonia. Alanine is degraded into α-
keto acid, ammonia and carbon dioxide by E. coli; acetic acid, ammonia, and
CO2 by Psuedomonas; and propionic acid, acetic acid, ammonia and CO2 by.
Clostridium nigrificans. Other nitrogenous compounds like amide, urea,
guianidine and creatine, etc. are also decomposed to ammonia, carbon dioxide
and other products.
Changes in Carbohydrates: Carbohydrates are preferred by the microorganisms
as energy yielding foods. They hydrolyse the polysaccharides to
monosaccharides before utilization such as to glucose which is then, oxidized
to CO2 and H2O. Anaerobically, these undergo decomposition involving one or
more types of fermentation.
• Alcoholic fermentation by yeast with ethanol and CO2 as products.
• Lactic fermentation by homofermentative lactic acid bacteria with lactic
acid or by heterofermentative lactic acid bacteria with lactic acid, acetic
acid, ethanol, glycerol and CO2 as chief products.
• Coliform type of fermentation by coliform bacteria with lactic acid, formic
acid, ethanol, CO2, hydrogen and perhaps aceton and butanediol as likely
products.
• Propionic acid fermentation by propionoic bacteria producing propionic
acid, acetic and succinic acid and CO2.
• Butyric- butyric isopropyl fermentation by anaerobic bacteria producing
butyric acid, acetic acid, CO2, H2 and in some cases, butylenes glycol,
butanol and 2-propanol.
They are present as salts and are oxidized by the microorganisms to carbonate
and cause the food medium to become alkaline. Organic acid aerobically are
oxidized to carbon dioxide and water as is done by the film yeast.
Changes in other compounds: Other compounds also undergo changes as
detailed here:
• Ethyl alcohol is oxidized to acetic acid.
• Glycoside is hydrolysed to sugars.
• Acetaldehyde is oxidized to acetic acid or reduced to ethanol.
• Protopectin are acted upon by pectinesterase – pectic acid + methanol
(water soluble) by hydrolysis of methyl ester. Polygalacturonases destroys
the linkage between galactouronic acid unit of pectin or pectic acid to yield
smaller chain and ultimately, free D-galacturonic acid, which may be
degraded to simple sugar.
Changes in Lipids: Fats present in the media are hydrolysed by lipase into
glycerol and fatty acid.Phospholipids may be degraded to their constituents
phosphate, glycerol, fatty acid, and nitrogenous base e.g. chlorine.
83
Introduction One or more such changes can be produced in the food undergoing spoilage.
The physical and sensory qualities of the food also undergo changes, thus
making the product unfit for human consumption.
4.6 MICROBIOLOGY OF FRESH FRUITS, VEGETABLES

AND THEIR PRODUCTS
4.6.1 Spoilage of Fruits and Vegetables

In general, fruits and vegetables are bulky, easily damaged mechanically,
consist largely of water which is readily lost and above all, are living
entities and must be kept so for their longivity. Thus, they are sensitive to
their environment viz., temperature, level of oxygen, carbon dioxide and
ethylene etc. Spoilage of fresh fruits and vegetables usually occurs during
storage and transport and while waiting to be processed into various
products. These also get contaminated with spoilage organsims either from
each other or when they are laid into the baskets, lugs, boxes etc. during
harvesting. Mechanical injuries during transportation further aggravate the
deterioration process. The decay of perishables may occur due to the physical
factors, action of their own hydrolytic enzymes or microbial contaminants
etc as discussed earlier. Since fruits and vegetables after picking are alive for
certain time thus, are sensitive to their environment, their rate of metabolism
is temperature dependent and they may be damaged by heat or cold or even by
levels of different gases in the atmosphere. Oxygen is taken in during
respiration and CO2 heat and water vapours are given-off. As fruits or
vegetables are detached from the mother plant, the continuity of the flow of sap
is totally disrupted but the respiration and water loss continues leading to
exhaustion of food reserve and moisture. The irreparable losses are caused
leading to deterioration and eventually spoilage. Spoilage is mainly of 2 types:
Abiotic spoilage and Biotic spoilage.
Abiotic spoilage: It is due to the different physical (wilting, caking and
melting etc.) and chemical changes in the product (hydrolytic action of
enzymes, oxidation of fats, putrefaction of proteins, browning reaction between
proteins and sugars). Temperature control is the major factor to provide
longevity to the fruits and vegetables.
Biotic spoilage: This includes the microbial action associated with bacteria,
yeasts and molds on vegetables and fruits and the normal processes of aging.
The species of microorganisms causing food spoilage largely depend upon
different factors e.g. kind and variety of fruits/vegetables, environmental
condition e.g. storage, temperature, relative humidity of the atmosphere and
various gas contents of the atmosphere etc. There are two types of microbial
spoilage: (a) Spoilage caused by plant pathogens which attack various parts of
the plant used as foods, (b) Spoilage caused by saprophytes. The most common
and general type of spoilage in fruits and vegetables are mildew are listed in
Table 4.4. Dry rots often lead to darkening and discoloring, and hardening of
the surface of vegetables and fruits. In microbial spoilage, the vegetables often
develop water soaked musky areas while the fruits generally have brown or
white colored patches.
84
Spoilage and
Table 4.4: The chief market diseases of some vegetables and fruits
Physical Changes in
Item Market diseases Food
Onions Bacterial soft rot, black mold rot, gray mold rot
Garlic Bacterial soft rot, black mold rot
Green beans Bacterial soft rot, mold rot, Rhizopus
Carrots Bacterial soft rot, black rot, Fusarium rot, gray mold rot,
watery soft rot
Beets Bacterial soft rot, black rot, blue mold rot, Fusarium rot
Lemons Alternaria rot, anthracnose, blue mold rots, stem-end

rots
Peaches Alternaria (or green mold rot), gray mold rot, black
mold rot
Apricots Blue mold rot, brown rot, Cladosporium rot, Rhizopus

rot
Bananas Anthracnose, Fusarium, Gleoporium, Pestalozia
Grapes Black mold rot, gray mold rot, Rhizopus rot, blue mold
rot
Strawberries Gray mold rot, leather rot (Phytophthora cactorum)

Rhizopus rot
Pears Black rot, blue mold rot, brown rot, gray mold, Rhizopus
rot
Potatoes Fusarium tuber rot, bacterial ring rot, bacterial soft rot
Cucumber Rhizopus soft rot, bacterial soft rot, blue mold rot, gray
mold rot
Cabbage Bacterial soft rot, gray mold rot, black rot, watery soft
rot
Cauliflower Bacterial soft rot, gray mold rot, black rot, watery soft
rot
Tomatoes Alternaria rot, bacterial canker, bacterial spot, gray mold

rot, green mold rot, Rhizopus rot
The composition of the fruit/vegetable, its pH and moisture content affect their
type of spoilage. Moisture content is usually expressed in terms of water
activity ‘aw’. Various microorganisms have different requirements for
moisture level (Figure 4.2). Amongst the microorganisms, spoilage can be
caused by bacteria, molds/yeasts etc. depending upon the pH of food.
85
Introduction Bacteria: Various groups of bacteria can attack different fruits and vegetables,
depending upon their composition such as lactic acid bacteria, acetic acid
bacteria, coliform bacteria and sporeforming bacteria. The food can be
preserved for longer time by prolonging the lag phase. This can be obtained by
avoiding the contamination of the food and turning the environmental
conditions e.g. temperature, moisture and pH unfavourable for the growth of
contaminants (microorganism). Thus, by lowering the storage temperature of
the fruits/vegetables, filling up the storage chamber with the inert gases will
definitely lead to longer shelf- life of the vegetables and fruits. pH is another
important factor governing the bacterial growth which range between pH 4-8.
Growth rate is lowered by a decrease in pH.
Yeasts: Yeasts are widely found in the environment. The yeast growth depends
largely upon the nature of fruit product. These are generally fermentative in
nature.
Molds: Molds are frequently associated with food products. Some of the molds
secrete toxic compounds (mycotoxins) like aflatoxins, patulin etc. Aflatoxin
has been detected in dried figs and fig paste while patulin is the most common
mycotoxin detected in the processed fruits. The mold Penicillium expansum
which causes apple rot and some other molds produce patulin. The mycotoxins
are deleterious to various animals and presumably the human beings also.
4.7 SPOILAGE OF PROCESSED FRUIT AND

VEGETABLE PRODUCTS
The spoilage of processed fruit and vegetable products is also of two types:
Abiotic spoilage and Biotic spoilage.
a) Abiotic spoilage: It is due to the different physical and chemical changes
in the product viz. putrification of proteins, browning reactions between
sugars and proteins and the physical changes of colour, caking and melting
etc. The temperature and humidity are the main factors responsible for
causing this type of spoilage. There is a relationship between moisture
content and relative humidity with respect to different causes of spoilage in
food products.
b) Biotic spoilage: It includes the actions associated with microorganisms,
damage caused by insects, rodents etc.
Manifestation of spoilage: The microbial deterioration of a processed food
product usually is manifested by alterations in the appearance, texture, colour,
odour, flavour or slime formation. The appearance includes colour changes,
visible growth of microorganisms, formation of pockets of gas or swelling of
cans and microbial growth especially that of molds on the surface of food
process (Plate 4.1). As some food products deteriorate, they tend to become
soft or mushy. Degradation of foods results in the formation of compounds
which have odours and flavours different from those of the fresh food.
86
Spoilage and
Physical Changes in
Food
Plate 4.1: Different spoiled products from fruits and vegetables. 1) Mold growth on
pickle, 2) Mold growth on jam, 3) Mold growth on juice, 4) Mold growth on
tomato crush, 5) Puffed can
Spoilage of Canned Fruits and Vegetables

Like other foods, canned fruit and vegetable products are also liable for
spoilage for various reasons. Number of microorganisms surviving the heat
process, storage temperature, suitability of the canned food to support the
growth of the microorganisms, pH of the food, oxygen tension etc. may affect
the spoilage of canned foods. Broadly, there are four causes for the spoilage
of canned fruit and vegetable products viz., microbiological, physical,
chemical and miscellaneous.
Microbiological spoilage: The spoilage caused by the growth of different
kinds of microorganisms may be affected by under processing, inadequate
cooling, infection resulting from leakage and pre-processing spoilage.
Under processing: Insufficient or improper heat treatment may result in
survival of certain microorganisms causing spoilage of food product during
subsequent storage. Some lot or a part of the lot may remain under processed
or not processed at all (gross under processing) by mistake. As a general rule,
only one type of microorganisms (bacteria or yeast or mold) are involved in
such spoilage. Most of these are facultative anaerobes unusually heat resistant
spore formers. Faulty operation during processing may also cause under
processing. This type of spoilage can be avoided by ensuring proper heat
transfer, proper retorting and proper stacking of cans in the retort.
Inadequate cooling: Immediately after processing, the cans are cooled using
cold water. It gives a sort of shock to the surviving microorganisms and kills
them. It also checks overcooking and hence, saves the food from textural
disintegration. If cooling is not proper, spoilage can occur. In addition, the
cans are cooled down (using fans also) upto 35-38oC only to allow the water to
evaporate readily from the can surface. It will check the rusting of cans during
storage, which may otherwise lead to some spoilage. It can be avoided by
proper cooling after processing.
87
Introduction Infection due to leakage: Post-processing contamination may take place if
there is leakage in the cans due to faulty seam, faulty lock seam or pinholes
due to corrosion from inside of the can or rusting of can from outside.
Here all the types of microorganisms can be present in the food. Lot of oxygen
can enter into the can. Contents of such type of cans are not suitable for
consumption. To avoid such kinds of spoilage, seam tests should be carried
out regularly while in operation, proper hygiene should be maintained and
handling should be proper. The cooling water should be chlorinated using 5-
7 ppm of chlorine.
Pre-process spoilage: If the raw material is already heavily contaminated
many microorganims may survive heat treatment and also the finished product
may not be of desirable flavour or quality. It may also be due to faulty
procedure during washing (of raw materials, cans and equipment) blanching
and filling of cans. Many respiratory gases can develop causing swelling of
cans during storage. To avoid such type of spoilage, proper testing of raw
materials, proper washing of raw material as well as equipment, chlorination of
water and proper sterilization of cans should be practiced.
Thermophillic spore forming anaerobic spoilage: The microbiological
spoilage of canned fruits and vegetables can be of different types.
Flat sour: It derives its name from the fact that the ends of the can of food
remain flat but the contents become sour. It is mostly found in non-acid foods
like canned vegetables by the action of microorganisms (flat sour bacteria). So
it cannot be detected without opening of can and culturing the microorganisms.
Bacillus stearothermophilus and Bacillus coagulans are the thermophilic
spore-forming bacteria responsible for flat sour type of spoilage. The latter are
found in tomato juices. The immediate source of the flat sour bacteria is
usually the plant equipment besides sugar, starch or soil.
TA spoilage: It is the short name for spoilage caused by thermophilic
anaerobes which do not produce H2S e.g. Clostridium thermosaccharolyticum.
It is a sugar-splitting obligate thermophilic, sporeforming anaerobe which
produces CO2 and H2, causing the swell and even bursting of cans. The spoiled
food usually have sour odour and the source of contamination could be the
plant equipment, sugar, starch and soil similar to that of flat source.
Sulphide or Sulphur Stinker spoilage: Such spoilage is caused by
Clostridium nigrificans, mostly found in low acid foods like peas and corn.
The spores of this bacterium are considerably less heat resistant and hence,
their appearance in canned foods is indicative of gross under processing. H2S
can be detected by its characteristic odour on opening the can and the organism
can also be detected in the form of black (FeS) colonies formed on iron
sulphite agar at 55oC.In case of peas, it is difficult to detect any marked
discolouration. The source of this organism includes sugar, starch, soil, manure
and the plant equipment.
Mesophilic spoilages: Some species of Bacillus and Clostridium, non-spore
forming bacteria and even yeasts or molds may spoil the under-processed
canned foods. The spore forming mesophilic thermoduric species of bacteria
include C. pasteurianum, C. butyricum, C. botulinum, C. sporogenes, B.
sublitis, B. polymyxa, etc. The non-spore forming mesophilic bacteria involved
in the spoilage of tomato products, pears and some other fruits were found to
be Lactobacillus and Leuconostoc. Other such genera may be Pseudomonas,
88
Micrococcus, Flavobacterium etc. which may come from water and leaks in Spoilage and
the cans. Physical Changes in
Food
Spoilage by yeasts: The yeasts have been found to spoil canned fruits, jams,
jellies, fruit juices, etc. underleakage or under-processing conditions. Film
yeasts like Candida, Pichia, Hansenula can grow on acid products like
saurkraut and pickles osmophilic yeasts like Saccharomyces rouxi, S. mellis
can spoil dry fruits, concentrated fruit juices and honey etc. Salt tolerant
yeasts like Torulopsis and Brettanomyces can grow in brine solution.
Spoilage by molds: The molds are the common spoilage organisms of home
canned foods like jams, jellies, marmalades. The common one are Aspergillus,
Penicillium, Byssochlamys fulva etc.
Spoilage by physical causes: Some physical deformation of the container can
lead to spoilage. Faulty technique in operation, under exhausting, over filling
and panelling or buckling of the cans can cause spoilage of processed food
products.
Faulty technique in operation: Just after retorting, if the pressure is released
at once instead of slow release, distortion of can body can occur. Joints or
seams may be distorted resulting in leakage. To avoid such kind of spoilage,
the pressure in the retort should be released slowly after retorting and standard
iron plates should be used for cans.
Under-exhausting: If the air entrapped in the tissues of canned fruit and
vegetables, and the filling medium is not expelled properly, adequate vacuum
may not develop which may consequently, impair the quality and appearance
of the product.
Over-filling: Over-filling of cans does not allow proper vacuum formation
after processing. It may also lead to flipper or springer type of spoilage. The
proper filling is essential to avoid this defect.
Panelling or buckling: It occurs in case of big sized cans. If there is very high
vacuum inside the can, atmospheric pressure can struck or force the can
inwards resulting in leakage type of spoilage. To check such spoilage, proper
vacuum should be created in the cans carefully.
Chemical spoilage: It includes reactions among ingredients, reactions between
can and ingredients, hydrogen swell etc. Mainly, it is due to H2S production,
presence of oxygen, acids etc. H2S is formed by the action of SO2 (added
through sugar or by decomposition of proteins) and H2 formed by fruit acid
acting on tin plate. If there is sufficient vacuum, H2 is absorbed after storage
for long time. Low acid foods have more H2 swells. Therefore, pH of the food
has important role in checking such spoilages. If the pH is near 4.0, it is
favourable for many chemical reactions. To avoid the spoilage, we can assure
proper vacuum (by hot filling) in the can and also adjust safe pH of the food
prior to canning. Rusting and corrosion and perforation of tin plates: After
cooling the processed cans in water, if some water remain on the surface of
cans, rusting can take place. Similarly, the hygroscopic nature of the labels
can also add to the rusting. In case of acid foods, there are more chances of
corrosion and perforation of tin plates. More the oxygen in the can, more is the
corrosion. Corrosion is more at higher than at lower temperatures. To avoid
corrosion and perforation of tin plates, proper exhausting should be done and
cans should not be cooled below 35oC in water or fans should be used to
89
Introduction evaporate water from the can body and non- hygroscopic in nature labels
should be used and be stored at relatively low temperatures.
Metallic contamination: The tannins of raw materials or spices used react
with exposed iron of the tin plate to form ferric tannate, a black product.
Similarly, the SO2 on reacting with H2 forms H2S that may further react with
iron content of the can to form iron sulphide thus, causing spoilage of the
processed food product. Also, on using the equipment made of copper or brass
after sometime, in spite of thoroughly cleaning, small traces of copper oxide
may remain there, which further form black copper sulphide on reacting with
H2S and discolour the product. To avoid such spoilage, proper exhausting and
proper selection of equipment, thorough washing of the raw material as well as
equipment.
External Appearance of Can
Flipper: A can with mild positive pressure is called a flipper. It may be an
initial stage of swell or hydrogen swell but more frequently, it is due to over-
filling or under exhausting, leakage or sealing at low temperature. The can ends
remain flat but when the sides of can are struck with some hard structure or if
the temperature of the contents is increased, bulging of ends take place.
Springer: A mild swell at one or both ends of a can is called a springer. One
end may also remain permanently bulged and other flat. Pressure on the bulged
end will bring it to normal but it will go to other end. Generally, the food in
such cans remains fit for consumption. The reasons are similar as for flipper
can.
Swells: In this case, both ends remain bulged. It may be a soft swell or hard
swell. In case of soft swell, the ends are not so hard. On applying some
pressure, the ends may go inward (normal) but do not remain normal on the
removal of pressure. Obviously, in hard swell, can ends are rigidly hard and
there is no effect of pressure except bursting or leakage of the can. The swell
occurs due to the production of H2 (formed by action of acids of food and tin
plate), CO2 or other gases (as a result of decomposition of contents by
microorganisms) involving both thermophiles or mesophiles. The food is not
fit for consumption and may even contain toxins produced by Clostridium
botulinum.
Leaker: A very small leak may appear in the can due to faulty seam, faulty
lock seam or pinholes resulting from corrosion from inside of the can or rusting
of the can from outside.
Breather: Tiny leak in the can may allow air to pass back and forth into the
can but not the microorganisms. The inside pressure of the can equals outside
pressure. The contents may be spoiled due to rusting of can caused by oxygen
in air passing through the tiny leak.
Buckled cans: Sometimes, the vacuum in big sized cans is so high that
atmospheric pressure can strike the can body resulting in deformation of can
leading to leakage of contents or contamination.
Spoilage of Fruit and Vegetable Juices
Fruit juice/squashes: The fruit juices are more spoiled by yeasts and molds
than by bacteria since they have lower pH while vegetable juices are
spoiled more by bacteria than yeasts and molds because of very high pH. If
90
the fruit and vegetable juices are not processed after extraction, they are Spoilage and
spoiled because of enzymatic changes and microbial actions. Apple and grape Physical Changes in
juices are spoiled by bacteria if the temperature of storage goes above 25oC. Food
Molds can grow on the surface of fruit juices if exposed to air. Most fruit juices
have sufficient sugar to favour the growth of yeasts. Deficiency of B group
vitamins discourages some bacteria. Concentrates of fruit and vegetable juices
favour the growth of yeasts and of acid bacteria and sugar tolerant
(Zygosacharomyces) species because of increased acidity and sugar
concentration. Fruit juice concentrates are fermented almost exclusively by
Saccharomyces rouxi, S. mellis, Torulopsis and Hansenula. Typical
fermentation products are ethanol and CO2. Heat treatment during canning of
these concentrates usually kills these microorganisms and freezing prevents the
growth of such organisms. In fruit squashes, if presevatives are not added in
proper concentration, some yeasts (e.g. Zygosaccharomyces) can spoil such
products.
Spoilage of canned fruit juices: The acidity is the single most important
factor affecting microbial spoilage of fruit juices. Most bacteria have an
optimum pH near 6.8 but may grow at pH values ranging from 4-8. Yeasts and
molds can grow at pH <2.
In canned foods, a pH of 4.5 is used as a borderline between acid and low
acid foods, that is foods not requiring and those requiring respectively, the
minimum botulinum cook (12 D). The typical spoilage flora of fruit juices is
represented by some Clostridia, Bacillus, members of Enterobacteriaceae,
lactic acid bacteria, Acetobacteriaceae, yeasts and molds. The spoilage is
characterised by lowering of pH (0.2-0.4 units), development of very high
volumes of hydrogen and CO2, and strong cheesy (butyric) odour. Three
types of Bacilli are usually involved in spoilage of fruit juices i.e. B.coagulans
(flat sour), B. macerans and B. polymyxa (both by storage at temperature
< 46oC).
The spoilage by enterobacteriaceae is characterised by the production of lactic
acid, acetic acid, formic acid, H2 and CO2. Lactic acid bacteria causing
spoilage of fruit juices include Lactobacillus, (L. plantarum, L. fermenti)
Leuconostoc mesenteroides, Streptococcus viscosum. Among the yeasts, the
most often involved species in juice fermentation belong to Saccharomyces,
Torulopsis, Candida, Pichia, Hansenula and Hanseniaspora.
Molds require O2 for development and so usually do not grow at the surface of
processed fruit products. However, Byssochlamys fulva and Penicillium
expansum, have been found in canned foods and the latter can grow under
vacuum also.
Carbonated beverages are usually not spoiled because of inhibitory effect of
CO2 on microorganisms. The acidity (resulting from carbonation and addition
of acids) also inhibits microorganisms. Since molds require air, they do not
grow on the carbonated beverages.
Spoilage of Jams, Jellies, Marmalades and Preserves
Jams and jellies have usually low water activity (0.75-0.86). In addition, due to
added acid, the pH is lowered and sometimes may have preservatives such as
benzoic acid. All these factors lower down the risk of spoilage. Also due to
heating, spoilage causing organisms (yeasts, molds) are eliminated until the
package is opened whereupon recontamination could be expected (Plate 1).
91
Introduction Usually, osmophilic yeasts such as Torulopsis, Xeromyces and many other
have been reported to spoil jams, jellies and preserves.
Spoilage of Frozen Fruits
Many fruits and fruit products are preserved by freezing including cherries,
fruit juice concentrates, purees (oB>45) and some sliced fruits. Usually, dry
sugar or syrup is added to fruit prior to freezing. The predominant
microorganisms are usually yeasts and molds besides lactic acid bacteria (in
orange juice concentrate).
Spoilage of Dried Fruits and Vegetables
In dried fruits (apples, apricots, dates, figs, peaches, prunes, resins etc.), a
number of microorganisms can be expected. Due to decreased water activity
(<0.65 in case of sun dried products), heat treatment during dehydration and
fumigation, the microorganisms may be killed or unable to cause spoilage.
But, spores of bacteria and molds are likely to be the most numerous. Dried
fruits may be spoiled due to the development of rancidity as concentrated
flavonoids may undergo oxidation. Dried or partially dried fruits (dates, figs
and prunes) are also susceptible to yeast spoilage i.e. Zygosaccharomyces.
4.8 PREVENTIVE MEASURES

While the fresh fruits and vegetables are spoiled by biological
(microbiological) and non-biological causes, their spoilage is checked by
adopting suitable preservative techniques. But the processed products do not
normally spoil unless the preservative technique applied is not proper or is not
applied properly or the product is stored improperly. There are a few
generalized preventive measures which can be adopted to avoid their spoilage.
• It is desirable to keep the initial microbial contamination as low as
possible. The commodities should be handled and stored to avoid further
contamination and create conditions to check the growth of
microorganisms.
• All efforts be made to apply the preservative technique, keeping in view the
various steps to avoid spoilage.
• Mechanical disruption of the processed product tissue should be prevented.
Equipments used for handling should be clean and free from contamination
and contamination from the soil microflora should be avoided. Dipping of
fruits and vegetables in solution of chlorine (50-125 ppm) removes the
adhering microflora.
• Inhibition of microbial growth can be achieved by storing the food at
low temperature or in inert atmosphere packaging.
• While packaging the processed product, it is absolutely essential that the
environment of packing should be microbes free or least contaminated and
away from stores to minimize the post-processing contamination.
• The chemical and microbiological quality of water is the single most factor
which can control the quality of the finished processed product. It should
conform to the prescribed standards of microbiological (indicator
microorganism) and chemical quality.
92
• The spoilage of canned products can be minimized especially leakage by Spoilage and
regularly checking the equipments used in canning (reformers, flanger, Physical Changes in
double seamer, retort). Food
• The quality of raw material is controllable factor which have profound

influence on the spoilage behaviour of processed product.
Check Your Progress Exercise 1 "

1. Why the food can get spoiled?
……………………………………………………………………………….
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2. List various causes of spoilage of food?
……………………………………………………………………………….
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3. Enumerate various principles used in preserving food?
……………………………………………………………………………….
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4. What are the major causes of spoilage of canned fruits and vegetable?
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93
Introduction ……………………………………………………………………………….
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5. Can dehydrated fruits and vegetables also get spoiled if so why?
……………………………………………………………………………….
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6. What is meant by acetification, putrefaction, rancidity, fermentation?
……………………………………………………………………………….
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7. What is meant by hard swell, Hydrgen swell and flat sour?
……………………………………………………………………………….
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8. What is meant by water activity?
……………………………………………………………………………….
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94
9. Can jams be spoiled? Spoilage and
………………………………………………………………………………. Physical Changes in
Food
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
10. Name a few microorganisms involved in spoilage of juices.
……………………………………………………………………………….
……………………………………………………………………………….
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11. Can the carbonated juices also get spoiled? If so how?
……………………………………………………………………………….
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……………………………………………………………………………….
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12. Enumerate various factors responsible for microbial spoilage.
……………………………………………………………………………….
……………………………………………………………………………….
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13. How would you classify the foods based on perishability?
……………………………………………………………………………….
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95
Introduction ……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
14. Classify the microorganisms according to their optimum temperature of
growth?
……………………………………………………………………………….
……………………………………………………………………………….
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……………………………………………………………………………….
……………………………………………………………………………….
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15. Name the bacterium responsible for causing flat sour.
……………………………………………………………………………….
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16. What is the relationship of initial number of microorganism with spoilage?
……………………………………………………………………………….
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17. Enlist five ways the spoilage can be prevented.
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96
Spoilage and
4.9 LET US SUM UP Associated Chemical/
Physical Changes in
The foods especially fruits and vegetable are a living commodities and are Food
therefore, liable for spoilage. The nature and kind of spoilage, however, depend
upon the type of food and the environment where it is kept or how it is handled
and stored. The causes of spoilage include contamination with microorganisms,
activity of microorganisms or their enzymes, activity of native enzymes of
food, infestation with rodents and influence of various external conditions
where the food is stored. Decomposition of food constituents such as proteins,
fats and carbohydrates and their interactions result into production of chemicals
with different quality and hence, not acceptable as a food. To prevent spoilage,
the foods are preserved either by giving low temperature during storage, or
heated in appropriate media, irradiated, dehydrated or preserved with
chemicals or made into fermented products. If such methods are not properly
employed the foods can get spoiled even when they are processed.
4.10 KEY WORDS

Spoiled food : A food is said to be spoiled if it has been
damaged or injured making it unsuitable for
human use.
Perishable foods : There are the foods which spoil readily unless
special preservation methods are used such as
meat, fish, most of fruits and vegetables, egg,
poultry and milk etc.
Non-perishable or
stable foods : These foods do not spoil until and unless they are
handled carelessly such as cereals, sugar etc.
Putrefaction : The anaerobic decomposition of the protein,
peptide or amino acid resulting in the production
of obnoxious odour is called as putrefaction.
Psychrophiles : Those microorganisms which have affinity for
low temperatures (8-10oC).
Mesophiles : Are the microorganisms which grow best at
medium temperatures (25-40oC).
Thermophiles : These microorganisms appear at higher
temperatures (50-55oC).
Under processing : It denotes insufficient or improper heat treatment
that may result in survival of certain
microorganisms causing spoilage of food
product during their subsequent storage.
Water activity (aw) : Moisture content is usually expressed in
terms of water activity.
Under-exhausting : If the air entrapped in the tissues of canned fruit
and vegetables and the filling medium is not
expelled properly, is called under-exhausting.
97
Introduction Panelling or buckling : It occurs in case of big sized cans when there is
very high vacuum inside the can, atmospheric
pressure can struck or force the can inwards
resulting in leakage type of spoilage.
Flipper : A can with mild positive pressure is called a
flipper.
Springer : A mild swell at one or both ends of a can is called
a springer. One end may also remain permanently
bulged and other flat.
Swells : In this case, both ends remain bulged. It may be a
soft swell or hard swell.
Leaker : A can with a very small leak due to faulty seam,
faulty lock seam or pinholes resulting from
corrosion from inside of the can or rusting of the
can from outside is called leaker.
Breather : It denotes tiny leak in the can that may allow air
to pass back and forth into the can but not the
microorganisms. The inside pressure equals
outside pressure.
Buckled cans : Sometimes, the vacuum in big sized cans is so
high that atmospheric pressure can strike the can
body resulting in deformation of can which may
further cause leakage of contents or
contamination.

EXERCISES
A food is a living commodity and respire and undergo various metabolic
changes which if unchecked can spoil the food or if it is not harvested at
proper maturity, is contaminated with dirt, or the microorganisms.
The major causes of spoilage are: the microorganisms or their enzymes, the
native enzymes of food, rodents, environmental factors and purely chemical
reactions.
• Delay or prevention of microbial decomposition of food.
• Delay or prevention of self decomposition of food.
• Prevention of damage to food caused by insects, rodents, birds and
mechanical causes.
98
4. Your answer should include the following points: Spoilage and
• Microbiological spoilage Physical Changes in
Food
• Spoilage by physical causes
Dried fruits can have a number of microorganisms but due to decreased
water activity, heat treatment during dehydration and fumigation, the
microorganisms may be killed so unable to cause spoilage, but, spores of
bacteria and molds may survive and cause spoilage.
Acetification: It is the process of conversion of ethanolic liquid into acetic
acid by the activity of acetic acid bacteria.
Putrefaction: The anaerobic decomposition of the protein, peptide or
amino acid results in the production of obnoxious odour which is called as
putrefaction.
Fermentation: It is the process in which the organic compounds are
converted into other organic compounds with generation of energy.
Rancidity: It implies the oxidation of fatty substances giving specific off-
odour.
In hard swells, both the ends of can remain bulged and rigid. In case of soft
swell, the ends are not so hard. On applying some pressure, the ends may
go inward (normal) but do not remain normal on the removal of pressure.
Water activity (aw): Moisture content is usually expressed in terms of
water activity.
Jams and jellies have usually low water activity (0.75-0.86) so have lower
risk of spoilage. However, if package is opened chances of
recontamination could be expected or if there is leakage the product could
be spoiled by osmophilic yeasts such as Zygosaccharomyces Torulopsis,
Xeromyces.
Fungi
Mucor,
Rhizopus,
Penicillium,
Aspergillus,
Alternaria, Cladosporium,
Byssochlamys
99
Introduction Bacteria
Clostridium butyricum
B. coagulans (flat sour)
E. coli
Lactobacillus,
Leuconostoc mesenteroides
Streptococcus viscosum
Yeast
Saccharomyces
Carbonated beverages are usually not spoiled because of inhibitory
effect of CO2 on microorganisms. The acidity (resulting from carbonation
and addition of acids) also inhibits microorganisms. Since molds require
air, they do not grow on the carbonated beverages but may develop at the
surface of uncarbonated soft drinks which contain air above the liquid
surface.
Following are some of the factors responsible for spoilage: Number and
kind of microorganisms, Suitability of temperature, Suitability of food, pH
of food and Presence of air
Perishable foods: are the foods which spoil readily unless special
preservation methods are used (meat, fish, most of fruits and vegetables,
egg, poultry milk).
Semi-perishable foods: Semi-perishable foods like waxed potatoes and
some varieties of apple, if handled and stored properly, shall remain
unspoiled for a fairly long period.
Non-perishable foods: are the foods that do not get spoiled until and
unless they are handled carelessly such as cereals, sugar.
Psychrophiles: These have affinity for low temperatures (8-10oC).
Mesophiles: These microorganisms grow best at medium temperatures
(25-40oC).
Thermophiles: These microorganisms appear at higher temperatures
(50-55oC).
Bacillus coagulans
The initial number of microorganisms present on the food has a direct
relationship with its spoilage. More the number of microorganisms present,
rapid is the spoilage.
100
17. Your answer should include the following points: Spoilage and
1) Keep the initial microbial contamination as low as possible and create Physical Changes in
Food
conditions to check the growth of microorganisms.
2) All efforts be made to apply the preservative technique.
3) Mechanical disruption of the processed product tissue should not occur.
4) Inhibit microbial growth by storing the food at low temperature or in
inert atmosphere packaging of dried fruits and vegetable products.
5) Equipments used for handling should be clean and free from
contamination.

1. Banwart, G.J. (1981) Indicator Organisms. In: Basic Food Microbiology.
AVI Publishing Co. Inc. CN, USA. p. 389.
2. Frazier, W.C. and Westhoff, D.C. (1996) Food Microbiology. 4th edn. Tata
McGraw Hill Publ. Co. Ltd., New Delhi.
3. Joshi, V.K., Pandey, A., Nigam, P. and Coccel (1998) Enterobacteriaceae,
coliform and E. coli. In: Encyclopedia of Food Microbiology R. Robinson,
C. Batt, P. Patel (eds.) Academic Press, London.
4. Lal, G., Siddappa, G.S. and Tandon, G.L. (1986) Spoilage in canned
foods. In: Preservation of fruits and vegetables. ICAR Publ., New Delhi,
p. 82.
5. Potter, N.N. (1987) Food Science. 3rd edn. CBS Publisher and Distributors,
New Delhi.
6. Sharma, A. (1998) Microbial Toxins. In: Biotechnology: Food
Fermentation Vol. I, V.K. Joshi and A. Pandey (eds.) Educational
Publishers & Distributors, New Delhi.
101
Concept,
UNIT 5 CONCEPT, DETERMINATION OF Determination of
Process Lethality
PROCESS LETHALITY Requirements and
Importance
REQUIREMENTS AND
IMPORTANCE
Structure
5.0 Objectives
5.1 Introduction
5.2 Classification of Foods According to pH
5.3 Relationship Between pH of Food and Heat Resistance of
Microorganisms
5.4 Heat Resistance of Microorganisms and Spores
5.5 Thermal Death Point
5.6 Thermal Death Time
5.7 Determination of Thermal Death Time
Glass Tube Methods
Decimal Reduction Time
Thermal Death Time Curve (TDT Curve)/Kinetics
12D Concept
5.8 Determination of Process Lethality Requirements at Low and High
Temperature
Heat Penetration
Cooling of Food after Heat Processing
Determination of Thermal Processes
5.9 Behaviour of Microorganisms under Freezing and Refrigeration
Environments
Growth of Microorganisms at Low Temperature
Effect of Freezing and Subfreezing Temperature on Microorganism
Factors Affecting Microorganisms during Freezing
Effect of Freezing on Constituents of Microbial Cells
5.10 Control of Microorganisms by Various Means
5.11 Principles Involved in Various Methods to Control Microbial Spoilage
of Food
5.12 Let Us Sum Up
5.13 Key Words
5.0 OBJECTIVES
After studying this unit, you should be able to understand:
• kind of foods based on their acidic reaction;
• relationship between pH of foods and heat resistance of microorganisms;
• difference in heat resistance of vegetative cells and spores of
microorganisms;
• what is thermal death time and how it is determined?;
• how microorganisms behave under freezing and refrigeration conditions?;
and
• the basic principle involved in various methods for controlling
microorganisms.
5
Controlling Organisms
5.1 INTRODUCTION
The foods, which we eat or drink are also excellent substrates (food) for
microorganisms, which are present in air, water, soil, utensils and even in raw
foods. Under suitable conditions of growth, particularly temperature and
moisture, the microorganisms multiply using these food items and produce
luxuriant growth. Many foods serve as carrier of various pathogenic and non-
pathogenic microorganisms, which may spoil the food by their growth, change
of chemical nature of food, release of unpleasant odour, production of various
harmful enzymes and toxins. Such foods are unfit for human consumption. For
these reasons, it is essential to prevent the entry and growth of microorganisms
in our food if present, by suitable processing. Before using a suitable process,
we should understand various factors which may influence the effectiveness of
a process.
5.2 CLASSIFICATION OF FOODS ACCORDING TO pH

Most foods are derived either from plants or from animals. In this course, we
are concerned with foods of plant origin and are known as vegetables or fruits
based on their use. These foods have different pH and are classified as low acid
foods, medium acid foods, acid foods and high acid foods.
a) Low acid foods
The foods having pH above 5.3 are called low acid foods. For example:
peas, corn, lima beans etc.
b) Medium acid foods
The foods which have pH between 4.3 and 5.3 are called medium acid
foods. For example: asparagus, beets, pumpkin, spinach etc.
c) Acid foods
Foods which have pH between 3.7 and 4.5 are called acid foods. For
example: pears, pineapple, tomatoes etc.
d) High acid foods
Foods having pH 3.7 or lower are included in this category. For example:
Berries and sauerkraut.
You must have noted that in general vegetables are low or medium acid foods
while fruits are acid or high acid foods.
Most foods are subjected to heat treatment or cooked before use. The heat
process is essential in the canning of foods to eradicate the microorganisms,
which may be present in the raw food or may enter from the environment
during processing; and may spoil the food if not eradicated.
The effect of pH of the food is complicated as the heating at high temperature
causes decrease in the pH of low or medium acid foods. Higher the original
pH, the greater the drop of pH by heating. Foods artificially adjusted to more
alkaline pH give increasing protection to spores against heat as pH increases
towards 9.
6
Concept,
5.3 RELATIONSHIP BETWEEN pH OF FOOD AND Determination of
HEAT RESISTANCE OF MICROORGANISMS Process Lethality
Requirements and
Importance
The pH of the foods influence the heat resistance of microorganisms. In
general cells or spores are most heat resistance in a substrate that is at near
neutrality. An increase in acidity or alkalinity hastens killing by heat.
However, a change towards acidic pH is more effective than a corresponding
change in alkalinity. This will be more clear from the Table 5.1, which shows
the effect of pH on heat resistance of spores of Bacillus subtilis. Therefore low
acid foods are heated under pressure (i.e. temperature above 100oC) while the
high acid foods are heated up to 100oC for making free from microorganisms.
Table 5.1: Effect of pH on heat resistance of spores of Bacillus subtilis in
1:15 M phosphate buffer (1000C)
pH Time of survival
(min)
4.4 2
5.6 7
6.8 11
7.6 11
8.4 7
5.4 HEAT RESISTANCE OF MICROORGANISMS AND

SPORES
The heat resistance of microorganisms varies widely within the species and
their forms:
• Thermophiles are more resistance than mesophiles and psychrophiles are
least resistance.
• Spores formers are more resistant than non-spore formers. Cocci are
usually more resistant than rods.
• The bacteria that clump considerably or form capasules are more resistant
to heat than those which do not.
• Cells high in lipid content are difficult to kill than cells having low lipid.
However, there are many notable exceptions to the above mentioned general
statements.
Higher the optimal temperatures for growth, the greater the resistance to heat.
Thermal death time of bacterial cells of a few microorganisms are exemplified
in Table 5.2.
7
Table 5.2: Thermal death time of bacterial cells
Bacteria Time Temperature

(min) (oC)
Gonococcus 2–3 50
Salmonella typhosa 4.3 60
Staphylococcus aureus 18.8 60
Escherichia coli 20-30 57.3
Streptococcus thermophilus 15 70-75
Lactobacillus bulgaricus 30 71
The heat resistance of microbial spores is much higher than the vegetative
cells, and vary with the species of microorganism and conditions during
sporulation. Resistance may vary from <1 min to 20 h at 100oC. Similar to
non-spore forming species, the spore forming species which have higher
optimal temperature for growth are more resistant to heat than those spore
forming species having lower optimal growth temperatures.
Simultaneous growth of two spores formers enhances the resistance of spores
having lower heat resistance, e.g. Clostridium perferingens growing with C.
sporogenes. Thermal death times of spores of a few microbial species are
given in Table 5.3.
Table 5.3: Thermal death times of bacterial species
Species Thermal death at 100oC

(min)
Bacillus anthracis 1.7
Bacillus subtilis 15-20
Clostridium botulinum 100-300
Clostridium calidotolerans 520
Bacillus coagulans (flat sour bacteria) > 1030
Above examples of thermal death times of vegetative cells as well as of spores

are at various concentrations of cells or spores in different substrates. These
values may change to lower or higher under different conditions.
What happens to enzymes in food by heat treatments?
Most foods and microbial enzymes are destroyed at 79.4oC, however some
may withstand higher temperatures, especially if high temperature for short
duration is employed. This is called pasteurization, which you will learn later
in this course.
5.5 THERMAL DEATH POINT (TDP)

Thermal death point is the lowest temperature at which all microorganism in a
liquid suspension are killed in 10 minutes.
8
Concept,
5.6 THERMAL DEATH TIME (TDT OR tD) Determination of
Process Lethality
The thermal death time is defined as the time required, at a given temperature, Requirements and
for heat killing of a population of a single species of microorganism in aqueous Importance
suspension. tD depends on the size of the population and on the pH of the
suspension. It is an important factor for controlling the microorganisms by heat
treatment or to determine the heat resistance of a microorganism.
5.7 DETERMINATION OF THERMAL DEATH TIME (tD)

The description of all the procedure and equipments/apparatus, used in the
determination of thermal death time is beyond the scope of this course.
However, a simple glass-tube method, used in canning industry is discussed
here.
5.7.1 Glass Tube Methods

A known population of cells of an axenic culture in a small volume (1 ml) of
buffer solution is sealed in small glass tube. The tubes are heated in a
thermostatically controlled bath to a selected temperature. The tubes are
selected periodically, cooled immediately to 0oC and the population of viable
cells is determined. In case of spores, the suspension is first pasteurized to kill
the vegetative cells, if present, before subjecting the spore suspension to dT
test. This is necessary as the lysed vegetative cells may have protective effect
on spore-population.
Care is also taken to break up the clumps and remove the growth medium by
centrifuging and washing. The volume of microbial suspension added to the
buffer is kept 1-2 percent to avoid the change in the composition of heating
substrate and the vials containing the suspension are brought to constant
temperature, usually 0oC before subjecting to heat treatment. If temperature
above 100oC is selected, oil bath instead of water bath is used. The test is
always made in multiple tubes. Viability of the surviving organism after heat
treatment should be checked on appropriate medium containing all the
nutrients, which support maximum growth of that organism.
5.7.2 Decimal Reduction Time

When a microbial population is heated, the cells die at a constant rate. For
example, suppose a population of 1 million (106) cells has been heated to a
high temperature for 1 minute and 90% has died. We are now left with 100,000
(105) cells. If the leftover population is heated for another 1 minute, 90% of the
population leaving 10000 (104) survivers. Thus the each one minute of heat
treatment will reduce 90% of the remaining population. This is shown in Table
5.4 and is known as decimal reduction time (DRT) and represented by D. It
can be defined as the time of heating at a temperature to cause 90% reduction
in the population of viable cells or spores.
9
Controlling Organisms Table 5.4: Microbial death rate at constant temperature
Time (min) Death min−1 Number of survivors

0 0 1,000,000
1 900,000 100,000
2 90,000 10,000
3 9,000 1,000
4 900 100
5 90 10
6 9 1
The D value (Decimal reduction time) may also be defined as the ‘time at
given temperature for the surviving population’ to be reduced by 1 log cycl.
Please refer Figure 5.1, if we extrapolate the times from 103 and 102, the time
difference is (3.5 – 2.5 = 1min is D). It means within 1 min initial population
will decrease by 90 per cent (from 1000 to 100, Difference 1000 – 100 = 900).
6
10
5
10
4
10
3
10
2
10
³D´
1
10
0
10
1 21 32 43 54 65
Time
Figure 5.1: A microbial death curve showing constant death rate of cells i.e. 90% per
minute D value may be calculated from the curve by extrapolating lines from
the Y axis and calculating the time difference.
5.7.3 Thermal Death Time Curve (TDT Curve)/Kinetics

The methods to construct TDT curve are: (i) The growth – no growth method
(ii) classical end point method and (iii) based on D values. We shall here
discuss the method based on D-values. D values can be calculated at different
temperatures (refer 5.7.2). As the temperature is increased, the D value
decreases. It means if we heat the sample at high temperature, it will take less
time to kill the microorganism in a given food sample. If we plot log D values
against temperature, we will get a straight line. From this we can derive
10
another important parameter in heat processing Z, the temperature change Concept,
Determination of
which results in a ten fold (1 log) change in D. Process Lethality
Requirements and
Z = (T2 – T1) Importance
D value for a known population of cells or spores of a microbial species at
several temperatures can be estimated. By plotting D values on the logarithmic
scale against temperature TDT curves can be constructed (Figure 5.2).
Time in minutes (Log D)
Temperature in Degrees Fahrenheit
Figure 5.2: TDT curve for spores of flat sour bacteria; 115,000 spores per ml in corn at
pH 6.1 (z = 19).
If we are interested to process the food item so that it may be free from any
spore or microorganisms, first we have to calculate D, Z and F values. F is the
time in minutes required to destroy the organism in a specified medium at
121oC. These values vary with the heat resistance and concentration of the test
organisms and with the medium in which it is heated. From the Z and F values
process times can be calculated.
5.7.4 12D Concept

Canned foods are susceptible to the spores of the organism Clostridium
botulinum, this organism causes botulism. As a safety measure, the canning
industry use the 12D heat treatment for low acid foods. In this process enough
heat is provided to reduce 1012 spores of C. botulinum to 1 spores per ml. It can
be explained as follows.
Assuming that D has a value of 0.21 minutes for spores of C. botulinum at
121oC and that out of 12 cans of food contains 1 spore. A heat process at
121oC for 2.52 min would reduce the spores to 1 spore in 1012 cans. The value
of 2.52 min has been arrived by the following formula:
Fo = D121 (log a – log b)
= 0.21 (log 1 – log 10−12)
= 0.21 × 12
= 2.52
11
5.8 DETERMINATION OF PROCESS LETHALITY
REQUIREMENTS AT LOW AND HIGH
TEMPERATURE
5.8.1 Heat Penetration

In heat/thermal processing of foods, the ratio of penetration of heat into a food
is very important, because every part of food in a container must get adequate
heat to prevent spoilage. The part which heats most slowly is the critical one;
and this is near the centre of container. Solids are heated by conduction while
liquids by convection. Foods which are semisolid are heated by combination of
both conduction as well as convection. Conduction is slow in foods and rapid
in metals.
The factor that determine the time required to bring the centre of the container
of food upto the sterilizing temperature are as follows:
i) Material of which the container is made
Glass has slower rate of penetration than metal (tin can).
ii) Size and shape of container
Large container takes long time than small container. Less the radius of
container faster the heating. For example: long slim cylinder will heat
faster than compact wide cylinder of the same volume.
iii) Initial temperature of food
For the foods with the higher initial temperature the average temperature
during heating is higher than in foods having lower initial temperature. A
high initial temperature is important in processing canned foods that heat
slowly. For example: pumpkin, cream style corn etc.
iv) Initial temperature of retort (steam sterilizer)
Fastest heating takes place in initially hot retort than retort having initially
low temperature. Therefore, preheated retort should be used.
v) Consistency of food, size and shape
Consistency of food, size of food pieces and even their shapes affect the
penetration of heat. The changes that takes place during heating (cooking
effect) also affect the heat penetration. Penetration of heat in large pieces
takes more time than in small pieces. This is applicable in foods, which
retain their shape and even size during heating. For example: peas, plum,
beats, whole grain corn etc.
Some foods become mushy or viscous during heating. In such foods
penetration is slow. For example: Sweet potatoes, pumpkin, etc.
Pieces that layer like asparagus layers or spinach layers interfere with
convection current
Sauces added on baked beans slow down heat penetration more than plain
sauce. Starch interferes increasingly as the concentration is raised. Sodium
chloride is never added in high concentration as it slows down rate of
12
heating. Rate of heat penetration also decreases with increasing
concentration of sugars; however this effect is counteracted some what by Concept,
Determination of
the marked decrease in the viscosity of sugar solutions. (Addition of sugar Process Lethality
and salt slow down the rate of heating) Requirements and
Importance
vi) Rotation and agitation
The rotation and agitation of the container of food during heat processing
hasten heat penetration, if food is in form of fluid. However, in some food
such operation may cause undesirable physical changes.
5.8.2 Cooling of Food after Heat Processing

The cooling operation involves the same principles of heat transfer as the
heating process. Rapid artificial cooling is recommended as slow cooling may
cause overcooking of the food and may allow the growth of thermophiles.
5.8.3 Determination of Thermal Processes

To determine the thermal processes data on the following two aspects are
required:
i) TDT curve for most heat-resistant organism likely to be present in food.
For example in low acid foods spores of Bacillus coagulanse (flat sour
organism), which is a thermophile, may be present.
ii) Heat penetration and cooling curves for the food when packed in specific
type of container of fixed size.
There are three methods to determine thermal processes:
¾ Graphical methods
¾ Formula method
¾ Nomogram method
The principle is similar for all the three methods; however the graphical
method is most simple and therefore explained here:
Graphical method to determine thermal processes
1. The TDT curve for the most heat resistance organism likely to be
encountered is determined in food being canned.
TDTs from this curve are converted to lethal rates for the various heating
temperatures. The lethal rate for a temperature is the reciprocal of the TDT.
If TDT is 400 min at 126.7oC to kill the spores in a food, the lethal rate
would be 1/400 i.e. 0.0025.
2. Heat penetration and cooling curve for the food and can size involved are
determined.
3. The lethal rates for different temperatures at the centre of can during the
length of heating and cooling process are plotted on the heat penetration or
cooling curves (see Fig. 5.3). In this figure the lethal rates are 0.01 units
and times are 10 minutes for a square. An area equivalent to 10 squares
under the lethality curve is unity. This means that the destruction of all the
spores or cells has been accomplished. If this area is less than unity (i.e.
less than 10 squares), the process is inadequate, and if more than
10 squares, it is greater than needed. The area beneath the curve is
13
Controlling Organisms measured by planimeter. In Figure 5.3 the heat treatment of 56 min at
126.7°C and 78 min at 121oC are adequate.
Figure 5.3: Equivalent lethality curves with retort at 126.7oC. (50,000 spores per ml)
5.9 BEHAVIOUR OF MICROORGANISMS UNDER

FREEZING AND REFRIGERATION
ENVIRONMENTS
Low temperatures are used to retard chemical reactions, and actions of food
enzymes and to reduce slow down or stop growth and activity of
microorganisms in food. The lower the temperature, the slower will be the
chemical reactions, enzyme and microbial growth; and temperature below
certain level will prevent the growth of a microorganism. Each microorganism
has a specific Cardinal temperature i.e. the minimal temperature and
maximal temperature at which it can grow and optimal temperature at which
the growth is fastest in the shortest time. As the temperature drops below the
optimal temperature towards the minimal, the rate of growth of organism
decreases and is slowest at the minimal temperature. Below minimal
temperature, the growth will stop but slow metabolic activity may continue.
Therefore cooling down of a food from normal temperature has different
effects on various microorganisms and slow down the growth of others;
however the extent would vary with different species and even strains of
microorganisms. A further decrease of 10oC would stop growth of more
organisms, and make still slower the growth of the others. Therefore storage at
low temperature influence the type of spoilage microorganisms which may
predominate as illustrated in Table 5.5.
Table 5.5: Growth rate of Pseudomonas fragii at various temperatures
Temperature (oC) Exponential growth rate

(generation h−1)
0.0 0.09
2.5 0.13
5.0 0.20
7.5 0.29
10.0 0.38
20.0 0.92
14
Concept,
5.9.1 Growth of Microorganisms at Low Temperature
Determination of
In general, freezing prevent the growth of most food born microorganisms and Process Lethality
Requirements and
refrigeration slow down growth rates except for Clostridium botulinum type E. Importance
Temperature below 5-6oC or less, effectively retard the growth of most food
spoilage microorganisms. However, some of the microorganisms may survive
at subfreezing temperature in frozen food. (Table 5.6 and 5.7)
Table 5.6: Microorganisms able to grow at subfreezing temperatures
Organisms Temperature (oC)

Molds
Claedosporium −6.7
Sporotrichum −6.7
Penicellium −4.0
Monilia −4.0
Yeast
Yeast (one strain) −34.0
Yeast (two strains) −18.0
Bacteria −5.0 to −17.8
Table 5.7: Different microorganisms able to grow in different frozen foods
Organisms Food Temperature (oC)

Bacteria Meat − 5.0
Cured meats −10.0
Fish −11.0
Vegetables −12.2
Ice cream −10.0
Yeast Meat − 5.0

Oysters −17.8
Molds Meat − 7.8
Vegetables − 7.8
Barries − 6.7
5.9.2 Effect of Freezing and Subfreezing Temperature on

Microorganisms
Feezing usually results in a considerable reduction in the number of viable
organisms in a food. The reduction in recoverable numbers can be the result of
lethal or sublethal effects.
a) Lethal effects
Though several cells of microorganisms are killed by freezing, a few may
remain viable with little or suspended metabolic activity. The lethal effects
15
Controlling Organisms are due to denaturation or flocculation of essential cell proteins or enzymes
possibly as a result of the increased concentration of solutes in the unfrozen
water or perhaps in part because of physical damage by ice crystal. Rapid
cooling of cells from an optimal temperature to 0oC is most injurious and
may lead to cell death due to cold shock. It is probably due to
crystallization of the liquids in the membrane which damage to the
permeability of the cell or due to the release of repair enzyme inhibitors,
e.g. ribonuclease inhibitors.
b) Sublethal effects
Freezing of food may cause cryo injury to the microorganisms present on
food. Such injured cells are referred as freeze injured, frost injured, cryo-
injured or metabolically injured. Such cells are not really dead and may
recover to start refunctioning, if repair time is permitted or additional
nutritional factors are added to the enumeration media. This fact of
cryoinjury is of great significance in the microbiological examination of
foods.
5.9.3 Factor Affecting Microorganisms during Freezing

1. The resistance or sensitivity of microorganisms vary with the kinds of
microorganisms, their form and growth phase. For example:
a) Thermophiles are most sensitive and psychrophiles most resistance.
b) Spore formers are more resistance than non-spore formers.
c) Bacteria in logarithmic phase are more sensitive than in stationary
phase.
d) Rods are more sensitive than cocci.
2. Microorganisms are classified on the basis of sensitivity to freezing:
i) Susceptible: Gram negative bacteria and vegetative cells of yeast and
molds.
ii) Moderately resistant: Gram positive streptococci and enterococci.
iii) Insensitive: Spore formers.
3. Freezing parameters:
i) Freezing rate
Rapid cooling upto 0oC is injurious to microbial cells.
ii) Freezing temperature
Freezing temperature between −4 to −10 is more injurious than −15 to
−30oC.
iii) Times of frozen storage
Maximum death of microorganisms occur during the freezing process.
Once the temperature is stabilized, the death is less and very low.
iv) Kinds of food
The composition of food influence the rate of killing of
microorganisms during freezing and storage. Sugar, salt, protein,
16
colloids, fats and other substance in foods provide protection to the Concept,
Determination of
microorganisms while low pH and high moisture hasten the death. Process Lethality
Requirements and
v) Slow defrosting of food cause death of microorganisms. Importance
vi) Freezing and thawing
Maximum casualty of microorganisms occur if foods are repeatedly
frozen and thawed.
5.9.4 Effect of Freezing on Constituents of Microbial Cells

With the lowering of temperature, water in cell gets frozen. As a result the
unfrozen fluids in cell gets concentrated with solutes (salts, proteins, nucleic
acids, etc). This may change the pH of cellular matter, concentrate electrolytes,
alter colloidal states, denature proteins and increase viscosity. Ice crystals also
form outside the cell due to the freezing of water molecules in food. These
extracellular ice crystals draw water from the cell causing dehydration or
concentration effect. Intracellular ice crystals due to the freezing of water may
rupture cell membrane and alter the permeability of cell membrane.
Intracellular ice crystals are more injurious than the extracellular ice crystals to
the microbial cells.
5.10 CONTROL OF MICROORGANISMS BY VARIOUS

MEANS
Most foods are either of plants or animal origin. Their spoilage is prevented by
controlling microbial growth on them by using various methods. Some
important methods are listed below:
1. Asepsis or preventing contacts with microorganisms.
2. Killing the microorganisms by heat treatment.
3. Keeping away from the microorganism e.g. maintenance of anaerobic
conditions in sealed or evacuated container.
4. Storage at low or ultra low temperature
5. Drying.
6. Increasing osmotic concentration in foods.
7. Mixing with preservative.
8. Change of pH
9. Mechanical destruction in industry: grinding or high pressure.
Usually a combination of more than one method is used to control the
microbial spoilage of food. For example: canned foods are preserved by heat
treatment followed by evacuation and sealing of can. Similarly, many
processed foods involve heating, mixing with preservative, evacuation of air
and sealing.
5.11 PRINCIPLES INVOLVED IN VARIOUS METHODS

TO CONTROL MICROBIAL SPOILAGE OF FOOD
The basic principle involved in various methods to control microbial spoilage
of foods are prevention or delay of microbial decomposition. These are
achieved by followings methods:
17
Controlling Organisms a) By keeping out microorganisms. For example: Aseptic condition during
processing of food.
b) By the removal of microorganisms: For example
• By removing the microbially infected portion of food, covering, skin
etc.
• Washing of raw food.
• Filteration.
c) By hindering the growth and activity of microorganisms by low
temperature, drying, anaerobic condition or chemical.
d) By killing the microorganisms: sterilization by heat or radiation.
5.12 LET US SUM UP

Human foods are excellent substrate for various microorganisms, which are
present in air, water, soil, raw foods, on the body of living and non-living
organisms. These microorganisms may harm the body if enter in the
body/tissue due to rupture or injury of outer covering layer. Many of these
microorganisms may enter the food during the processing and remain either
dormant for long time till get the right conditions for multiplication, if
consumed, cause unpleasant odour or taste making the food unfit for
consumption. Such spoilage is called microbial spoilage of food.
Prevention of microbial spoilage of foods depend upon the kind of foods. The
acidity or pH of the food influence heat resistance of microorganisms.
Microbial cells are more resistance to heat at pH near to neutrality and
therefore, low acid foods are heated under pressure to kill the microorganisms,
where as the high acid foods are heated upto 100oC for short duration to make
free from organisms.
The thermal death time (TDT) which is defined as the time required to kill a
known population of microorganisms at a certain temperature by heat. This
value differ for different microorganisms and also on various environmental
factors. TDT values are of great significance in heat control of
microorganisms.
The death of microbial cells is at constant rate and is expressed in D-value i.e.
decimal reduction time. D-values of a microbial species at several temperatures
are estimated and plotted against temperature to construct curve, which are
used to determine the actual time required for killing microbes in a food at a
particular temperature. In practice 12D concept is used in canning industry.
This is based on the reduction of 1012 spores ml−1 of Clostridium botulinum in
low acid food to 1 spore ml−1 by heat treatment.
Low temperature under freezing and refrigeration reduce microbial growth and
at 0oC most of the microorganisms stop the growth. However a few exceptions
of molds, yeast and bacteria, which may grow up to −6 to −38oC have been
encountered.
Freezing may have lethal or sub-lethal effect on microbial cell. In sub-lethal
effect the freezing injury makes the microbial cell unable to multiply, however
under favourable conditions, such cells may get repaired and grow.
18
The various method for controlling microorganisms are asepsis, heat treatment, Concept,
Determination of
anaerobic conditions, storage at low temperature, drying, increasing osmotic Process Lethality
concentration, mixing with preservatives, change of pH, irradiation and Requirements and
mechanical destruction by grinding or high pressure. The basic principle Importance
involving in these methods are; prevention or delay of microbial
decomposition by keeping out or removing the microorganisms or creating
suboptimal conditions for survival and growth of microorganisms or by killing
the microorganisms.

1. What are acid foods?
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
2. Based on acidic reaction of food, which foods require more heating time to
kill the microorganisms?
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
3. Which of the two is more resistant to heat and why? Escherichia coli or
Clostridium botulinum.
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
19
Controlling Organisms 4. Define thermal death time.
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
5. Define thermal death point.
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
6. What do you understand by decimal reduction time?
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
7. Why TDT curves are constructed?
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
8. What is 12D concept?
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
20
9. Thermal processing of food is affected by the …………………. of food. Concept,
Determination of
………………………………………………………………………………. Process Lethality
Requirements and
………………………………………………………………………………. Importance
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
10. Which method is most common and simple to determine thermal
processes?
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
11. Will all microorganisms stop growing, if the food is stored at 0°C?
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
12. What is cryoinjury?
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
13. Should the food be cooled rapidly to 0oC to kill microbial cells?
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
21
Controlling Organisms 14. Intracellular ice cause ……………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
15. Microorganisms in food can be controlled by ……………………………...
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
16. Spores are more resistant to heat than ………………………………………
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
17. Which of the following microorganisms is insensitive to freezing:
i) Escherichia coli
ii) Vibrio cholerae
iii) Clostridium botulinum
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
18. Freezing of food is one of the important method to prevent their spoilage
on what principle it is based?
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
22
19. Most microorganisms should be killed but the enzyme should not be Concept,
Determination of
destroyed. What method of heat processing should be applied? Process Lethality
Requirements and
………………………………………………………………………………. Importance
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
20. If heat treated food is cooled slowly, what kind of microorganisms during
storage may grow.
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
5.13 KEY WORDS

TDP : Thermal death point is the lowest temperature at
which all microorganisms in a liquid suspension
are killed in 10 minutes.
TDT : Thermal death time is defined as the time

required, at a given temperature, for heat killing
of a population of a single species of
microorganism in aqueous suspension.
D Value : Decimal reduction time is defined as the time of

heating at a temperature to cause 90% reduction
in the population of viable cells or spores.
12D Concept : A process in which enough heat is provided to

reduce 1012 spores of clostridium botulinum to
1 spores per ml in canned food.

EXERCISES
"
Food which have pH between 3.7 and 4.5. Example: pear, pineapple and
tomatoes.

Low acid foods.
23
Controlling Organisms 3. Your answer should include the following points:
Clostridium botulinum as it is spore former.

Thermal death time is the minimum time required to kill a population of
microorganisms in liquid suspension at certain temperature.

Thermal death point is the minimum temperature required to kill
population of microorganimsm in liquid in 10 min.

By any killing agent, the death of microorganisms is at a constant rate.
Suppose a population of 106 cells are subjected to heat treatment, 90% of
them will die in first minute. Of the remaining population, 90% will die in
next one minute and so on until all the population die.

TDT curves are constructed to determine the thermal process time at
different temperature to kill the microorganisms in a food.

It is a concept of time required to reduce 1012 spores of Clostridium
botulinum in 1 ml suspension to 1 spore by heating to certain temperature.
It is used in canning industry.

Thermal processing is affected by the consistency of food.

Graphic method.

No; psychrophiles will continue growing.

Injury to microbial cells during freezing.

Yes.

Rupture of cell membrane leading to the leakage of cell constituent.

Sterilization
24
16. Your answer should include the following points: Concept,
Determination of
Vegetative cells Process Lethality
Requirements and
Importance
Clostridium botulinum

Reducing or suspending the metabolic activity and growth.

Pasteurization

Thermophiles

1. Adams, M.R. and Moss, M.O. (1995) Food Microbiology. Low cost Indian
addition published by New Agro International (P) Limited Publishers, New
Delhi 398p.
2. Frazier W.C. and Westhofl D.C. (1967) Food Microbiology. Tata McGraw
Hill Publishing Co., New Delhi 540p.
3. Jay, J.M. (1970) Modern Food Microbiology. Van Nostrand Reinhold Co.,
London. 328p.
25
UNIT 6 THERMAL CONTROL OF
MICROORGANISMS
Structure
6.0 Objectives
6.1 Introduction
6.2 Thermal Preservation of Foods
6.3 Heat Preservation Processes
Sterilization
Commercially Sterile Food Products
6.4 Pasteurization
Low Temperature Long Time (LTLT)
High Temperature Short Time (HTST)
Ultra High Temperature (UHT) Processing Treatments
6.5 Preservation by Moist Heat
Thermal Death Time (TDT)
D-Value
Z-Value
12-D Concept
F-Value
6.6 Microbiology of Thermally Processed Food
Spoilage by Thermophilic Bacteria
Spoilage by Mesophilic Organisms
6.7 Let Us Sum Up
6.8 Key Words
6.0 OBJECTIVES
After studying this unit, you should be able to:
• make you understand the principals of food preservation using high

temperatures;
• explain various processes for thermal preservation;
• describe the terms associated with heat preservation; and
• discuss the causes of spoilage of heat processed products and common
spoilage organisms.
6.1 INTRODUCTION
The minute living organisms, not visible to the naked eye and classified as
microorganisms, are virtually everywhere. Those of primary medical interest
are bacteria, viruses, spirochetes, rickettsia, molds, and yeasts. They flourish in
the soil of the farms that grow our grains, fruits and vegetables, on the hides
and feathers of our meat animals and on the fins and organs of the seafood we
eat. Though there are innumerable genera and species of each class of
microorganisms, not all are of medical significance or involved in disease
processes. Many of these organisms can be beneficial. In fact the
predominance are composed of those that are necessary to food production,
friendly environments, and metabolic processes, examples being cheese/wine
26
production, decomposition of organic matter, and digestion of food. Lactic acid Thermal Control of
bacteria in the dairy industry, yeasts in the baking and brewing industries, Microorganisms
molds for specialty cheeses are examples of “domesticated” microorganisms.
But in a many cases these microscopic flora create serious problems in our
food supply. These problems fit into two categories. Food spoilage occurs
when the food becomes unpalatable as the result of microbial growth. Products
develop undesirable flavors, odors, appearances or textures via microbial
action. The other, more dangerous problem is food poisoning, which occurs
when the organisms present in food cause human illness or death. The
microorganisms either produce a toxin or cause an infection, generally
intestinal, when consumed. Those organisms that spoil product are typically
called spoilage organisms, while those that can make people sick are referred
to as pathogens. Therefore, to avoid both of these problems we need to
understand the techniques which prevent their growth.
Food preservation has been around for a long time. The technique of food
preservation may vary but the goal of food preservation has been the same i.e.
to keep the food in a stable condition over a period of time so that it will
not spoil or make people sick. There are various ways of food preservation,
including chemical preservation, modified atmospheres, irradiation, low
temperature preservation, preservation by drying and high temperature
preservation.
6.2 THERMAL PRESERVATION OF FOODS
The most common method of killing microorganisms is to subject them to a

heat treatment. High temperatures act by killing vegetative cells and also
spores and denaturing the food enzymes. It may also act to destroy toxins
produced by certain microorganisms.
The heat treatment used depends on the following factors. In order to safely
preserve foods using heat treatment, the following must be known:
• What time-temperature combination is required to inactivate the most heat

resistant pathogens and spoilage organisms in one particular food? The
higher the temperature, the less time needed and vice versa. Heat
destruction of microorganisms is a gradual phenomenon the longer is the
treatment time at lethal temperatures, the larger is the number of
microorganisms killed. As higher is treatment temperature the shorter is the
time required to kill microorganisms and lower is heat induced damage to
food products.
• What are the heat penetration characteristics in one particular food,

including the can or container of choice if it is packaged?
• What are the types of micro-organisms present in the food material? The
thermal death time of different microorganisms vary widely with the
species. Different foods will support growth of different pathogens and
different spoilage organisms so the target will vary depending upon the
food to be heated.
• What is the concentration of the microorganisms? The higher the

concentration, the more time is needed.
27
Controlling Organisms • What is the state of the microorganism? Spores are more resistant than
vegetative cells. Organisms that have been stressed are more susceptible to
heat.
• What is effect of heat on the product? Obviously, the temperatures required

to kill microorganisms affect most food products.
• The degree of heat penetration also must be considered. Preservation

processes must provide the heat treatment which will ensure that the
remotest particle of food in a batch or within a container will reach a
sufficient temperature, for a sufficient time, to inactivate both the most
resistant pathogen and the most resistant spoilage organisms if it is to
achieve sterility or "commercial sterility", and to inactivate the most heat
resistant pathogen if pasteurization for public health purposes is the goal
• What is the effect of various environmental factors, such as pH and salts or

solutes. Food acidity / pH value has a tremendous impact on the target in
heat preservation/ processing.

1. Differentiate between spoilage organisms and pathogens?
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2. What is food preservation?
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3. Write various ways of food preservation?
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28
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4. List important factors which need consideration in order to safely preserve Thermal Control of
foods using heat treatment? Microorganisms
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6.3 HEAT PRESERVATION PROCESSES

The most common type of food preservation by high temperatures is cooking.
However, there are many more processes that involve the use of temperature
above that of ambient air.
6.3.1 Sterilisation
By sterilisation we mean complete destruction of micro-organisms. Because of
the resistance of certain bacterial spores to heat, this frequently means a
treatment of at least 121° C (250° F) of wet heat for 15 minutes or its
equivalent. It also means that every particle of the food must receive this heat
treatment. If a can of food is to be sterilized, then keeping it at 121° C or retort
for the 15 minutes will not be sufficient because of relatively slow rate of heat
transfer through the food in the can to the most distant point. In such cases
time needs to be increased.
6.3.2 Commercially Sterile Food Products

Sterile means free of life of every kind and is actually achieved under very
limited conditions. The control of microorganisms in medicine, industry,
sanitation, food, and feed service involves the acceptance that sterilization is
most often not achievable without destroying or severely damaging the
product. Only Low Acid Foods [LAF], having pH higher than 4.6, must be
sterilized, because all microorganisms are able to grow in LAF. More acid
products [pH equal/lower than 4.6] do not allow the growth of pathogenic
spore forming bacteria. Then Sterilization is not required. Hence Commercial
Sterility is a term commonly used in the canning industry meaning the
condition achieved by the application of heat sufficient to render the processed
product free from viable microorganisms (including those of known public
health significance), capable of growing in the food under normal non-
refrigerated temperatures at which the food is likely to be held during
distribution and storage.
The process was developed by Nicolas Appert and published in 1810. All
vegetative organisms that could grow in the food and cause spoilage under
normal handling and storage conditions are destroyed. However commercial
sterile foods may contain a small number of heat resistant bacterial spores, but
they will not multiply under normal handling and storage conditions. Types of
commercially sterile processes include canning, bottling, and aseptic
processing. Commercial sterilization must make sure the numbers of surviving
spores are at an acceptable level. The acceptable number of spores will depend
on what type of damage they are capable of causing if they start to grow.
29
Controlling Organisms Check Your Progress Exercise 2
# Note: a) Use the space below for your answer.
1. What is sterilization?
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2. What is commercial sterility? How commercial sterilization is different
from sterilization?
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6.4 PASTEURIZATION
In the previous section you have read about strerilzation and commercially
sterile foods. Now we will discuss milder heat treatment i.e. pasteurization. It
is one type of preservation by heat that most people are familiar with. It is
process of heating a liquid, particularly milk, to a temperature between 55 and
70 degrees C (131 and 158 degrees F), to destroy harmful bacteria. This
process is named after the French chemist Louis Pasteur, who devised it in
1865 to inhibit fermentation of wine. Pasteur's aim was to destroy bacteria,
molds, spores etc. He discovered that the destruction of bacteria can be
performed by exposing them to certain minimum temperature for certain
minimum time and the higher the temperature the shorter the exposure time
required. Through this process, all of the bacteria (such as E.coli, Lysteria, and
Salmonella) are not destroyed, it still exists in pasteurized products, but in very
low concentrates. Refrigeration keeps the bacteria from further growth, very
low. There are other bacteria that aren't harmful to humans, but they produce
acids that turn the milk sour. They are called lactophilic because they consume
the lactose in milk and produce acids. The extent of the pasteurization
treatment required is determined by the heat resistance of the most heat-
resistant enzyme or microorganism in the food. For example, milk
pasteurization is based on Mycobacterium tuberculosis and Coxiella burnetii.
These two organisms are the most heat resistant of pathogens that are not spore
forming. Milk is a product that most people know is pasteurized. It is
pasteurized by heating at a temperature of 63 degrees C (145 degrees F) for 30
minutes, rapidly cooling it, and then storing it at a temperature below 10
degrees C (50 degrees F).
30
Pasteurization is a comparatively low order of heat treatment, generally at a Thermal Control of
temperature below the boiling point of water. The more general objective of Microorganisms
pasteurization is to extend product shelf-life from a microbial and enzymatic
point of view. Pasteurization is frequently combined with another means of
preservation - concentration, chemical, acidification, etc. Blanching is a type of
pasteurization usually applied to vegetables mainly to inactivate natural food
enzymes. Depending on its severity, blanching will also destroy some
microorganisms.
Depending upon time and temperature treatment there are three kinds of
pasteurization processes.
6.4.1 Low Temperature Long Time (LTLT)

Where pasteurization time is in the order of minutes and related to the
temperature used; two typical temperature/time combinations are as following:
63°C to 65°C for 30 minutes or 75° C over 8 to 10 minutes. Pasteurization
temperature and time will vary according to:
• nature of product; initial degree of contamination;
• pasteurized product storage conditions and shelf life required.
In LTLT pasteurization it is possible to define three phases:
• heating to a fixed temperature;
• maintaining this temperature over the established time period
(= pasteurization time);
• cooling the pasteurized products: natural (slow) or forced cooling.
This is a typical batch method where a quantity of milk is placed in an open vat
and heated to 63°C and held at that temperature for 30 min. Sometimes filled
and sealed bottles of milk are heat-treated in shallow vats by that method and
subsequently cooled by running water.
6.4.2 High Temperature Short Time (HTST)

HTST pasteurization is characterized by a pasteurization time in the order of
seconds and temperatures of about 85° to 90° C or more, depending on holding
time. Typical temperature/time combinations are as follows:
• 88° C for 1 minute
• 100° C for 12 seconds
• 121°C for 2 seconds.
While bacterial destruction is very nearly equivalent in low and in high
pasteurization processes, the 121°C/2 seconds treatment give the best quality
products in respect of flavour and vitamin retention. This is the most widely
used process. The “hold time” is typically 125°C to pasteurize milk. This
process is a continuous method and a “hold tube” is used. The “hold tube” is
the tubing in the system that transports the milk after the point where the
product is heated. The tubing is sized so that it takes 15-20 seconds for the
product to travel all the way through it. When it reaches the end, if the
temperature is at 125°C or hotter, it is considered pasteurized. It is then cooled
and put in storage. The warm milk passes through the cooling section where it
is cooled to 4° C or below by coolant on the opposite sides of the thin, stainless
steel plates. The cold, pasteurized milk passes on to a storage tank filler for
packaging. 31
Controlling Organisms 6.4.3 Ultra High Temperature (UHT) Processing Treatments
In this method, milk is exposed to a brief, intense heating, normally to
temperatures in the range 135-140 °C but for a very short time, a second or
less. The treatment kills all microorganisms that would otherwise spoil the
product. The process depends upon a fairly complicated sterilizer/aseptic
filling design. The two stages of effective heat sterilization followed by aseptic
filling represent an integral system. Frequently the packaging material for UHT
milk is cardboard which must be chemically sterilized prior to the filling
operation
This method is used mainly for coffee creamers and boxed juices with the
exception of Europe. They pasteurize milk in this way. After this is done,
there is no need to refrigerate, because it sterilizes the product. Sometimes the
products can have a "cooked" taste that can be detected after being brought to
such a high temperature.
Industrial applications of pasteurization process are mainly used as a means of
preservation for milk and fruits and vegetable juices and specially for tomato
juice.

1. Name the scientist who invented Pasteurization. In which food material this
process is used the most?
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2. Why pasteurization of milk is important?
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32
3. What are the factors responsible for microbial inactivation during Thermal Control of
pasteurization? Microorganisms
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4. Does pasteurization kill all the bacteria in the product?
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5. Name the organisms on which milk pasteurization time and temperature is
based?
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6. Why HTST pasteurization is better treatment than LTLT?
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6.5 PRESERVATION BY MOIST HEAT

Moisture levels of the food material are a definite influencing factor in the
shelf life of food. Moist heat readily kills viruses, bacteria, and fungi by
33
Controlling Organisms denaturing enzymes whereas dry heat kills by oxidation of cell contents. There
is a correlation between the percent of water and the effectiveness of heat to
kill microorganisms. Moist heat is a more effective sterilizing agent than dry
heat because the moisture increases the rate of heat penetration. Moist heat
requires less heat (temperature or time) than dry heat (121°Cfor 10 min of
moist heat is equivalent to about 30 min at 200°C dry heat).
For this reason a lot of sterilization procedures use super heated steam that
provides moist heat. Temperature over 1000C requires heating under elevated
pressure, (like in a pressure cooker) 1210C require 100 kpa extra pressure. It is
important that no air pockets are allowed to develop when a product being
sterilized with steam. In air pockets food is exposed to dry heat and thus the
time /temperature is not enough. Moist heat denatures proteins which destroys
essential enzyme activities.
Endospores are much more resistant to heat than are vegetative cells. For this
reason, moist heat sterilization is aimed at ensuring that endo spores are killed.
Terms Associated with Heat Preservation
Scientists use different terms to refer the effect of moist heat on the
preservation of food. These terms include thermal death time, D-value, and z-
value.
6.5.1 Thermal Death Time (TDT)
Thermal death time is the amount of time that is necessary to kill a specific
number of microbes at a specific temperature. This value is obtained by
keeping temperature constant and measuring the time necessary to kill the
amount of cells specified.
6.5.2 D-Value
The term D-value refers to decimal reduction time. This is the amount of time
that it takes at a certain temperature to kill 90% of the organisms being studied.
Thus after an organism is reduced by 1 D, only 10% of the original organisms
remain. The population number has been reduced by one decimal place in the
counting scheme. When referring to D-values it is proper to give the
temperature as a subscript to the D. For example, a hypothetical organism is
reduced by 90% after exposure to temperatures of 149°C for 2 minutes, Thus
the D-value would be written as D300F = 2 minutes. Several parameters help us
to do thermal calculations and define the rate of thermal lethality. The D-value
is a measure of the heat resistance of a microorganism. It is the time in minutes
at a given temperature required to destroy 1 log cycle (90%) of the target
microorganism. (Of course, in an actual process, all others that are less heat
tolerant are destroyed to a greater extent). For example, a D-value at 72°C of 1
minute means that for each minute of processing at 72°C the bacteria
population of the target microorganism will be reduced by 90%. D-values vary
according to the temperature, species of microorganisms, number of initial
population, and other factors that may affect thermal resistance. In the
illustration below, the D-value is 14 minutes (40-26=14 min.) and would be
representative of a process at 72°C.
34
Thermal Control of
Microorganisms
(40-26
=14)
Figure 6.1: The D-value
6.5.3 Z-Value
The Z-value reflects the temperature dependence of the reaction. It is defined
as the temperature change required to change the D-value by a factor of 10.
While the D-value gives us the time needed at a certain temperature to kill an
organism, the Z-value relates the resistance of an organism to differing
temperatures. In the illustration below the Z-value is 10°C.
Figure 6.2: The Z-value
The Z-value allows us to calculate a thermal process of equivalency, if we

have one D-value and the Z-value. So, if it takes an increase of −12°C to move
the curve one log, then our Z-value is 10. So then, if we have a D-value of
35
Controlling Organisms 4.5 minutes at 66°C, we can calculate D-values for 71°C by reducing the time
by 1 log. So, our new D-value for 71°C is 0.45 minutes. This means that each -
12°C increase in temperature will reduce our D-value by 1 log. Conversely, a -
12°0C decrease in temperature will increase our D-value by 1 log. So, the D-
value for a temperature of 60°0C would be 45 minutes.
Reactions that have small Z-values are highly temperature dependent, whereas
those with large Z-values require larger changes in temperature to reduce the
time. A Z-value of 10°C is typical for a spore forming bacterium. Heat induced
chemical changes have much larger Z-values that microorganisms, as shown
below:
Z (°C) D121(min)
bacteria 5-10 1-5

enzyme 30-40 1-5
vitamins 20-25 150-200
pigment 40-70 15-50
Figure 6.3 illustrates the relative changes in time temperature profiles for the
destruction of microorganisms. Above and to the right of each line the
microorganisms or quality factors would be destroyed, whereas below and to
the left of each line, the microorganisms or quality factors would not be
destroyed. Due to the differences in Z values, it is apparent that at higher
temperatures for shorter times, a region exists (shaded area) where pathogens
can be destroyed while vitamins can be maintained. The same holds true for
other quality factors such as colour and flavour components. Thus in milk
processing the higher temperature, shorter time (HTST) process (72°C/16 sec)
is favoured compared to a lower temperature longer time (batch or vat) process
since it results in a slightly lower loss of vitamins and better sensory quality.
Figure 6.3: The relative changes in time temperature profile
36
Alkaline phosphatase is a naturally-occurring enzyme in raw milk which has a Thermal Control of
similar Z value to heat-resistant pathogens. Since the direct estimation of Microorganisms
pathogen numbers by microbial methods is expensive and time consuming, a
simple test for phosphatase activity is routinely used. If activity is found, it is
assumed that either the heat treatment was inadequate or that unpasteurized
milk has contaminated the pasteurized product.
6.5.4 12-D Concept

Canned foods are susceptible to the spores of the organism Clostridium
botulinum. This is the organism that causes botulism. Their bacterial spores
can survive many heat treatment processes. However, in modern food
production, canned foods are subjected to a time/temperature process that will
reduce the probability of the survival by the most heat-resistant C. botulinum
spores by 12 logs or 12-D at 250F (the temperature used in the calculation of
most commercial 12-D processes is 250F, and the D-value for this organism at
250F is 0.21 minutes). This process is based on the assumption of the number
of surviving spores in one can. If it is assumed that a container had one million
spores per can the heat treatment needed to reduce the number to one in one
million i.e. from 106 to 10−12 involves a reduction of twelve decimal places i.e.
from 1,000,000 to 0.0000001
6.5.5 F-Value
If we assume that there are 10 surviving spores in one can, then we can
calculate the time for a 12-D process to occur by using the following formula:
• F0 = D250F (log a − log b), where a = initial population and b = final
population.
• So F0 = (0.21min.) (log 101 − log 10−11), we move down 12 log values
(1 − (−11)) = 12.
• So, F0 = (0.21min.) (1 − (−11)), or 0.21 × 12 = 2.52 minutes.
Simply put, (D-value at 250F) × (12) results in a 12-D process.
The killing effect of a time / temperature combination is referred to as the F-
value.
F = 1 is heat killing effect equivalent to 1 min at 121°C.
The F-value required to achieve a 12D cook depends on the resistance of the
particular type of bacteria. One of the most resistant species is Bacillus
stearothermophilus which is 5 or 6 time more resistant than C. botulinum.
A 12-D cook for Cl. botulinum may require an F value of 2.52
A 12-D cook for B. stearothermophilus may require F = 18
From food safety angle, the microorganisms of greatest concern are Salmonella
sp., Clostridium perfringens, Staphylococcus aureus, Listeria monocytogenes,
Campylobacter sp., and E. coli, all of which have much lower z values and
consequently should achieve a 12D process in a shorter time. Bacillus of the
most heat resistant strains of bacteria known.
37
Controlling Organisms Check Your Progress Exercise 4
# Note: a) Use the space below for your answer.
1. Why moist heat is a more effective sterilizing agent than dry heat?
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2. Define D-value and Z-value. How these terms are inter-related?
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3. What is the principal of 12D concept?
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4. Define F-value?
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38
Thermal Control of
6.6 MICROBIOLOGY OF THERMALLY PROCESSED Microorganisms
FOOD
As you can now very well understand the heat is an important way of
preserving foods. Still some thermally processed foods undergo spoilage due to
chemical or biological reasons. The most important chemical spoilage of
canned foods is the hydrogen swell produced as a result of action of food acids
with the metals. Biological spoilage of thermally processed foods by
microorganisms may result either from the survival of organisms after the heat
treatment or leakage of the container permitting entrance of the
microorganisms. Surviving organisms may be vegetative cells or spore formers
depending upon the heat treatment. Acid foods are processed at temperature
around 100OC which result in the killing of all vegetative cells of bacteria,
yeasts and molds. Only bacterial spores may survive stearothermophilus is a
non-pathogenic organism that has been shown to be one but these do not grow
in acid foods. On the other hand, meat, vegetables and milk are processed at
low temperatures. This may eliminate vegetative cells but not the spores,
which germinate later and cause spoilage. Microorganisms that enter through
leaks during cooling need not necessarily be heat resistant.
6.6.1 Spoilage by Thermophilic Bacteria

Under processing of low acid foods result in spoilage by thermophilic
(microorganisms which require high temperature, more than 45°C for their
growth) bacteria such as Bacillus coagulance, Bacillus stereothermophilus.
These microbes produce heat resistant endospores than can survive 121°C for
4-5 minutes. These organisms produce acid without gas. This is known as flat
sour spoilage. Some times In Low and medium acid foods the cans swell due
to production of carbon dioxide and hydrogen by Clostridium
thermosaccharolyticum. This is known as thermophilic spoilage. Sulphide
spoilage is caused by Clostridium nigrificans in low acid foods. Spores of this
bacterium are not very heat resistant and their presence is indicative of under
processing. Spoilage is indicated by the the presence of H2S and blackening of
material. Sources of all these material are generally, the plant equipment,
sugar, starch, soil etc.
6.6.2 Spoilage by Mesophilic Organisms

Mesophilic microorganisms are those microorganisms which grow best at
temperature 25-45°C. Spoilage of canned foods by mesophilic organisms is
indicative of under processing and is caused by species of Bacillus,
Clostridium, Yeast and fungi. Clostridium butyricum and C. pasteurianum
produce a butyric acid type of fermentation in acid or medium acid foods with
swelling of the container by the production of CO2 and H2. Other species of
Clostridia may produce H2S causing can to swell. These putrefactive
anaerobes (Micro organism that grow in the absence of oxygen) generally
grow in low acid foods such as peas corn, meat, poultry etc. but some times
may also spoil medium acid foods.
Some Bacilli such as Bacillus subtilis and B. mesentroides have been found to
grow in poorly evacuated cans of sea foods, meat and milk. The gas forming
Bacilli (B.polymyxa, B. macerans) are also reported to cause spoilage of
canned peas, spinach, peaches and tomatoes.
39
Controlling Organisms The presence of non spore forming bacteria in canned food is an indicative of
leak or under processing. Streptococcus thermophilus, Pseudomonas,
Micrococcus and proteus have also been reported to cause spoilage of
thermally processed products. Molds, yeast and their spores are destroyed at
pasteurization temperature. Their presence is indicative of under processing or
leakage. Spoilage of canned fruits and fruit products by yeasts may result in
CO2 production and spoilage of cans. Film yeast and fungi grow on the surface
and cause degradation of the product.

1. What are the probable reasons for biological spoilage of thermally treated
foods?
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2. Name two thermophilic microorganisms responsible for flat sour spoilage.
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3. What is thermophilic spoilage?
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40
4. What do you understand about sulphide spoilage of low acid foods? Thermal Control of
Microorganisms
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5. Name some mesophilic bacteria responsible for spoilage of thermally
treated foods.
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6.7 LET US SUM UP

Thermally processed foods are those that have been i) heated in hermetically
sealed container ii) have been filled hot into a container which is then closed
and cooled. The purpose of these processes is to destroy pathogenic
microorganisms and those that might grow and cause spoilage of the particular
food. The food that are commercially sterile are those that will not support
microbial growth when exposed to the usual temperatures during storage,
transport and marketing. However, they may not be completely free of
microorganisms. Pasteurization is heat treatment to inactivate some
microorganisms. Thermally processed foods may get spoiled due to under
processing or leakage.
6.8 KEY WORDS

Thermophilic : Microorganisms which grow at temperature
above 45°C.
Mesophilic : Microorganisms which grow at temperature 25-
45°C.
Under processing : Lower time or temperature treatment.
41
EXERCISES
Check You Progress Exercise 1
• Microorganisms that spoil product are typically called spoilage

organisms.
• Microorganisms that can make people sick are pathogens.
• to keep the food in a stable condition over a period of time

• to prevent it from spoilage or making people sick.
• chemical preservation
• modified atmospheres
• irradiation
• low temperature preservation
• preservation by drying
• high temperature preservation.
• Time-temperature combination
• heat penetration characteristics of particular food
• the type of micro-organisms present in the food material
• the thermal death time of different microorganisms
• type of food
• concentration of the microorganisms
• state of the microorganism
• effect of heat on the product
• the degree of heat penetration
• food acidity / pH value.
• complete destruction of micro-organisms.
• the condition achieved by the application of heat sufficient to render the

processed product free from viable microorganisms
• capable of growing in the food under normal non-refrigerated
temperatures at which the food is likely to be held during distribution
and storage
42
• Unlike sterilization here the food is not completely free of Thermal Control of
microorganisms. Microorganisms
• process created by Louis Pasteur

• aimed to destroy bacteria, molds, spores etc.
• discovery about the destruction of bacteria by exposing them to certain
minimum temperature for certain minimum time
• the higher the temperature the shorter the exposure time required.
• process applied to milk.
• Public Health Aspect − to make milk and milk products safe for human
consumption by destroying all bacteria that may be harmful to health
(pathogens)
• Keeping Quality Aspect − to improve the keeping quality of milk and
milk products. Pasteurization can destroy some undesirable enzymes
and many spoilage bacteria. Shelf life can be 7, 10, 14 or up to 16 days.
3. Your answer should include the following point:
• extent of microbial inactivation depends on the combination of

temperature and holding time.
• Through pasteurization all of the bacteria are not completely destroyed,

it still exists in pasteurized products, but in very low concentrates.
• Refrigeration keeps the bacteria from further growth, very low.
• thermal death time studies for the most heat resistant pathogens found
in milk
• Coxiella burnetii and Mycobacterium tuberculosis are the most heat
resistant non spore forming pathogens.
• bacterial destruction is very nearly equivalent in LTLT and in HTST

pasteurization processes but
• HTST treatment give the best quality products in respect of flavour and
vitamin retention.
43
Controlling Organisms Check You Progress Exercise 4
• moisture increases the rate of heat penetration.

• Moist heat requires less heat (temperature or time) than dry heat
• 121°C for 10 min of moist heat is equivalent to about 30 min at 200°C
of dry heat.
• D-value is the amount of time that it takes at a certain temperature to

kill 90% of the organisms being studied.
• Z value is defined as the temperature change required to change the D-
value by a factor of 10.
• D-value gives us the time needed at a certain temperature to kill an
organism
• Z-value relates the resistance of an organism to differing temperatures.
• process based on the assumption of the number of surviving spores in

one can.
• canned foods subjected to a time/temperature process that will reduce
the probability of the survival of the most heat-resistant C. botulinum
spores by 12 logs i.e. from 1,000,000 to 0.0000001.
• killing effect of a time / temperature combination.
• Under processing or leakage may be the cause of spoilage of thermally

treated foods.
• Bacillus coagulance and Bacillus stereothermophilus.
• In Low and medium acid foods the cans swell due to production of
carbon di oxide and Hydrogen by Clostridium thermosaccharolyticum.
• caused by Clostridium nigrificans in low acid foods.

• Spores of Clostridium nigrificans are not very heat resistant and their
presence is indicative of under processing.
44
• Spoilage is indicated by the the presence of H2S and blackening of Thermal Control of
material. Microorganisms
• Sources of all these material are generally, the plant equipment, sugar,
starch, soil etc.
• Clostridium nigrificans
• C. pasteurianum
• Bacillus subtilis
• B. mesentroides
• B.polymyxa
• B. macerans
• Streptococcus thermophilus
• Pseudomonas
• Micrococcus and Proteus.

1. Adams, M.R. and Moss, M.O. (2000) Food Microbiology. Royal Society of
Chemistry, Cambridge, U.K.
2. Jay, J.M. (2000) Modern Food Microbiology, Van Nostrand Company,
New York.
45
UNIT 7 DRYING − CONTROLING OF

MICROORGANISMS
Structure
7.0 Objectives
7.1 Introduction
7.2 Principles
7.3 Mechanisms of Dehydration
7.4 Theory of Drying
Heat Transfer Theory
Mass Transfer Theory
7.5 Importance of Water Activity (aw)
7.6 Microorganisms Associated with Dried Fruits and Vegetables
7.7 Microbiology of Dried Foods
Microbiology of Fresh Fruits and Vegetables
Microbiology of Dried Fruits and Vegetables
Before Reception at the Processing Plant
In the Plant before Drying
During the Drying Process
After Drying
7.8 Survival of Microorganisms in Dried Foods
Survival at Freezing Temperatures
Survival at Moderate Temperatures
Survival at Elevated Temperatures
7.9 Microbial Spoilage of Dried Foods
7.10 Let Us Sum Up
7.11 Key Words
7.12 Answers to Check Your Progress
7.0 OBJECTIVES
• know the importance of dehydrated fruits and vegetables;

• have an idea about the drying/dehydration theories of fruits and
vegetables;
• have the knowledge about the microorganisms evolved in dried products;
• know the different kinds of microorganism associated with dried foods;
and
• know about the microbial spoilage of dried fruits and vegetable products.
7.1 INTRODUCTION
Drying or dehydration is accomplished by the removal of water from the fruits
and vegetables below a certain level at which enzyme activity and growth of
microorganisms is affected adversely. The dried fruits and vegetable are called
as high sugar high acid foods or high value low volume foods. These dried or
concentrated products save energy, money and space in shipping, packaging,
storing and transportation. Dehydration or drying process usually involves
heating, in which water is removed from solid or near solid substances. The
term drying is generally used for drying of the produce under the influence of
non-conventional energy sources like sun and wind. Dehydration on the other
46
Drying − Controlling of
Microorganisms
hand refers to the process of removal of moisture by the application of
artificial heat under controlled conditions of temperature, relative humidity
and air flow. The sun drying is a slow process and thus, not suitable for many
high quality products. Generally, it lowers the moisture contents below about
15% which is too high for storage stability of numerous products.
7.2 PRINCIPLES
The basic principle in the process of drying or dehydration is the removal of
sufficient moisture to protect the product from spoilage. The process reduces
the amount of available moisture i.e. the water activity (aw ) and hence, product
becomes shelf-stable and is preserved for quite a long period. Moisture-solid,
relationship in fruits and vegetables are more complex than in inorganic
materials as the matter in fruits and vegetables exhibits an energetic retention
of moisture and the moisture is bound to the solid. The solid skeleton consists
essentially of numerous cells joined together to provide a network of
capillaries, some of them are very fine. First the moisture in the larger
capillaries has to be evaporated then only the moisture in the finer capillaries
can be removed. The cell walls act as semi-permeable membranes for the
diffusion of moisture which is mainly held osmotic ally. Finally, there is a
small amount of moisture adsorbed on the skeletal frame in multi molecular
layers. In order to dehydrate any product specific requirements need to be
fulfilled so that the product retains as much as possible, its original
characteristics.

1. What do you mean by drying and dehydration?
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
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………………………………………………………………………………
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2. What are the main objective of drying?
………………………………………………………………………………
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………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
3. What is the role of air in the process of drying or dehydration?
………………………………………………………………………………
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47
………………………………………………………………………………
………………………………………………………………………………
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4. How the fruits & vegetables are more complex than inorganic?
………………………………………………………………………………
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7.3 MECHANISM OF DEHYDRATION

The changes during dehydration can be largely explained in terms of heat and
mass transfer phenomena. A cue of food in the course of dehydration loses
moisture from its surface and develops dried layer with remaining moisture
confined to its centre. From the centre to the surface a moisture gradient will
be stabilized. The outside dried layer acts as an insulation barrier against rapid
heat transfer into the food pieces, this is further decreased by air voids formed
by evaporating water. In addition to less driving force from decreased heat
transfer, the centrally remaining water also now has further to travel to get out
of the food piece than did surface moisture at the start of drying. In addition,
as the food dries it approaches its normal equilibrium relative humidity, as it
does; it begins to pick up molecules of water vapour from the drying
atmosphere as fast as it loses them. When these rates are equal drying ceases.
7.4 THEORY OF DRYING

There are two steps involved in drying & dehydration.
7.4.1 Heat Transfer Theory

Transfer of heat consists of transferring of molecular or atomic motion from
one region to another. There are three broad mechanisms by which such
transfer can occur, conduction, convection, and radiation. In conduction, the
energy is transmitted from particle to particle by a process of direct contact.
Transfer of heat by convection involves bulk mixing of fluids of different
temperatures. Radiation is the transfer of energy from a radiating source
through space which may or may not be occupied by matter. It is by radiation
that we receive all our energy from the sun.
7.4.2 Mass Transfer Theory

The removal of moisture from a food product involves simultaneous heat and
mass transfer. Heat transfer occurs within the product structure and is related
to the temperature gradient between the product surface and the water surface
at some location within the product. As sufficient thermal energy is added to
the water to cause evaporation, the vapours are transported from the water
48
Microorganisms
surface within the product to the product surface. The gradient causing
moisture –vapour diffusion is vapour pressure at the liquid water surface, as
compared with the vapour presser of air at the product surface. The heat and
the mass transfer within the product structure occurs at the molecular level,
with heat transfer being limited by thermal conductivity of the product
structure, while mass transfer is proportional to the molecular diffusion of
water vapour in air. The rate of moisture diffusion can be estimated by the
expression for molecular diffusion. The mass flux for moisture movement is a
function of the vapour pressure gradient as well as the mass diffusion for
water vapour in air, the distance for water vapour movement within the
product structure and temperature. The transport of vapour from the product
surface to the air and the transfer of heat from the air to the product surface is
a function of the existing vapour pressure and temperature gradients,
respectively, and the magnitude of the convective coefficient at the product
surface.

1. In the food product how the changes are occurred.
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
2. What are the main mechanisms by which heat can transfer?
………………………………………………………………………………
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………………………………………………………………………………
………………………………………………………………………………
3. How the evaporation is occurred during drying?
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
4. What are the functions of vapour pressure and temperature gradient?
………………………………………………………………………………
………………………………………………………………………………
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49
………………………………………………………………………………
………………………………………………………………………………
7.5 IMPORTANCE OF WATER ACTIVITY (aw)

Water activity (aw) is defined as the ratio of the vapour on the aqueous
solution to that of pure water at the same temperature i.e.
aw = Vapour pressure of solution at ToC
Vapour pressure of pure water at ToC.
Water activity is also equal to the equilibrium relative humidity (ERH);
Equilibrium relative humidity
aw =
100
The aw has a major role to play in microbiological spoilage and chemical
changes produced in the food. The principles of water and microorganisms
relation includes:
1) Water activity, rather than water content, determines the lower limit of
available water for microbial growth. Most bacteria do not grow below aw
0.91 and most molds cease to grow at water activity of 0.80. Some
xerophylic fungi have been reported to grow at water activities of 0.65, but
the range of 0.70 – 0.75 is generally considered their lower limit.
2) Environmental factors affect the level of water activity required for
microbial growth. The less favourable the other environmental factors
(nutrients, pH, oxygen pressure, temperature) the higher becomes the
minimum a w at which microorganisms can grow.
3) Some adaptation to low water activities occurs, particularly when aw is
depressed by addition of water soluble substances (principle of IMF –
Intermediate Moisture Food), rather than by water crystallization (frozen
foods) or water removal (dehydrated foods).
4) When water activity is depressed by solutes. The solutes themselves may
have effects which complicate the effect of aw per se. For instance, at a
given aW microbial growth is less effectively depressed by glycerol than by
sodium chloride. More recent (IMF – Intermediate Moisture Food) have
resulted in the following additional findings.
a) Water activity modifies sensitivity of microorganisms to heat, light and
chemicals. In general organisms are most sensitive at high water
activities (i.e. in dilute solution) and minimum sensitivity occur in an
intermediate moisture. Minimum water activities for production of
toxins are often higher than those for microbial growth. The
phenomenon may represent an important safety factor in the
distribution of dehydrated and intermediate moisture foods.
b) The effect of water on chemical reactions in foods are more
complicated than are its effect on microbial growth. It plays one or
more of the following roles; a) as a solvent for reactant and for
products, b) as a reactant (e.g. in hydrolysis reactions) c) as a product
of reactions and d) as a modifier of the catalytic or inhibiting activities
of other substances (e.g. water in activities some metallic catalysts of
lipid per oxidation).
50
Microorganisms
All microorganisms have an optimal and minimal water activity for growth.
Adjusting the aw of a product by addition of solutes or the removal of water,
to a point below the minimal aw of the normal spoilage flora results in a
microbiological stable product. Many of the products contain viable
microorganisms and spores, which are not able to germinate because of the
restrictative aw. In fabrication of a product with a reduced aw other factors
which would affect the growth of microorganisms present need to be
considered, since the aw on microorganisms is influenced by pH, oxygen level,
temperature, nutrient content, and possibly food preservative, either natural or
added.
Water activity (aw) influences the physical, chemical and microbiological
properties of many substances. The shelf life of foods, their colour, stability,
taste, texture, vitamin content, aroma, mold formation and microbiological
growth properties are influenced directly by the aw value. aw measurement is
required to meet standards like FDA – Food Drug Act, USDA – United State
Department of Agriculture, GMP – General Manufacturing Practices,
HACCP – Hazard Analysis and Critical Control Points, and BIS 15000 –
Bureau of Indian Standards: The foods types and range of aw is discussed as
given below.
Aw range Upper limit values for Foods in this range
micro-organisms
1.00-0.95 Pseudomonas, Perishable (fresh) food and fruit in
Escherichia, Proteus, tins, Vegetables, meat, fish and
Shigella, Clesiella, milk, cooked sausage, backed
Bacillus, Clostridium, bread, food with a content up to
perfringens, some yeast 40% weight sucrose or 7%
common salt.
0.95-0.91 Salmonella, Vibrio Some cheese (cheddar, Swiss,
parahaemoliticus, C. Muenster, and Provolone) smoked
botulinum, Serratia, meat (ham) some fruit juice
Lactobacillus, concentrates, food with a 55%
Pediococcus, some weight sucrose (saturated) or 12%
mold, yeast common salt.
0.91-0.87 Many types of yeast Matured sausages (salami), cake,
(Candida, Torulopsis, dry chesses, margarine, and food
Hansenula), with a 65% weight sucrose
Micrococcus (saturated) or 15% common salt.
0.87-0.80 Most types of mold Most fruit juice concentrates,
(mycotoxic Penicillia), sweetened milk, chocolate syrup,
Staphylococcus aureus, maple and fruit syrup, flour, rice,
most Saccharomyces pulses with a water content 15-
(bialii) spp. , 17%, fruit cakes traditional smoked
Deboryamyces hams.
0.80-0.75 Most types hallophilic Marmalade, jam, fruit jelly,
bacteria, mycotoxic marzipan, glace fruit, some types
aspergilli of marshmallow.
0.75-0.65 Xerophylic mold Rolled oats with a 10% water
(Aspergillus chevalier, content, naught, fondant,
A. candid us, Wallemia marshmallows, grouts, molasses,
semi), Saccharomyces raw sugar, some dried fruit, and
bisporus nuts.
51
0.65-0.60 Osmophyilc yeast Dried fruit with 15-20% water

(Saccharomyces rouxi), content, some types of toffee,
some mould caramel, honey
(Aspergillums
echinulatus, Monascus
bisporus)
0.5 Noodles, Spaghetti, pasta. etc. with
about 12% water content, spices
with about 10% water content
Egg powder with about 5% water
No microbial growth content
0.4
Biscuits, crackers, bread crust,
cookies, etc. with about 3-5%
0.3 water content
Powder milk with about 2-3%

water content, dried vegetables/
0.2 fruit with about 5% water content,
cornflakes with about 5% water
content fruit cake, rustic, crackers,
biscuits

1. Name some of products which have the aw between 0.65-0.60.
………………………………………………………………………………
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2. Name the microorganisms which are found at aw between 0. 65-0.60.
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7.6 MICROORGANISMS ASSOCIATED WITH DRIED

FRUITS AND VEGETABLES
Microorganisms are associated, in a variety of ways, with all of the food we
eat. They may influence the quality, availability, and quantity of our food.
Naturally occurring foods such as fruits and vegetables normally contain some
microorganisms, and may be contaminated with additional organisms during
handling. Food serves as a medium for the growth of microorganisms, and this
growth may cause the food to undergo decomposition and spoilage. Food may
52
Microorganisms
also carry pathogenic microorganisms and as a result transmit diseases. Dried
foods has been used for centuries and they are more common throughout the
world than frozen foods. Growth of all microorganisms can be prevented by
reducing the moisture content of their environment below a critical level. The
critical level of moisture is determined by the characteristics of the particular
organisms and the capacity of the food item to bind water so that it is not
available as free moisture.

1. In what way microorganisms affect the products.
………………………………………………………………………………
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2. How the growth of microorganisms can be arrested.
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7.7 MICROBIOLOGY OF FRUITS AND VEGETABLE
7.7.1 Microbiology of Fresh Fruits and Vegetables

Fruits and vegetables are normally susceptible to infection by bacteria, fungi
and viruses. Microbial invasion of plant tissue can occur during various stages
of fruits and vegetables development and hence to the extent that the tissues
are infected the likelihood of spoilage is increased. A second factor
contributing to the microbial contamination of fruits and vegetables pertains to
their post harvest handling. Mechanical handling is likely to produce breaks in
the tissue which facilitates invasion by microorganisms. The pH of fruits is
relatively acid ranging from 2-3 for lemons to 5.0 for bananas. This resists
bacterial growth but does not retard fungal growth. The range for vegetables is
slightly higher pH 5.0 to 7.0 and hence they are more susceptible than fruits to
attack by bacteria.
7.7.2 Microbiology of Dried Fruits and Vegetables

The microorganisms on most of the dried fruits vary a few hundred per gram
of fruits to thousands, and in whole fruits they are mostly on the outer
53
surfaces. Spores of bacteria and molds are likely to be most numerous. When
part of the fruit has supported growth and sporulation of mold before or after
drying, mold spores may be present in large numbers. The number on the
vegetable just before drying may be high because of contamination and
growth after blanching and the percentage killed by the dehydrating process
usually is less than with the more acid fruits. If drying trays are improperly
loaded, souring of such vegetables as onions or potatoes by lactic acid bacteria
with marked increase in number of bacteria which may take place during the
drying process. The risk may be grater of the fruits and vegetables which are
not blanched before drying like onion. Microbial counts on dried vegetables
range from negligible to millions per gram. A number of genera of bacteria
found on dried vegetables includes: Escherichia, Enterobactor, Bacillus,
Clostridium, Micrococcus, Pseudomonas, Streptococcus, Lactobacillus and
Leuconostoc. Of these, Lactobacillus and Leuconostoc species are
predominant in many samples of dehydrated vegetables.
Dried fruits become musty of molds and dried vegetables soft or slimy if kept
in a damp atmosphere in unsealed containers. Hence, proper sealing and
storing of containers at ambient temperature and in a dry place is important.
Dried fruits and vegetables should be packed in moisture proof containers.
Higher density polythene (HDP) package of multiple aluminium foil are
utilized for packing of dried vegetables.
The bacteria, such as Bacillus sp., Clostridium sp., Micrococcus sp,
Streptococcus sp., and Pseudomonas sp. are common as soil and water born.
Bacteria capable of causing food poisoning such as Salmonella sp. and
Clostridium botulinum, are not found in dehydrated foods as in case of
dehydrated onion where the microbial load is influenced by the following
factors:
1. The load and types of microorganism present on the raw material.
2. Pre-treatment given to the material
3. Time lag between preparation and dehydration
4. Drying time and temperature
5. Moisture content of the finished product
6. In plant sanitation,
7. Packaging and storage conditions of the finished product.
One of the important types of microbiological spoilage in onion during
dehydration is fermentation and souring, which are undesirable and make the
product sub-standard. Sour onions have characteristics sour taste commonly
associated with vegetable tissues undergoing lactic acid fermentation. Onion
slices do not dry properly. Pink discolouration and off taste are indications of
spoilage. Bacteria which predominate in fresh onions include representatives
of the genera: Lactobacillus, and Aerobactor. Fresh onions juice sterilized by
filtration suppressed the growth of Bacillus subtilis and E. coli. but did not
prevent the growth of Lactobacillus brevis and Aerobactor aerogens. In good
quality of dehydrated onion many aerobic bacilli and other soil and water born
bacteria are not found which may be due to the toxicity of constituents
present in fresh onions.
To check the souring and fermentation as well as to reduce the microbial load
to the minimum the following points should be closely watched:
1. Onion bulbs selected for dehydration should be free from disease and
blemish
54
Microorganisms
2. Onions should be thoroughly washed after peeling in 3-5 ppm chlorine
water.
3. The cut slices should be dried immediately under controlled conditions so
that the finish product can be obtained in the minimum time having
moisture at 6-7 percent.
4. Sanitary conditions and workers hygiene in the factory should be
controlled and
5. Proper packing room facilities and nitrogen gas packaging are important
for the storage of finished product
The number of microorganisms and their kinds vary at different stages of
processing such as
7.7.3 Before Reception at the Processing Plant

The microbiology of foods before their reception at the processing plant is
likely to be similar whether the foods are to be dried, chilled, frozen, canned
or otherwise processed. Fruits and vegetables have soil and water organisms
on them when harvested, plus their own natural surface flora and spoiled parts
contain the microorganisms causing the spoilage. Growth of some of these
organisms may take place before the foods reach the processing plant if
environmental conditions permit. Thus piled vegetables may raise temperature
and stimulate the growth of slime- forming, flavour harming, or even rot-
producing organisms.
7.7.4 In the Plant before Drying

Growth of microorganisms that begun on foods before they have reached the
drying unit may continue up to time of drying. Also equipment and workers
may contaminate the food. Some of the pre-treatment reduce number of
organisms while other may increase them, but the foods may be contaminated
after these treatments. The grading, selection, and sorting of fruits and
vegetables, influences kinds and number of microorganisms. The elimination
of spoiled fruits and vegetables or of spoiled parts reduced number of
organisms in the product to be dried.
Washing of fruits and vegetables removes soil and other adhering materials
and serves to remove microorganisms. There also possibility of adding
organisms if the water is of poor quality.
Peeling fruits or vegetables, especially with steam or lye, and slicing or cutting
reduces the number of organisms if equipment is adequately cleaned and
sterilized.
Dipping in dilute alkali as applied to certain fruits before sun drying may
reduce the microbial population.
Blanching or scalding vegetables reduces the bacterial numbers greatly, as
much as 99 percent in some instances. Sulphuring of fruits and vegetables
also causes a great reduction in number of microorganisms and serves to
inhibit growth in the dried product.
7.7.5 During the Drying Process

Heat applied during a drying process causes a reduction in total number of
microorganisms, but the effectiveness varies with the kinds and numbers of
organisms originally present and the drying process employed. Usually all
55
yeasts and most bacteria are destroyed, but spors of bacteria and molds
commonly survives, as do vegetative cells of a few species of heat resistant
bacteria, improper conditions during drying may even permit the growth of
microorganisms. More microorganisms are killed by freezing than by
dehydration during the freezing- drying process.
7.7.6 After Drying

If the drying process and storage conditions are adequate there will be no
growth of microorganisms in the dried foods. During storage there is a slow
decrease in number of organisms, the microorganisms that are resistant to
drying will survive best: therefore the percentages of such organisms will
increase. Especially resistant to storage under dry conditions are the spores of
bacteria and molds, some of the micrococci, and micro bacteria. There may be
some opportunity for contamination of the dried food during packaging and
other handling subsequent to drying.
Special treatment given to some dry foods will influence microbial numbers
e.g. sweating of dry fruits to equalize moisture may permit some microbial
growth. Pasteurization of dry fruits will reduce number of microorganisms.
Some products are re-packaged for retail sale, e.g. figs in the near east, are
subjected to contamination. The microbial content and the temperature of
water used to rehydrate for dried foods also affect the number of
microorganisms if rehydration done in water at 50°C the number of
microorganisms will be more and number of microorganisms is almost
eliminated when the product is rehydrated at 85 to 100°C.

1. Write the name of microorganisms which spoil attack to the fruits and
vegetables.
………………………………………………………………………………
………………………………………………………………………………
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2. What are the factors which affect the microbial load in the finished
products?
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………………………………………………………………………………
………………………………………………………………………………
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56
Microorganisms
3. Write few points which help to check the incidence of souring,
fermentation, and microbial load.
………………………………………………………………………………
………………………………………………………………………………
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4. What are the factors which reduce the microorganisms in fresh fruits and
vegetables?
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
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………………………………………………………………………………
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5. Write few treatments which given to dehydrated fruits for control of
microorganisms.
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………………………………………………………………………………
7.8 SURVIVAL OF MICROORGANISMS IN DRIED

FOODS
The survival of microorganisms in dried foods can be markedly affected by aw

level. There are important interaction between aw and such factors as pH,
oxygen and food composition. For many foods, deterioration during storage in
the dry state is least at the relatively low aw levels. Survival of pure cultures of
vegetative bacteria equilibrated to a range of aw levels after freeze drying have
shown clearly the increase in survival that accompanies reduction in aw level
to 0.1 -0.2. Survival of Salmonella newport at 0.0 aw, after freeze –drying in
papain digest, was nearly maximum in vacuum, but was very poor when
stored in air. Although Pseudomonas fluorescense proved more susceptible to
death on storage, the qualitative response to aw was similar. Death of bacteria
during storage at reduced aw levels is greatly influenced by the nature of
solution from which they had been dried.
7.8.1 Survival at Freezing Temperatures
57
Freezing and frozen storage may reduce greatly the viability of populations of
sensitive microorganisms. The latter include the vegetative cells of yeasts and
molds and most gram negative bacteria. Gram positive bacteria, especially
cocci, are more resistant, and for these reasons enterococci are frequently
claimed to be more suitable than Escherichia coli as indicators of fecal
contamination in frozen foods. Many fungal spores also show this level of
resistance, however bacterial spores are least affected by freezing.
The rate of freezing influences the survival, because of it influence the size of
ice crystals and hence the degree of mechanical damage caused to cellular
structures. Rapid freezing is less damaging than slow freezing. It is the range
of temperature between freezing point of a food and its eutectic, influences the
aw of food, but not its overall solute concentration. The eutectic means that the
solute remain in equilibrium in frozen food. A frozen food held at -20oC has
an aw of 0.823, irrespective of its composition. Although composition of food
does not control aw, it can have a marked influence on survival of
microorganisms frozen in food. Sugars, sugar alcohol, glycols, and proteins,
may have protective effect. The added sucrose have effect on the survival of
Torula sp. in frozen orange juice.
7.8.2 Survival at Moderate Temperatures
Many sterile foods are microbiologically stable in the moderate or room

temperature range. The majority of dried or concentrated foods, owe their
stability to reduced level of aw. However, if rehydrated before consumption,
regain the ability to support microbial growth, so that the capacity of
contaminating organisms to survive the period of low aw storage is of obvious
relevance. Studies on survival of pure cultures of vegetative bacteria
equilibrated to range of aw levels after freeze drying have shown clearly the
increase in survival that accompanies reduction in aw level to 0.1-0.2. In dried
foods the bacteria during storage at reduced aw levels is greatly influenced by
the nature of solution from which they had been dried. While non-reducing
sugars are protective, and reducing sugars accelerate bacterial inactivation.
7.8.3 Survival at Elevated Temperatures

Microorganisms vary in heat resistance, the more resistant bacteria (e.g.
Bacillus stearothermophillus) producing spores with decimal reduction times
in neutral foods as long as 4 minutes at 121°C. or 40 minutes at 110°C. Yeast
ascospores are only slightly more heat resistant than vegetative yeast cells.
The qualitative effect of moisture upon microbial heat resistant is well known-
moist heat is a much more effective sterilizing agent than dry heat and,
wherever, practicable, steam sterilization is preferred as being much more
rapid than hot air (dry ) sterilizing. Water activity is also likely to be
significance in the heat treatment of foods in the intermediate moisture range,
Pasteurization temperature for salmonellae, staphylococci, and yeast (50-
60°C) death rates are lowest in the aw range 0.75- 0.85 in glycerol adjusted
solutions. Osmophilic yeasts respond similarly to salmonellae when heated in
sucrose solution with decimal reduction times increasing as aw decreases from
0.995 to 0.85. These organisms are more heat sensitive than salmonellae.
7.9 MICROBIAL SPOILAGE OF DRIED FOODS

Spoilage of dried fruits and vegetables by insects
58
Microorganisms
The presence of water in fruits and vegetables is mandatory to undergo
microbial spoilage. All organisms require water for carrying on their life
processes. If the microorganisms cannot acquire the water it either dies or its
further growth is arrested. Potential spoilage of a dried fruit, then depends
upon how available water is to the spoilage microorganisms, It is therefore the
thin demarcation line of water activity which establishes dehydration as a
good preservative techniques. The degree to which water is available to the
microorganisms is expressed by the term water activity, (aw,) that is the
vapour pressure of the solution divided by the vapour of the solvent.
Removing the water from the fruit and vegetables is to reduce its availability
to the microorganisms. In a moist solid substance , the water vapour pressure
is lower than the vapour pressure of free water at the same temperature
because, in a solid substance, water reacts with polar group such as –CO -, -
NH, -OH. Still further vapour pressure inside of capillaries (between plant
cells) is lower than the vapour pressure of a plane surface of water. As the
solutes present in the fruit are dissolved in water the vapour pressure is
depressed. Certain osmophilic yeasts and certain xerophilic molds and fungi
are able to live and proliferate at water activities of low values. These are the
microorganisms responsible for the spoilage of dried fruit and vegetables.
Bacterial growth of generally impossible when aw is reduced below 0.90. The
growth of normal yeast is generally impossible when the aw is reduced below
0.88. The growth of normal molds is generally impossible below 0.80. Each
organism has its own characteristics optimum aw at which growth will occur.
Molds are the most troublesome group of microorganisms will grow at aw
values below 0.70.
Dried fruits and vegetables are also subjected to insect attack when not dried
and stored properly. Insect not only consume food stuffs but also leave much
debris which spoils the appearance of the product. These insect can be killed
either by heating or by fumigation. In heat treatment, dried fruits are dipped in
boiling water or in dilute solution of salt (NaCl) –NaHCO3) and then, redried
at 54-65oC. Dried vegetables may be heated directly without preliminary
dipping. Fumigation with ethylene oxide inside the storage chamber also
reduces attack by insects.
Dried fruits become musty or moldy and dried vegetables soft or slimy if kept
in a damp atmosphere in unsealed containers. Hence, proper sealing and
storing of containers at ambient temperature and in a dry place is important.
Dehydrated fruits and vegetable potential defects and means to prevent them
are given below:
Defects Causes Prevention
Molding High product moisture, Reduce water content

above equilibrium down to optimum
relative humidity values, pack in hermitic
corresponding to water air tight package.
activity aw = 0.70.
Infestation Presence in dried Storage room

products of larva or disinfection with toxic
insects. gases. Fumigation of
packed products and of
packages. Disinfection
59
by heat (60-65oC) of
products before
packing.
Considering the variety of natural food substances and the methods by which
each is handled during processing , it is apparent that practically all kinds of
microorganisms are potential contaminants. The type of food substance and
the method by which it processed and preserved may favour contamination by
certain groups of microorganisms. Most foodstuffs serve as good media for
the growth of many different microorganisms, and microorganisms’ changes
in appearance, flavour odor, and other qualities of foods. These degradation
processes may be descrived as follow;
Putrefaction
Protein foods + proteolytic microorganisms → amino acids + amines =

ammonia + hydrogen sulphide.
Fermentation
Carbohydrate foods + carbohydrate-fermenting microorganisms → acids =

alcohol = gases.
Rancidity
Fatty foods + lipolytic microorganisms→ fatty acids = glycerol.
Some microorganisms discolour foods as a result of pigment production.

Slimes may be developed in or on foods by microorganisms capable of
synthesizing certain polysaccharides.
60
Microorganisms

1. At low moisture content how the growth of microorganisms is check.
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
2. List the microorganisms which are responsible to spoil the dried fruits and
vegetables.
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
3. Write the level of aw at which the growth of bacterial, yeast and mould is
impossible.
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
4. What are the changes occur in the food products when they are attack by
microorganisms.
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
7.10 LET US SUM UP
61
The reduction in weight and bulk by drying and dehydration of the commodity
can result in economies in cost of containers, shipping and distribution of
dehydrated products. The dehydration also result in the production of
convenience products e.g. instant coffee instant milk and instant mashed
potatoes. The production of dried fruits and vegetable products are less costly
as there is a minimum of labour and processing equipment required. These
products also require less space for storage then fresh canned or frozen fruits
and vegetables.
Microorganisms are associated, in a variety of ways, with all of the food we
eat. They may influence the quality, and availability of our food. Naturally
occurring foods such as fruits and vegetables normally contain some
microorganisms, and may be contaminated with additional organisms during
handling. Food can serve as a medium for the growth of microorganisms, and
this growth may cause the food to undergo decomposition and spoilage. The
microorganisms on most of the dried fruits vary a few hundred per gram of
fruits to thousands, and in whole fruits they are mostly on the outer surfaces.
Spores of bacteria and molds are likely to be most numerous. When part of the
fruit has supported growth and speculation of mold before or after drying,
mold spores may be present in large numbers. Microbial counts on dried
vegetables range from negligible to millions per gram. A number of genera of
bacteria found on dried vegetables includes: Escherichia, Enterobactor,
bacillus, Clostridium, Micrococcus, Pseudomonas, Streptococcus,
Lactobacillus and Leuconostoc. Of these, Lactobacillus and Leuconostoc
species are predominant in many samples of dehydrated vegetables.
Water activity (aw) influences the physical, chemical and microbiological
properties of many substances. The shelf life of foods, their colour, stability,
taste, texture, vitamin content, aroma, mold formation and microbiological
growth properties are influenced directly by the aw value. The survival of
microorganisms in dried foods can be markedly affected by aw level, there are
important interaction between aw and such factors as pH, oxygen and food
composition. Certain osmophilic yeasts and certain xerophilic molds and fungi
are able to live and proliferate at water activities of low values. These
microorganisms are responsible when water activity (aw) is reduced below
0.88, and the growth of normal yeast is generally impossible below the water
activity (aw) of 0.88.
7.11 KEY WORDS

Water activity : Water activity (aw) is the ratio of vapour pressure
of food (P) and pure water (po) and expressed by
aw = p/po.
Dehydration : Removal of moisture under controlled conditions
of temperature, air flow and humidity.
Drying : Drying of the product under the source of non-
conventional energy sources like sun and wind.
Blanching : Partial pre-treatment in which vegetables are
heated in water or in steam to inactivate enzyme
before processing.
Sulphuring : Exposing the fruits to the fumes of burning
sulphure inside of closed chamber.
62
Microorganisms
In-package desiccant : Packaging of the dried products with a material
like calcium oxide or silica gel.
Sorption isotherms : Water sorption isotherms is a graphical
presentation of data which shows the water
relationship of food.
Preservation : Methods to hold food for a longer period than
generally kept at ambient conditions. Food is
safe, nutritive and free from and microbial
infection.
ERH : Equilibrium Relative Humidity.
Osmotic dehydration : Removal of water through a membrane from
higher concentration to lower concentration.
Sweating : Process for holding the dried fruits and
vegetables in to bins or package for equalization
of moisture
Pasteurization : Pasteurization of fruits and vegetable products
by heat process below 100°C.
Rancidity : Discolouration of food products.
Processing : The application of heat to the fruit and
vegetables after hermetic (air tight) sealing in
containers is called the processing.
Spoilage : The food which has been damaged or injured
which make the food undesirable for human use.
Rehydration ratio : Reconstitution ratio is the quantity of water
replaced by dehydrated foods.
Reverse osmosis : Reverse osmosis means movement of water
through the membrane by applying pressure on
the solute side of the membrane in excesses of
the osmotic pressure.

EXERCISES "
• Drying of commodity in the sun with non conventional sources of

energy like sun and wind is called drying.
• Drying the commodity under controlled conditions like temperature,
relative humidity and air flow is called dehydration.
• To reduce the weight and bulk.

• To reduce the water activity.
63
• To convey the heat to the product.

• To let out the moisture from the product.
• They exhibits an energetic retention of moisture.

• They bound the moisture to the solid content.
• Due to heat transfer to the product.

• Due to mass transfer out the product.
• Heat transfer through conduction.

• Heat transfer through convection.
• Heat transfer through radiation.
• By addition of thermal energy to the product.

• By transfer the heat to the product and water surface.
• Transfer of vapour from product surface to the air.

• Transfer of heat from the air to the product.
• Dried fruits
• Some types of toffee
• Honey
• Osmophyile yeast
• Aspergillus echinulatus
• Monascus bisporus
• In the form of quality

• In the form of availability
• In the form of quantity
64
Microorganisms
• By reduce the moisture content of the product.

• By reduce the moisture content of their environment below critical
level.
• Some of bacteria
• Some of fungi
• Some of viruses
• Load and types of microorganisms in raw material.

• Time leg between preparation and drying.
• Moisture content in the finished product.
• Bulbs should be free from diseases and blemish

• Dried the cut slices immediately under control condition
• Pack and stored under proper conditions
• Grading
• Selection
• Sorting
• Sweating
• Pasteurization
• By arrest the growth of the microorganisms.

• By destroys the microorganisms.
2 Your answer should include the following points:
• Osmophilic yeast
• Xerophilic molds
• Fungi
• For bacterial growth aw below, 0.90
65
• For yeast the aw below , 0.88

• For mold growth the aw below, 0.80
• Changes in appearance
• Changes in flavour
• Changes in odor
• Changes in quality

1. Attas, R.M., Dogra, K.M., Brown, A.E. And Piller, L. (1989)
Microbiology − Fundamentals and Applications, 2nd Ed. MacMilan
Publishing.
2. Banwart, G.J. (1979) Basic Food Microbiology. The AVI Publishing
Company, Inc, Westport, Connecticut.
3. Christian, J.H.B., and Stewart, B.J. (1973) Survival of Staphylococcus
aureus and Salmonella newport in dried foods. As influenced by water
activity, and oxygen. Microbiol. Saf. Food. Proc. Int. symp. Food
microbiological. 8th 1972, pp 107-119.
4. Frazier, W.C. and Westhoff, D.C. (1996) Food Microbiology. Tata
McGraw- Hill Publishing Company Limited. New Delhi.
5. Pelczar, M.J., Chan, E.C.S. and Krieg, N.R. (1993) Microbiology −
Concepts and application. McGraw Hill Inc. New York.
6. Potter, N.N. and Hotchkiss, J.H. (1996) Food Science. 5th Edition. CBS
Publishers and Distributors, New Delhi.
7. Powar, C.B. and Daginawola, H.F. (1986) General Microbiology. Vol II.
Himalya Publishing Company, Bombay.
8. Purohit, S.S. (1994) Microbiology −Fundamental and Application. 5th
Ed. Agro Botanical publishers, Bikaner, India.
9. Ranganna, S. (1979) Manual of Analysis of Fruit and Vegetable
Products. Tata MacGraw Hill Publishing Company Limited New Delhi.
10. Tauro, P., Kapoor, K.K. and Yadav, K.S. (1986) An Introduction to
Microbiology. Wile Eastern Limited, New Delhi.
66
Chemicals for
UNIT 8 CHEMICALS FOR CONTROLING Controlling
Microorganisms
MICROORGANISMS
Structure
8.0 Objectives
8.1 Introduction
8.2 Use of Various Food Additives and Chemical Preservatives
Types of Additives
Role of Food Additives
Preservatives
Acidulants
Control of Psychotropic Contamination in Food
8.3 General Considerations in the Selection of Chemical Food Additives
Desirable Properties of Food Preservatives
Mode of Action of Food Additives
Factors Affecting the Antimicrobial Activity of Food Additives
Precautions to be taken for Using Food Additives
Adverse Effects of Using Food Additives
8.4 Developed and Added Preservatives
Acids Produced during Fermentation
Alcohol
Bacteriocins
8.5 Let Us Sum Up
8.6 Key Words
8.0 OBJECTIVES
This unit introduces you to the concept of preservation of food with chemicals.
After going through this unit you will be able to know how chemical reactions
causing spoilage are prevented or delayed by use a wide range of chemical
additives.
• know the various classes of chemical additives used in the food industry;
• explain how these chemicals help to prevent the spoilage of food;
• know the permitted and non permitted chemical additives;
• discuss the general considerations required in the selection of food
preservatives; and
• that apart from certain added preservatives there are some naturally
occurring preservative factors in food.
8.1 INTRODUCTION
In Unit 1, you read about the various types of microorganisms that are
important in the food industry. In this unit, we shall tell you how the spoilage
of food can be prevented or delayed which are caused due to these
microorganisms or some other chemical reactions. This unit highlights the
various classes of chemical preservatives that have been approved for the use
67
Controlling Organisms in food and their use. The various aspects to be considered for the selection of
chemical additives (food additives), their mode of action and the adverse
reactions resulting due to the consumption of the additives is also elaborated in
this unit. The unit also deals with the developed additives, namely acids,
alcohol and bacteriocins.
8.2 USES OF VARIOUS FOOD ADDITIVES AND

CHEMICAL PRESERVATIVES
For centuries, man has recognized the effects of food additives and has used
whatever was available-marigold for colour, wood ashes for leavening, the
lining of calf stomachs for cheese making etc. today, thousands of compounds
are used as food additives, whose chemical identity and structure are known.
The use of food additives is imperative in the complex and integrated society
in which we live. Additives have provided protection against food spoilage
during storage, transportation, distribution or processing. Also, with the
present degree of urbanization, it would be impossible to maintain food
distribution without the processing and packing and packing with which many
additives are involved.
Additives permit the variety of foods that we deem desirable and which
certainly are objectively important in maintaining important nutrition.
Vitamins and minerals are important in maintaining good nutrition. Many of
these chemical additives can be manufactured so that foods can be “fortified”
or “enriched”.
There is then the need for the use of food additives to maintain the nutritional
quality of food, to enhance the stability with resulting reduction in waste, to
make food more attractive and to provide efficient aids in processing, packing
and transport. The amount of food additives used should be kept to a minimum
and it should conform to a standard of purity and be safe. Over 3000 different
chemical compounds are used as food additives. They are categorized into
different groups which will be discussed below.
According to WHO a food additive is defined as a substance or mixture of
substances other than the basic foodstuff, which is present in food as a result of
any aspect of production, processing, storage and packaging. The term does
not include chance contaminants- thus the former refers to intentional food
additive while latter is incidental un-intentional food additive.
Intentional food additives could be nutritive, freshness maintenance, sensory
and processing aids; preservatives, antioxidants, emulsifiers, stabilizers,
maturing agents, colours, special sweeteners, nutrient supplements, flavouring
compounds and natural flavouring materials.
8.2.1 Types of Additives

• Acidity regulators, used to alter and control the acidity or alkalinity levels
for different desired effects, which can include preservation, added/altered
tartness, colour retention and to assist raising agents.
• Acids, used to control to what degree other substances function and/or to

impart a sharp taste. Assists in the release of carbon dioxide in raising
agents and can have a preservative effect.
68
• Anti-caking agents, used to ensure the free flow in products such as dried Chemicals for
milks, icing sugar and table salt. Controlling
Microorganisms
• Anti-foaming agents, used to reduce or prevent foaming (frothing) on
boiling and to reduce scum forming.
• Antioxidants, used to protect food against deterioration caused by exposure

to air (oxidation), such as fat rancidity, flavour deterioration or colour
changes.
• Bleaching agents, used to artificially whiten flour.
• Buffers, see acidity regulators.
• Bulking agents, used to increase volume without significantly adding to

the energy levels of the food. Normally used in diet foods but can also be
used to pad out expensive ingredients. Not usually digested and acts as a
source of dietary fibre (roughage).
• Carriers and carrier solvents, used to modify a food additive (by

dissolving, diluting or dispersing etc.), without changing its function, to
enable easier use or handling.
• Emulsifiers, used to aid in the formation and maintenance of the dispersion

of two or more substances which would normally separate and not
normally mix, such as oil and water. Milk, mayonnaise and salad dressings
are typical oil in water emulsions, butter and margarine water in oil
emulsions.
• Emulsifying salts, used to disperse protein so reducing the stringiness in

cooked cheese.
• Firming agents, used to make or retain firmness or crispness in fruit and

vegetables and to strengthen gels.
• Flour improvers, used to enhance the elastic properties and aid the
development of dough. Also accelerates the effect of bleaching agents.
• Foaming agents, used to provide a uniform dispersion of gas in a food.
• Gelling agents, used to form a jelly so providing texture to a product.
• Glazing agents, used to produce a protective coating or to impart a

polish/sheen on the surface of a food such as confectionery or citrus fruit.
• Humectants, used to retain moisture in foods by absorbing water from the

air to prevent drying out.
• Modified starch, used for various functions including adding texture,

adding bulk, stabilizing and as a thickener.
• Packaging gases, used to replace air in the packaging of foodstuffs

susceptible to oxidation but not necessarily shown on food labels.
69
Controlling Organisms • Preservatives, used to extend the shelf-life of products by preventing the
growth of microorganisms which could otherwise cause food decay and, in
some cases, food poisoning.
• Propellants, a gas or volatile liquid used to expel foodstuffs from aerosols.
• Raising agents, used to increase the volume of doughs and batters by

promoting gas release (aeration).
• Releasing agents, used to prevent foodstuffs sticking to machinery, molds,

packaging etc. but not necessarily shown on food labels even though some
may remain in the food.
• Sequestrants, used to combine with trace metals in the environment to

render them inactive.
• Stabilizers, used to maintain the physical state of a food and to stabilize,

retain or intensify the existing colour of a food, particularly emulsions, and
therefore often used with emulsifiers.
• Sweeteners, there are two different types of sweeteners:

1. Intense sweeteners − these have a sweetness many times that of sugar
and are therefore used at very low levels. They are used in products
such as diet foods, soft drinks and table top sweeteners;
2. Bulk sweeteners − these have a similar sweetness to sugar and are
used at comparable levels. Unlike intense sweeteners they also provide
bulk (although their main function is to provide sweetness). They are
used in products such as sugar-free confectionery and foods for
diabetics.
• Thickeners, used to increase viscosity, modify texture and impart stability.
8.2.2 Role of Food Additives

Food additives help to enhance the consumer acceptability, help in maintaining
or improving the nutritional quality, enhance stability or keeping quality by
acting as antimicrobial agents with the resulting reduction in waste and
prevention of chemical and biological deterioration, make food more attractive
and provide sufficient aids in the food products for improving texture, colour
and flavour, check spoilage by inactivating microorganisms and maintain
safety of foods, facilitate preparation and help to improve palatability of the
product.
It helps to enhance the shelf life of food or food products. It has been estimated
that we consume about 5 kilograms of food additives as preservatives, colours,
bleaches, flavours, emulsifiers and stabilizers every year in the food we eat.
This not only results in extra work for our body to remove them, but frequently
trigger asthma attacks; rashes; respiratory disturbances; hyperactivity in
children, and in some people, an abnormal sensitivity to prescribed
medications, particularly aspirin. Below are some common additives found in
refined foods, and well-worth avoiding by those susceptible to their effects.
70
Acceptable daily intake (ADI) for various preservatives Chemicals for
Controlling
Preservative ADI Microorganisms
(mg/kg body wt/day)
Acetic acid including its Na/K salts No limit
Sodium diacetate 0-15
Benzoic acid including its Na/K salts 0-5
Formic acid 0-3
Hexamethylene tetramine 0-0.15
Para hydroxy benzoic acid esters 0-10
Lactic acid and its salts No limit
Propionic acid and its salts No limit
Natamycin/pimaricin 0-0.3
Na NO3 and KNO3 0-5
NaNO2 and KNO2 0-0.2
Sorbic acid including its Na/K/Ca salts 0-2.5
SO2, Na2 SO3, NaHSO3, Na/K metabisulphite 0-0.7
8.2.3 Preservatives
Preservatives are substances which are capable of inhibiting, retarding or
arresting the process of fermentation, acidification or other decomposition of
food or of masking any of the evidence of putrefaction but it does not include
salt, sugar, vinegar, glycerol, alcohol, spices, essential oils etc. Sulphur dioxide
(including sulphites) and benzoic acid (including benzoates) are among the
principle preservatives used in the food processing industry. The permitted
quanitity of sulphur dioxide and benzoic acid is given in the following tables.
Food additives and their usage concentrations
Food additives Concentration (%) Foods

Antioxidant : Not exceeding 0.02% of Rasogolla and Vadas
Butylated Hydroxy the total fat content and
Anisole (BHA) Whole and partially
0.01% of the finished
skimmed milk powder
product
0.02 Margarine
Colours 0.02 Most foods
Flavour : Monosodium 0.05 Meat product, soup
glutamate powder
Anticaking agent: 2 Table salt, onion
Aluminum silicate powder, garlic powder,
soup powder
Sweetening agent : 100 ppm Carbonated non-
Saccharin alcoholic drinks
Sequestrant : Ethylene 33-800 ppm Canned carbonated
Diethyle Tetra Amino beverages, salad
Acetic Acid (EDTA) dressings and margarine
71
Controlling Organisms Classes of preservatives
CLASS I:
Common salt, sugar, dextrose, spices, vinegar or acetic acid, honey
CLASS II:
Benzoic acid and its salts, sulphur dioxide and the salts of sulphurous
acid, nitrites and nitrates, sorbic acid and its salts, propionic acid and its
salts, lactic acid and its salts.
Sulphur dioxide
Sulphur dioxide and its derivatives have been extensively used in foods
as a food preservative. It acts both as an antioxidant and reducing agent
and prevents enzymatic and non-enzymatic reactions, leading to
microbial stability. The common used forms are sulphur dioxide gas
and sodium, potassium and calcium salts of sulphite, bisulphite or
metabisulphite. It is like a biocidal and biostatic agent and is more
active against bacteria than molds and yeasts.
Sulphite or metabisulphite sprays or dip with or without added citric
acid provides effective control of enzymic browning in pre-peeled and
pre-sliced potatoes, carrots, mushroom and apples.
Sodium benzoate
It was the first chemical preservative permitted in foods by the FDA,
and it continues in wide use today in a large number of foods.
Benzoates have greatest activity at low pH. As used in acidic foods,
benzoates act essentially as a mold and yeast inhibitor.
In foods such as fruit juices, benzoates may impart disagreeable tastes
at the maximum level of 0.1 per cent. The taste has been described as
being ‘peppery ‘or burning.
Permitted quantity of benzoic acid in food
Processed food Permitted quantity of

Benzoic Acid (ppm)
Non-alcoholic wines, squashes, crushes, fruit 600
syrups, cordials, fruit juices and barley water (to
be used after dilution)
Jams, marmalades, preserves, canned cherry, 200
fruit jelly
Sweetened mineral water and sweetened ready 120
to serve beverages
Brewed ginger beer 120
Pickles and chutneys 250
Tomato and other sauces 750
Danish tinned caviar 50
Tomato puree and paste 750
Syrups and sherbets 600
Fat spread 1000
72
Quantity of sulphur dioxide permitted in food Chemicals for
Controlling
Processed food Permitted Microorganisms
quantity of SO2
(ppm)
Sausages and sausage meat containing raw meat, cereals 450
and condiments
Fruit, fruit pulp or juice (not dried) for conversion into
jams or crystallized glace or cured fruit or other products
a) cherries 2000
b) straw berries and raspberries 2000
c) other fruits 1000
Fruit juice concentrate 1000
Dried fruits
Apricots, peaches, apples, pears and other fruit 2000
Raisins and sultanas 750
Other non-alcoholic wines. squashes, crushes, fruit 350
syrups, cordials, fruit juices and barley water (to be used
after dilution)
Jams, marmalade, preserves, canned cherry and fruit jelly 40
Crystallized glaced or cured fruit (including candied peel) 150
Fruit and fruit pulp not otherwise specified in the 350
schedule
Plantation white sugar, cube sugar, dextrose, gur, jaggery 70
or misri
Khandsari(s) and Bura 150
Refined sugar 40
Corn flour and similar starches 100
Corn syrup 450
Canned rossogullas 100
Gelatin 1000
Beer 70
Cider 200
Alcoholic wines 450
Sweetened mineral water/ready to serve beverages 70
Pickles and chutneys made from fruits or vegetables 100
Dehydrated vegetables 2000
Syrups and sherbets 350
Dried ginger 2000
Hard boiled sugar confectionery 350
Dry mixes of rossogullas 100
73
Controlling Organisms 8.2.4 Acidulants
Sour or acidic taste of a food is attributed to the acidic components present in

the food. Many processed foods and beverages, however, need the addition of
acids to impart characteristic taste and flavour to the final food product. The
intensity of sourness and ability to reduce pH vary among the organic group of
acidulants in the decreasing order as follows:
Fumaric > tartaric > malic > acetic > citric > lactic > gluconic acid
Commonly used acidulants include acetic, adipic, citric, fumaric, lactic, malic,
phosphoric and tartaric acids. Citric acid is the most versatile and widely used
food acidulant.
Main foods in which acidulants occur or added to food
Acid Main food
Acetic acid Pickles, sauces, relishes, fermented vegetables

and fruits, vinegar, wheat bread, cheeses and
creams, apple juice, grapefruit juice.
Adipic acid Beet juice, guava, papaya, raspberry, pork fat,

dairy foods, gelatin and desserts, puddings,
beverages, jams and jellies, snack foods,
condiments
Citric acid Oranges, lemons, grapefruit, black currants,

gooseberries, pineapple, raspberries,
strawberries
Fumaric acid Confectionery, powdered gelatins, desserts,

cheese cake, jams, and jellies
Glucono-delta-lactone Cured meats, frankfurters, salami, sausages,

dessert mixes, bakery mixes, processed
cheese, fish products, spice preparation
Lactic acid Fresh meat, yogurt, cheese, bread, pickles,

sauces, relishes, fermented foods, buttermilk,
wines, beer
Malic acid Watermelon, plum, apple, cherry, peach, pear,

grape, gooseberry, pineapple
Phosphoric acid Cola beverages, jams and jellies, bread dough,

cake, flour
Tartaric acid Grapes, tamarind, pineapple, mulberries,

gherkins, wines
74
Check Your Progress Exercise 1 " Chemicals for
Controlling
Microorganisms
1. Define food additives. What are intentional and unintentional food
additives?
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
2. Briefly discuss the functions of food additives.
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
8.2.5 Control of Psychotropic Contamination in Food

Increasingly all types of consumers are demanding minimally processed foods
that are high in quality, nutritionally superior and easy to prepare. Food
processors have met this demand by developing refrigerated foods with
extended shelf life. The most important bacteriological problem in processed
food products today is deterioration due to contamination by psychotropic
microbes, as storage at low temperature is favourable for the growth of high
levels of psychrotrophic microorganisms. Psychotrophs are bacteria, yeasts
and molds that grow although slowly, at refrigeration temperature (below 7°C)
but grows optimally at temperatures above refrigeration, e.g. 25-30°C. Their
maximum growth temperature is 30-35oC. Several pathogens such as
Aeromonas hydrophila, Clostridium botulinum, Listeria spp., Yersinia
enterocolitica, some strains of Bacillus cereus, enteropathogenic Escherichia
coli and Vibrio parahaemolyticus can grow at refrigeration temperature. These
bacteria may enrich in food during cold if storage times are long enough. Some
of these pathogens can cause illness when even few cells are ingested.
The control of psychrotrophs in food products should start from the very
beginning of raw material procurement up to processing and storage of the
finished product. The following steps are of considerable importance.
Processors need to select high-quality raw materials with low levels of
microorganisms, especially psychrotrophs. Fabrication of raw materials by
using clean and sanitized machinery and equipments into finished products
75
Controlling Organisms under hygienic conditions is also important. Apart from this regular check on
quality of water supply and proper chlorination of water used in the food
industry should be done. Appropriate use of salt and other ingredients which
reduces the aw to 0.98 or below will lengthen the lag phase of most bacteria
and will further reduce the rate of any subsequent growth. Most recent studies
have shifted to the use of lactic acid bacteria that produces bactericidal
chemicals called bacteriocins to slow or inhibit the growth of psychrotrophic
organisms. By lactic acid bacteria a wide variety of food borne pathogens are
either inhibited or killed, and many spoilage organisms are affected in similar
ways, especially Gram-negative psychrotophs.
Thus the effective control of microbial contaminants must begin on the farm
and be followed through to the retail store. Clean equipment and packages, use
of approved food additives and chemical preservatives of Generally
Recognized As Safe (GRAS) status in proper concentration, limited time of
storage, low holding temperatures for raw materials and the finished product,
effective laboratory control and attention to good manufacturing practices
which will slow the outgrow of psychrotrophs will help the food plant to
produce with good yield, good flavour, long shelf –life and high sales appeal.
8.3 GENERAL CONSIDERATIONS IN THE SELECTION

OF CHEMICAL FOOD PRESERVATIVES
Antimicrobial preservatives added to foods can be grouped as follows:
1. Those added preservatives not defined as such by law: natural organic
acids (lactic, malic, citric etc.) and their salts, vinegar, sodium chloride,
sugars, spices and their oils, woodsmoke, carbon dioxide and nitrogen
2. Substances generally recognized as safe (GRAS) for addition to foods:
propionic acid and sodium and calcium propionate, caprylic acid, sorbic
acid and its salts, sulphur dioxide, sodium nitrite
3. Chemicals considered to be food additives, which would include all that
are not included in the first two categories
4. Chemicals proved safe and approved by the Food and Drug Administration
8.3.1 Desirable Properties of Food Preservatives

There are seven requirements for food preservatives:
• No toxicity problems.
• Microbiocidal rather than microbiostatic properties.
• Must be stable in foods (especially if only microbiostatic).
• The spectrum of activity should correspond to the spectrum of micro-
organisms likely to appear in the food.
• Must not stimulate the development of resistant strains of microorganisms.
• Chemicals used therapeutically are not recommended as food additives.
• An assay procedure should be available.
76
Categories of Antimicrobial Food Additives Added to the Food: Chemicals for
Controlling
Following chemicals and biochemicals are used in food preservation: Microorganisms
• Naturally present or formed in the food, chemicals added to the food,

bacteriocins e.g. lactoperoxidase enzyme, lysozyme, lactoferrin, nisin etc.
• Chemicals with antimicrobial properties of salts of organic acids, like
citric, benzoic, propionic and ascorbic. Chemical preservatives (sulphur
dioxide and sulphites, parabens etc.), nitrites and nitrates.
• Chemicals with multifunctional properties added to the food, one property
being antimicrobial e.g. spices and essential oils, salt, sugar, antioxidants,
vinegar etc.
8.3.2 Mode of Action of Food Additives

• Alteration of cell wall permeability.
• Alteration of colloidal nature of protoplasm.
• Damage of the cell wall.
• Damage of proteins.
• Inhibition of enzyme activity.
• Disruption of cytoplasmic membrane.
• Bacteriostatic or bactericidal action (toxicity of the antimicrobial agent
towards microorganisms).
• Interference with synthetic processes.
8.3.3 Factors Affecting the Antimicrobial Activity of Food Additives
Many factors must be considered for the selection of a specific antimicrobial
food additive for a specific food. These factors are as follows:
• Physical and chemical properties of the antimicrobial agents (such as water
solubility, hydrophobic lipophyllic balance, boiling point, ability to ionize
and potential interaction with food constituents). The activity of the
antimicrobial is reduced as a result of reaction with lipids, proteins or
carbohydrates.
• Composition of food, its pH/ acidity and nutritional value.
• Type of preservation system other than chemicals used in the food.
• Characteristics and number of microorganisms.
• Initial contamination by microbes prior to preservation/processing.
• Type and concentration of chemical used.
• Time and temperature of food storage.
• Cost and toxicity of the antimicrobial.
8.3.4 Precautions to be taken for Using Food Additives

• Food additives must be thoroughly tested before use. Foods containing
physical hazards such as stones, seeds, glass fragments or metal pieces
must be thoroughly checked.
• FDA approved food additives should be used. Improper use of some of
them may prove to be harmful to human health.
77
Controlling Organisms • Use of additives should not be permitted if:
¾ They fail to serve the interest of consumers.
¾ They are used to mark the effect of faulty processing and handling
techniques.
¾ They are used to deceive the consumers.
¾ Their use results in a significant reduction in the nutritive value of the

foods.
¾ Additives should be used in a controlled way so as to maximize
benefits and prevent abuses.
Status of some additives and acceptable dietary intake (ADI)
Material Status
Amaranth (red ozo dye) Carcinogenic, but still WHO/FAO-prescribed an

ADI* for Amaranth
Saccharin Bladder cancer. However, still permitted in soft

drinks to the extent of 100ppm. WHO/FAO-
prescribed ADI from 2.5-5.0 mg/kg of body
weight.
Cyclamate Bladder cancer. WHO/FAO-prescribed ADI of 4

mg/kg of body weight for cyclamate.
Brominated vegetable oil Banned in India and UK
Hydrogen peroxide Used for extending shelf-life of milk, is

repeatedly turned down on grounds that it would
have undesirable consequences on milk collection
practices.
Gallate, Phenols Permitted in most countries, but in India there use

required specific permission which is not granted.
Nitrates and nitrites Give rise to nitrosamine which are carcinogenic

but still used in our country within the
permissible limit.
Sulphites Banned in USA
*ADI-Acceptable Daily Intake (mg/kg body weight/day)
8.3.5 Adverse Effects of Using Food Additives

Although these additives are regarded as GRAS (generally recognized as safe),
their increased used may also lead to various health problems viz. acidity,
dyspepsia, digestive disorders etc.
78
Food additive Often used in Common reactions Chemicals for
Controlling
name Microorganisms
Tartrazine drinks, cakes, snacks, asthma; hyperactivity;
(colour) ice-cream, confectionery aspirin sensitivity
Sunset yellow drinks, packet soups, hyperactivity; allergies;
(colour) dessert, biscuits, aspirin sensitivity
confectionery, ice-cream
Cochineal cakes, confectionery, hyperactivity
(colour) ice-cream
Azorubine packet soups, sauces, asthma; hyperactivity;
(colour) jams, desserts (jellies) asprin sensitivity
Indigotine tablets, capsules, ice nausea; skin rashes;
(colour) cream, biscuits allergies; high blood
pressure
Brilliant blue tinned peas, bacon- hyperactivity
(colour) flavored snacks
Caramel (colour) drinks, sauces, soups, hyperactivity
cakes, pickles, vinegar
Benzoic acid confectionery, cheeses, asthma; hyperactivity;
(preservative)
Sulphur dioxide beer, wine, soft drinks, asthma; hyperactivity
(preservative) dried fruit, cordials
Sodium bisulphite wine, beer, soft drinks, asthma; destroys vitamin
(preservative) juices, cordials B1; hyperactivity
Sodium nitrite cured meats, some hyperactivity; adverse
(preservative) cheeses reactions in children;
potentially carcinogenic
Propyl gallate oils, margarine, salad gastric and skin irritant
(antioxidant) dressings
Tert-butyl fats, oils, margarine, nausea; delirium
hydroquinone packet chips
(antioxidant)
Butylated fried snacks, soft drinks, hyperactivity; asthma;
hydroxyanisole edible oils, margarine, adverse reactions; allergies;
(antioxidant) chewing gum increases cholesterol levels
Carageenan ice-cream, jellies, cake allergies; intolerances
(thickener) decorations, cheese,
(emulsifier) salad dressings
Mannitol icecream, confectionery, allergies; diarrhoea, nausea
(emulsifier) low calorie foods
Monosodium prepacked meals, snacks, hyperactivity; asthma;
glutamate (MSG) Chinese cooking adverse reactions; allergies;
(flavour enhancer) aspirin sensitivity
79
Disodium 5' flavoured crisps, instant skin rashes; not easily
ribonucleotide noodles, party pies broken down by body
(flavour enhancer)
Aspartame diet drinks, diabetic allergies; headaches;
(sweetener) confectionery, ice cream nervous disorders
8.4 DEVELOPED AND ADDED PRESERVATIVES

Developed preservatives include those synthesized naturally, by various
microorganisms during fermentation and growth and metabolism.
8.4.1 Acids Produced during Fermentation

Food fermentations may serve either or both of two purposes: (1) to produce
new and desired flavours and physical characteristics and hence a different
food product and (2) to help preserve the food. The preservatives produced in
foods by microbial action are the most part acids (chiefly lactic) and alcohol.
The preservative effect of these substances nearly always is supplemented by
one or more additional preservative agents, such as low temperature, heat,
anaerobic conditions, sodium chloride, sugar or added acid.
Developed acidity plays an important part in the preservation of sauerkraut,
pickles, green olives, fermented milk, cheese and certain sausages and in
various fermented foods of plant origin. Development of full amount of acidity
from the sugar available may be permitted in the pickle and green olive
fermentations, or the fermentation may be stopped by chilling or canning
before the maximum acidity is attained in other fermentations. The
approximate acidity developed in some of these products, expressed as lactic
acid, is sauerkraut, 1.7 per cent: dill pickles and green olives, 0.9 per cent and
fermented milks, 0.6 to 0.85 per cent.
8.4.2 Alcohol
The alcohol content of beer, ale, fermented fruit juices and distilled liquors has
a preservative effect but was not produced primarily for that purpose.
8.4.3 Bacteriocins
Many natural products have been found to have efficient preservative effect
and their application in food is catching up fast due to the increased awareness
about their nutritional and health benefits. These are termed as bio-
preservatives as they act on harmful spoilage and pathogenic microbes and
prevent their growth in foods.
Bacteriocins constitute an important segment of these biopreservatives.
Technically speaking, the bacteriocins are proteinaceous antimicrobial
compounds that kill or inhibit closely related bacteria and also are capable of
exhibiting a wide inhibitory spectrum against spoilage and pathogenic bacteria.
Various microorganisms such as the lactic acid bacteria (comprising species of
Lactobacillus, Lactococcus, Leuconostoc and Pediococcus) and species of
Corynebacterium, Propionibacterium, Enterococcus, Bacillus and Escherichia
have been reported to produce bacteriocins or bacteriocin-like inhibitory
substances. Lactic acid bacteria have been shown to produce sufficient
quantities of bacteriocins in various cultured and fermented food preparations
80
(dahi, yogurt, cheese etc.) to prevent the growth of harmful bacteria. Chemicals for
Alternatively the purified bacteriocin preparation can also be added directly to Controlling
the food. So far, researchers have extensively tried out only two bacteriocins, Microorganisms
namely nisin and pediocin as biopreservatives in various food systems of
which nisin is the only bacteriocin that has been approved as a GRAS food
additives.
Nisin is a well known and most widely used bacteriocin produced by
Lactococcus lactis subsp. lactis (formerly Streptococcus lactis). It has been
used in processed cheese, pasteurized milks, flavored milk and various other
dairy products, in addition to canned foods and alcoholic beverages. The
recommended doses of nisin used varies from 100-150 IU/g depending on the
type of food. Nisin has sporostatic activity. This results in significant energy
savings in canning processes by way of low heat application. So it is useful for
the non-thermal preservation of foods. Nisin also has a great potential for use
in brewing industry. It also finds application in low pH foods. In many
European countries nisin has affirmed GRAS status in 1998 by Food and Drug
Administration (FDA) for use as an antimicrobial agent.
Besides nisin, several other bacteriocins produced by lactic acid bacteria
include pediocin PA-I and pediocin AcH produced by Pediococcus acidilacti,
sakacin A from Lactobacillus sake, plantaricin from Lactobacillus plantarum,
acidophilicin LA-I from Lactobacillus acidophilus and helveticin J produced
by Lactobacillus helveticus and so on. The Pediococci which are used as
starter cultures in certain vegetable and meat fermentations have also been the
subject of recent investigation with regard to their bacteriocin-producing
ability.
Advantages of using bacteriocins: The bacteriocins offer several advantages
over the preservatives that are presently being used in several foods. They do
not have any ill effect on the health of the consumer so they are safe to use and
the inhibitory effect of bacteriocins on the growth of microorganisms exhibits
the potential to inactivate microorganisms in foods.

1. Define bacteriocins. Give a few examples and possible uses.
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
2. What are the desirable characteristics of food additives?
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
81
Controlling Organisms 3. List down the mode of action of food additives.
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
8.5 LET US SUM UP

Chemical additives that control microorganisms are not only a technological
and functional tool in hands of food technologists but also aid in restoring the
nutrients lost through processing. The availability of chemical/food additives
has allowed the production of numerous out-of-season foods and a variety of
new food products. Additives have increased the development of convenience
foods, snack foods, low-calorie and health promoting (functional) foods, exotic
foods and a variety of food substitutes. The present day consumer demands
high quality, convenient and minimally processed foods. Some products can
be formulated with ingredients such as organic acids, chemical preservatives,
nitrite, bacteriocins, high concentrations of salt, carbon dioxide etc. that are
barriers to microbial growth and can also inhibit there growth due to their
antibacterial and antifungal properties. The role of chemical additives thus,
becomes all the more important, hence to be selected judiciously, keeping in
view their toxicological and biochemical role in food, before they are
recommended and they have become an integral part of food industry for day
to day life for the production of various processed products. They help to
assure a food supply with the safety, variety, appeal, wholesomeness and
affordability we have become accustomed to.
8.6 KEY WORDS

Food Additive : Food additive is defined as a substance or
mixture of substances other than the basic
foodstuff, which is present in food as a result of
any aspect of production, processing, storage and
packaging. The term does not include chance
contaminants.
Bacteriocin : Proteinaceous antimicrobial compounds that kill
or inhibit closely related bacteria and also are
capable of exhibiting a wide inhibitory spectrum
against spoilage and pathogenic bacteria.
Nisin : Widely used bacteriocin produced by
Lactococcus lactis subsp. lactis (formerly
Streptococcus lactis).
GRAS : Substances generally recognized as safe
ADI : The Acceptable Daily Intake (ADI) is defined as
an estimate of the amount of a food additive,
expressed on a bodyweight basis that can be
82
ingested on a daily basis in the diet over a Chemicals for
lifetime without appreciable risk to health. Controlling
“Without appreciable risk” means the practical, Microorganisms
in view of the actual level of knowledge,
certainty that no harm will result, even after a
lifetime of exposure to the chemical additive
concerned. The ADI is usually given as a range
of 0-x milligrams per kilogram of bodyweight per
day.

EXERCISES
"
1. • Food additive is a substance or mixture of substances other than the
basic foodstuff, which is present in food as a result of any aspect of
production, processing, storage and packaging.
• Intentional food additives are added deliberately to food and could be
nutritive, freshness maintaining, sensory and processing aids;
preservatives, antioxidants, emulsifiers, stabilizers, maturing agents,
colours, special sweeteners, nutrient supplements, flavouring
compounds and natural flavouring materials.
• Unintentional food additives are chance contaminants which may get
incorporated into food during any step of processing and are not
desirable.
2. Food additives help to:
• Enhance consumer acceptability.
• Help improve or maintain the nutritional quality.
• Enhance stability and prevent deterioration.
• Make food more attractive and palatable.
• Maintain the safety of foods.
1. • Bacteriocins are proteinaceous antimicrobial compounds produced by
bacteria, that kill or inhibit closely related spoilage and pathogenic
bacteria eg: Nisin, Pediocin, Acidophillin etc.
• They are used in processed cheese, pasteurized milks, flavored milks
and various other dairy products, canned foods and alcoholic drinks,
brewing industry etc.
2. Food additives should be non-toxic, economical, must be stable in foods,
should be microbiocidal rather than microbiostatic, should have broad
antimicrobial spectrum, must prevent growth of resistant strains and an
assay procedure should be available to detect them.
83
Controlling Organisms 3. Action of food additives is by:
• Altering cell wall permeability of bacteria.
• Altering its protoplasm.
• Damaging proteins and cell wall.
• Inhibition of enzyme activity of cell.
• Disruption of cell membrane and interfering with cell synthesis
processes.

1. Benedict, L.F. (2000) Inhibition of pathogenic microorganisms by carbon
dioxide. The magazine Louisiana agricultural experiment station, 43(2),
1-24.
2. Branen, A.L., Davidson, P.M, Salminen, S. and Thorngate III, J.H. (eds.)
(2002) Food Additives. Marcel Dekker. Inc. New York. pp 938.
3. Jay, J.M. (1992) “Modern Food Microbiology.’” 4th Ed. Chapman & Hall,
New York. The Society for Food Science and Technology.
4. Kumar, C.G. (1998) Bacteriocins: New food biopreservatives. Processed
Food Industry, 2(1), 17-19.
5. Kundu, S. and Misra, A.K. (2004) Consequences of psychrotrophic
contamination and growth in food. Indian Food Industry, 23(4), 61-65.
6. Srivastava, R.P. and Kumar, S. (1998) Fruit and Vegetable Preservation –
Principles and Practices. International Book Distributing Co. Lucknow.
pp.444.
84
Food Intoxications
UNIT 10 FOOD INTOXICATIONS

Structure
10.0 Objectives
10.1 Introduction
10.2 Natural Toxins
10.3 Mycotoxins
Aflatoxin
Ochratoxin
Patulin
Alternaria Toxins
Citrinin
Penicillic Acid
Sterigmatocystin
Fusarium Toxins
10.4 Botulism
Occurrence
Types and Symptoms
Diagnosis
Food Implicated in Botulism
Conditions Necessary for Outbreak
Prevention and Control
10.5 Staphylococcal Food Poisoning
Occurrence
Symptoms
Diagnosis
Food Incriminated
Prevention and Control
10.6 Let Us Sum Up
10.7 Key Words
10.0 OBJECTIVES
After reading this unit, you will be able to:
• discuss the causal organisms responsible for food borne intoxications;
• explain the toxins production by mold and bacteria (Clostridium botulinum
and Staphylococcus aureus);
• the microbial toxins produced, the foods associated in intoxication,
symptoms of the disease, diagnosis, conditions necessary for outbreak and
preventive measures required will also be discussed; and
• know the naturally present toxins in the food products will also be
accounted for.
After reading this unit you will be able to distinguish between food borne
intoxications caused by the various microbiological agents and their preventive
measures.
10.1 INTRODUCTION
23
Food Poisoning
We have already studied about the food borne diseases and their classification.
Food borne intoxications are basically food borne illness caused due to
ingestion of toxin produced my microorganisms (mycotoxins, bacterial toxins).
Natural toxins present in food may also result in food poisoning in humans.
Food poisoning is also caused by consuming old, used, residual, fermented,
spoiled, contaminated, toxic and bacteria infested food.
10.2 NATURAL TOXINS

Some plants and animals originate food contain toxic substances. Some pulses
and legumes contain a number of toxic substances such as protease inhibitors,
lathyrogens, and flavism causing agents, cyanogens, haemaglutinins and
paponins which are discussed below:
a) Trypsin inhibitor is a proteinous in nature. It suppresses the release of amino
acid. It thus interferes with the normal growth of animals fed with such
pulses.
b) Haemglutinins are also proteins. They impair the absorption system.
c) Cynogenic glycosides cause cyanide poisoning on hydrolysis of the
glycoside by the enzyme α-glucosidase, hydrogen cyanide is liberated. A
cyanides content of 10-20 mg per 10 gm of pulses is considered safe. Many
legumes excepting limabean (Phaseolus lunatus) contains cyanide within
these limits.
d) Saponins are glycosides of high molecular weight. This has been reported in
soyabean, swordbean and jackbean. Toxic saponins cause nausea and
vomiting and can be removed by soaking the beans prior to cooking.
e) Alkaloids are known to occur in the seeds of many legumes but they are
relatively innocuous.
f) Some compounds present in pulses appear to bind iodine thus producing a
state of iodine deficiency in the thyroid and eventually goitre.
g) Lathyrism is a disease that paralyses the lower limbs. The disease is
associated with consumption of kesari dal regularly as high as 300g daily.
In lathyrism, the toxic substances interfere with formation of normal
collagen fibers in the connective tissue.
h) A hemolytic factor in Vicia faba causes flavism. It is caused by eating
broadbeans or by inhaling pollen of its flowers. Flavism is hemolytic
anemia. In several cases, death may occur within 24-48 hours of the onset of
the attack.
i) Oxalic acid, a constituent of rhubarb, spinach and beet may cause oxalic
poisoning in certain individuals.
j) Some poisonous substances may also be present in some cereals and
vegetables e.g. protease inhibitor in cereals and potatoes, saponins in
spinach and asparagus and goitrogens in rapeseed mustard, cabbage and
related species. Goitrogens cause hypothyroidism and thyroid enlargement
k) Tissues of certain marine animals contain toxic substances, which cause
adverse responses when eaten. Some algae like Gymnodinium and
Gonyaulax are toxic. Heating does not destroy these substances.
24
Food Intoxications
l) Algal or Planktonic Fish Poisonings: Fish poisoning can result from the
ingestion of fish or shellfish that have fed upon algae toxic to human beings.
Paralytic shellfish poisoning is caused by ingestion of shellfish such as
scallops, clams and mussels which have consumed toxic dinoflagellates.
Symptoms appear within 10min after ingestion and include gastrointestinal
distress, parasthesia of the lips and fingertips followed by ataxia, muscular
uncoordination and ascending paralysis. Death may occur within 2 to 12
hours from cardiovascular collapse or respiratory failure. The human lethal
dose of toxin is considered to be 3 to 4 mg.
Prevention: Soaking, heating or fermentation of pulses can reduce or eliminate
most of the toxic factors in them. Heat causes denaturation of the proteins
responsible for trypsin inhibition and haemagglutination and of the enzyme
causing hydrolysis of cyanogenic glycosides. Fermentation also destroys toxic
factors and yield more digestible products of high nutritive value.
10.3 MYCOTOXINS
Fungi are a very diverse group of organisms and have a significant impact on
the production, spoilage and safety of food. Molds have not only served to
synthesize antibiotics but also to produce some foods. Fermented foods such as
some cheese, soy sauce, miso, tempeh and other oriental delicacies are prepared
with the help of molds.
It is well documented that some molds produce toxic substances. Some fungi
elaborate the toxin in large macroscopic fruiting bodies; for example, the toxin
produced by certain species of Amanita, a poisonous mushroom. Other fungi
always grow and sporulate as parasites on living host plants, and sometimes
will do so only on a specific host. Claviceps is an example of this group of
fungi and it produces mycotoxins. In contrast to fungi that are parasitic on
living plants another group of fungi is saprophytic and causes destruction of
dead plants and animal material. There is abundance of the spores of these
molds in atmosphere and are found to inhabit stored grain and dried products
and hence have been referred to as “storage fungi”. These molds include
Cladosporium, Fusarium, Penicillium, Aspergillus and Alternaria.
Mycotoxins are secondary metabolites produced by molds on foodstuffs that
causes illness or death when ingested by man or animals. The primary
metabolites are those that are essential for growth whereas secondary
metabolites are formed during the end of the exponential growth phase and
have no apparent significance to the producing organism relative to growth. The
mycotoxins commonly encountered in food are around one million times less
toxic than most lethal of the botulism toxin. But long term chronic toxicity is of
special concern because several of the mold metabolites are carcinogenic and
influence the immune response of a number of animal species. The syndrome
resulting from ingestion of toxin in a mold contaminated food is referred to as
mycotoxicosis.
At the beginning of the last century, two major mycotoxicosis caused
considerable suffering and mortality. They were alimentary toxic aleukia
(ATA) in Russia, caused by consumption of corn contaminated with T-2 toxin
produced by Fusarium sporotrichoides and yellow rice disease in Japan,
associated with Penicillium islandicum. More recently, outbreaks of
aflatoxicosis caused by consumption of corn contaminated with Aspergillus
flavus were reported from India involving approximately 1000 people of whom
nearly 100 died.
25
Food Poisoning
Several very important mycotoxins such as the sporidesmins, slaframine and

tremorgens are associated with animal feeds and forages which affect the
quality of meat and other animal products. It is also seen that mycotoxins
present in animal feed get into human foods because they pass through the food
chain in either their original or metabolized form.
When we store foods under inappropriate conditions they are susceptible to
mold growth. Many mycotoxigenic species are able to produce several
mycotoxins. It is likely, therefore, that contaminated foods will contain a
cocktail of toxins that can interact synergistically.
Some major mycotoxins found in foods
Mycotoxin Major Foods Common Producing

Species
Aflatoxins Corn, groundnuts, figs, Aspergillus flavus, A.
tree nuts parasiticus
Aflatoxin M1 Milk, milk products (secreted by cow after
metabolism of Aflatoxin
B1B
Deoxynivalenol Cereals Fusarium graminearum,

F. culmorum
Fumonisin Corn Fusarium moniliforme
Ochratoxin Corn, cereals, coffee Penicillium verrucosum,
beans Aspergillus ochraceus
Patulin Apple juice Penicillium expansum
Sterigmatocystin Cereals, coffee, beans, Aspergillus versicolor
cheese
Zearalenone Corn, barley, wheat Fusarium graminearum
10.3.1 Aflatoxin
Aflatoxins are the most widely studied of all mycotoxins. Knowledge of their
existence dates from 1960, when more than 100,000 turkey died in England
after eating peanut meal imported from Africa and South America. From the
poisonous feed were isolated Aspergillus flavus and a toxin produced by this
organism that was designated aflatoxin (Aspergillus flavus toxin- A-fla-toxin).
These compounds are highly substituted coumarins, and at least 18 closely
related toxins are known. Aflatoxin B1 is the most important of this large family
of compounds and is produced by Aspergillus flavus, A. parasiticus and
A.nominus. The toxicity of the six most potent aflatoxins decreases in the
following order: B1>M1>G1>B2>M2≠G2.
Occurrence: Aflatoxigenic molds can occur in warmer parts of the world and
aflatoxicosis maybe produced in a wide range of tropical and subtropical food
commodities such as figs, tree nuts and cereals. The most important crops are
corn and groundnut, but it can also occur in temperate crops such as wheat.
Although the production of aflatoxin initially was considered to be a problem in
post harvest crops stored at inappropriate temperatures and water activities, it is
now known that these compounds can be present in the field before harvest.
A.flavus and A. parasiticus may infect healthy plants at a very early stage.
26
Food Intoxications
Biological effects: Aflatoxins are acute hepatotoxins and are known to be

carcinogenic in some animal species as rat. Aflatoxin B1 is acutely toxic to our
species and is responsible for liver necrosis. The toxicological effect of the
aflatoxins are influenced by their metabolism after intake into their body
(Figure 10.1).
Aflatoxin
B1
Aflatoxin M1
Milk
Aflatoxin
epoxide
Dihydroxy
aflatoxin
Carcinogenic
Acute
toxicity
Figure 10.1: Representation of the metabolism of aflatoxin and its biological effects
When cows eat feed containing aflatoxin, aflatoxin M1 and M2 is excreted in the
milk. Although M1 and M2 are less toxic than the parent compound B1, M1
retains its toxic and carcinogenic ability in many animals. The LD50 of AFB1
for rats by the oral route is 1.2mg/kg and 1.5 to 2.0 mg/kg for AFG1.
Control: Because aflatoxins are potentially widespread in occurrence and have
an insidious combination of acute and chronic toxicity, it is prudent to control
their presence in food. Many countries have legislation establishing maximum
tolerance levels. Chemically treating the aflatoxin contaminated commodities
like nuts maybe possible or to use technologically sophisticated equipment to
sort and discard the contaminated units. It may also be possible to control the
production of aflatoxin in the field by an integrated programme of agricultural
management that may include plant breeding, improved irrigation and
replacement of aflatoxigenic strains by non- aflatoxigenic strains of A. flavus.
27
Food Poisoning

1. What are mycotoxins? How are they harmful?
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
2. Give the biological effects of aflatoxin.
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
3. List the various natural toxins present in food.
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
10.3.2 Ochratoxin
Occurrence: Ochratoxin A is a phenylalanyl derivative of a substituted
isocoumarin produced by Penicillium verrucosum in temperate climate and by
several species of Aspergillus in warmer and tropical parts of the world.
Penicillium verrucosum is especially associated with stored cereals although it
has also been isolated from meat and fish, however the occurrence of
ochratoxin A in meat products is usually due to transmission into muscle,
kidney and blood in animal fed on contaminated animal feed such as barley.
Ochratoxin may also be transferred from dietary intake into milk. Aspergillus
ochraceus is common on coffee beans, spices, soybeans, groundnut, rice and
corn. Ochratoxin is heat resistant and is not destroyed by roasting or
autoclaving though the ochratoxin producing fungi are capable of growth and
mycotoxin production at temperature below 10ºC.
28
Food Intoxications
Biological effects: It is associated with the chronic progressive kidney disease

in humans known as Balkan endemic nephropathy. There is increased evidence
that it can also be considered as a carcinogen with genotoxic property as well as
a potent nephrotoxin. The availability of improved methods of analysis has
demonstrated that ochratoxin is quite widespread in foods and its presence in
human body fluids confirms that there is a significant exposure with the human
population. Ochratoxin is immunosuppressive and inhibits protein biosynthesis.
Ochratoxin A has been classified by the International Agency for Reseach on
Cancer (IARC) as a possible human carcinogen. Low doses as 70μg/kg body
weight can induce kidney tumors in male rats. Its oral LD50 in rats is 20 to 22
mg/kg, and it is both hepatotoxic and neprotoxic.
Control: Once ochratoxin A has been formed in a food, it is difficult to remove
by most forms of food processing. Cooking with or without previous soaking
removes a significant amount of ochratoxin from beans but does not lead to
total destruction. Beans still contain 16% to 60% of the initial ochratoxin
contamination and it seems probable that the material may have leached out
rather than destroyed.
10.3.3 Patulin
It is a toxic and antibiotic metabolite produced by several species of Penicillin,
Aspergillus and Paeciliomyces but the most important in the context of human
food production is P. expansum, a soft rot pathogen of apple and pears. Patulin
is an unsaturated lactose and is sensitive to sulphur dioxide and is unstable in
alkali but stable in acid.
Occurrence: It is found in a range of foods based on fruits. Presence of patulin
in fruit juice is a indication that the juice was extracted from poor quality fruit
which is undesirable and should be avoided with good manufacturing practices.
This mycotoxin has also been found in moldy bread, sausage, fruits (including
bananas, pears, pineapples, grapes and peaches) and other products.
Biological activity: Patulin has an acute oral LD50 in rodents of about 30-50
mg/kg and has been shown to be teratogenic, immunotoxic and neurotoxic and
to cause gastrointestinal disturbances in rats. Patulin is quite rapidly excreted
from animals. It causes chromosomal aberrations in animal and plant cells and
is a carcinogen.
Control: In apples molded by Penicillium expansum, most of the patulin is
confined to the region of damaged tissue and simply removing the lesions
reduces the toxin by 90%, but if 1cm around the lesion is also removed, no
patulin is detectable in rest of the apple. Ascorbic acid has been reported to
reduce levels of patulin. Although pasteurization (using high temperature, short
time treatment of ten seconds at 90ºC) causes some reduction in patulin in fruit
juices, it is only of the order of 20%, which is not sufficient to make a badly
contaminated food product acceptable.
10.3.4 Alternaria Toxins

Several species of Alternaria (A.citri, A. alternata, A.solani and A. tenuissima)
produce toxic substances that have been found in apples, tomatoes, blueberries
and others. The toxins produced include alternariol, alternariol monomethyl
ether, altenuene, tenuazonic acid and altertoxin-I.
10.3.5 Citrinin
29
Food Poisoning
This mycotoxin is produced by Penicillin citrinum, P.viridicatum and other

fungi. It has been recovered from polished rice, moldy bread, country cured
hams, wheat, oats, rye and other similar products. It is a known carcinogen.
10.3.6 Penicillic Acid

This mycotoxin has biological properties similar to patulin. It is produced by a
large number of fungi, including many Penicillia as well as members of the
A.ochraceus. One of the best producers is P.cyclopium, it has been found in
corn, beans and other field crops. Its LD50 in mice by subcutaneous route is 100
to 300 mg/kg and it is a proved carcinogen.
10.3.7 Sterigmatocystin
These mycotoxins are structurally and biologically related to the aflatoxins, and
like the latter, they cause hepatocarcinogenic activity in animals. Among the
organisms that produce them are Aspergillus versicolor, A. nidulans,
A.rugulosus. The LD50 for rats by intra-peritoneal injection is 60 to 65 mg/kg.
10.3.8 Fusarium Toxins

Another important genus of mycotoxin producers is Fusarium, many species of
which produce members of the trichothecene family of mold metabolites like
deoxynivalenol, neosolaniol and T-2 toxin etc.
Deoxynivalenol
Deoxynivalenol (DON) is a far more common, but much less toxic,
trichothecene and is produced by species such as F. graminearum and F.
culmorum. LD50 of DON is 70mg/kg. The trichothecenes are remarkably stable
compounds, and DON will survive both dry milling and wet milling processes
of corn. The baking of bread has relatively little effect on trichothecenes such as
DON.
Zearalenone
It was first isolated as the agent responsible for vulvovaginitis in pigs, has very
little acute toxicity, but there should be some concern about chronic exposure to
a compound known to be estrogenic. It may be produced, together with DON
and other trichothecenes, in a wide range of cereals including corn, barley and
wheat.
Moniliformin
It was first obtained from a strain of Fusarium moniliforme isolated from
southern leaf blight- damaged corn seed as a water soluble toxin. The LD50 for
mice has been reported to be 20.9 mg/kg for females and 29.1 mg/kg for males.
At toxic doses moniliformin causes rapid death without obvious overt cellular
damage, although acute degenerative lesions in the myocardium are reported.
Fumonisins
The most recently characterized mycotoxins of any major significance in
human health are the fumonisins produced by species of Fusarium, such as F.
moniliforme. Like a number of mycotoxins, the fumonisins are relatively heat
stable and would not be significantly destroyed by drying processes for corn or
heat treatments used for the production of maize derivatives. Fumonisin B1 is
water-soluble is known to be responsible for equine encephalomalacia, porcine
pulmonary edema syndrome and hepatic cancer in rats and maybe involved in
30
Food Intoxications
the epidemiology of esophageal carcinoma in humans in southern Africa and

parts of China.
The range of regulatory limits for mycotoxins
Mycotoxin Regulatory Limit (μg/kg)

Aflatoxins in foods 0-50
Aflatoxin M1in milk 0-0.5
Deoxynivalenol in wheat 1000-4000
Ochratoxin A in foods 1-300
Patulin in apple juice 20-50
T-2 Toxin 100
Zearalenone 30-1000
10.4 BOTULISM
Botulism (Latin botulus, sausage) is a neuro-paralytic disease caused by the
ingestion of food containing the neurotoxin produced by Clostridium
botulinum.
10.4.1 Occurrence
Clostridium botulinum is an anaerobic, Gram-positive, spore forming, rod that
produces the potent neurotoxin. The organism and its spores are widely
distributed in nature and occur in both cultivated and forest soils, bottom of
streams, lakes and coastal waters and in the intestinal tracts of fish and
mammals and in viscera of shellfish.
On the basis of the serological specificity of their toxins, seven types of
Clostridium botulinum are recognized: A, B, C, D, E, F and G. Types A, B, E, F
and G cause disease in humans; type C causes botulism in fowls, cattle, mink
and other animals and type D is associated with forage poisoning of cattle.
Being a saprophyte, the organism seldom grows or produces toxic in the live
animal; it can do so only by growing in food. The toxins are simple heat labile
proteins and can be destroyed if heated at 80ºC for 10 minutes or longer.
C.botulinum does not produce the fully toxic molecule; instead a progenitor
toxin is activated to its full toxicity by enzymes. The progenitor toxin is
hydrolyzed to the highly toxic derivative toxin and is carried to target nerves
where it binds to the synapses of motor neurons and prevents the release of the
neurotransmitter acetylcholine. As a consequence, muscles do not contract in
response to motor neuron activity and flaccid paralysis results.
10.4.2 Types and Symptoms

Different types of botulism are recognized: adult, infant and wound. A very
small amount (a few nano grams) of toxin can cause illness.
Adult Botulism
Symptoms of botulism may develop anywhere between 12 and 72 hours after
the ingestion of toxin containing foods. Symptoms include nausea, vomiting,
fatigue, dizziness and headache, dryness of skin, mouth and throat, constipation,
lack of fever, paralysis of muscles, double vision and finally respiratory failure
31
Food Poisoning
and death. The duration of the illness is from 1 to 10 or more days depending
upon host resistance and other factors.
Infant Botulism
In the adult form of botulism, preformed toxins are ingested; in infant botulism,
viable botulinal spores are ingested and upon germination in the intestinal tract,
toxin is synthesized. It is confined to infants under a year of age. High number
of spores are found in the feces of infants during the acute phase of the disease.
It appears that ingested endospores, which maybe present in honey or other
baby foods, germinate in the infants intestine. C.botulinum then multiplies and
produces the exotoxin. The infant becomes constipated, listless, generally weak
and eats poorly. Death may result from respiratory failure.
Wound Botulism
It is the rarest form of botulism. The illness results when C.botulinum by itself
or with other microorganisms infects a wound and produces toxins which reach
other parts of the body via the bloodstream. Foods are not involved in this type
of botulism.
10.4.3 Diagnosis
Although botulism can be diagnosed by clinical symptoms alone, differentiation
from other diseases maybe difficult. The most direct and effective way to
confirm the clinical diagnosis of botulism in the laboratory is to demonstrate the
presence of toxin in the serum or feces of the patient or in the food which the
patient consumed.
10.4.4 Foods Implicated in Botulism

The types of foods involved in botulism vary according to food preservation
and eating habits in different regions. Any food that is conducive to outgrowth
and toxin production, that when processed allows spore survival, and is not
subsequently heated before consumption can be associated with botulism.
Almost any type of food that is not very acidic (pH above 4.6) can support
growth and toxin production by C.botulinum. Botulinal toxin has been
demonstrated in a considerable variety of foods, such as canned corn, peppers,
green beans, soups, asparagus, mushrooms, spinach, tuna fish ham, sausage and
smoked and salted fish.
10.4.5 Conditions Necessary for Outbreak

The following conditions are necessary for an outbreak of botulism:
1. Presence of spores of C.botulinum of type A, B or E in foods being
consumed or being processed in some other way
2. A food in which the spores can germinate and the clostridia can grow and
produce toxin
3. Survival of the spores of the organism eg: because of inadequate heating in
canning or inadequate processing otherwise
4. Environmental condition after processing that will permit germination of the
spores and growth and toxin production by the organism
5. Insufficient cooking of the food to inactivate the toxin
6. Ingestion of the toxin-bearing food
32
Food Intoxications
10.4.6 Prevention and Control

The prevention and cure of botulism involves:
1. Strict adherence to safe food-processing practices by the food industry
2. Educating the public on safe home-preserving (canning) methods for foods
3. Not feeding honey to infants younger than 1 year of age
4. Not tasting any processed food having a questionable odor
5. Recommended treatment for botulism includes early administration of
botulinal antitoxin and intensive supportive care (including mechanical
breathing assistance).
"
1. What are the causative organism and the foods associated with botulinal
food intoxication?
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
2. Differentiate between adult and infant botulism.
……………………………………………………………………………….
……………………………………………………………………………….
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……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
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……………………………………………………………………………….
3. List down the various mycotoxins associated with food.
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
33
Food Poisoning
……………………………………………………………………………….
……………………………………………………………………………….
10.5 STAPHYLOCOCCAL FOOD POISONING

Staphylococcal food poisoning results from consumption of food containing
enterotoxin produced by enterotoxigenic strains of Staphylococcus aureus. It is
caused by ingestion of improperly stored or cooked food (particularly foods
such as ham, processed meats, chicken salad, pastries and ice cream) in which
S.aureus has grown.
S.aureus is a Gram-positive coccus, very resistant to heat, drying and radiation.
If the bacteria are allowed to incubate in certain foods, they produce heat-stable
enterotoxin that render the food dangerous. Six different enterotoxins have been
identified and are designated as A, B, C, D, E and F.
10.5.1 Occurrence
Staphylococci exist in air, dust, sewage, water, milk and food or on food
equipment, environmental surfaces, humans and animals. Humans and animals
are the primary reservoirs. Staphylococci are present in the nasal passages and
throats and on the hair and skin of 50 percent or more of healthy individuals. A
wide range of foods maybe involved in Staphylococcal food poisoning
including ham, turkey, chicken and chicken salad, baked products, especially
filled pastries, table ready-meats (sausage etc.), precooked frozen foods and
dairy products.
S.aureus cells are relatively more resistant than many gram negative food
spoilage organisms. Human intoxication is caused by ingesting enterotoxins
produced in food by strains of S.aureus, usually because the food has not been
kept hot enough (60ºC, or above) or cold enough (7.2ºC, or below). In frozen
foods they may survive at –10ºC. In general, survival of S.aureus is best in
foods that contain high concentration of sugars, eggs and buffering component
such as phosphates and protein. Salt concentration less than 9.5%, temperature
more than 20ºC and a pH in the range 6-8 are favourable for growth and
enterotoxin formation.
10.5.2 Symptoms
A toxin dose of less than one micro gram in contaminated food will produce
symptoms of staphylococcal intoxication. This toxin level is reached when S.
aureus populations exceed 100,000 per gram. Symptoms of staphylococcal food
poisoning usually develop with 1-6 hours of ingestion of contaminated food.
Typical symptoms include severe abdominal pain, diarrhoea, vomiting,
sweating, headache, prostration, nausea and sometimes a fall in body
temperature. The mortality rate of staphylococcal food poisoning is negligible
among healthy individuals.
10.5.3 Diagnosis
Diagnosis is based on the symptoms or laboratory diagnosis of the bacteria
from leftover foods and from the stool cultures of victims. Enterotoxin maybe
detected in foods by animal toxicity tests.
10.5.4 Foods Incriminated
34
Food Intoxications
Foods that are frequently incriminated in staphylococcal food poisoning include

meat and meat products, poultry and egg products, egg, tuna, chicken, potato
and macaroni, bakery products like cream-filled pastries, cream pies, chocolate
eclairs, sandwich fillings, milk and dairy products. Foods that require
considerable handling during preparation and that are frequently involved in
staphylococcal food poisoning.
10.5.5 Conditions Necessary for Outbreak

The following conditions are necessary for an outbreak of staphylococcal food
poisoning:
1. The food must contain enterotoxin producing staphylococci.
2. The food must be a good culture medium for growth and toxin production
by the staphylococci.
3. The temperature must be favourable for growth of the cocci and enough
time must be allowed for production of enterotoxin.
4. The enterotoxin bearing food must be ingested.
10.5.6 Prevention and Control

Staphylococcal food poisoning can be prevented by:
1. Avoiding contamination of food with S.aureus.
2. Prevention of growth of staphylococci by adequate refrigeration of foods
and adjustment of more acid pH.
3. Killing staphylococci in susceptible foods by heating rapidly to 65-70ºC for
12-15 minutes.
4. Good personnel hygiene- exudates from skin lesions (pimples, boils) and
nasal discharges of food handlers are rich sources of staphylococci and
should be avoided.
5. Prolonged storage at room temperature of filled pastries, meat, salads and
similar products that receive only a minimal heat treatment should be
avoided.

1. What are the conditions favouring the outbreak of Staphylococcal food
poisoning?
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
35
Food Poisoning
……………………………………………………………………………….
……………………………………………………………………………….
2. Give the preventive and control strategies of Staphylococcal food
poisoning.
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
10.6 LET US SUM UP

The unit deals with the food borne intoxications which are caused by ingestion
of toxins produced from molds (Aspergillus niger, Penicillium sp.) or bacteria
(C.botulinum and S. aureus). An effort has been made to point out the major
conditions required for outbreak of the disease along with the diagnosis and the
preventive measures required to prevent the outbreak of disease.
10.7 KEY WORDS

Food Borne
Intoxication : It is the food borne disease caused due to
ingestion of food containing the toxin.
Mycotoxins : These are secondary metabolites produced by
filamentous fungi (molds) on food and feedstuffs
that cause illness and death when ingested by man
or animals.
Aflatoxin : This is the mycotoxin elaborated by Aspergillus
flavus.
Ochratoxin : Mycotoxin produced by Penicillium verrucosum.
Patulin : Mycotoxin produced by species of Penicillin,
Aspergillus and Paeciliomyces.
Citrinin : Mycotoxin produced by Penicillin citrinum.
Botulism : Food borne intoxication caused due to
consumption of food containing toxin, produced
by Clostridium botulinum.
Staphylococcal Food
Poisoning : Food borne intoxication caused due to
consumption of food containing toxin, produced
by Staphylococcus aureus.
36
Food Intoxications
LD50 Dose : Lethal Dose of toxin at which minimum 50% of

the population may get infected by the toxin.
Zearalenone : Mycotoxin produced by Fusarium graminearum
and F. tricinctum.
10.8 ANSWERS TO CHECK YOUR PROGRESS "

EXERCISES
1. • Mycotoxins are toxins (secondary metabolites) produced by filamentous
fungi on food and feedstuffs that cause illness when ingested
• Cause hepatotoxicosis, carcinogenesis, liver cirrhosis etc.
2. Biological effects of aflatoxin include hepatotoxicosis, carcinogenesis and
liver cirrhosis.
3. Natural toxins in foods: Trypsin inhibitor, haemaglutenins, cyanogenic
glycosides, saponins, alkaloids, goitrogens .
1. • Botulism is caused by the bacteria Clostridium botulinum.
• All low acid foods can support growth and toxin production by
Clostridium botulinum.
• Unheated foods are causative agents e.g.: canned foods (canned corn,
peppers, green beans, soups), sausages, smoked fish etc.
2. • Adult Botulism is prevalent amongst adults whereas Infant Botulism is
prevalent in infants of less than one year of age.
• Caused due to ingestion of viable spores of C. botulinum.
• Symptoms in adults: Nausea, vomiting, fatigue, dizziness, headache,
dryness of skin and respiratory failure
• Symptoms in infants: Constipation, weakness and loss of appetite and in
severe cases death
3. Mycotoxins associated with foods are:
• Aflatoxins (produced by Aspergillus flavus)
• Ochratoxin (produced by Penicillium verrucosum)
• Patulin (P. expansum)
• Citrinin (Penicillin citrinum)
• Alternaria toxins (Alternaria sp.)
• Penicillic acid (P.cyclopium)
• Sterigmatocystin (Aspergillus versicolor)
• Fusarium toxins (Fusarium sp)
1. Conditions necessary for outbreak of Staphylococcal food poisoning:
37
Food Poisoning
• Presence of viable staphylococcal bacteria in the food

• Growth and toxin production in food
• Toxin containing food must be ingested into the body
2. To prevent and control Staphylococcal food poisoning:
• Avoid contamination of foods
• Kill organism by heating, refrigeration
• Personnel hygiene
• Adequate cooking
• Proper storage

1. Defigueiredo, M.P. and Splittstoesser, D.F. (1976) Food Microbiology:
Public Health and Spoilage Aspects, AVI Publishing Co. Inc., Westport,
Connecticut. pp 492.
2. Jay, J. (1996) Modern Food Microbiology, CBS Publishers, New Delhi.
pp701.
38
Bacterial Food
Infections
UNIT 11 BACTERIAL FOOD INFECTIONS

Structure
11.0 Objectives
11.1 Introduction
Zoonotic Diseases
11.2 Salmonellosis
11.3 Escherichia coli gastroenteritis
11.4 Bacillus cereus gastroenteritis
11.5 Cholera
11.6 Vibrio parahaemolyticus gastroenteritis
11.7 Shigella dysentery
11.8 Campylobacteriosis
11.9 Yersiniosis (Yersinia enterolytica infection)
11.10 Listeria monocytogenes infection (Listeriosis)
11.11 The Most Important Point to Remember to Wash you Hand
11.12 Let Us Sum Up
11.13 Key Words
11.0 OBJECTIVES
• describe major bacteria causes food born infections
• explain the mode of transmission of the food borne infection, symptoms
and preventive measures.
11.1 INTRODUCTION
Food infection occurs when a pathogen enters the gastrointestinal tract and
multiplies. Microorganisms can penetrate into the intestinal mucosa and grow
there, or they can pass through other systemic organs. Infections are
characterized by a delay in the appearance of gastrointestinal disturbance while
the pathogen increases in numbers or affects invaded tissue. There is also
usually a fever, one of the body’s general responses to an infective organism.
Foodborne infections remain a major public health problem. The Council for
Agricultural Science and Technology estimated in its 1994 report, Foodborne
Pathogens: Risks and Consequences, that as many as 9,000 deaths and 6.5 to
33 million illnesses in the United States each year are food-related.
11.1.1 Zoonotic Diseases

The World Health Organization defines Zoonoses (Zoonosis, sing.) as "Those
diseases and infections which are naturally transmitted between vertebrate
animals and man".
Mode of transmission: Feces, urine, saliva, blood, milk, via aerosol, oral,
contact with bedding or animals, etc.
39
Food Poisoning
Approximately 150 zoonotic diseases are known to exist. Wildlife serves as a

reservoir for many diseases common to domestic animals and humans. Persons
working with wildlife should be alert to the potential for disease transmission
from animals. Generally, disease is more easily prevented than treated. Many
zoonotic diseases are so common in nature, so rare in humans, or so mild in
their symptoms, that wild animals pose a minimal health risk to people.
Zoonotic diseases include:
• Those which can be transmitted directly from animals to humans (e.g.
rabies).
• Diseases that can be acquired indirectly by humans through ingestion,
inhalation or contact with infected animal products, soil, water, or other
environmental surfaces which have been contaminated with animal waste
or a dead animal (e.g. salmonellosis, leptospirosis, anthrax). Campylobacter
infection is mainly found in chicken meat. Listeriosis and E.coli
gastroenteritis are two other common infections caused by zoonotic agents.
All these will be discussed one by one.
• A disease which has an animal reservoir, but requires a mosquito or other
arthropod to transmit the disease to humans (e.g. St. Louis encephalitis,
Rocky Mountain spotted fever).
11.2 SALMONELLOSIS
Salmonellosis is the most reported zoonotic disease in European countries.
Salmonellosis (Salmonella gastroenteritis) results from the ingestion of foods
that contain significant numbers of viable cells of the members of the genus
Salmonella. It is the most frequently occurring food borne infection.
Salmonella are small gram negative, motile, non-spore forming rods that
ferment glucose, usually with gas, but usually do not ferment lactose or
sucrose. They are widely distributed in nature, with humans and animals being
their primary reservoir. Generally large number of salmonellae typically 106 to
109 bacterium must be ingested to cause illness.
Occurrence: The initial source of the bacteria is the intestinal tract of animals
such as birds, reptiles, farm animals, humans and occasionally insects. As
intestinal form, the organisms are excreted in feces from which they maybe
transmitted by insects and other living creatures to a large number of places,
polluted water and contaminated food. The organism may get transferred from
actual infected cases of the disease or from carriers. A carrier is defined as a
person or an animal that repeatedly sheds bacteria, usually through feces,
without showing any signs or symptoms of the disease. Infected rodents, rats
and mice may contaminate unprotected foods with their feces and thus spread
Salmonella bacteria. Flies may play an important role in the spread of
Salmonella, especially from contaminated fecal matter to foods. Humans
acquire the bacteria from contaminated food such as beef products, poultry,
eggs, egg products or water.
Symptoms: The susceptibility of humans varies with the species and strains of
the organism and the total number of bacteria ingested. A longer incubation
period usually distinguishes salmonellosis from staphylococcus poisoning:
40usually 12-36 hours for the former and about 2-4 hours for the latter. The
Bacterial Food
Infections
principle symptoms of a salmonella gastroenteritis infection are nausea,
vomiting, abdominal pain and diarrhoea that usually appear suddenly. This
may be preceded by a headache and chills. Other evidences of the disease are
watery, greenish-fowl-smelling stools, prostration, muscular weakness,
faintness, usually a moderate fever, restlessness, twitching and drowsiness. The
mortality is less than 1%. Intesibility may vary from slight discomfort and
diarrhoea to death in 2 to 6 days. About 0.2 to 5.0% of the patients may
become carriers of the Salmonella organism. During the acute phase of the
disease, as many as one billion salmonellae can be found per gram of feces.
Associated foods: Raw meats, poultry, eggs, milk and dairy products, fish,
shrimp, coconut, sauces and salad dressings, cake mixes, cocoa, peanut butter
and chocolate.
The food must contain or become contaminated with the Salmonella bacteria.
These bacteria must be there in considerable numbers i.e., food should be a
good culture media, temperature favourable and enough time allowed for
appreciable growth.
The viable organism must be ingested.
Prevention of Outbreak: The control of food borne salmonella infection
requires the following:
1. Preventing food contamination by human carriers, especially food handlers.
2. Avoiding the use of animal products from domestic livestock that are
grossly infected with salmonellae.
3. Avoiding the use of food ingredients that contain salmonellae.
4. Processing all foods susceptible to Salmonella contamination at time-
temperature schedules sufficient to destroy the organism. Heating foods so
that all portions reach 66ºC for 12-15 minutes will assure destruction of
even most resistant Salmonella types.
5. Refrigerating all foods susceptible to Salmonella contamination and
avoiding prolonged holding of these foods at room temperature.
11.3 ENTEROPATHOGENIC ESCHERICHIA COLI

Escherichia coli is generally regarded as part of the normal flora of the human
intestinal tract and that of many animals. Serotypes of E. coli which have been
implicated in human diarrhoeal diseases or food poisoning outbreaks and have
been designated enteropathogenic E. coli (EEC). They grow over a wide range
of temperatures, 20-40ºC with a minimum growth temperature at 10ºC and an
optimum at 37ºC. Heating at 65ºC for 15-20 minutes is lethal. The pH range for
growth is 4.2-8.50, with an optimum in the range of pH 7.2-7.5. E. coli will
grow in the presence of 5.0% salt at 37ºC but 10% is inhibitory.
Symptoms: The E. coli gastroenteritis syndrome is caused by the ingestion of
106-1010 viable cells/g that must colonize the small intestine and produce
enterotoxin. The syndrome is characterized primarily by non-bloody, watery
diarrhoea without inflammatory exudates in stools. Incubation time of disease
41
Food Poisoning
is around 2 days after eating the contaminated food and may last for 8 days.
Common symptoms included are cramps, chills, vomiting, aches and headache.
Associated Foods: E. coli is the etiologic agent of food poisoning involves
variety of foods such as cream pie, mashed potatoes, cream puffs and creamed
fish. Other E. coli food poisoning outbreaks have been attributed to the
consumption of milk, cheese, ice cream, meats, fish and macaroni. E. coli is
relatively sensitive to destruction by drying or freezing but some survivors may
exist for extended periods.
“Enteropathogenic” strains colonize in the small intestine and cause acute
gastroenteritis in newborns and in infants up to two years of age.
“Enteroinvasive” strains invade the epithelial cells of large intestine and cause
diarrhoea in older children and adults. “Enterotoxigenic” (enterotoxin
producing) strains produce one or both of two different toxins:a heat stable
toxin (ST) and a heat labile toxin (LT). Both toxins cause diarrhoea in adults
and infants. Enterotoxigenic strains of E. coli are often associated with
Travellers’ diarrhoea, a common disease contracted by tourists when visiting
developing countries. Diagnosis of travellers’ disease is based on the past
travel history and symptoms. Laboratory diagnosis is by isolation of the
bacteria from feces. Treatment is with fluid and electrolytes. Other strains of E.
coli which are usually harmless in their normal habitat (the intestine) can cause
disease when they gain access to other sites or tissues. These diseases include
urinary tract infections, septic infections, bacteremia, meningitis, pulmonary
infections, abscesses, skin and wound infections.
Prevention and Control: Involves avoiding contaminated food and water that
have high coliform counts, avoiding unpasteurized juices, washing fresh fruits
and vegetables thoroughly before eating raw, using adequate cooking
procedures for destruction and prompt refrigeration. Most people recover from
E. coli infection within 5-10 days without treatment. Antibiotics and
antidiarrhoeal drugs are usually not helpful.
11.4 BACILLUS CEREUS GASTROENTERITIS

Bacillus cereus is not a common cause of food poisoning. It is a Gram positive,
aerobic, spore forming rod shaped bacteria normally present in soil, dust and
water. The bacterium has a minimum growth temperature around 4-5ºC, with
maximum around 48-50ºC. Optimum pH range for growth is 4.9 to 9.3.
Symptoms: Extremely large numbers (108 per gram) of viable cells of
B.cereus must be ingested to develop signs and symptoms of the syndrome.
The bacterial cells produce intoxication characterized by acute abdominal pain,
flatulence and watery diarrhoea. Headache and dizziness are common,
dehydration and prostration may occur but nausea, vomiting, fever and chills
are rare. The illness appears within 6-15 hours after consumption of food and
the symptoms usually last less than 24 hours.
Associated Foods: Vehicle foods consist of cereal dishes that contain corn and
corn starch, mashed potatoes, vegetables, minced meat, liver sausage, milk,
cooked meat. Food mixtures such as sauces, puddings, soups, pastries and
salads have frequently been incriminated in outbreaks.
42
Bacterial Food
Infections
11.5 CHOLERA
Cholera is caused by the gram negative, V. cholerae, which is acquired by
ingesting food or water contaminated by fecal material from patients or carriers
(shellfish and plankton may be the natural reservoir).
Symptoms: Once the bacteria enter the body, the incubation period is from
several hours to three or more days. An infective dose of around one million
organisms should be ingested to cause illness. The bacteria adhere to the small
intestine wall, where they secrete the cholera enterotoxin, choleragen. As a
result, there is hyper secretion of water and chloride ions, while inhibiting
absorption of sodium ions. The patient experiences an outpouring of fluid and
electrolytes with associated abdominal muscle cramps, vomiting, fever and
watery diarrhoea. The diarrhoea can be so profuse that a person can lose 10-15
liter of fluid during the infection. Death may result from the elevated
concentration of blood proteins, caused by reduced fluid levels, which leads to
circulatory shock and collapse. Onset of the illness is generally sudden, with
incubation periods varying from 6 hours to 5 days.
Associated Foods: Cholera is generally a disease spread by poor sanitation,
resulting in contaminated water supplies. Sporadic cases occur when shellfish
harvested from fecally polluted coastal waters are consumed raw.
Diagnosis: Cholera can be confirmed only by the isolation of the causative
organism from the diarrheic stools of infected individuals.
Prevention: Following recommendations are there to prevent cholera
outbreak:
• Drink only water that you have boiled or treated with chlorine or iodine.
Other safe beverages include tea and coffee made with boiled water and
carbonated, bottled beverages with no ice.
• Eat only those foods that have been thoroughly cooked and are still hot, or
fruit that you have peeled yourself.
• Avoid undercooked or raw fish or shellfish.
• Make sure all vegetables are cooked, avoid salads.
• Avoid foods and beverages from street vendors.
A simple thumb rule is “Boil it, cook it, peel it, or forget it”.
Control: Individuals infected with cholera require oral rehydration therapy
with NaCl plus sucrose, sodium bicarbonate and potassium chloride to
stimulate water uptake by the intestine. The antibiotics of choice are a
tetracycline or aproflaxin. The most reliable control methods are based on
proper sanitation, especially of water supplies. The mortality rate without
treatment is often over 50%. Medical treatment to prevent dehydration prevents
all complications.
43
Food Poisoning
#
1. Briefly discuss the Salmonella food infection.
……………………………………………………………………………….
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2. How do you prevent food borne infection?
……………………………………………………………………………….
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11.6 VIBRIO PARAHAEMOLYTICUS GASTROENTERITIS

While most other known food poisoning syndromes may be contracted from a
variety of foods, V. parahaemolyticus gastroenteritis is contracted almost solely
from seafood. It can grow in presence of 1-8% NaCl, in pH range 9.8-11.0
with 7.6-8.6 being optimum.
Symptoms: A total of greater than one million organisms may cause disease.
Symptoms of intoxication which range from mild to severe and fatal, include
abdominal pain, which maybe intense; a burning sensation of the stomach;
vomiting and diarrhoea with watery stools and sometimes bloody discharges;
fever. The mean incubation period is in range of 3-76 hours after the ingestion
of the organism.
Associated foods: Vehicle foods for outbreak are raw, improperly cooked, or
cooked, recontaminated seafoods, such as, oysters, shrimps, crabs, lobsters,
clams and related shellfish. Cross-contamination may lead to other foods as
vehicles. Improper refrigeration of seafoods contaminated with this organism
will allow its proliferation, which increases the possibility of infection.
Diagnosis: Diagnosis of gastroenteritis caused by this organism is made by
culturing the organism from the diarrheic stools of an individual.
44
Bacterial Food
Infections
Prevention: Consumption of raw or improperly cooked seafoods should be
avoided as they are susceptible to infection by V. parahaemolyticus.
11.7 SHIGELLOSIS
Shigellosis or bacterial dysentery, is caused by facultatively anaerobic, gram-
negative, non-spore forming, rod-shaped organisms belonging to the genus
Shigella within the family enterobacteriacae. In general, shigellosis is a self-
limiting disease, lasting 5 to 6 days if untreated, however in young
malnourished children, the elderly and the immuno compromised (eg, AIDS
patients), the disease may be fatal. It is estimated that shigellosis is responsible
for the death of 500,000 children worldwide each year. There are many points
of similarity between Shigella and Salmonella. They dwell primarily in the
gastrointestinal tract, with optimum temperature of 37ºC, grow both aerobically
and anaerobically they grow freely in warm, bland, moist foods. But unlike
salmonellae, the shigellae have no flagella and thus are non-motile. The species
involved are Shigella sonnei, S. dysenteriae, S. flexineri and S. boydii. As few
as 10cfu of S. dysenteriae are known to initiate infection in susceptible
individuals. The illness caused by Shigella accounts for less than 10% of the
reported outbreaks of food borne illness in US. The organisms tolerate salt
concentration of 5-6% and are relatively heat sensitive.
Occurrence: Poor personal hygiene is a common factor in food borne
shigellosis, with shellfish, fruits and vegetables, chicken and salads being
prominent among vehicle foods. The prominence of these foods is due to the
fecal-oral route of transmission. Outbreaks have been also traced to foods such
as chocolate pudding, salads.
Symptoms: Pathogenicity involves the release of lipopolysaccharide endotoxin
which infects the intestinal mucosa. Shigellosis ranges from fairly mild to very
severe and fatal. The onset is usually abrupt, requiring from 1-7 days of
incubation, but sometimes requiring as many as 14 days. Symptoms are
abdominal pain and cramps caused by inflammation of mucus surface of large
intestine, nausea, diarrhoea, vomiting, elevated temperature. The mortality
associated with S. dysenteriae infection is around 20% but it is much lower
with other species. In severe instances, excessive diarrhoea leads to electrolytic
imbalance in the bloodstream and ulceration in large intestine. There may be
kidney failure, jaundice and persistent internal bleeding. The infection is
localized and organs other than the large intestine are not invaded.
Diagnosis: Serological identification of culture isolated from stool helps to
diagnose the disease.
Prevention and Control: The control of Shigella food borne infection is
similar to that of salmonellae; avoiding contamination of foods by animal or
human carriers or their excrement, thorough cooking and prompt cooling.
Proper personal hygiene should be maintained. In severe cases of shigellosis,
dehydration of the body may necessitate intravenous replacement of fluid with
electrolytes. Ampicillin antibiotic can decrease the duration of the disease.
11.8 CAMPYLOBACTERIOSIS
It is caused by Campylobacter jejuni, a Gram negative rod. It is a
microaerophillic organism, which means it has a requirement for reduced levels
of oxygen. It is often isolated from healthy cattle, chickens, birds and even
45
Food Poisoning
flies. It is also sometimes present in non-chlorinated water sources such as

streams and ponds. The bacteria cause between 5 and 14 per cent of all
diarrhoeal illnesses worldwide. C. jejuni primarily affects children under 5
years old and young adults (15-29 years old).
Symptoms: C. jejuni infection causes diarrhoea, which may be watery or
sticky and can contain blood and fecal leukocytes (white cells). Other
symptoms often present are fever, abdominal pain, nausea, headache and
muscle pain. The illness usually occurs 2-5 days after ingestion of the
contaminated food or water. Illness generally lasts 7-10 days, but relapses are
not uncommon (about 25% of cases). Most infections are self-limiting and are
not treated with antibiotics. The infective dose of C. jejuni is considered to be
400-500 bacteria.
Associated Foods: C. jejuni frequently contaminates raw chicken. Survey
shows that 20-100% of retail chickens are contaminated. Many healthy
chickens carry these bacteria in their intestinal tracts. Raw milk is also source
of infections. The bacteria are often carried by healthy cattle and by flies on
farms. Non-chlorinated water may also be a source of infection.
Prevention: the various ways to prevent campylobacteriosis are:
• Wash hands before preparing foods.
• Wash hands immediately after handling raw poultry or other meat.
• Proper cooking of chicken to internal temperature of 170ºC.
• Drink pasteurized milk and chlorinated water.
• Wash hands after handling pet feces or visiting zoos.
Control: If a person is suffering from campylobacteriosis, he can take an
antibiotic such as ciproflaxin or azithromycin. Erythromycin also helps to treat
the diarrhoea. Those having diarrhoea should take plenty of water.
1. Give the causative organism and symptoms of Bacillary Dysentry.
……………………………………………………………………………….
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2. What are the symptoms and foods associated with campylobacteriosis?
……………………………………………………………………………….
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46
Bacterial Food
Infections
11.9 YERSINIOSIS (YERSINIA ENTEROLYTICA

INFECTION)
In the genus Yersinia, 11 species are recognized, including Y. pestis, the cause
of plague. The species of primary interest in foods is Y. enterolytica.
Occurrence: Y. enterolytica is widely distributed in the terrestrial environment
and in lake, well and stream waters which are sources of warm-blooded
animals. It is more animal adapted and is found more often among human
isolates than the other species.
Animals from which Y. enterolytica has been isolated include cats, birds, dogs,
beavers, guinea pigs, rats, camels, horses, chickens, deer, cattle, swine, fish and
oysters. It is widely believed that swine constitutes the single most common
source of Y. enterolytica in humans.
In addition to gastroenteritis, this organism has been associated with human
pseudoappendicitis, mesenteric lymphadenitis, reactive arthritis, colon and
neck abscesses and cholecystis. It has been recovered from urine, blood,
cerebrospinal fluid and the eyes of infected individuals. It is also recovered
from the stools of gastroenteritis victims.
Associated Foods: The organism has been isolated from cakes, vacuum-
packaged meats, seafood, vegetables, milk and other food products. It has been
isolated also from beef, lamb and pork. Of all sources, swine appears to be the
major source of pathogenic for humans.
Symptoms: Symptoms of the gastroenteritis syndrome develop several days
following ingestion of contaminated foods and are characterized by abdominal
pain and diarrhoea. Children appear to be more susceptible than adults and the
organism may be present in stools for up to 40 days following illness. A variety
of systemic involvements may occur as a consequence of the gastroenteritis
syndrome.
The usual symptoms, including severe abdominal pain, fever and diarrhoea
occur 24 to 36 hours after consumption of the product. The abdominal
discomfort is quite specific and usually manifests itself as a sharp pain in the
lower right quadrant of the abdomen. For this reason it has frequently been
described as pseudoappendicitis.
Although the organism has been isolated from many foods, there have been
relatively few food-borne outbreaks attributed to Y. enterolytica. The isolation
from pasteurized milk is probably the result of post pasteurization
contamination. The unique characteristic of the organism is its ability to grow
at commercial refrigeration temperatures, i.e. less than 5ºC.
11.10 LISTERIA MONOCYTOGENES INFECTION

(LISTERIOSIS)
Listeria monocytogenes is a gram-positive, motile, non-sporing rod. It is widely
distributed in nature and can be found on decaying vegetation and in soils,
animal feces, sewage, silage and water. In cattle it can result in abortion and
mastitis and the infected animals shed the organism in milk. Other infected
organisms including sheep and chicken can serve as source of the organism in
the food supply.
47
Food Poisoning
Syndrome: Listeriosis in humans is not characterized by a unique set of

symptoms since the course of the disease depends upon the state of the host.
Non-pregnant healthy individuals who are not immunosuppressed are highly
resistant to infection by L. monocytogenes. When susceptible adults contract
the disease, meningitis and sepsis are the most commonly recognized
symptoms. Pregnant females who contract the disease may not present any
symptoms, but when they do, they are typically mild and influenza-like.
Abortion, premature birth or still birth is often the consequence of listeriosis in
pregnant females. When a newborn is infected at the time of delivery,
listeriosis symptoms typically are those of meningitis and they begin at 1 to
4 weeks after birth, although a four week incubation has been recorded. The
usual incubation time in adults ranges from one to several weeks.
Since L. monocytogenes can grow over the temperature range of about 1º to
45ºC and the pH range 4.1 to around 9.6, it maybe expected to survive in foods
for long periods of time.
Some of the ways in which L. monocytogenes is disseminated throughout the
environment, along with the many sources of the organism to humans, are
illustrated below.
ANIMAL SECRETION AND

EXCRETION
Feces, uterine and vaginal
secretion, placenta, membranes
Aerial Aerial
contamination contamination
ANIMAL ENVIRONMENT MAN

Soil—Water
ase/Colonization Disease/Colonization
Sewage Vegetable
Sludge
(cabbage, plants,
water
silage)
ANIMAL PRODUCTS
Milk, meat, carcass, dairy
products
Figure 13.1: Ways in which L. monocytogenes is disseminated in the environment,

animals, foods and humans
48
Bacterial Food
Infections
1. What is yersiniosis? Give its symptoms.
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2. How is L. monoctogenes infection transmitted?
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Tips on Foodborne Illness Prevention for Consumers

Salmonella, Escherichia coli, Shigella, Campylobacter, Listeria, and the list
goes on. Interesting names for little organisms that can cause big bad health
problems. Bacterial foodborne diseases have become an acute public health
crisis in this country, responsible for about 6.5 to 81 million illnesses and 9,000
deaths per year. While it is unrealistic to think there would ever be a zero level
of contamination in our food supply, 90% - 95% of all foodborne bacterial
illness is preventable. You cannot see, taste, or smell most bacterial foodborne
problems, so here's what you can do to safeguard your family.
In General:
• Don't ever consume products such as unpasteurized milk or unpasteurized
apple cider or other foods made with unpasteurized products. Thoroughly
cook all meat, poultry and fish products. Meats are thoroughly cooked at
71ºC, poultry at 82ºC, and fish when they easily flake with a fork.
• Break open any hamburger patties (other ground meat dishes such as
meatballs, too) prior to ingestion to make sure there is no pink meat and
that the juices run clear.
When Dining Out
• Hot food should be served hot and cold food should be served cold or send
it back.
• Talk to the restaurant manager. Find out how much importance they place
on sanitary and bacterial issues. Do they routinely use thermometers? Do
49
Food Poisoning
they own and routinely use a thermocouple which is a special thermometer

used to accurately test temperatures of thin food? If you receive
undercooked food (especially ground meat) express your displeasure and
nicely inform them of the risks. Let them know how important food safety
is to you.
When Shopping
• Take great care to avoid dripping raw juice from meat, poultry or fish onto
your hands or other foods, especially produce. Raw juices often contain
bacteria.
• Shop for cold and frozen foods last and take them immediately home to the
refrigerator or freezer. Use ice chests in your car during transport,
especially in the summer months or when running errands.
• Buy food only if it is in good condition. Frozen foods should be solid,
refrigerated case food should be well-chilled, and canned goods should be
free of dents and bulging lids. Point out any problems to the store manager.
When in your Kitchen
• Always wash your hands in hot soapy water before food preparation and
after handling raw meat, poultry or fish.
• Keep your refrigerator's temperature as cold as possible without freezing
your milk or produce (approximately 4°C). Keep your freezer's temperature
cold enough to keep frozen food rock hard (approximately -18°C). If you
are ever in doubt, temperatures can be checked with an appliance
thermometer.
• After shopping, put any fresh meat, poultry, or fish, which won't be use
within the next few days directly into the freezer.
• Thaw frozen food in the refrigerator or in a microwave followed by
immediate cooking. Do not thaw food at room temperature on the
counter.
• Take great care to avoid dripping of raw meat, poultry, or fish juices onto
or into other foods in the refrigerator. Use plates, platters or containers
under them if necessary.
• Never put cooked food back on a plate/container which has had fresh juices
on it. For example, when barbecuing take out an extra platter to put the
grilled food on.
• Use non-porous plastic cutting boards for preparation and cutting of meat,
poultry, and fish.
• Wash all cutting boards surfaces, platters, and containers which fresh meat,
poultry, and fish have come in contact with, in hot, soapy water thoroughly
before using for other foods.
• Avoid cross contamination by washing kitchen towels after contact with
raw juices and by replacing sponges often. Use paper towels wherever you
can. A good disinfectant for utensils and countertops is one tablespoon
household bleach in few litres of water.
50
Bacterial Food
Infections
• When using eggs, cook them until firm. Don't use recipes calling for only
partially cooked eggs. For example, raw cookie dough could be dangerous.
When using the microwave remember there can be cold spots, so stir and rotate
food for thorough cooking.
11.11 THE MOST IMPORTANT POINT TO REMEMBER

IS TO WASH YOUR HAND
Preferably with antibacterial soap for at least 20 seconds....
• before food preparation
• after fresh meat, poultry, or fish handling and before you then touch
other food, eat, or prepare baby bottles
• after using the bathroom
• after changing diapers
• after helping toddlers in the bathroom
• after cleaning the bathroom
• after handling pets, cleaning litter boxes or dog runs
PLEASE REMEMBER: Foodborne bacterial illness can be very
contagious. This is called secondary transmission where a person gets ill not
from ingesting the contaminated food but from coming in close contact with
someone who has. Secondary transmission has been documented in the home,
in day care centres, in preschools, in schools, in hospitals, and in senior citizen
facilities. When someone you know has diarrhoeal illness, use extreme sanitary
measures, to guard against the spread of the disease. The use of anti-diarrhoeal
medication for treatment of foodborne bacterial diarrhoea is not recommended
and in some cases can be harmful. If symptoms are severe, see your doctor. If
food poisoning is suspected, call your local health department. Your prompt
action could help prevent someone else from getting ill.
11.12 LET US SUM UP

In this unit, we attempted to familiarize you with certain outbreaks of bacteria
which are responsible for food borne infections. We hope that you will be able
to know the various bacteria which cause food borne infections. You will also
be able to differentiate in the symptoms of the diseases caused and list the main
reasons for the outbreak of the infections. This unit would also have helped you
to know the information regarding the prevention of the outbreaks of the
disease.
11.13 KEY WORDS

Food Infection : Food borne disease caused due to ingestion of
large number of viable organisms which cause
disease.
Zoonotic Diseases : Those diseases and infections which are naturally
transmitted between vertebrate animals and man.
51
Food Poisoning
Salmonellosis : Food borne infection caused by Salmonella

gastroentritis.
Enterotoxigenic
E. coli : E. coli which produce toxins in the intestine.
Cholera : Food borne infection caused by Vibrio cholarae.
Shigellosis : Food borne infection caused by Shigella sonnei,
S. dysenteraei.
Yersiniosis : Food borne infection caused by Yersinia
enterolytica.
Listeriosis : Food borne infection caused by Listeria
monocytogenes.

EXERCISES
1. • Salmonella food infection is caused by Salmonella gastroenteritis.
• Transmitted by fecal contamination of foods.
• Incubation period 12-36 hours.
• Symptoms: nausea, vomiting, diarrhoea, abdominal pain and green
watery stools.
• About 106-109 organisms must be ingested to cause infection.
• Salmonellosis prevented by: prevention of contamination of food by
Salmonella, avoiding intake of contaminated food and by destruction of
organism at 66ºC for 12-15 min.
2. Prevention of Food borne infections:
• Avoid consumption of contaminated foods and water
• Eat properly cooked foods
• Wash raw fruits and vegetables properly
• Proper hygiene
1. • Bacillary dysentery is caused by Shigella sp. (Shigella sonnei, S.
dysenteraei).
• Incubation period is about 1 to 7 days.
• Causes abdominal pain, cramps, inflammation of intestinal mucousa,
diarrhoea, vomiting, nausea and fever.
• In severe cases; intestinal bleeding, electrolytic imbalance, ulceration,
kidney failure and jaundice.
2. • Campylobacteriosis incubation period: 2-5 days after ingestion of
contaminated food.
52
Bacterial Food
Infections
• Symptoms: Diarrhoea containing blood, fever, abdominal pain, nausea,
headache and muscle pain.
• Transmission of infection by raw chicken, raw milk and non-
chlorinated water.
1. • Yersiniosis caused by the bacteria Yersinia enterolytica.
• Causes severe abdominal pain, fever and diarrhoea.
2. • Listriosis caused by Listeria monocytogenes infection.
• Transmitted by animal excretions (fecal matter) and secretions, infected
vegetables, aerial contamination, sewage, sludge, polluted water, rivers
and infected animal products like milk and milk products, meat, fish
etc.

1. Ayers, J.C., Mundt, J.O., Sandine, W.E. (1980) Microbiology of Foods,
W.H Freeman and Co., San Franscisco. pp708.
2. Frazier, W.C. and Westoff, D.C. (1988) Food Microbiology, Tata McGraw-
Hill Publishing Co., New Delhi. pp 539.
3. Pelczar, M.J., Chan, E.C.S., Kreig, N.R. (1997) Microbiology, Tata
McGraw-Hill Publishing Co Ltd, New Delhi. pp 918.
53
Food Born Diseases
UNIT 9 FOOD BORN DISEASES
Structure
9.0 Objectives
9.1 Introduction
9.2 Types of Food Borne Diseases
9.3 Human Diseases
9.4 Chemical Contamination of Foods
9.5 Non-bacterial Microbiological Contamination of Food
Viruses (Hepatitis A Virus, Polio Virus, Norwalk and Norwalk-like Virus)
Rickettsia
Food Borne Parasites (Trichinellosis, Amoebiasis, Giardiasis, Ascariasis)
9.6 Investigation of Food Borne Disease Outbreak
9.7 Let Us Sum Up
9.8 Key Words
9.0 OBJECTIVES
• know the food borne diseases;
• learn to differentiate between food borne intoxications and food borne
infections;
• explain that the food borne diseases may be caused due to microorganisms,
both bacterial and non bacterial or chemicals;
• explain why investigation of food borne disease outbreak is necessary; and
• describe how the investigation of food borne disease outbreak is carried
out.
9.1 INTRODUCTION
Food borne disease (FBD) is caused by consuming contaminated foods or
beverages. In addition poisonous chemicals or other harmful substitutes can
cause food borne diseases if they are present in food. More than 250 different
food borne diseases have been described. A classification of food borne
diseases is given in Figure 9.1. Food borne diseases may be intestinal diseases
but can be other type as well.
9.2 TYPES OF FOOD BORN DISEASES

With regard to their epidemiology, they can be divided into two major
categories (Figure 9.2):
i) Food borne Intoxication: Examples include botulism or staphylococcal
food poisoning, the causative microorganism produces an exotoxin in food:
when a person consumes the food, the toxin is ingested and gives rise to
disease.
ii) Food borne Infections: the causative organisms are ingested: these
subsequently grow within the body and cause damage.
5
Food Poisoning Both infections and intoxications often cause diarrhoea. Severe diarrhoea
accompanied by blood or mucus is called dysentery. Both types of digestive
system diseases are also frequently accompanied by abdominal cramps, nausea
and vomiting. Diarrhoea and vomiting are both defensive mechanisms
designed to rid the body of harmful material.
The general term gastroenteritis is applied to disease causing inflammation of
the stomach and intestinal mucosa.
FOOD-BORNE DISEASES
POISIONINGS INFECTIONS
Chemical Intoxications Enterotoxigenic Invasive
Sporulation Growth and lysis
Poisonous Poisonous Microbial Intestinal Systemic Other

plant tissues animal intoxications mucosa tissues
tissues
Algal Mycotoxins Bacterial Muscle Liver

toxins toxins
Enterotoxins Neurotoxins Interferes with

carbohydrate
metabolism
Figure 9.1: Classification of food borne diseases
6
Food Born Diseases
FOOD-BORNE DISEASES: BACTERIAL
Intoxications Infections
1. Staphylococcal intoxication 1. Salmonellois: enterotoxin
(staphyloccocal enterotoxicosis): and cytotoxin of Salmonella
an enterotoxin produced by spp.
Staphylococcus aureus
2. Clostridium perfrigens illness:
an enterotoxin released during
sporulation of Clostridium
perfringens type A in the
2. Botulism: a neurotoxin produced by intestinal tract
Clostridium botulinum
3. Bacillus cereus gastroenteritis:
an exo-enterotoxin released
during lysis of Bacillus cereus
in the intestinal tract
4. Enteropathogenic Escherichia
coli infection: Several serotypes
of E. coli, some invasive and
some enterotoxigenic
5. Others: Yersiniosis, Shigellosis

Vibrio parahaemolyticus
Figure 9.2: Bacteria responsible for food borne intoxications and infections
9.3 HUMAN DISEASES

Microorganisms that produce infectious diseases in human are categorized as
pathogens, because they have the ability to injure body tissues and/or alter
body functions.
In general, pathogenic microorganisms express their disease-producing
properties through two kinds of mechanisms: (1) invasion of tissues (invasive
microorganisms) and (2) production of toxins (toxigenic microorganisms).
Invasive pathogens have the ability to produce and excrete one or more kinds
of extracellular enzymes resulting in injury to host tissues. Toxigenic
microorganisms produce toxins of two types:
• Exotoxins − produced within certain kinds of bacteria and excreted into
their surrounding environment. They are proteins in chemical composition
and are relatively specific in terms of damage to the host.
• Endotoxins − are complex polysaccharides cell wall components of certain
kind of bacteria. They are not released until the cell disintegrates. They are
relatively heat stable, less specific in their actions and less potent than
exotoxins.
7
Food Poisoning The severity and distribution of any infectious disease are influenced by the
manner in which the agent, the host and factors within the environment
interact.
Environment
Indirect
Interaction
Agent Host
(Microorganism) (Animals, Human,
Arthropods)
Figure 9.3: Generalized version of the triangle of causation for any kind of
communicable disease
The main causes of reported food borne diseases are due to foods being
mishandled. Foods that are implicated are usually “potentially hazardous”
which are capable of supportive growth of disease-causing microorganisms.
The various factors leading to FBD are listed in Table.
Table 9.1: Factors leading to reported food borne diseases (Ranked by
% number of outbreaks)
40% Improper cooling of foods

21% Time lapse between preparing to serving
20% Infected persons touching foods
16% Inadequate cooling
16% Improper hot storage
12% Inadequate reheating
11% Contaminated raw food
7% Cross-contamination
7% Improper cleaning
4% Use of leftovers
9.4 CHEMICAL CONTAMINANTS OF FOOD

Poisoning by consumption of chemicals is rather uncommon and usually is
characterized by appearance of the symptoms within a short period of time
after the poisonous food is eaten. Various chemical contaminants include:
i) Fumigants are used to sterilize food under conditions in which steam
heating is impractical. Ethylene oxide is a commonly used fumigant, which
reacts with food constituents and destroys essential nutrients. It reacts with
inorganic chloride to form ethylene chloro hydrine, which is toxic.
8
ii) Various solvents are used for the extraction of oil from oilseeds. But Food Born Diseases
solvents like trichloro ethylene react with the foodstuff being processed
with the formation of toxic products.
iii) Smoking of meat and fish for preservation and flavouring is an old
practice. This processing contaminates the food with polycyclic
hydrocarbons such as benzopyrene, many of which are carcinogenic.
iv) Metals are one of the many unintentional contaminants of food. When
present beyond small quantities they are toxic. They find their way into
food through air, water, soil, industrial pollution and other routes.
Antimony, arsenic, cadmium, chlorinated hydrocarbons, copper, cyanide,
fluoride, lead, selenium, mercury and zinc in foods have been blamed for
food poisoning. Poisonous chemicals may enter foods from utensils e.g.
from cheap enamelled utensils which contain antimony. Lead and arsenic
residues from fruit sprays maybe on the surface of fruits but usually in
harmless amounts, especially after washing. Symptoms of lead poisoning
include weakness, dental caries, nausea, pains and paralysis. A major
source of tin contamination is tin plate, which is used for containers of all
types of processed foods. Canned foods if acidic and foods stored in tins
after opening, change in colour, or develop a metallic flavour that is
unacceptable. Insecticides, pesticides, growth regulators, fungicides and
growth stimulators are essential in modern agriculture for the production of
adequate quantities of sound food. They include insecticides like lead
arsenate, organophosphate compounds (malathion), dinitro compounds.
Toxicity levels for human vary.
"
1. What are food borne diseases?
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
2. Differentiate between exotoxins and endotoxins.
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
9
Food Poisoning 3. List the various chemical contaminants in food.
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
Table 9.2: Toxicity of some metals in different foods
Metal Type of Food Toxic Effect

Arsenic Fruits sprayed with lead Dizziness, chills, cramps,
arsenate paralysis leading to death
Barium Foods contaminated with rat Violet peristalsis,
poison (barium carbonate) muscular twitching and
convulsions
Cadmium Fruit juice, soft drinks in Excessive salivation and
contact with cadmium kidney damage, prostrate
plated vessels cancer, multiple fractures
Cobalt Water, beer Cardiac failure
Copper Acid foods in contact with Vomiting, diarrhea,
tarnished copper ware and abdominal pain
brass utensils
Lead Processed foods Paralysis, brain damage
Mercury Mercury fungicide treated Paralysis, brain damage
seed grains or mercury and blindness
contaminated fish
Tin Canned foods Colic, vomiting.
Photophobia
Zinc Foods stored in galvanized Dizziness, vomiting
iron ware
Table 9.3: Toxicity in food due to various pesticides and chemicals
Name of Pesticide Type of Food Toxic Effect

Pesticides All type of raw, cooked, Acute or chronic poisoning
processed, canned foods causing damage to liver,
kidney, brain, nerves
leading to death
Diethylstil bestrol Meat of still bestrol fed Teratogenesis,
animals carcinogenesis
Antibiotics Meat of animals fed Drug resistance, hardening
antibiotics of arteries, heart diseases
10
Table 9.4: Permissible limits of some metals in foods Food Born Diseases
Name of Foodstuff PPM

metal
Arsenic 1. Milk 0.10
2. Beverages 0.50
3. Soft Drinks 0.50
4. Ice creams and frozen confections 0.50
5. Dehydrated onions 2.0
6. Dried spices and herbs 5.0
Copper 1. Beverages, soft drinks 7.0
2. Tomato ketchup 50.0
3. Cocoa powder 70.0
4. Tomato puree, paste, juice powder 100.0
5. Sugar confectionary 5.0
Lead 1. Beverages and soft drinks 0.50
2. Fruits and vegetable juices 1.0
3. Ice cream, frozen confections, 1.0
canned fish, meat, dehydrated
vegetables
Tin 1. Processed and canned foods 5.0
2. Hard boiled sugar confectionary 5.0
Zinc 1. Beverages 5.0
2. Fruit products 50.0
3. Hard boiled sugar confectionary 5.0
9.5 NON-BACTERIAL MICROBIOLOGICAL

CONTAMINATION OF FOOD
Microbiological contamination of food may include food borne illness due to
bacteria or non bacterial sources such as mycotoxins, viruses, rickettsias,
parasitic worms or protozoa or from the consumption of food contaminated
with toxic substances. The food borne infections caused by bacteria will be
dealt with in Unit 3 in detail.
9.5.1 Viruses
Much less is known about the incidence of viruses in foods than about bacteria
and fungi because they do not grow in culture media as do bacteria and fungi,
they do not replicate in foods thus found in low numbers. It is noted that
virtually any food can serve as a vehicle for virus transmission. The most
common food source of gastroenteritis causing virus is shellfish.
11
Food Poisoning Hepatitis A virus
There are more documented outbreaks of hepatitis A traced to foods than any
other viral infection. The virus causes hepatitis (jaundice) and leads to
inflammation of liver. The incubation period for infectious hepatitis ranges
from 15 to 45 days and lifetime immunity usually occurs after an attack. The
fecal-oral route is the mode of transmission, and raw or partially cooked
shellfish from polluted waters is the most common vehicle food. Shellfish are
able to concentrate the numbers of bacteria or viruses during their normal
feeding, which is to filter and remove particles from the water. The infectious
hepatitis virus has been shown to be stable during refrigerated storage of
shellfish. In addition to shellfish, raw milk, potato salad, sandwiches and cold
meat cuts are also probable sources of the virus. Symptoms of jaundice include
loss of appetite, yellowing of eyes, nails and skin (due to presence of bile
pigments) and gastrointestinal disorder. Proper cooking, hygiene, sanitation
and personnel cleanliness help to prevent virus attack.
Polio Virus
There are a large number of reported food-borne outbreaks of polio in India. It
is most common in children up to 5 years. Milk is the most probable food there
causes spread of polio virus. The virus reproduces in the intestinal tract, from
there it invades the motor cells of the central nervous system. Initial symptoms
are gastrointestinal, headache, muscle pain and paralysis. The paralytic
symptoms range from sub clinical to fatal. Preventive measures include
immunization of children, proper processing (pasteurization) of milk, hygienic
conditions and use of potable water.
Norwalk Virus and Norwalk-like Viruses
These viruses, also known as small round structured viruses or caliciviruses,
are an important cause of gastrointestinal illness throughout the United States.
Members of this category of viruses are typically named for the location in
which they were first identified, for example, Hawaii, Snow Mountain,
Montgomery County and Oklahoma. The Norwalk virus is the prototype for
this group of viruses – there are at least 11 other related viruses – hence the
name “Norwalk-like virus.”
Symptoms: The signs and symptoms of Norwalk-like viruses include nausea,
vomiting, diarrhea, abdominal pain, muscle aches, headache, tiredness and
low-grade fever. Symptoms typically last 24 hours to 48 hours and subside on
their own. There are no known long-term effects after recovery from this
infection.
Transmission of viruses: Humans are the only source for these viruses. These
viruses do not multiply outside the human body. The viruses are present in the
feces of infected persons and can be transmitted to others when hands are not
thoroughly washed after having a bowel movement.
9.5.2 Rickettsias
Rickettsias maybe considered as degenerative bacteria since they represent a
form of life closely resembling bacteria except that they cannot be cultivated
outside of living cells. Like the viruses they are obligate parasites. Many of the
major human rickettsial diseases are by bites from fleas, lice or ticks.
Examples of human rickettsial diseases include epidemic typhus, rickettsial
12
pox, Rocky mountain spotted fever and Q fever. Cows infected with rickettsia Food Born Diseases
of Q fever, Coxiella brunettii excrete contaminated milk which result in human
infections. Hence milk is pasteurized at a minimum temperature of 62.8ºC for
30 minutes to ensure its destruction.
9.5.3 Food Borne Parasites

Trichinosis
Trichinella spiralis causes trichinosis, which results from the consumption of
raw or incompletely cooked pork containing the encysted larvae.
Symptoms: One or two days after ingestion of heavily encysted meat,
trichinae penetrate the intestinal mucosa, producing nausea, abdominal pain,
diarrhea and sometimes vomiting. The symptoms may persist for several days.
The larvae then attack the skeletal muscles, muscle pain (paralysis) is the
universal symptom accompanied in difficulty in breathing, chewing and
swallowing. After six months of initial infection, pain, swelling and fever
occur.
Prevention and control: Chief method for prevention of trichinosis is the
treatment of pork (or other meat) to ensure the destruction of any trichinae that
maybe present by cooking of pork till at least 58.3ºC, quick freezing or storage
at -15ºC or lower for not less than 20 days, irradiating or processing of sausage
and similar meat products properly by salting, drying, smoking and
refrigeration. Also trichinosis can be controlled by avoiding feeding of infected
meat scraps to swine and by preventing the consumption of infested tissue by
other animals.
Amoebiasis
Entamoeba histolytica is a single celled parasitic animal – a protozoa, that
infects predominantly humans and other primates and causes amoebiasis.
Diverse mammals such as dogs and cats can become infected but usually do
not shed cysts (the environmental survival form of the organism) with their
feces, thus do not contribute significantly to transmission. The active
(trophozoite) stage exists only in the host and in fresh feces; cysts survive
outside the host in water and soil and on foods, especially under moist
conditions on the latter. When swallowed they cause infections by excysting
(to the trophozoite stage) in the digestive tract (amoebiasis).
Symptoms: Infections that sometimes last for years may be accompanied by 1)
no symptoms, 2) vague gastrointestinal distress, 3) dysentery (with blood and
mucus). Most infections occur in the digestive tract but other tissues may be
invaded. Complications include 4) ulcerative and abscess pain and, rarely, 5)
intestinal blockage. Onset time is highly variable. It is theorized that the
absence of symptoms or their intensity varies with such factors as 1) strain of
amoeba, 2) immune health of the host, and 3) associated bacteria and, perhaps,
viruses. The amoeba's enzymes help it to penetrate and digest human tissues; it
secretes toxic substances.
Diagnosis: The ingestion of one viable cyst can cause an infection. Human
cases are diagnosed by finding cysts shed with the stool; various flotation or
sedimentation procedures have been developed to recover the cysts from fecal
matter; stains (including fluorescent antibody) help to visualize the isolated
cysts for microscopic examination. Since cysts are not shed constantly, a
13
Food Poisoning minimum of 3 stools should be examined. In heavy infections, the motile form
(the trophozoite) can be seen in fresh feces. Serological tests exist for long-
term infections. It is important to distinguish the E. histolytica cyst from the
cysts of nonpathogenic intestinal protozoa by its appearance.
Transmission: Amebiasis is transmitted by fecal contamination of drinking
water and foods, but also by direct contact with dirty hands or objects as well
as by sexual contact.
Giardiasis
Giardia lamblia (intestinalis) is a single celled animal, i.e., a protozoa, that
moves with the aid of five flagella. Organisms that appear identical to those
that cause human illness have been isolated from domestic animals (dogs and
cats) and wild animals (beavers and bears). A related but morphologically
distinct organism infects rodents, although rodents may be infected with
human isolates in the laboratory. Human giardiasis may involve diarrhea
within 1 week of ingestion of the cyst, which is the environmental survival
form and infective stage of the organism.
Symptoms: Normally illness lasts for 1 to 2 weeks, but there are cases of
chronic infections lasting months to years. Chronic cases, both those with
defined immune deficiencies and those without, are difficult to treat. The
disease mechanism is unknown, with some investigators reporting that the
organism produces a toxin while others are unable to confirm its existence. The
organism has been demonstrated inside host cells in the duodenum, but most
investigators think this is such an infrequent occurrence that it is not
responsible for disease symptoms. Mechanical obstruction of the absorptive
surface of the intestine has been proposed as a possible pathogenic mechanism,
as has a synergistic relationship with some of the intestinal flora.
Diagnosis: Giardia lamblia is frequently diagnosed by visualizing the
organism, either the trophozoite (active reproducing form) or the cyst (the
resting stage that is resistant to adverse environmental conditions) in stained
preparations or unstained wet mounts with the aid of a microscope. A
commercial fluorescent antibody kit is available to stain the organism.
Organisms may be concentrated by sedimentation or flotation; however, these
procedures reduce the number of recognizable organisms in the sample.
Associated foods: Giardiasis is most frequently associated with the
consumption of contaminated water. Outbreaks have been traced to food
contamination by infected or infested food handlers, and the possibility of
infections from contaminated vegetables that are eaten raw cannot be excluded.
Cool moist conditions favor the survival of the organism.
Prevalence: Giardiasis is more prevalent in children than in adults, possibly
because many individuals seem to have a lasting immunity after infection. This
organism is implicated in 25% of the cases of gastrointestinal disease and may
be present asymptomatically. This disease afflicts many homosexual men, both
HIV-positive and HIV-negative individuals. This is presumed to be due to
sexual transmission. The disease is also common in child day care centers,
especially those in which diapering is done.
14
Ascariasis Food Born Diseases
Humans worldwide are infected with Ascaris lumbricoides and Trichuris

trichiura; the eggs of these roundworms (nematode) which are "sticky" and
may be carried to the mouth by hands, other body parts, fomites (inanimate
objects), or foods. Ascariasis and trichuriasis are the scientific names of these
infections. Ascariasis is also known commonly as the “large roundworm”
infection and trichuriasis as “whip worm” infection.
Diagnosis: Infection with one or a few Ascaris sp. may be unapparent unless
noticed when passed in the feces, or, on occasion, crawling up into the throat
and trying to exit through the mouth or nose. Infection with numerous worms
may result in a pneumonitis in the lungs, where the larvae break out of the
pulmonary capillaries into the air sacs, ascend into the throat and descend to
the small intestine again where they grow, becoming as large as 31 × 4 cm.
Vague digestive tract discomfort sometimes accompanies the intestinal
infection, but in small children with more than a few worms there may be
intestinal blockage because of the worms' large size. Not all larval or adult
worms stay on the path that is optimal for their development; those that wander
may locate in diverse sites throughout the body and cause complications. Both
infections are diagnosed by finding the typical eggs in the patient's feces; on
occasion the larval or adult worms are found in the feces or, especially for
Ascaris sp., in the throat, mouth, or nose.
Associated foods: The eggs of these worms are found in insufficiently treated
sewage-fertilizer and in soils where they embryonate (i.e., larvae develop in
fertilized eggs). The eggs may contaminate crops grown in soil or fertilized
with sewage that has received nonlethal treatment; humans are infected when
such produce is consumed raw. Infected food handlers may contaminate a wide
variety of foods.
Prevention: Both infections may self-cure after the larvae have matured into
adults or may require anthelmintic treatment. In severe cases, surgical removal
may be necessary. Allergic symptoms (especially but not exclusively of the
asthmatic sort) are common in long-lasting infections or upon reinfection in
ascariasis.
"
1. What are the non-bacterial food borne disease causing agents?
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
15
Food Poisoning 2. What is the causative organism for jaundice and how is it transmitted?
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
3. List the food-borne parasites. What are the symptoms of the disease cause
by them?
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
9.6 INVESTIGATION OF FOOD BORNE DISEASE

OUTBREAK
Analysis of an outbreak involves field analysis as well as laboratory analysis
and is undertaken by Ministry of Health. From public health point of view the
objectives of the investigation of outbreak are:
1. Prevention and control
Identification of contaminated foods
2. Knowledge of disease causation
Observe the track record of various illnesses-causing agents
3. Administration guidance
Assessment of trends to justify regulations, decisions/actions
This requires the location and the identification of the causative agent,
establishing of the means of transmission, demonstration of opportunity for
growth of the pathogen.
Personnel Involved in Investigation
The team to investigate an outbreak of food borne disease consists of a person
in charge, a field group and a laboratory group. The field group interviews
persons, both ill and healthy, who consumed the suspected foods, physicians
and nurses who are treating the victims, and personnel at the place of exposure
to the disease; collects samples of suspected foods and transits them to the
laboratory, collects specimens from patients or food handlers when such
16 sample is indicated; inspects the premises where the foods were stored,
prepared and served; fills out appropriate reports on these activities, and makes Food Born Diseases
it available for laboratory staff. The laboratory group makes microbiological
and chemical tests as indicated by reports of the field group and the nature of
the suspected food and records its findings on appropriate report blanks. The
person in-charge or a qualified epidemiologist then can interpret the data from
all sources to determine the cause and source of disease outbreak.
Steps of Investigation
Field Analysis
a) Gathering information
The field group inspects the place or places where the suspected meal,
meals or beverages were prepared and consumed and then record the
results in appropriate forms. Information sought includes the menu for
meals, source and method of preparation of each item on the menu,
methods of storage of perishable foods, health of employees serving or
preparing foods and their health history. The information obtained is
recorded on a form entitled “Case History Questionnaire” as given.
Performa
Name of Person Age Time of First Foods Consumed

affected Symptom
The Performa indicate the most likely age group affected and the type of
food being consumed by them. Also we get an idea of the type of food
through which the infection could have probably been caused.
b) Sample collection
Samples of left over food and /or beverage served at suspected meal are
aseptically collected by means of sterile sampling devices in sterile
containers, labelled and transited to laboratory under refrigerated
conditions.
c) Collection of specimens from human sources
Specimens maybe obtained from patients with food illnesses or from food
handlers to ascertain the ultimate source of the pathogen that entered the
food. Culture from nose, throat or skin lesions, fecal or blood samples may
be done.
Laboratory Analysis
The sample received from the field are analyzed microbiologically and
sometimes for chemical contamination also. The report is then sent to the in-
charge, the epidemiologist (who is expert in dealing with epidemics).
17
Food Poisoning Performa for Laboratory Report
Etiological Clinical Laboratory Complete Preventive

Agent Symptoms Investigation Investigation Measures
(Causative (of affected (Food
Agent of people) responsible for
Spoilage) Outbreak)
Eg: For Investigation of Infection due to Asepsis, prevent
Bacillus food, stools and accidental inadequate
gastroenteritis faeces. Isolation of contamination or treatments,
the microorganism otherwise proper storage
conditions
Interpretation of Report
This is done by the epidemiologist and conclusion is taken out for cause of the
outbreak. Complete investigation of food borne disease outbreak is given as:
i) Name of the reporting officer
ii) Name of the local authority who is analyzing the complete outbreak
iii) Area/place of outbreak
iv) Date and time when suspected meal was taken
v) Number of persons’ affected
vi) Number of people at risk indicating those who have developed symptoms)
vii) Incubation Period
viii) Symptoms
ix) Occupational/age group
x) Details of suspected meal (complete analysis of food)
xi) Foods which are eaten by affected persons
xii) Number of meal sittings and time
xiii) Methods of cooking
xiv) Time and temperature of storage of cooking
xv) General notes regarding the facilities and equipments
Minimum Infrastructure/ Materials Required
• Field Kit for field analysts to collect sample, medical equipment, sterile
containers, sampling device, sterilized thermometer, lamps of alcohol,
sterile wrapping foil, tapes for sealing, sterile paper, towels, ice boxes,
insulated boxes for carrying samples.
• Laboratory- Facility to find the total plate count and types of
microorganisms, glassware, pipettes, flask, media, laminar flow, stains.
Chemicals, specific test kits for enumerating specific organisms.
• Data interpretation infrastructure.
WHO’S Golden Rule for Safe Food Production
1. Choose foods processed for safety.
2. Cook foods thoroughly.
18
3. Eat cooked foods immediately. Food Born Diseases
4. Store cooked foods carefully.

5. Reheat cooked foods thoroughly.
6. Avoid contact between raw and cooked foods.
7. Wash hands repeatedly.
8. Keep all kitchen surfaces meticulously clean.
9. Protect foods from insects, rodents and other animals.
10. Use pure water.
"
1. What are the main objectives for which investigation of any outbreak is
done?
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
2. Explain the various steps involved to carry out any investigation.
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
3. State WHO’s Golden rule for safe food production.
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
19
Food Poisoning
9.7 LET US SUM UP
In this unit on food borne disease, we have examined what are the food borne
disease and how to differentiate with food borne infections and intoxications.
This includes study of various contaminants of food, namely, bacterial, non
bacterial and toxic chemicals. We have also studied the importance of the
investigation of food borne disease outbreak. The steps involved in the
investigation were also studied. We have seen that food borne diseases maybe
caused due to contaminated food and beverages. They are a common,
distressing and sometimes life-threatening problem for millions of people all
around the world. We also studied in the end the related golden rules for the
prevention of such food borne disease outbreaks. Hence following good
manufacturing practices (GMP’s) and giving publicity to an outbreak and the
explanation of its cause maybe helpful in educating and warning the public and
avoiding further outbreaks.
9.8 KEY WORDS

Food Borne Disease : Disease caused by the consumption of
contaminated foods or beverages
Food Borne
Intoxication : Food borne disease caused due to ingestion of
toxin already present in food.
Food Borne Infection : Food borne disease caused due to ingestion of
causative organisms into the body where they
multiply, grow and cause disease.
Exotoxin : Proteinaceous toxin produced by bacteria and
excreted into surrounding environment
Endotoxin : Toxin produced by bacteria and excreted into
surrounding environment after the cell lysis. Are
mainly composed of polysaccharides.

EXERCISES
1. Food borne diseases are caused by consuming contaminated foods or
beverages e.g., food borne infection and food borne intoxication.
2. Exotoxins are proteinaceous toxins produced by bacteria and excreted into
surrounding environment.
Endotoxins are toxins produced by bacteria and excreted into surrounding
environment after the cell lysis (breaking of cell membrane), which are
mainly composed of polysaccharides.
3. Chemical contaminants in food include fumigants, solvents like hexane,
metallic contaminants such as antimony, arsenic, cadmium etc.,
insecticides, pesticides and growth regulators etc.
20
Check Your Progress Exercise 2 Food Born Diseases
1. Non-bacterial food borne disease causing agents include viruses, rickettsias

and parasites (Trichinella, Entamoeba, Giardia, Ascaris)
2. Jaundice is caused by Hepatitis A virus. through faecal-oral transmission.
Foods associated are raw or partially cooked shellfish, raw milk, potato
sandwiches and cold meat cuts.
3. Food borne parasites include:
a) Trichinells spiralis (causes Trichinosis) characterized by nausea,
abdominal pain, diarrhea and vomiting. Severe attack may cause
paralysis, fever and severe pain.
b) Entamoeba histolytica (causes amoebiasis) results in gastrointestinal
distress, dysentery, ulcers and intestinal blockage.
c) Giardia lamblia (causes giardiasis) results in severe gastroenteritis and
intestinal problems.
d) Ascaris lumbricoides causes ascariasis, resulting in digestive tract
disturbance, intestinal disorder and blockage.
1. Objectives of food borne disease outbreak investigation are:
• To prevent and control contamination of foods.
• To know the disease causing agents.
• To develop guidelines and take regulatory decisions.
2. Three steps in the investigation of any food borne disease outbreak:
• Field Analysis: Collection of samples and data pertaining to the causes
of outbreak in the particular area.
• Laboratory Analysis dealing with the isolation of the causative
organism from the various collected samples.
• Identification and the Interpretation of Report involving the
compilation of all facts and figures and stating the cause of outbreak
and the possible causative measures to be taken.
3. WHO’s Golden rule for safe food production states that:
• Choose foods processed for safety
• Cook foods thoroughly
• Eat cooked foods immediately
• Store cooked foods carefully
• Reheat cooked foods thoroughly
• Avoid contact between raw and cooked foods
• Wash hands repeatedly
• Keep all kitchen surfaces meticulously clean
• Protect foods from insects, rodents and other animals
• Use pure water
21
Food Poisoning
1. A manual entitled ‘Procedures to Investigate Food borne Illnesses’ (1976)
has been written by the Committee on communicable Diseases Affecting
Man in the International Association of Milk, Food and Environmental
Sanitarians and provides excellent information on such food borne diseases
and their outbreaks. A complete listing of diseases transmitted by foods,
including etiologic agents, nature of the organism, incubation period, signs
and symptoms, source or reservoir, epidemiology, foods involved and
control measures can be found in Center for Disease Control (1976a).
2. Frazier, W.C. and Westoff, D.C. (1988) Food Microbiology, Tata
McGraw-Hill Pub. Co., New Delhi. pp. 539.
th
3. Purohit, S.S. (1994) Microbiology-Fundamental and Application. 5 edn.
Agro Botanical Publishers, Bikaner, India.
4. Srivastava, R.P. and Kumar, S. (1994) Fruits and Vegetable Preservation,
International Book Distribution Co., Lucknow.
22
Chemical
UNIT 12 CHEMICAL
Structure
12.0 Objectives
12.1 Introduction
Need for Food Preservation
Techniques of Food Preservation
12.2 Charactistics of Chemical Preservatives
12.3 Classification of Preservatives
Antioxidant Preservatives
Preservatives that Targets Enzymes
Preservatives from Natural Products
Traditional Chemical Food Preservatives
12.4 Antimicrobial Preservatives
Organic Acids and Esters
Gaseous Chemical Food Preservatives
Nitrites and Nitrates
12.5 General Rules for Chemical Preservation
12.6 Let Us Sum Up
12.7 Key Words
12.0 OBJECTIVES
• know about different types of chemical preservatives; and

• define the essential chemical preservative limits for the various foods.
12.1 INTRODUCTION
In this unit we will make you aware about the characteristics of various
chemical preservatives with special stress on antimicrobial food preservatives.
We will also brief you about different chemical preservatives permitted in
processed products along with maximum levels of antimicrobials permitted in
foods. You will also learn about various factors which determine/ influence the
action of chemical food preservatives.
12.1.1 Need for Food Preservation

Unless you grow all your food in your own garden and prepare all your meals
from scratch, it's almost impossible to eat food without preservatives added by
manufacturers during processing.
Food preservation is a method of preparing food so that it can be stored for
future use. Because most foods remain edible for only a brief period of time,
people since the earliest ages have experimented with methods for successful
food preservation. Among the products of early food conservation were cheese
and butter, raisins, pemmican, sausage, bacon, and grain. Scientific
investigations pointed that food spoilage was mainly caused by
microorganisms widely distributed in the environment. Therefore, food
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Safe Chemicals and preservation depends on rendering conditions unfavourable for microbial
Microbial Limits for growth
Different Foods
12.1.2 Techniques of Food Preservation
The techniques of food preservation can be separated into two groups:
• physical
• chemical
Physical methods of preservation rely on killing the microorganisms present,
or at least stopping their growth for long enough to allow the food to be safely
consumed. The physical methods include canning, freezing, drying, gamma
irradiation, ultraviolet or high intensity white light, ultra high pressure and
filtration.
Chemical food preservatives are substances which, under certain conditions,
either delay the growth of microorganisms without necessarily destroying
them. These are added in very low quantities and which do not alter the
organoleptic and physico-chemical properties of the foods at or only very little.
These work either as direct microbial poisons or by reducing the pH to a level
of acidity that prevents the growth of microorganisms.
12.2 CHARACTERISTICS OF CHEMICAL

PRESERVATIVES
The Food, Drug, and Cosmetic Act permit for the use of chemical
preservatives in foods if the chemical is:
1. Generally recognized as safe (GRAS) for such use; or if a food additive is
covered by food additive regulations prescribing conditions of safe use.
2. Not used in such a way as to conceal damage or inferiority or to make the
food appear better or of greater value than it is.
3. Properly declared on the label of the food in which used.
4. It should be food grade.
5. It should perform its intended function.
6. It should be used in accordance with good manufacturing practices and,
where applicable, in accord with existing food additive regulations.
According to rules, a food manufacturer must get approval from Government
regulatory authorities before using a new preservative, or before using a
previously approved preservative in a new way or in a different amount. In its
petition for approval, the manufacturer must demonstrate that the preservative
is safe for consumers, considering:
• the probable amount of the preservative that will be consumed with the
food product, or the amount of any substance formed in or on the food
resulting from use of the preservative
• the cumulative effect of the preservative in the diet
• the potential toxicity (including cancer-causing) of the preservative when
ingested by humans or animals.
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• A preservative may not be used to deceive a consumer by changing the Chemical
food to make it appear other than it is. For example, preservatives that
contain sulfites are prohibited on meats because they restore the red colour,
giving meat a false appearance of freshness.
• The food additive regulations require the preservative to be of food grade
and be prepared and handled as a food ingredient.
• The quantity added to food must not exceed the amount needed to achieve
the manufacturer's intended effect.
Regulations about the use of nitrites demonstrate the scrutiny given to the use
of additives. Nitrites, used in combination with salt, serve as antimicrobials in
meat to inhibit the growth of bacterial spores that cause botulism, a deadly
food-borne illness. Nitrites are also used as preservatives and for flavouring
and fixing colour in a number of red meat, poultry, and fish products. Since the
original approvals were granted for specific uses of sodium nitrite, safety
concerns have arisen. Nitrite salts can react with certain amines (derivatives of
ammonia) in food to produce nitrosamines, many of which are known to cause
cancer. A food manufacturer wanting to use sodium nitrites must show that
nitrosamines will not form in hazardous amounts in the product under the
additive's intended conditions of use. For example, regulations specify that
sodium nitrite, used as an antimicrobial against the formation of botulinum
toxin in smoked fish, must be present in 100 to 200 parts per million. In
addition, other antioxidants, such as sodium ascorbate or sodium erythorbate,
may be added to inhibit the formation of nitrosamines.

1. What are chemical preservatives?
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2. Name three important characteristics of chemical preservatives?
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Safe Chemicals and 3. Name important facts that a manufacturer must demonstrate to regulatory
Microbial Limits for authorities for getting a new preservative formulation approved?
Different Foods
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12.3 CLASSIFICATION OF PRESERVATIVES

Preservatives can be categorized into following types:
1. Antimicrobials that inhibit growth of bacteria, yeasts, or molds.
2. Antioxidants that slow air oxidation of fats and lipids, which leads to
rancidity.
3. Antienzymatic that blocks the natural ripening and enzymatic processes
that continue to occur in foodstuffs after harvest.
4. Preservatives from natural products.
5. Traditional preservatives
We will discuss the first type of preservatives in detail but before that brief
description of other types is given below for the sake of awareness.
12.3.1 Antioxidant Preservatives

As antioxidants, they keep foods from becoming rancid, browning, or
developing black spots. Rancid foods may not make you sick, but they smell
and taste bad. Antioxidants suppress the reaction that occurs when foods
combine with oxygen in the presence of light, heat, and some metals.
Antioxidants also minimize the damage to some essential amino acids--the
building blocks of proteins--and the loss of some vitamins.
Antioxidant preservatives, such as butylated hydroxytoluene, butylated
hydroxyanisole, tert-butylhydroquinone, and propyl gallate, stop the chemical
breakdown of food that happens in the presence of oxygen. Unsaturated fatty
acids in oils and lipids are particularly susceptible to autooxidation. In this
process, a free radical initiates peroxide formation at fatty acid double bonds.
The chain reaction propagates to other double bonds, and aldehyde, ketone,
and acid-termination products eventually build up to create the rancid off-
flavors characteristic of oils and fats gone bad. Antioxidant preservatives sop
up the free radicals that help initiate and propagate these reactions.
12.3.2 Preservatives that Targets Enzymes

These are preservatives that target enzymes in the food itself that continue to
metabolize after harvest. The enzyme phenolase, for example, goes to work as
soon as an apple or potato is cut. It browns the exposed surface. Acids such as
8 citric acid and ascorbic acid (vitamin C) inhibit phenolase by making the pH
uncomfortably low for the enzyme. Metal-chelating agents such as EDTA Chemical
(ethylenediamine tetraacetic acid) can remove the metal cofactors that many
enzymes need. Chelators also make it difficult for bacterial and fungal
enzymes to carry on.
12.3.3 Preservatives from Natural Products

Some of the newest antimicrobials have been found in microorganisms
themselves as they form their own chemical defenses when competing with
each other for space and nutrients. For example, nisin and natamycin, the
cheese preservatives called bacteriocins − are harvested from microorganisms.
In the U.S., nisin is used to inhibit outgrowth of Clostridium botulinum spores
(the cause of botulism) and toxin formation in pasteurized process cheese
spreads with fruits, vegetables or meats at levels not exceeding good
manufacturing practice. Current good manufacturing practice in this case is the
quantity of the ingredient that delivers a maximum of 250 p.p.m. of nisin in the
finished product. Nisaplin-brand nisin is also approved for liquid egg products,
dressings, and sauces. In other countries it is also used in fresh and recombined
milk, fermented beverages like beer, canned foods, frozen desserts, and high
moisture/reduced fat foods. Nisin is considered effective at controlling a wide
range of gram-positive organisms including: Listeria enterococcus, Bacillus
sporothermodurans, and clostridium. Used alone, it is not effective on gram-
negative bacteria (like E. coli), yeasts, and molds. However, research suggests
that it may be useful against some gram-negative bacteria when used in
conjunction with other preservatives.
12.3.4 Traditional Chemical Food Preservatives
Traditional chemical food preservatives and their use in fruit and vegetable
processing technologies are common salt and sugar.
Common salt used in brined vegetables. There is no limit for their use.
Sugars (sucrose, glucose, fructose and syrups): foods preserved by high sugar
concentrations such as jellies, preserves, syrups, juice concentrates. It acts by
interaction of sugar with other ingredients or processes such as drying and
heating. There is no limit for their use.

1. Name two anti-oxidant preservatives?
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Safe Chemicals and 2. How does ascorbic and inhibit phenolase enzyme?
Microbial Limits for
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3. Name two preservatives of microbial origin?
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4. Is there any limit for traditional food preservatives?
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12.4 ANTIMICROBIAL PRESERVATIVES

Usually accepted chemical food preservatives are detailed in Table 12.1.
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Table 12.1: Commonly used antimicrobial chemical food preservatives Chemical
Agent Acceptable Commonly Typical usage

Daily intake used levels
(mg/Kg body (%)
weight)
Sorbic acid fruits; vegetables;
25 0.05-0.2 pickled products; jams,
Potassium sorbate
jellies
Benzoic acid Vegetable pickles;
5 0.03-0.2 preserves; jams; jellies;
Sodium benzoate
semi-processed
products
Propionic acid Bakery goods, cheese
10 0.1-0.3 spread, fruits,
Sodium
vegetables
propionate
Methyl paraben Bakery goods, fruit
10 0.05-0.1 products; pickles;
Ethyl paraben
sauces
Propyl paraben
Lactic acid Fermented meat, dairy
No limit No limit and vegetable products,
sauces and dressings,
drinks.
Citric acid No limit No limit fruit juices; jams; other
sugar preserves
Acetic acid No limit No limit vegetable pickles; other
vegetable sauces,
chutney
Sodium nitrite 0.2 0.01-0.02 Meat products
Sulphur dioxide 0.7 0.005-0.2 fruit juices, dried /
dehydrated fruits and
vegetables, semi-
processed products
12.4.1 Organic Acids and Esters

Sodium Benzoate and Benzoic Acid
Benzoic acid is the compound with the antimicrobial properties, and is found
naturally in cranberries, prunes, greengage plums, cinnamon, ripe cloves and
apples. Sodium benzoate produces benzoic acid once it is dissolved in water.
Sodium benzoate is the sodium salt of benzoic acid and is preferred over
benzoic acid in many food applications because it is 180 times more soluble in
water. There is a marked pH effect for this preservative: the lower the pH, the
more effective it is. Sodium benzoate will only work if the food product has a
pH below 4.5; that is, if the food is naturally acidic or has been acidified.
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Safe Chemicals and For example, at pH 3.0 you only need approximately 0.05% of the compound
Microbial Limits for to achieve the same antimicrobial effect as pH 4.0 and 0.1% benzoate.
Different Foods
Optimum functionality occurs when the pH is between 2.5 and 4.0.
Sodium benzoate is used in fruit products, jams, relishes, beverages, dressings,
salads, pie and pastry fillings, icings, olives and sauerkraut, and is effective
against yeasts, some bacteria (food borne pathogens but not spoilage bacteria)
and some molds. Sodium benzoate is a white granular or crystalline powder,
odorless, inexpensive (at the usage level) and should be stored in a cool, dry
place in watertight containers, if possible. It should be used at low levels to
avoid possible off-flavours in some products. The maximum level allowable by
law is 0.1%.
Sorbates
This family of compounds are available as sorbic acid, potassium sorbate,
sodium sorbate or calcium sorbate. Sorbic acid is the compound with the
antimicrobial properties but its salts (sorbates) are used in many cases due to
differences in solubility.
Potassium sorbate is the potassium salt of sorbic acid, and is much more
soluble in water than the acid. It is a white crystalline powder, inexpensive (at
the usage level), with basically no noticeable flavour at normal usage
concentrations. In wine processing, sorbates are used to prevent
refermentation. Maximum level allowable by law is 0.1%. It produces sorbic
acid once it is dissolved in water and is the most widely used food preservative
in the world. It is effective up to pH 6.5 but effectiveness increases as the pH
decreases. It has about 74% of the antimicrobial activity of the sorbic acid, thus
requiring higher concentrations to obtain the same results that pure sorbic acid
provides. It is effective against yeasts, molds, and select bacteria, and is widely
used at 0.025 to 0.10 % levels in cheese, dips, yogurt, sour cream, bread,
cakes, pies and fillings, baking mixes, doughs, icings, fudges, toppings,
beverages, margarine, salads, fermented and acidified vegetables, olives, fruit
products, dressings, smoked and salted fish, confections and mayonnaise.
It is important to know that the addition of sodium benzoate and/or potassium
sorbate to a food product will raise the pH by approximately 0.1 to 0.5 pH
units depending on the amount, pH, and type of product. Additional adjustment
of the pH might be needed to keep the pH at a safe level.
In many food products, sorbate and benzoate are used together to provide
greater protection against a wider variety of microorganisms. This only makes
sense if the pH of the product is below 4.5.
Propionic acid
Propionic acid occurs naturally in strawberries, apples, violet leaves, grains. It
is produced during the fermentation of some cheeses such as Swiss cheese, in
concentrations as high as 1%, thus inhibiting the growth of molds. The acid is
effective against bread molds and the spores of the bacterium Bacillus
mesentericus, which cause an inedible condition in baked goods called rope. It
is an oily liquid, soluble in water, with a slight pungent, disagreeable, rancid
odour. It is also corrosive and flammable, thus requiring special handling.
Propionic acid and its salts, sodium and calcium propionates, are approved in
the United States as GRAS (Generally Recognized As Safe) substances for
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food use. Their antimicrobial action is directed to molds and rope bacteria, Chemical
with almost no effect on yeast, thus making them an ideal choice for products
that use commercial yeast as an ingredient.
Like other preservatives, propionates effectiveness is affected by the pH of the
food, with 5.5 pH being the upper effective limit. They are used mainly as
mold and rope inhibitors in bread; although they are also useful in cheese, non-
alcoholic beverages, confections, fillings, frostings, fresh dough, pizza crust,
puddings, gelatins, jams, jellies and some meat products.
The sodium and calcium salts are transparent and white crystals with a mild
cheese like flavour. The sodium form is more soluble in water than the calcium
salt. Sodium propionate is recommended in baked products that use baking
powder and baking soda instead of yeast as the leavening agent, because the
presence of calcium ions (if you were to use calcium propionate) disrupts the
leavening process. Calcium propionate is preferred in baked foods that use
yeast, such as breads and rolls, because the nutritional value is increased by the
added calcium.
Typical usage level of propionic acid and propionates is 0.1 to 0.4 %. Federal
regulations limit the maximum level for flour, white bread and rolls at 0.32%
based on the weight of the flour; for whole wheat products at 0.38% based on
the weight of the flour; and for cheese products at 0.3 %.
It is important to know that the addition of sodium and calcium propionate to a
food product will raise the pH by approximately 0.1 to 0.5 pH units depending
on the amount, pH and type of product. Additional adjustment of the pH might
be needed to keep the pH at a safe level.
Parabens
The parabens are esters of para-hydroxibenzoic acid. The two most common
esters are methyl and propyl parabens, which are approved for food use in the
United States under the GRAS classification. The maximum concentration
allowed is 0.1 %. They are most active against yeasts and molds.
Parabens are white powders with faint odour and fair solubility in water at
room temperature. The solubility is greatly increased by heating the water to
71.1°C-82.2°C. Methyl paraben is more soluble in water but less effective
against molds than propyl paraben. To balance these differences, mixtures of 2
to 3 parts of methyl paraben with 1 part propyl paraben are normally used.
Important advantages of parabens are their effectiveness at higher pH values,
from 3 up to 8, and stability to high and low temperatures, even to steam
sterilization. Despite these properties, parabens are not as widely used as other
antimicrobial agents, probably due to higher cost and flavour objections.
Applications include bakery products (formulated without yeast), beverages,
flavour extracts, food colours, fruit products, jams, jellies, preserves
(artificially sweetened), gelatin, marinated and smoked fish, pickles, salad
dressings, syrups, wine and olives.
Lactic acid
This acid is the main product of many food fermentations; it is formed by
microbial degradation of sugars in products such as sauerkraut and pickles.
The acid produced in such fermentations decreases the pH to levels
unfavourable for growth of spoilage organisms such as putrefactive anaerobes
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Safe Chemicals and and butyric-acid-producing bacteria. Yeasts and molds that can grow at such
Microbial Limits for pH levels can be controlled by the inclusion of other preservatives such as
Different Foods
sorbate and benzoate.
Acetic acid
Acetic acid is a general preservative inhibiting many species of bacteria, yeasts
and to a lesser extent molds. It is also a product of the lactic-acid fermentation,
and its preservative action even at identical pH levels is greater than that of
lactic acid. The main applications of vinegar (acetic acid) includes products
such as pickles, sauces and ketchup.
12.4.2 Gaseous Chemical Food Preservatives

Sulphur dioxide and sulphites
Sulphur dioxide (SO2) has been used for many centuries as a fumigant and
especially as a wine preservative. It is a colourless, suffocating, pungent-
smelling, non-flammable gas and is very soluble in cold water (85 g in 100 ml
at 25°C).
Sulphur dioxide and its various sulphites when dissolved in water at low pH
yield sulphurous acid, bisulphite and sulphite ions. The various sulphite salts
contain 50-68% active sulphur dioxide. A pH dependent equilibrium is formed
in water and the proportion of SO2 ions increases with decreasing pH values.
At pH values less than 4.0 the antimicrobial activity reaches its maximum.
Sulphur dioxide is used as a gas or in the form of its sulphite, bisulphite and
metabisulphite salts which are powders. The gaseous form is produced either
by burning Sulphur or by its release from the compressed liquefied form.
Metabisulphite are more stable to oxidation than bisulphites, which in turn
show greater stability than sulphites.
Mode of action
Sulphites inhibit microbial growth through a number of actions. They react
with the energy rich compound, adenosine triphosphate; inhibit some
metabolic pathways; and block cellular transport systems. Other antimicrobials
alter microbial membrane or cell wall permeability or destroy the genetic
material. In addition to its antimicrobial action, sulphur dioxide inhibits
degradation reactions in fruits. It keeps raisins and other dried fruits from
losing their light colour by blocking both enzymatic browning and a
nonenzymatic browning reaction between reducing sugars and amino acids
called the Maillard reaction. The reaction darkens raisins, alters their flavour,
and reduces essential amino acid levels.
Uses
Sulphites are used to prevent or reduce discolouration of light-coloured fruits
and vegetables, such as dried apples and dehydrated potatoes. These are added
to sun-dried tomatoes, dried apricots, dried sweet potatoes, balsamic vinegar,
red wine vinegar, lemon juice, and Hawaiian coconut syrup. These are also
commonly used to lengthen the life of fruit juices. They are also used in wine-
making because they inhibit bacterial growth but do not interfere with the
desired development of yeast. Sulphites are also used in other ways, such as for
bleaching food starches and as preventives against rust and scale in boiler
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water used in making steam that will come in contact with food. Some Chemical
sulphites are used in the production of cellophane for food packaging.
Precautions
FDA prohibits the use of sulphites in foods that are important sources of
thiamin (vitamin B1), such as enriched flour, because sulphites destroy the
nutrient. It causes severe allergic reactions, especially in asthmatics though, for
the majority of the population, they are safe.
According to FDA sulphites used specifically as preservatives must be listed
on the label, regardless of the amount in the finished product. Sulphites used in
food processing but not serving as preservatives in the final food must be listed
on the label if present at levels of 10 parts per million or higher.
According to FDA the use of sulphites on fruits and vegetables intended to be
eaten raw is banned. These were used to maintain the colour and crispness of
fresh produce.
Carbon dioxide (CO2)
CO2 is a colourless, odourless, non-combustible gas, acidic in odour and
flavour. In commercial practice it is sold as a liquid under pressure (58 kg per
cm³) or solidified as dry ice.
Carbon dioxide is used as a solid (dry ice) in many countries as a means of
low-temperature storage and transportation of food products. Beside keeping
the temperature low, as it sublimes, the gaseous CO2 inhibits growth of
psychrotrophic microorganisms and prevents spoilage of the fruits and
vegetables, etc.
It is used as a direct additive in the storage of fruits and vegetables. In the
controlled/ modified environment storage of fruit and vegetables, the correct
combination of O2 and CO2 delays respiration and ripening as well as retarding
mold and yeast growth.
The final result is an extended storage of the products for transportation and for
consumption during the off-season. The amount of CO2 (5-10%) is determined
by factors such as nature of product, variety, climate and extent of storage.
Chlorine
The various forms of chlorine constitute the most widely used chemical
sanitizer in the food industry. These chlorine forms include chlorine (Cl2),
sodium hypochlorite (NaOCl), calcium hypochlorite (Ca(OCl)2) and chlorine
dioxide gas (ClO2).These compounds are used as water adjuncts in processes
such as product washing, transport, and cooling of heat-sterilised cans; in
sanitising solutions for equipment surfaces, etc.
Important applications of chlorine and its compounds include disinfection of
drinking water and sanitation of food processing equipment.
12.4.3 Nitrites and Nitrates

Nitrites and nitrates are used mainly among the packaged meats. Sodium
nitrate is added to meats such as ham, bacon, hot dogs and smoked fish.
Nitrates break down in the body to nitrites and this stops the growth of
bacteria, especially the bacteria that cause botulism poisoning. They are the
15
Safe Chemicals and food industry's primary chemical defense against the bacterium Clostridium
Microbial Limits for botulinum.
Different Foods
It also stabilizes the red colour in cured meat and stops it turning grey. Nitrates
readily convert to nitrites, which then react with the protein myoglobin to form
nitric oxide myoglobin. During cooking, this is converted to
nitrosohemochrome, a stable, pink pigment. They also impart a pink, fresh hue
to cured meat. This chemical stabilises the red colour of the meat and gives an
appearance of fresh meat. That is why nitrites are a favourite preservative of
meat processors even though its excess use is restricted in many countries.
Precaution
Nitrite salts can react with certain amines in food to produce nitrosamines,
which are known to cause cancer. Addition of Sodium ascorbate or sodium
erythorbate inhibits nitrosamine formation and reduces the problem of
nitrosamines.
The use of nitrite and nitrate has decreased greatly because of refrigeration and
restrictions on the amounts used. Even though nitrite and nitrate cause only a
small risk, it is always better to have fresh meat and meat product.

1. Name four important organic acids used as chemical preservatives?
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2. What precautions should be observed before using SO2 as preservative?
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3. How does nitrates impart red colour to meat?
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Chemical
12.5 GENERAL RULES FOR CHEMICAL
PRESERVATION
• Chemical food preservatives have to be used only at a dosage level which
is needed for a normal preservation and not more.
• “Reconditioning” of chemical preserved food, e.g. a new addition of

preservative in order to stop a microbiological deterioration already
occurred is not recommended.
• The use of chemical preservatives MUST be strictly limited to those

substances which are recognised as being without harmful effects on
human beings’ health and are accepted by national and international
standards and legislation.
Factors which determine/ influence the action of chemical food
preservatives
a) Factors related to micro-organisms:
As a general rule it is possible to take the following facts as a basis:
• Sulphur dioxide and its derivatives can be considered as an “universal”
preservative as; they have an antiseptic action on bacteria as well as on
yeasts and molds;
• Benzoic acid and its derivatives have a preservative action which is
stronger against bacteria than on yeasts and molds;
• Sorbic acid acts on molds and certain yeast species; in higher dosage
levels it acts also on bacteria, except lactic and acetic ones;
• The initial number of microorganisms in the treated product determines
the efficiency of the chemical preservative.
• The efficiency is less if the product has been contaminated because of
preliminary careless hygienic treatment or an incipient alteration.
Therefore, with a low initial number of microorganisms in the product,
the preservative dosage level could be reduced.
b) Factors related to the product:
• Chemical composition of the product.
• pH value of the product: the efficiency of the majority of chemical
preservatives is higher at lower pH values, i.e. when the medium is
more acidic.
• Physical presentation and size which the product is sliced to: the
chemical preservative’s dispersion in food has an impact on its
absorption and diffusion through cell membranes on microorganisms
and this determines the preservation effect. Therefore, the smaller the
slicing of the product, the higher the preservative action. Preservative
dispersion is slowed down by viscous foods (concentrated fruit juices,
etc.)
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Safe Chemicals and c) Miscellaneous factors
Different Foods • Temperature: chemical preservative dosage level will be established as
a function of product temperature and characteristics of the micro-flora;
• Time: at preservative dosage levels in employed in industrial practice,
the time period needed in order to obtain a "chemical sterilisation" is a
few weeks for benzoic acid and shorter for sulphurous acid.

1. What is general rule about the dosage level of chemical preservative?
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2. Name one universal preservative?
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……………………………………………………………………………….
3. How does particle size of product affect the efficiency of preservative?
……………………………………………………………………………….
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……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
……………………………………………………………………………….
12.6 LET US SUM UP

In this unit you have learnt about the chemical preservatives. Herein we have
discussed the characteristics of approved chemical preservatives. Various types
of preservatives grouped according to their mode of action have been
discussed. More stress has been laid on antimicrobial preservatives owing to
their importance. Factor influencing the effectiveness of various preservatives
have also been discussed. We hope that after reading this unit you will become
18
more conscious about reading the label of processed product before consuming Chemical
it.
12.7 KEY WORDS

Food Spoilage : Undesirable change in flavours, odours
appearances or texture of food.
Preservation : Safeguarding
Preservative : Additive
Anti microbial : Which act against microorganisms.
Anti oxidant : Which removes the oxygen.
GRAS : Generally recognized as safe.

EXERCISES
"
Chemical food preservatives are substances which, under certain

conditions, either delay the growth of microorganisms without necessarily
destroying them. These are added in very low quantities and which do not
alter the organoleptic and physico-chemical properties of the foods at all or
only very little.
i) It should have GRAS (Generally recognized as safe) status.

ii) It should be properly declared on the label of the food in which used.
iii) It should be used only at approved dosage level.
In its petition for approval, the manufacturer must demonstrate that the
preservative is safe for consumers, considering:
• the probable amount of the preservative that will be consumed with the
food product, or the amount of any substance formed in or on the food
resulting from use of the preservative
• the cumulative effect of the preservative in the diet
• the potential toxicity (including cancer-causing) of the preservative
when ingested by humans or animals.
19
Safe Chemicals and Check Your Progress Exercise 2
Different Foods
The two important antioxidant preservatives are:
• butylated hydroxytoluene
• butylated hydroxyanisole
Ascorbic acid (vitamin C) inhibit phenolase by making the pH

uncomfortably low for the enzyme.
• nisin
• natamycin
There is no limit for traditional food preservatives like salt and sugar.
The four important organic acids used as chemical preservatives are:
• Benzoic Acid
• Sorbic acid
• Propionic acid
• Acetic acid
1. Sulphites should not be used in foods that are important sources of

thiamin (vitamin B1), such as enriched flour, because sulphites destroy
the nutrient.
2. It should not be consumed by asthmatic patients because it may cause
severe allergic reactions.
Nitrates readily convert to nitrites, which then react with the protein
myoglobin to form nitric oxide myoglobin. During cooking, this is
converted to nitrosohemochrome, a stable, pink pigment. That is how they
impart a pink, fresh colour to cured meat.
20
Check Your Progress Exercise 4 Chemical
Chemical food preservatives have to be used only at a dosage level which

is needed for a normal preservation and not more.
Potassium meta bisulphate
• The smaller is the slicing of the product, the higher is the preservative
action.
• Preservative dispersion is slowed down by viscous foods
1. Adams, M.R. and Moss, M.O. (2000) Food Microbiology. Royal Society
of Chemistry, Cambridge, U.K.
2. Branen, L.A. and Davidson, P.M. (1983) Antimicrobials in Food. Marcel
Dekker, New York.
3. Igoe, R.S and Hui, Y.H. (1996) Dictionary of Food Ingredients − 3rd ed.
Chapman & Hall, New York.
New York.
5. Lewis, R.J. (1989) Food Additives Handbook. Van Nostrand Reinhold,
New York.
21
Safe Chemicals and
Different Foods
UNIT 13 MICROBIAL
Structure
13.0 Objectives
13.1 Introduction
13.2 Microbiological Profile of Harvested Fruits and Vegetables
Sources of Microorganisms on Fresh Fruits and Vegetables
Factors Affecting Type and Number of Microorganism on Fresh Fruits and
Vegetables
Human Pathogens Associated with Fresh Fruits and Vegetables
13.3 Standards for Water for Human Consumption
Sources of Contaminants in Drinking Water
Contamination Due to Harmful Microorganisms
Drinking Water Standards
13.4 Microbiology of Canned Fruits
History of Canning
Basic Principal of Canning
Spoilage of Canned Products
Clostridium Botulinum A Major Threat in Canned Products
13.5 Microbiological Standards for Processed Foods
Purpose of Microbiological Standards
Sampling
Microbiological Assessment
Categories of Food Based on Microbial Quality
13.6 Let Us Sum Up
13.7 Key Words
13.0 OBJECTIVES
After studying this unit, you should be able to explain:
• fresh fruits and vegetables;

• canned and processed food products; and
• drinking water.
13.1 INTRODUCTION
In this unit we will make you aware about the types of micro flora present on
the surface of fresh fruits and vegetables, the sources of these microorganisms
and the characteristics of pathogenic microorganisms. We will also brief you
about quality standards for drinking water and the human pathogen present in
contaminated water. Canning is an established way of food preservation. You
will learn about the history of canning, spoilage of canned products with
special reference to Clostridium botulinum. After that we will brief you about
the microbial limits for processed foods.
13.2 MICROBIOLOGICAL PROFILE OF HARVESTED

FRUITS AND VEGETABLES
22
Microbial
The consumption of fresh fruits and vegetables is increasing as consumers

strive to eat healthy diets. Global trade in fruits and vegetables and changing
horticultural practices have enabled this year-round abundance to be possible,
as well as adding new varieties of fresh produce to the market. During the last
few decades pre-prepared minimally processed fruits and vegetables have
become popular among the consumers. These products include pre-washed
pre-cut salads items, grated vegetables, prepared fruit salads, or fruit
combinations. Most of these products are generally eaten raw without further
processing. Some products are packed in modified atmospheres to provide
extension of shelf life both in relation to the potential acceptable quality and
safety of the product.
Since minimum processing is required for fresh and fresh-cut fruits and
vegetables, which omits any effective microbial elimination step, results in
food products that naturally would carry microorganisms, some of which may
be potentially hazardous to human health.
13.2.1 Sources of Microorganisms on Fresh Fruits and Vegetables
Fruits and vegetables can become contaminated whilst growing in fields, or
during harvest, handling, processing, distribution and use. However, there are
certain factors, which contribute to the microbiological contamination of these
products with pathogens. Table 13.1 lists the sources of pathogenic
microorganisms on fresh produce and conditions that influence their survival
and growth.
Contamination can arise as a consequence of treating soil with organic
fertilizers such as manure and sewage sludge and from irrigation water.
Manure, bio-solids and irrigation water should be of a quality that does not
introduce pathogens to the treated commodity. The potential of organic
farming to contaminate fruits and vegetables with pathogens has to be
investigated. Harvesting at the appropriate time and storing the harvested
products under controlled conditions will help to retard growth of post-harvest
spoilage and pathogenic microorganisms. Humid and warm storage conditions
encourage the growth of microbial contaminants. The use of additional post-
harvest procedures could reduce the contamination level of fruits and
vegetables. Washing with water of potable quality can reduce the microbial
load. Although a wide range of different agents are available for disinfecting/
sanitizing fresh produce their efficacy is variable and none is able to ensure
elimination of pathogens. Fruits and vegetables carry a natural non-pathogenic
epiphytic micro flora. During growth, harvest, transportation and further
processing and handling the produce can, however, be contaminated with
pathogens from human or animal sources. The microbial composition of the
different forms of organic fertilizer will vary depending on its origin and
further treatment. The quality of the water used for irrigation and as a carrier
for plant protection products, fertilizers and frost protection products has to be
related to the potential risk it can cause at a later stage. Technologies for
irrigation are important for the control of spreading microbiological hazards.
The use of drip irrigation instead of flooding or spray irrigation should reduce
waterborne contamination and aerosols. However, heavy rains and wind may
provide other opportunities for the transfer of microorganisms from soil to
plant surfaces.
23
Safe Chemicals and
Different Foods Table 13.1: Sources of pathogenic microorganisms on fresh produce and
conditions that influence their survival and growth
Pre-harvest
• Soil
• Irrigation water
• Green or inadequately composted manure
• Air (dust)
• Wild and domestic animals
• Human handling
• Water for other uses (for example, pesticides, foliar treatments,
growth hormones)
Post-harvest
• Human handling (workers, consumers)
• Harvesting equipment
• Transport containers (field to packing shed)
• Air (dust)
• Wash and rinse water
• Sorting, packing, cutting and further-processing equipment
• Ice
• Transport vehicles
• Improper storage (temperature, physical environment)
• Improper packaging (includes new packaging technologies)
• Cross contamination (other foods in storage, preparation and
display areas)
• Improper handling after wholesale or retail purchase
• Cooling water (for example, hydro cooling)
13.2.2 Factors affecting Type and Number of Microorganism on Fresh

Fruits and Vegetables
Fruits and vegetables normally carry a non-pathogenic epiphytic micro flora.
The majority of bacteria found on the surface of plants are usually Gram-
negative and belong either to the Pseudomonas group or to the
Enterobacteriaceae. Many of these organisms are normally non-pathogenic
for humans. The numbers of bacteria present will vary depending on seasonal
and climatic variation and may range from 104 to 108 per gram. The inner
tissues of fruits and vegetables are usually regarded as sterile. However,
bacteria can be present in low numbers as a result of the uptake of water
through certain irrigation or washing procedures. If these waters are
contaminated with human pathogens these may also be introduced. The
survival or growth of contaminating microorganisms is affected by intrinsic,
extrinsic and processing factors. Factors of importance are nutrient
composition, pH, presence of scales and fibers, redox potential, temperature
and gaseous atmosphere. Mechanical shredding, cutting and slicing of the
produce open the plant surfaces to microbial attack. About two thirds of the
spoilage of fruits and vegetables is caused by molds. Members of the genera
Penicillium, Aspergillus, Sclerotinia, Botrytis and Rhizopus are commonly
24
Microbial
involved in this process. The spoilage is usually associated with cellulolytic or

pectinolytic activity, which causes softening of tissues, and weakening of
plant structures. These structures are important barriers to prevent growth in
the products by contaminating microbes.
13.2.3 Human Pathogens Associated with Fresh Fruits and Vegetables

However, risk profile surveys on the microbiological contamination of fruits
and vegetables eaten raw demonstrates, potential for a wide range of these
products to become contaminated with microorganisms, including human
pathogens. The range of microorganisms associated with outbreaks linked to
fresh produce encompasses bacteria, viruses and parasites. Most of the
reported outbreaks have been associated with bacterial contamination,
particularly members of the Enterobacteriaceae. Of these, Salmonella and
Escherichia coli O157 are of particular concern. Outbreaks of illness caused
by bacteria, viruses and parasites have been linked epidemiologically to the
consumption of a wide range of vegetables and, to a lesser extent fruits.
Surveillance of vegetables has indicated that these foods can be contaminated
with various bacterial pathogens, including Salmonella, Shigella, E. coli
O157:H7, Listeria monocytogenes and Campylobacter. Table 13.2 shows the
characteristics of some microbial pathogens that have been linked to outbreaks
of fresh fruits and vegetable associated illness.
Table 13.2: Characteristics of some microbial pathogens that have been
linked to outbreaks of produce associated illness
Microorganism Typical Symptoms Infectious Source

Incubation Dose
Period (Number
of cells)
BACTERIA
Clostridium 12 to 36 h Nausea, vomiting, Intoxication Soil, lakes,
botulinum fatigue, dizziness, growth and streams,
dryness of mouth toxin decaying
and throat, muscle production vegetation,
paralysis, in food reptiles
difficulty in
swallowing,
double or blurred
vision, drooping
eyelids, and
breathing
difficulties
Escherichia coli 2 to 5 d Bloody diarrhoea, 10 to 1000 Animal
O157:H7 abdominal pain. feces,
Can lead to especially
hemolytic uremic cattle, deer
syndrome and and human;
kidney failure cross
especially in contaminati
children and the on from raw
elderly meat
Salmonella spp. 18 to 72 h Abdominal pain, 10 to Animal and
diarrhoea, chills, 100,000 human
fever, nausea, feces; cross
vomiting contaminati
on from raw
25
Safe Chemicals and
Different Foods meat,
poultry, or
eggs
Shigella spp. 1 to 3 d Abdominal pain, ~10 Human
diarrhoea, fever, feces
vomiting
Listeria 1 d to 5 or Febrile Unknown Soil, food
monocytogenes more wk gastroenteritis in dependent processing
healthy adults; upon health environ-
may lead to of ments
spontaneous individual
abortion or
stillbirth in
pregnant women;
severe septicemia
and meningitis in
neonates and
immuno-
compromised
adults; mortality
may be 20 to 40%
PARASITES
Cryptosporidium 1 to 12 d Profuse watery ~30 Animal and
spp. diarrhoea, human feces
abdominal pain,
anorexia,
vomiting
Cyclospora spp. 1 to 11 d Watery diarrhoea, Unknown, Others?
nausea, anorexia, probably specific
abdominal low environment
cramps (duration al sources
7 to 40 d) unknown at
this time
VIRUSES
Hepatitis A 25 to 30 d Fever, malaise, 10 to 50 Human
anorexia, nausea, feces and
abdominal pain, urine
jaundice, dark
urine
Norwalk/ 12 to 48 h Vomiting Unknown, Human
Norwalk-like diarrhoea, probably feces,
virus malaise, fever, low vomitus
nausea,
abdominal cramps
However, multiplication of the pathogen is also essential for causing any

damage. Some microorganisms cause illness only when ingested in high
numbers (for example, Clostridium perfringens), while in other cases, the
infectious dose is thought to be dependent upon the susceptibility of the
individual (most infectious agents). Illness due to Staphylococcus aureus,
Bacillus cereus, or Clostridium botulinum is a result of the production of
toxins in the food, and it is the toxins that are responsible (sometimes in the
absence of viable cells) for symptoms of the disease. These toxins are only
produced by multiplying cells. This requires favourable growth conditions.
26
Microbial

"
1. What are the sources of microbial contamination on fresh fruits and
vegetables?
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
2. What are the factors that affect the survival and growth of microorganisms
on fruits?
………………………………………………………………………………
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3. Name few fungi responsible for spoilage of fresh fruits and vegetables?
………………………………………………………………………………
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4. Name two bacteria associated with outbreaks linked to fresh fruits and
vegetables?
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13.3 STANDARDS OF WATER FOR HUMAN

CONSUMPTION
Clean water is one of the most important needs of our bodies. It is a sad fact
that something as essential to life as clean drinking water can no longer be
granted to us. According to research articles and news, most tap and well
water now are not safe for drinking due to heavy industrial and environmental
27
Safe Chemicals and
Different Foods pollution. We have reached to a point that, all sources of our drinking water,
including municipal water systems, wells, lakes, rivers, and even glaciers,
contain some level of contamination.
13.3.1 Sources of Contaminants in Drinking Water

Several contaminants occur in nature that may present a health risk if they are
found in drinking water. The various pollutant /contaminants are bacteria,
viruses, uranium, radium, nitrate, arsenic, chromium and fluoride. Other
sources of contamination are a result of human activity such as manufacturing
or agriculture, or individual misuse. The following activities may cause
harmful microorganisms and chemicals to enter the well water owner's water
supply.
• Leakage from waste disposal, treatment, or storage sites.
• Discharges from factories, industrial sites, or sewage treatment facilities.
• Leaching from aerial or land application of pesticides and fertilizers on
yards or fields.
• Accidental chemical spills.
• Leakage from underground storage tanks.
13.3.2 Contamination due to Harmful Microorganisms

The most common and widespread health risk associated with drinking water
is microbial contamination, either directly or indirectly, by human or animal
excreta and micro-organisms contained in faeces.
The pathogenic agents involved include bacteria, viruses and protozoa, which
may cause diseases that vary in severity from mild gastroenteritis to severe
and sometimes fatal diarroea, dysentry, hepatitis or typhoid fever. Most of
them are widely distributed through out the world. Fecal contamination of
drinking water is only one of the several faeco-oral mechanisms by which they
can be transmitted from one person to another, or in some cases, from animal
to people. The human pathogens potentially transmitted in drinking water are
Bacteria viz. Escherichia coli,Salmonella, Shigella, Vibrio cholera, Yersinia
enterocolitica, Campylobacter jejuni; viruses viz, Adenoviruses, Enterovirus,
Hepatitis A, Hepatitis E, Norwalk virus, Rotavirus, and small round viruses;
The parasites, Giardia, Cryptosporidium, Entamoeba histolytica and
Drancunculus. The removal of these agents from drinking water should be
given top priority.
Apart from the above said pathogens of high health significance, there are
some more organisms that are present in environment and not normally
regarded as pathogen, may caue disease opportunistically. When such
organisms are present in water they cause infection predominantly among
people whose local or general defence mechanisms are impaired. Those most
likely to be at risk include the very old, the very young and patients in the
hospitals,e.g. those with burns or immunosuppressive therapy, and those
suffering from acquired immunodeficiency syndrome(AIDS). Water used by
such patients for drinking or bathing, if it contains excessive number of these
agents, may produce a variety of infections involving the skin and mucous
membrane of the eye, ear, nose and throat. Pseudomonas, Flavobacterium,
Acinetobacter, Klebsiella and Serratia are examples of such opportunistic
pathogens. Legionella infects the lung. These organisms while clearly of
medical importance, acquire public health significance only under certain
conditions. There removal from drinking water may therefore be given
moderate priority.
28
Microbial
13.3.3 Drinking Water Standards

Microorganisms, including pathogenic organisms, may enter water supplies at
every stage of the collection and distribution cycle. Emphasis should be
placed on the need for an active watershed protection program, including an
emergency plan for responding to major pollution events such as spills or
contamination. Major quality requirements for drinking water are listed in
Table 13.3.
Table 13.3: Drinking water standards
Micro-organism Requirement
Cryptosporidium System must remove 99% of Cryptosporidium
Giardia lamblia 99.9% killed
Heterotrophic Plate Not more than 500 colonies per ml.
count (HPC)
Total Coliform Must not be detectatable in any 100 ml sample. In case
of large supplies where sufficient samples are
examined must not be present in 95% of the samples
taken through out any 12 months period.
Fecal Coliform or No fecal coliform is allowed.
E.coli
Turbidity At no time can turbidity go above 5 NTU

(Nephlometric turbidity unit)
Viruses 99.99% killed / inactivated
Let us know in brief about the above contaminants.

Coliform bacteria
These are common in the environment and are generally not harmful.
However, the presence of these bacteria in drinking water is usually a result of
a problem with the treatment system or the pipes which distribute water, and
indicates that the water may be contaminated with germs that can cause
disease. Fecal Coliform and E.Coli are bacteria whose presence indicates that
the water may be contaminated with human or animal wastes. Microbes in
these wastes can cause short-term effects, such as diarrhoea, cramps, nausea,
headaches, or other symptoms.
Turbidity has no health effects. However, turbidity can interfere with
disinfection and provide a medium for microbial growth. Turbidity may
indicate the presence of disease causing organisms. These organisms include
bacteria, viruses, and parasites that can cause symptoms such as nausea,
cramps, diarrhoea, and associated headaches.
Cryptosporidium is a parasite that enters lakes and rivers through sewage and
animal waste. It causes cryptosporidiosis, a mild gastrointestinal disease.
However, the disease can be severe or fatal for people with severely weakened
immune systems.
Giardia lamblia is a parasite that enters lakes and rivers through sewage and
animal waste. It causes gastrointestinal illness (e.g. diarrhoea, vomiting,
cramps).
29
Safe Chemicals and
Different Foods Hence, it is important that our drinking water does not contain any
concentration of microorganisms, parasites or any other substance which
constitutes a potential human health risk and it meets the minimum
requirements (microbiological and chemical parameters and those relating to
radioactivity) laid down by the directives.

1. Name some common diseases caused due to contaminated drinking water?
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
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………………………………………………………………………………
2. Name various pollutant/Contaminants of water?
………………………………………………………………………………
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………………………………………………………………………………
3. List some important human pathogen transmitted by drinking water?
………………………………………………………………………………
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4. Name some opportunist pathogens in drinking water?
………………………………………………………………………………
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13.4 MICROBIOLOGY OF CANNED FOODS
30
Microbial
Foods are processed for convenience and safety. Food processing involves
procedures such as: drying, canning, freezing, and pasteurization. For
example, drying is a process by which water is removed from the product,
causing the product to become dehydrated. Since microorganisms need water
to grow, without moisture, they can’t flourish. Canning is a process where
foods are put into a container and given a high heat treatment to make the
product sterile. The process of canning, be it vegetables, meat, or seafood,
makes food safe because all dangerous microorganisms are destroyed. The
canning process was developed to preserve food safely and for long periods of
time.
13.4.1 History of Canning

The canning process dates back to the late 18th century in France when the
Emperor Napoleon Bonaparte, concerned about keeping his armies fed,
offered a cash prize to whoever could develop a reliable method of food
preservation. Nicholas Appert conceived the idea of preserving food in
bottles, like wine. After 15 years of experimentation, he realized if food is
sufficiently heated and sealed in an airtight container, it will not spoil. An
Englishman, Peter Durand, took the process one step farther and developed a
method of sealing food into unbreakable tin containers, which was perfected
by Bryan Dorkin and John Hall, who set up the first commercial canning
factory in England in 1813. As more and more of the world was explored, and
as provisioning armies took on greater importance, the demand for canned
foods grew. Thomas Kensett, who emigrated to the United States, established
the first U.S. canning facility for oysters, meats, fruits and vegetables in New
York in 1812. More than 50 years later, Louis Pasteur provided the
explanation for canning’s effectiveness when he was able to demonstrate that
the growth of microorganisms is the cause of food spoilage.
13.4.2 Basic Principal of Canning

The basic principles of canning have not changed dramatically since Nicholas
Appert and Peter Durand developed the process. Heat sufficient to destroy
microorganisms is applied to foods packed into sealed or “airtight” containers.
The canned foods are then heated under steam pressure at temperatures of
116-121°C. The amount of time needed for processing is different for each
food, depending on the food's acidity, density and ability to transfer heat. For
example, tomatoes require less time than green beans, while corn and
pumpkin require far more time. Processing conditions are chosen to be the
minimum needed to ensure that foods are commercially sterile, but retain the
greatest flavour and nutrition.
13.4.3 Spoilage of Canned Products
Heated canned foods may undergo spoilage either due to chemical or
biological reasons. The most common spoilage of canned foods is the
hydrogen swells produced as a result of action of food acid with the metal can.
Such spoilage occurs mostly due to imperfect tinning and lacquering of
interior of the can used for canning acidic foods. Biological spoilage of
canned foods by the microorganism may result either from the survival of the
organisms after the heat treatment or leakage of the container permitting
entrance of the microorganisms, Surviving organisms may be vegetative cells
or spore formers depending upon the heat treatment. Acid foods are processed
at a temperature around 100OC which result in killing of all vegetative cells of
bacteria yeast and molds.
31
Safe Chemicals and
Different Foods 13.4.4 Clostridium botulinum a Major Threat in Canned Products
Growth of the bacterium Clostridium botulinum in canned food may cause
botulism − a deadly form of food poisoning. These bacteria exist either as
spores or as vegetative cells. Botulism is an intoxication that is caused by
the ingestion of a virulent nerve toxin produced by the growth of the gram
positive, obligate anaerobe, spore-former Clostridium botulinum. This
bacterium appears to be a normal inhabitant of the soil, hence is ready
contamination of most foods. The spores can survive harmlessly in soil and
water for many years. When ideal conditions exist for growth, the spores
produce vegetative cells which multiply rapidly and may produce a deadly
toxin within 3 to 4 days of growth in an environment consisting of:
• a moist, low-acid food
• a temperature between 4°C and 49°C
• less than 2 percent oxygen
It is able to grow in absence of oxygen in a wide variety of foods and in so
doing produces a protein neural toxin, two to three grams (an amount
equivalent to the quantity of salt in the average salt shaker on your table) of
which would be sufficient to kill human being. However, the organism will
not grow in the presence of oxygen or nitrate salts and it does not produce the
toxin at a pH below 4.7. Only one strain, which is found associated with
marine organisms, is able to produce the toxin at refrigerator temperature. The
toxin is destroyed by boiling it at 100oC for 10 to 15 min. However, the spore
requires a temperature of 121oC for 15 min to kill it. The toxin acts by binding
to nerve junctions and destroying the nerve. The symptoms, which occur
usually within 12 to 36 hours, but which can take up to 8 days to appear,
classically consist of double vision, dizziness, inability to speak, breathe or
swallow. Death often occurs due to the inability to breath. The only treatment
is the injection of antitoxin to the several varieties of the toxin. This treatment
is only effective against free toxin, as once the toxin has bound to the nerves
the damage is irreversible. The entire canning process is built around
ensuring that all spores of this bacterium contaminating any canned food
are destroyed in the sterilization process. Industry has a sterling record in
that deaths from commercial-botulism are very rare. This is influenced by the
fact that once a product is known to contain botulism toxin none of that
product is ever again purchased by a customer. The majority of botulism
poisonings occur in HOME-CANNED FOODS prepared by grandma or
your favourite aunt. A rule of thumb is “READ THE CANNING
DIRECTIONS” and if you think a food might contain the botulism toxin never
tastes even the smallest drop of it!
Some interesting additional information about this disease is:
• Never feed raw honey to a child under the age of two because the
botulism spores can grow in the immature gut and produce the toxin. This
can not occur in the adult due to our gut micro flora which is absent in
infants.
• The botulism toxin is being used to treat certain neurological conditions
where nerves that shouldn't fire do. In these cases tiny quantities of the
botulism toxin is injected into the nerve, which the toxin kills and cures
the condition.
• Ducks and chickens often die from botulism poisoning by eating rotting
material in which the bacterium has grown. However, vultures, which as
32
Microbial
you know, eat disgusting rotten, stinking carrion, are immune to the toxin
through evolution.
Botulinum spores are on most fresh food surfaces because they grow only in
the absence of air, they are harmless on fresh foods. Botulinum spores are
very hard to destroy at boiling-water temperatures; the higher the canner
temperature, the more easily they are destroyed. Therefore, all low-acid foods
should be sterilized at temperatures of 115°C to 121°C, attainable with
pressure canners operated at 10 to 15 PSIG. PSIG means pounds per square
inch of pressure as measured by gauge. At temperatures of 115°C to 121°C,
the time needed to destroy bacteria in low-acid canned food ranges from 20
to 100 minutes. The exact time depends on the kind of food being canned,
the way it is packed into jars, and the size of jars. The time needed to safely
process low-acid foods in boiling-water canner ranges from 7 to 11 hours; the
time needed to process acid foods in boiling water varies from 5 to 85
minutes.

1. What is Canning?
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2. Name the person who for the first time conceived the idea of canning?
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3. Name the factor important in deciding canning time?
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4. Why is Closteridium botulinum a major threat in canned products?
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33
Safe Chemicals and
Different Foods ………………………………………………………………………………
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13.5 MICROBIOLOGICAL STANDARDS FOR

PROCESSED FOODS
By now you know that microbiological hazards are one of the biggest threats
to food safety. With better understanding in microbiology and food safety,
safety limits have been set for a range of pathogenic microorganisms in foods.
This subunit presents the recommended microbiological guidelines for some
ready-to-eat food. According to the Codex Alimentarius Commission of the
United Nations, an international food standard setting authority, the functions
of
microbiological guidelines include formulation of design requirements,
indication of required and expected microbiological status of the food
commodities, and the verification of efficacy of hygienic practice. These
guidelines stipulate the safety limits of nine major food borne pathogens such
as Salmonella species, Listeria monocytogenes, E coli O157 and Vibrio
cholerae, as well as providing a classification of microbiological quality of
ready-to-eat food for reflecting the hygienic status of the food concerned.
13.5.1 Purpose of Microbiological Standards

Microbiological Guidelines are criteria indicating the microbiological
condition of the food concerned so as to reflect its safety and quality. These
standard lists the maximum permissible levels of food borne microorganisms
that pose a risk to human health in nominated foods, or classes of foods. They
can be introduced to the food industry to observe voluntarily or stipulated in
legislation for compliance.
13.5.2 Sampling
The statistical validity of a microbiological examination increases with the
number of field samples analysed. For regulatory purposes, a minimum of 5
sample units from a lot is generally specified for examination. The size of the
samples taken should also be adequate to enable appropriate microbiological
analyses to be undertaken. A minimum sample size of 100g or ml is
commonly required. A lot is defined as a quantity of food or food units
produced and handled under uniform conditions. This may be restricted to a
food item produced from a particular production line or piece of equipment
within a certain time period (not exceeding 24 hours).
13.5.3 Microbiological Assessment

There are three major components under microbiological assessment of any
food.
Aerobic colony count is a count of viable bacteria based on counting of
colonies grown in nutrient agar plate. This is commonly employed to indicate
the sanitary quality of foods. The incubation condition of ACC used in this
guideline is 30° C for 48 hours.
34
Microbial
Indicator organism Counts refers to the selected surrogate markers. The

main objective of using bacteria as indicators is to reflect the hygienic quality
of food. E. coli is commonly used as surrogate indicator. Its presence in food
generally indicates direct or indirect fecal contamination. Substantial number
of E. coli in food suggests a general lack of cleanliness in handling and
improper storage. Specific pathogens Counts refer to bacteria that may cause
food poisoning. Mechanisms involved may be toxins produced in food or
intestinal infection. Nine specific bacterial pathogens are included in this set
of guidelines. The symptoms of food poisoning vary from nausea and
vomiting (e.g. caused by S. aureus), through diarrhoea and dehydration
(Salmonella spp. and Campylobacter spp.) to paralysis and death in the rare
cases of botulism. The infectious doses vary from less than 10 to more than
106 organisms.
13.5.4 Categories of Food based on Microbial Quality

For assessment of hygienic quality, food items are grouped into five categories
taking into account the raw ingredients used, and the nature and degree of
processing before sale. The microbiological assessment of ready-to-eat food on
the above three components will lead to the classification of the food quality
into one of the following four classes:
a) Class A: the microbiological status of the food sample is satisfactory.
b) Class B: the microbiological status of the food sample is less than
satisfactory but still acceptable for consumption.
c) Class C: the microbiological status of the food sample is unsatisfactory.
This may indicate a sub-optimal hygienic conditions and microbiological
safety levels. Licensees of food premises should be advised to investigate
and find out the causes and to adopt measures to improve the hygienic
conditions. Taking of follow-up samples to verify the improvement may be
required.
d) Class D: the microbiological status of the food sample is unacceptable.
The food sample contains unacceptable levels of specific pathogens that is
potentially hazardous to the consumer. In addition to giving advice to
the licensee of the food premises as stated in (c) above, warning letters as
well as other enforcement actions should be considered. Microbiological
limits in respect of the above components are summarized in the table
13.4.
Table 13.4: Guideline levels for determining the microbiological quality of
ready-to-eat foods
Microbiological Quality (CFU per gram)

Criterions
Class A Class B Class C Class D
Satisfactory Marginal Unsatisfactory Potentially
Hazardous
Aerobic colony count (ACC)[ 30OC/48 hr]
Food 1 <103 103-<104 ≥104 N/A

Category
2 <104 104-<105 ≥105 N/A
35
Safe Chemicals and
Different Foods (Food items <105
3 105-<106 ≥106 N/A
detailed in
Table 5) 4 <106 106-<107 ≥107 N/A
5 N/A N/A N/A N/A
Indicator organisms ( Apply to all food categories)
E.coli (Total) <20 20 -< 100 ≥100 N/A
Pathogens (Apply to all food categories)
Campylobacter not detected N/A N/A Present in

spp in 25g 25 g
Escherichia coli not detected N/A N/A Present in

0157 in 25g 25 g
Listeria not detected N/A N/A Present in

monocytogenes in 25g 25 g
Salmonella spp not detected N/A N/A Present in

in 25g 25 g
Vibrio cholerae not detected N/A N/A Present in

in 25g 25 g
Clostridium <20 20<100 100<103 ≥103

perfringens
Staphylococcus <20 20<100 100<104 ≥104

aureus
Vibrio <20 20<100 100<104 ≥104

parahaemolyticus
Bacillus cereus <103 103-<104 104-<105 ≥105

N/A Not applicable
The desired microbiological quality of the some food samples is summarized

in Table 13.5.
36
Microbial
Table 13.5: Food category table for aerobic colony count assessment
Food group Food item Category

Meat Beefburgers and kebabs 1
Dim sum 2
Pate (meat, seafood or vegetable) 3
Poultry (unsliced) 2
Preserved meat 4
Salami and fermented meat products 5
Sausages 2
Smoked meat 5
Siu-mei & lo-mei 3
Sliced meat (ham and tongue) (cold) 4
Sliced meat (beef, haslet, pork, poultry, etc.) 3
(dried)
Steak and kidney / meat pies 2
Tripe and other offal 4
Seafood Crustaceans 3
Pickled fish 1
Other fish (cooked) 3
Oysters (raw) 5
Seafood meals 3
Shellfish (cooked) 4
Smoked fish 4
Dessert Cakes, pastries, slices and desserts – with dairy 3
cream
Cakes, pastries, slices and desserts – without 2
dairy cream
Cheesecake 5
Mousse / dessert 1
Tarts, flans and pies 2
Trifle 3
Savoury Bean curd 5
Cheese-based bakery products 2
Fermented foods 5
Flan / quiche 2
Dips 4
Mayonnaise / dressings 2
Samosa 2
Satay 3
Spring rolls 3
Vegetable Coleslaw / salads (with or without meat) 3
Fruit and vegetables (dried) 3
Fruit and vegetables (fresh) 5
Rice 3
37
Safe Chemicals and
Different Foods Vegetables and vegetable meals (cooked) 2
Dairy Cheese 5
Yoghurt 5
Ready-to-eat Pasta / pizza 2
meals Meals (others) 2
Sandwiches With salad 4
and Without salad 3
filled rolls
Sushi & Fish fillet and fish roe sashimi / sushi 3
sashimi Sashimi other than fish fillet and fish roe 4
Controlling microbes
Control of microbes in processed products primarily depend upon good
manufacturing practices and one of the most effective way to ensure this is by
application of HACCP. HACCP stands for Hazard Analysis of Critical
Control Point. HACCP is a preventive system for assuring production of safe
food. It is a process that identifies food safety hazards associated with a
product and process and strictly manages and monitors the Critical Control
Points (CCP’s) designed to control the hazard as a way of ensuring the process
is in control and that the safest product possible is being produced. It requires
establishment of hazard, identification of critical control points, effective
monitoring follow up and evaluation. For a food processor it is necessary to
know the microbial quality of the raw material, the processing environment,
and the packaging component. This also requires validation of all processing
stages designed to destroy both the pathogens and the spoilage agents and the
efficacy of preservative system.

1. What is codex Alimentarius?
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2. Why do we need microbial standards?
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38
Microbial
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3. How many sample units from a lot are generally specified for
examination?
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4. What are the major components under microbiological assessment of any
food?
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5. Define Aerobic colony count?
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6. What does the presence of indicator organism in food reflect?
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7. Name the specific pathogens to be observed for microbial assessment of

any food?
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39
Safe Chemicals and
Different Foods ………………………………………………………………………………
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13.6 LET US SUM UP

In this unit you have learnt about the microbial quality limits for various foods
(raw and processed). Herein we have discussed the sources of microorganisms
on fresh fruits and vegetables and in drinking water. Various Food and water
borne harmful microorganisms including indicator bacteria, viruses and
pathogens have been discussed briefly. Clostridium botulinum is an important
organism for canning point of view. Hence, it has been dealt in more detail.
WE HOPE that after reading this unit you will become more conscious about
the microbial quality of food that you are going to eat.
13.7 KEY WORDS

Spoilage
microorganism : Microorganism which spoil the product by
developing undesirable flavours, odours and
changing food appearances or textures via
microbial action.
Pathogenic : Microorganism which may infect plants, animals

and man and make them sick.
Hazardous : Harmful
Epidemic : Out break of infectious disease
Gastroenteritis : Inflamatory change of lining of stomach caused

by microorganism ingested with food and water.
Hepatitis : Virus Hepatitis A
Parasites : Life on, within or at the expense of other

organisms.
Toxin : Poison
Intoxication : Toxin production
13.8 ANSWERS TO CHECK YOUR PROGRESS "

EXERCISES
• Sources of microorganisms on fresh fruit are air, orchard soil,

irrigation water, harvesting device, storing and packaging containers,
handling personnel etc.
40
Microbial
• The survival or growth of contaminating microorganisms is affected

by intrinsic, extrinsic and processing factors.
• Factors of importance are nutrient composition, pH, presence of scales
and fibers, redox potential, temperature and gaseous atmosphere.
• Mechanical shredding, cutting and slicing of the produce open the
plant surfaces to microbial attack.
• Members of the genera Penicillium, Aspergillus, Sclerotinia, Botrytis

and Rhizopus are commonly involved in spoilage of fresh fruits and
vegetables.
• Most of the reported outbreaks have been associated with bacterial

contamination.
• Members of the Enterobacteriaceae. Of these, Salmonella and
Escherichia coli O157 are of particular concern.
• Gastroenteritis
• Diarroea
• Dysentry
• Hepatitis
• Typhoid fever
• Microbes
• Radionuclide
• Inorganics
• Volatile organics
• Disinfectants
• Disinfection by products etc.
• The human pathogens potentially transmitted in drinking water are

Bacteria viz. Escherichia coli,Salmonella, Shigella, Vibrio cholera,
Yersinia enterocolitica, Campylobacter jejuni.
• Viruses viz, Adenoviruses, Enterovirus, Hepatitis A, Hepatitis E,
Norwalk virus, Rotavirus, and small round viruses
• Parasites viz. Giardia, Cryptosporidium, Entamoeba histolytica and
Drancunculus.
• Pseudomonas
41
Safe Chemicals and
Different Foods • Flavobacterium
• Acinetobacter
• Klebsiella
• Serratia
• Legionella.
• Process where foods are put into a container and given a high heat
treatment to make the product sterile.
• Nicholas Appert
• Food’s acidity
• Density
• Ability to transfer heat.
• Growth of the bacterium Clostridium botulinum in canned food may

cause botulism − a deadly form of food poisoning.
• Codex Alimentarius is an international food standard setting authority.
• Lists the maximum permissible levels of food borne micro-organisms

that pose a risk to human health in nominated foods, or classes of
foods.
• Indicate the microbiological condition of the food concerned so as to
reflect its safety and quality.
• Minimum of 5 sample units from a lot is generally specified for

examination.
• Aerobic colony count

• Indicator organism Counts
• Specific pathogens Counts
42
Microbial
• Count of viable bacteria based on counting of colonies grown in

nutrient agar plate.
• Commonly employed to indicate the sanitary quality of foods.
• The presence of indicator organisms in food reflect the hygienic

quality of food.
• Campylobacter spp.
• Escherichia coli 0157
• Listeria monocytogenes
• Salmonella spp
• Vibrio cholerae
• Clostridium perfringens
• Staphylococcus aureus
• Vibrio parahaemolyticus
• Bacillus cereus

1. Adams, M.R. and Moss, M.O. (2000) Food Microbiology. Royal Society
of Chemistry, Cambridge, U.K.
New York.
43
Preparation of
Media
EXPERIMENT 1 PREPARATION OF MEDIA

Structure
1.1 Introduction
Objectives
1.2 Experiment
Principle
Requirements (Equipment/Machinery/Instrument and Chemicals/Material)
Procedure
Observations
Result
1.3 Precautions
1.1 INTRODUCTION
Microorganisms require certain basic nutrients and physical factors for the
sustenance of life as do all other living organisms. However, their particular
requirements may vary greatly. Nutritional needs of microbial cells are
supplied in the laboratory through a variety of media to detect their presence.
Microbiological media for the evaluation of spoilage and detection of bacteria,
yeasts and molds in foods are mentioned in this chapter.
Objectives
After studying and performing this experiment, you should be able to:
• learn that microorganisms are ubiquitous and in nature, they do not
segregate themselves by species but exist a mixture of many other cell
types; and
• learn to make different media which are suitable for a particular genera of
microorganisms to produce their discrete colonies.
1.2 EXPERIMENT
1.2.1 Principle
Many special purpose media are needed to facilitate recognition, enumeration
and isolation of different types of microorganisms. To meet these needs, the
microbiologists has developed numerous media which on the basis of their
function may be classified as follows:
A) Selective media
These media provide nutrients that enhance the growth and predominance
of a particular type of bacterium and do not enhance (and may even
inhibit) other types of organisms that may be present. For instance, a
medium in which cellulose is the only carbon source, will specifically
select for, or enrich the growth of cellulose-utilizing organisms when it is
inoculated with a soil sample containing many kinds of bacteria.
B) Differential media
Certain reagents or supplements, when incorporated into culture media,
may allow differentiation of various kinds of microorganisms. For
example, if a mixture of bacteria is inoculated on to a blood containing
5
Practical Manual ⎯
Food Microbiology
agar medium (blood agar) some of the bacteria may hemolyze (destroy)
the red blood cells, others do not. Thus one can distinguish between
hemolytic and non-hemolytic bacteria on the same media.
1.2.2 Requirements (Equipment/Machinery/Instrument/Chemicals/

Material)
• Autoclave (Portable)
• Balance
• Heating mantle/ water bath
• pH meter
• Laminar air flow
• Stirrer
• Pipettes
• Distilled water
• Media (nutrient agar, potato dextrose agar, violet red bile agar, plate count
agar)
• Test tubes
• Beakers
• Cotton plugs
• pH paper
• Measuring cylinder
1.2.3 Procedure
The preparation of microbiological media usually involves the following
steps:
1. Carefully weigh the proper amount of the dehydrated base medium or the
correct proportion of constituent ingredients and dissolve in appropriate
volume of distilled water and heat. Composition of the media is as given in
Annexure-1.
2. Determine the pH of the medium, and adjust if necessary with dilute acid
or alkali.
3. If a solid medium is desired, add agar (1.5-2%) and boil the medium to
dissolve the agar.
4. Distribute the medium into tubes or flasks. The amount of medium
distributed per container should be limited so that no point within the
volume of the medium is more than 2.5 cm from the top surface of the
container.
5. Autoclave at 121°C for 15 minutes. Some media (or specific ingredients)
that are heat labile are sterilized by filtration.
1.2.4 Observations
Observe the pH using the pH meter or colour indicator solutions. At the next
laboratory period observe for any contamination to ensure proper sterilization.
Discard any contaminated flasks and wash them in disinfectant solution.
1.2.5 Results
After performing this experiment you will observe that suitable media for
growth of bacteria is nutrient agar (pH 6.8-7.0) or plate count agar (pH 7.0),
whereas for yeasts and moulds it is potato dextrose agar (pH 5.6) and malt
agar (pH 5.4).
6
Preparation of
Media
1.3 PRECAUTIONS
• Adjust the pH of the media accurately, to provide favourable condition of
growth for the microorganisms. pH of the medium may change during
sterilization and because of possible browning reactions, it is important not
to exceed the recommended time and temperature.
• Prepare medium in such quantities that if stored, it will be used before loss
of moisture through evaporation that becomes evident.
• To prevent contamination and excess evaporation of water from a medium
in flask and tubes during storage, optionally fit aluminium foil or plastic
with loose rubber bands before autoclaving in order to allow air to escape
and to prevent the container from bursting.
• Avoid over loading autoclaves so that the rate of air exhaust and heating is
not appreciably delayed. The autoclave should reach 121°C (15 psi)
slowly but within 10 min. after starting the air exhaust operation.
• Flask or test tubes should be plugged with cotton or capped with paper.
• After sterilization gradually reduce the pressure within the autoclave
(using no less than 15min) since liquids may be at a temperature above
their boiling point at atmospheric pressure. If the pressure is lowered too
rapidly, liquids may boil over and come out from the container.
• Used plates, pipettes, tubes etc. should be routinely decontaminated by
autoclaving for 30 minute at 121°C.
• Media should be stored at 2-8°C in a dry, dust free area and should not be
exposed to direct sunlight.
7
Practical Manual 
Food Microbiology
EXPERIMENT 2 MICROSCOPIC STAINING
TECHNIQUES
Structure
2.1 Introduction
Objectives
2.2 Experiment
Principle
Procedure
Observations
Result
2.3 Precautions
2.1 INTRODUCTION
Visualization of microorganisms in the living state is most difficult, not only
because they are minute but also because they are transparent and practically
colourless when suspended in an aqueous medium. To study their properties
and to differentiate microorganism into specific groups for diagnostic
purposes, biological stains and staining procedures in conjunction with light
microscopy have become major tools in microbiology. Chemically a stain may
be defined as an organic compound containing a benzene ring, a chromophore
(chemical group that imparts colour to benzene), and an auxochrome (chemical
compound that helps in binding to cells).
Objectives
• learn the practical and theoretical basis of chemical staining;
• describe manipulative technique of smear preparation;
• explain procedures for simple staining and negative staining; and
• perform differential staining procedures such as the Gram’s staining, acid
fast staining and spore staining.
2.2 EXPERIMENT
2.2.1 Principle
Staining by various dyes provides contrast between microorganisms and their
background, permitting differentiation among various morphological types and
internal structure such as cell wall, vacuoles or nuclear bodies. It also enables
the microbiologist to use higher magnifications.
Numerous staining techniques are available for visualization, differentiation
and separation of bacteria in terms of morphological characteristics and
cellular structures. A summary of commonly used procedures and their
purpose is outlined in Figure 2.1.
8
Microscopic Staining
Techniques
Simple Staining For visualization of morphological shape
Use of single stain (cocci, bacilli and spirilli) and
arrangement (chains, clusters, pairs and
tetrads)
Types of Gram’s Stain
staining Separation into groups
techniques Acid-fast stain
Differential Flagella stain

staining Visualization of Capsule stain
Use of two structures Spore stain
Contrasting stains Nuclear stain
Figure 1: Staining techniques
2.2.2 Requirements (Equipment /Machinery/Instrument and Chemicals/

Material)
• Bunsen Burner
• Microscope
• Test tube shaker
• Inoculating needle
• Cover slips
• Glass slides
• Sterilized test tubes
• Wash bottles
• Microbial cultures
• Distilled water
• Stains
• Immersion oil
• Tissue paper
2.2.3 Procedure
Preparation and fixation of bacteria for staining
Prior to staining, you must “fix” the material to be observed that is make it
stick to the glass slide upon which is to be stained. If a preparation is not fixed,
the film of cells will wash off during the staining procedure. Purpose of
fixation is also to kill the microorganism and coagulate the protoplasm of the
cell so as to fix it on glass surface (Figure 2.2).
The fixing technique, although not difficult, requires adequate care in its
preparation. Follow these basic rules meticulously:
1. Preparation of glass slides: Clean slides are essential for preparation of
microbial smears. Grease or oil from fingers on slides must be removed by
washing the slides with soap and water, followed by a water rinse. After
cleaning dry the slides and place them on laboratory towels until ready for
use.
9
Practical Manual  From liquid media From solid media
Food Microbiology
(a) Place on to two loopfuls of the cell (a) Take one drop of water on the loop and
suspension on the clean slide. place it on the centre of the slide.
(b) With a circular movement of the loop, (b) Transfer a small amount of the bacterial
spread the suspension into a thin area inoculum from the slant culture into the
approximately 5-7mm. drop of water. Spread both into a think
area approximately 10 mm.
Fixation
(c) Allow the smear to air dry. (c) While holding the slide at one end,
quickly pass the smear over the flame
of the Bunsen burner two to three times.
Figure 2.2: Bacterial smear preparation
2. Preparation of smear: Avoidance of thick, dense smears is absolutely

essential. A good smear is one that, when dried, appears as a thin whitish
layer or film. Those made from broth cultures or cultures from a solid
medium require variations in technique.
• Broth cultures: One or two loopful of suspended cells should be
applied directly to the glass slide with a sterile inoculating loop and
spread evenly over a small area.
• Cultures from a solid media: Organisms cultured in a solid medium
produce thick, dense surface growth and are not amenable to direct
10
transfer to the glass slide. These cultures must be diluted by placing a
loopful of water on the slide in which the cells will then be emulsified. Microscopic Staining
Suspension is accomplished by spreading the cells in a circular motion Techniques
in the drop of water with the needle tip. At this point, the smear must be
allowed to dry completely. Do not blow or wave it in the air.
3. Heat fixation: Unless fixed on the glass slide, the bacterial smear will
wash away during the staining procedure. This is avoided by heat fixation,
during which the bacterial proteins are coagulated and fixed to the glass
surface. Heat fixation is performed by the rapid passage of the air-dried
smear two or three times over the flame of the Bunsen burner.
Staining with basic dyes
Herein, the bacterial smear is stained with a single basic stain. The bacterial
nucleic acid and certain cell wall components carry a negative charge that
strongly attract and bind to the cationic (negatively charged) chromogen. The
purpose of simple staining is to elucidate the morphology and arrangement of
bacteria.
The most commonly used basic stains are methylene blue, crystal violet and
carbol fuchsin. Note that exposure time for staining cells to these dyes differs
for each of these stains; carbol fuchsin requires 15-30 seconds, crystal violet
20-60 seconds and methylene blue 1-2 minutes for fresh cultures. For old
cultures more time is required for staining.
General staining
Procedure for staining with different dyes:
1. Prepare bacterial smear of the organisms. Note: All smears must be heat
fixed prior to staining.
2. Flood the smear with any one of the stains, using the appropriate exposure
time.
3. Wash the stained preparation with tap water to remove excess stain. During
this step, hold the slide parallel to the stream of water; in this way you can
reduce the loss of organisms from the preparation.
4. Dry the slide using blotting paper.
5. Examine the stained preparation under the oil-immersion objective of the
microscope.
Observe closely for significant difference in cell size, shape and arrangements.
Negative or indirect staining
1. Place a small drop of nigrosin close to one end of a clean slide.
2. Using sterile technique, place a loopful of inoculum from the mixed culture
in the drop of nigrosin and mix.
3. With the edge of the second slide held at above 30° angle and placed in
front of the bacterial surface, push the mixture to from a thin smear.
4. Air dry. Do not heat fixed slide.
5. Examine the slide under oil-immersion objective of the microscope.
11
Practical Manual  Differential staining
Food Microbiology
Gram’s stain
1. Prepare smear of the bacterial culture. Air-dry and fix these preparations
with heat.
2. Flood smear with crystal violet and let stained for 30 seconds.
3. Rinse with water.
4. Cover the film with Gram’s Iodine instantly and let stained for 1 min.
5. Wash with tap water.
6. Decolorize with 95% alcohol. For a thin smear, 10-20 second is long
enough.
Caution: Do not over-decolorize. Add reagent drop by drop until crystal
violet fails to wash from smear.
7. Rinse with water.
8. Counter stain with safranine for 20-30 seconds.
9. Rinse with water and blot dry.
10. Examine under the oil-immersion objective.
Table 1: Steps in the gram’s stain
Step Procedure Results

Gram + Gram -
Initial Stain Crystal Stain for 30 Stains purple Stains purple
seconds
Mordant Iodine for 30 Remains purple Remains purple
seconds
Decolourization 95% ethanol for Remains purple Becomes
10-20 seconds colourless
Counterstain Safranine for 20-30 Remains purple Stains pink
seconds
Acid fast stain

1. Prepare a smear of bacterial culture.
2. Allow to air dry and heat fix in usual manner.
3. Flood smear with carbol fuchsin and place on a warm hot plate, allowing
the preparation to steam for 5 minutes. Caution: Do not allow stain to
evaporate, replenish stain as needed. Also prevent stain from boiling by
adjusting the hot plate to a proper temperature.
4. Wash with tap water. Heated slides must be cooled prior to washing.
5. Decolorize with acidic alcohol (95% ethyl alcohol containing 2.5% HNO3)
for 10-30 seconds, a carbol fuchsin fails to wash from smear.
12 6. Wash with water.
7. Counter stain with methylene blue for 2 min. Microscopic Staining
Techniques
8. Wash smear with tap water and blot dry.
9. Examine under the oil immersion objective.
Structural stain
Endospore stain
1. Prepare smear, air dry and fix with heat.
2. Flood smear with malachite green and place on a warm hot plate, allowing
the preparation to steam for 2-3 minute.
Caution: Do not allow stain to evaporate; replenish stain as needed.
Prevent the stain from boiling by adjusting the hot plate at a proper
temperature.
3. Cool slide and wash with water.
4. Counter stain with safranine for 30 min.
5. Wash with water and blot dry.
6. Examine under oil immersion objective.
2.2.4 Observations
In the space provided:
1. Draw a representative field for each organism
2. Describe the morphology of the organism with reference to their shape
(bacilli, cocci, spirilli) and arrangements (chains, clusters, pairs)
Stain Methylene blue Gram Stain Carbol Fuschin

Drawing of a
representative
field
Cell morphology
Arrangement
Cell colour
2.2.5 Results
Staining the microorganisms makes them contrast in colour with their
surroundings so that they are more readily visible. Certain stains can also be
used to identify certain structures of the cell which would otherwise be unseen.
13
Food Microbiology 2.3 PRECAUTIONS
• Clean, dry glass slide must be taken to prepare a smear.
• Thick dense smears should be avoided.
• The smear should be properly heat fixed on the slide to avoid its washing
off during staining procedure.
• Do not heat fix in case of negative staining.
• Do not over decolorize in case of Gram’s staining.
• Do not allow stain to evaporate while acid staining technique. Replenish
stain as needed.
14
Culturing and
EXPERIMENT 3 CULTURING AND Identification of
Microorganisms
IDENTIFICATION OF
MICROORGANISMS
Structure
3.1 Introduction
Objectives
3.2 Experiment
Principle
Procedure
Observations
Result
3.3 Precautions
3.1 INTRODUCTION
Microorganisms are ubiquitous. They are found in soil, air, water, food,
sewage and body surfaces. In short, every area of our environment is replete
with them. When grown on a variety of media, microorganisms will exhibit
differences in the microscopic appearance of their growth. These differences,
called cultural characteristics, are used as basis for separating microorganisms
into taxonomic groups. The cultural characteristics for all non-microorganisms
are contained in Bergy’s Manual of Systemic Bacteriology with their
morphological characteristics.
Objectives
• differentiate microorganisms into bacteria, yeasts and moulds; and

• know the different forms / shapes of microorganisms.
3.2 EXPERIMENT
3.2.1 Principle
The microorganisms can be divided into bacteria, yeasts and moulds on basis
of the difference in their morphological, cultural and physiological
characteristics.
Bacteria
Among the major characteristics of bacterial cells are their size, shape,
structure and arrangement. These characteristics constitute the morphology of
the cell. Bacteria are very small, most being approximately 0.5 to 1.0
micrometers in diameter. They are unicellular, have cell wall and cytoplasm
but the nucleous is not well developed. The shape of a bacterium is governed
by its rigid cell wall. Typical bacterial cells are spherical (cocci), straight rods
(bacilli) or rods that are helically covered (spirilla).
15
Practical Manual  Different patterns for arrangement for identification purposes are monococci,
Food Microbiology
diplococci, streptococci, tetrads, staphylococci and sarcinae (Figure 3.1). Cocci
generally reproduce by binary fission. Rod shaped bacteria may be sporulating
type like Bacillus species and Clostridium species which produce endospores
or they are non-sporulating like Lactobacillus species (Figure 3.2). Bacteria
may be both motile (having flagella) or non-motile (no flagella).
A. Diplococci
Diplococcus
pneumoniae
B. Steptococci
Streptococcus
agalactiae
C. Tetracocci
Gaffkya
tetragena
E. Sarcinae
Sarcina
lutea
D. Staphylococci
Staphyloccus
aureus
Figure 3.1: Characteristic arrangements of cocci
Figure 3.2: Types of rod-shaped bacteria. A) Clostridium sporogenes; B) Pseudomonas

sp.; C) Bacillus megaterium; and D) Salmonella typhi
16
Fungi Culturing and
Identification of
Fungi is a group of eukaryotic organisms. They comprise of yeasts and Microorganisms
moulds. Whereas moulds are filamentous and multicellular, yeasts are
unicellular.
Yeasts
In general yeast cells are larger than most bacteria. Yeasts vary considerably in
size ranging from 1-5 micrometer in width and from 5-30 micrometer in
length. They are commonly egg-shaped, but some are elongated and some
spherical. Yeasts lack flagella and other means of locomotion (Figure 3.3).
Saccharomyces sp. (a
common bread yeast)
Figure 3.3: Yeast cell
Moulds
The thallus of moulds consist essentially of two parts: the mycelium and the
spores. The mycelium is a complex of several filaments called hyphae.
Filaments are made up of cells arranged end to end, branched and intertwined.
Cells are like cells of higher plants in that they have visible nuclei, cell wall of
varying thickness and cytoplasm. Mycelia in some fungi are divided into
individual cells separated by cross walls and each cell containing a nucleus
(Figure 3.4 and 3.5).
Sporangium
Sporangiophore
Hyphae
Figure 3.4: Mucor sp.
17
Food Microbiology
STOLON
Figure 3.5: Rhizopus sp.
3.2.2 Requirements (Equipment/ Machinery/ Instrument and Chemicals/

Material)
• Compound microscope
• Bunsen burner
• Immersion oil
• Glass slides
• Inoculating needle
• Cover slips
• Tissue paper
• Microbial culture
• Distilled water
3.2.3 Procedure
1. Prepare the required media (broth or agar) for culturing the
microorganisms
2. Place a small amount of media into test tubes, plug and sterilize them in an
autoclave.
3. In case of solid media tubes, cool them in an incline position (slants)
4. When the medium is cold and solid, inoculate the surface of the medium
using pre-sterilized needle. Move the needle gently on the agar surface in a
snakelike motion from the butt to the top. In case of broth tubes, inoculate
in the liquid media
5. Incubate both culture tubes at 30°C for few days.
6. In case of solid media, scoop out the mass of surface growth in which
organism grows and put on clean, dry slide. From liquid broth, place a drop
of culture on slide.
7. Observe under microscope.
18
3.2.4 Observations Culturing and
Identification of
In the chart provided: Microorganisms
1. Draw several cells from a typical microscopic field as viewed under each
magnification.
2. Give the total magnification for each objective.
3. Observe spores or conidia and their arrangement.
Bacteria Yeast Mould

Drawing of a
representative
field
Magnification --------------------- --------------------- --------------------
3.2.5 Results
Different types of spoilage have been encountered caused by various
microorganisms. The type of microorganism proliferating depends on the
composition of the material. The different spoilage microorganisms include
bacteria, yeasts and moulds that can be observed and identified under a
microscope by studying the morphological characteristics. These organisms
vary in size, shape, colour, growth habit and mode of reproduction.
3.3 PRECAUTIONS
1. Use clean glass slides for smear preparation.
2. Thick, dense smears should be avoided.
3. Sterilize the inoculating needle before inoculation to avoid contamination.
4. The agar tubes should be properly sterilized.
5. Do not place the cotton plugs on ground during experiment.
6. Carefully view the characteristics of the microorganisms so as to
differentiate them correctly.
19
Food Microbiology
EXPERIMENT 4 ASEPTIC CULTURE

TECHNIQUE
Structure
4.1 Introduction
Objectives
4.2 Experiment
Principle
Observations
4.3 Precautions
4.1 INTRODUCTION
In previous experiments you learned that microorganisms thrive pretty much
everywhere. It is far too easy to contaminate your lab cultures and
experiments with stray microorganisms from the air, the countertop, or your
tools. It is also possible to expose your surroundings or yourself to a possible
pathogen. In this lab exercise, you will learn to transfer microbiological
cultures from one medium to a second sterile medium without contamination
of the culture, sterile medium, or the surroundings.
Objectives
• know how to handle microorganisms, tubed media, plated media, and

inoculating tools such as loops, needles, or swabs etc.;
• learn how to transfer bacteria from test tubes or broth and agar; and
• learn how to transfer bacteria from Petri plates.
4.2 EXPERIMENT
4.2.1 Principle
Aseptic technique is a method that prevents the introduction of unwanted

organisms into an environment. In order to protect sterile broth, media, plates,
slants etc. from contamination we must practice aseptic i.e. sterile techniques
to protect our material from contamination. By using aseptic technique only
sterile surface touches other sterile surface and exposure to the non sterile
environment is minimized.
Though, observing aseptic technique is the most important instruction for any
microbiology experiment, some common circumstances will be discussed in
this practical to make you aware of aseptic techniques.
Specific Aseptic Techniques
20
Aseptic Culture
Technique
A) Sterilization of inoculation loop
The inoculation loop is sterilized by passing it at an angle through the
flame of a gas burner until the entire length of the wire becomes orange or
red hot. In this way all contaminants on the wire are incinerated. Never lay
the loop down once it is sterilized or it may again become contaminated.
Allow the loop to cool a few seconds to avoid killing the inoculum.
B) Transferring bacteria from broth culture to fresh broth
Requirements
• Bunsen burner.
• Inoculation needle.
• Trypticase Soy Broth cultures of Bacillus subtilis, Escherichia coli and
Micrococcus luteus and Mycobacterium phlei − referred to as Tubes A.
• Sterile Trypticase Soy Broth tubes (4 -one for each microorganism) –
referred to as Tubes B.
• Glass Marking pen.
Procedure
1. Turn on the Bunsen burner.
2. Vortex culture suspensions of Bacteria given (Tubes A).
3. Place culture suspensions tube near sterile broth tubes (tubes B). Label
sterile tubes with name of microorganism and date.
4. Sterilize the inoculation loop as explained above.
5. While holding inoculation loop between thumb and first two fingers of
right hand, pick up tube A with left hand and open the cap/cotton plug
with last two fingers of right hand.
6. Flame the lip of test tube A.
7. Place the sterile loop into culture A and take loopful of culture.
8. While still holding the inoculum in your right hand, pick up tube B
with left hand and open the cap/cotton plug with last two fingers of
right hand.
9. Flame the lip of test tube B gently
10. Place the loop containing droplet of culture in tube B and gently swirl
it to transfer the microbes into sterile broth..
11. Take out the loop and continue to hold it in your hand.
12. Flame the lip of test tube B gently and replace the cap/plug which
should be still in your right hand. Place tube B back in the test tube
rack. Like wise plug the tube A and place in a test tube rack.
13. Sterilize the inoculation loop in flame.
14. Repeat the procedure with all bacterial cultures.
Results
21
Food Microbiology
1. Draw and describe the growth seen in each of the four broth cultures.
Bacillus Escherichia Micrococcus Mycobacterium

subtilis coli luteus phlei
Growth = Growth = Growth = Growth =
Figure 4.1: Growth of bacterial culture
C) Streaking plating bacteria

a) From broth culture to sterile medium plate
b) From one petridish to fresh sterile medium plate
Requirements
• Bunsen burner.
• Inoculation needle.
• Trypticase Soy Broth cultures of bacteria (Bacillus subtilis and
Escherichia coli) to be transferred (Tubes A).
• Trypticase Soy Agar plate cultures of bacteria (Bacillus subtilis and
Escherichia coli) to be transferred.
• Sterile petridish having Trypticase Soy Agar medium (4 no. Two for
each bacterium).
• Glass Marking pen.
Procedure
Removing inoculum from a broth culture
1. Label the plates and tubes.
2. Turn on the Bunsen burner.
3. Loosen the top of the bottle/ Tube containing the inoculum.
4. Hold the loop in the right hand.
5. Flame the loop and allow to cool.
6. Lift the bottle/test tube containing the inoculum with the left hand.
7. Remove the lid/cotton wool plug of the bottle/test tube with the little
finger of the left hand.
22
Aseptic Culture
Technique
8. Flame the neck of the bottle/test tube.
9. Insert the loop into the culture broth and withdraw. At all times, hold
the loop as still as possible.
10. Flame neck of the bottle/test tube.
11. Replace the lid/cotton wool plug on the bottle/test tube using the little
finger. Place bottle/test tube on bench.
Removing inoculum from a plate culture
1. Sterilize the inoculating loop in the flame of a gas burner.
2. Lift the lid of the culture plate slightly and stab the loop into the agar
away from any growth to cool the loop.
3. Scrape off a small amount of the organisms and close the lid
Transferring the inoculum into a petri plate
1. Partially lift the lid of the Petri dish containing the solid medium.
2. Hold the charged loop parallel with the surface of the agar; smear the
inoculum backwards and forwards across a small area of the medium
3. Remove the loop and close the Petri dish.
4. Flame the loop and allow it to cool. Turn the dish through 90º
anticlockwise.
5. With the cooled loop streak the plate from area A across the surface of
the agar in three parallel lines. Make sure that a small amount of
culture is carried over.
7. Flame the loop and allow to cool. Turn the dish through 90º
anticlockwise again and streak from B across the surface of the agar in
three parallel lines.
9. Flame the loop and allow to cool. Turn the dish through 90º
anticlockwise and streak loop across the surface of the agar from C
into the centre of the plate
10. Remove the loop and close the Petri dish. Flame the loop.
11. Seal and incubate the plates inoculated with Bacillus subtilis and
Escherichia coli at 37°C upside down (lid on the bottom) to prevent
condensing water from falling down on the growing colonies and
causing them to run together in inverted position.
Results
23
Food Microbiology
Figure 4.2: Streak plate technique
Expressing results
Bacterial colonies contain millions of cells and exhibit diverse morphologies;
however, all isolated colonies produced on streak plates arise from a single
bacterial cell. When evaluating colony morphology, use specific terms to
describe the shape, elevation, colony margin shape, and surface texture
(Figure 4.3). Colony size and colour are also useful features that are noted. All
of these characteristics may be useful in the initial identification of unknown
bacteria. Colonies that have different morphologies may be considered to
contain different bacterial species. However, colonies that appear to be similar
in morphology are not always the same bacterial species.
Common colony shapes
Figure 4.3: Terminology used to describe colony morphology
4.2.2 Observations
Obtain your streak plate from the incubator and visually examine the different
regions:
1. Notice a dilution effect as you move from region to region.
2. Look for isolated individual colonies present.
3. Note different types of colony morphologies present.
4. Measure the size (diameter or length) and record the colony colour in
Table 1.
24
Aseptic Culture
Technique
Table 1: Colony characteristics of two bacterial colonies isolated using
streak plating
Colony Size Colour Shape Margin Surface Elevation

(mm)
1.
2.
4.3 PRECAUTIONS
• Operations must not be started until all requirements are within immediate
reach and must be completed as quickly as possible.
• Carry out all microbiological operations in a laminar flow hood.
• Wear gloves and lab coat to protect yourself but also to prevent dry skin
and microorganisms from contaminating your samples.
• Use plugs made of non-absorbent cotton wool in test tubes and pipettes to
prevent microorganisms from passing in or out and contaminating either
the culture or the environment. The cotton wool must remain dry because
this filtration property is lost if the cotton wool becomes moist – hence the
use of non-absorbent cotton wool.
• For use in test tubes a plug should be properly made to ensure that it can
be held comfortably without being dropped and its shape and form are
retained while being removed from and returned to a test tube several
times.
• Disinfect all surfaces prior to use with a disinfectant solution.
• Swab down the working surface liberally with 70% ethanol.
• Periodically spread a solution of 70% ethanol over the exterior of gloves to
minimize contamination. Replace them if torn.
• In case of any spill, spread a solution of 70% alcohol and swab
immediately with non-linting wipes.
• Discard gloves after use and do not wear them when entering any other lab
area.
• Bring into the work area only those items needed for a particular
procedure.
• Leave a wide clear space in the centre of the hood (not just the front edge)
to work on. Do not clutter the area to prevent blockage of proper air flow
and to minimize turbulence.
• Swab with 70% alcohol all glassware (medium bottles, beakers, etc.)
before placing them inside the hood.
• Arrange the work area to have easy access to all of it without having to
reach over one item to get at another (especially over an open bottle or
flask).
• Use sterile wrapped pipettes and discard them after use into a biohazard
waste container.
25
Food Microbiology
• Check that the wrapping of the sterile pipette is not broken or damaged.
• Vessels must be open for the minimum amount of time possible and while
they are open all work must be done close to the Bunsen burner flame
where air currents are drawn upwards.
• Discard any contaminated material immediately.
• Never perform mouth pipetting. Pipetting aids must be used.
• When handling sterile containers with caps or lids, place the cap on its side
if it must be laid on the work surface.
• Make sure not to touch the tip of the pipette to the rim of any flask or
sterile bottle.
• Clean the work area when finished by wiping with 70% alcohol.
26
Visual and
EXPERIMENT 5 VISUAL AND Microscopic
Examination of Raw
MICROSCOPIC and Processed
Product
EXAMINATION OF RAW
AND PROCESSED
PRODUCT
Structure
5.1 Introduction
Objectives
5.2 Experiment
Principle
Requirements
Procedure
Observations
Results
5.1 INTRODUCTION
Isolation and identification of microbial food contaminants help to understand
how infectious agents enter and spread through the food chain. There is a need
to estimate the risk that food borne pathogens pose to human health in a
national and international context and to identify possible interventions to
reduce or eliminate these risks.
Objectives
• take visual observations of food samples
• visualize food-borne micro-organisms under the microscope.
5.2 EXPERIMENT
5.2.1 Principle
The potential for food to become contaminated with chemical substances or
microorganisms starts from the time it is harvested and continues right through
until the time it is eaten. The examination of food samples is one of the most
important tasks:
Initial record of specimen as received
• Examine the specimen carefully for information such as to how it was
received, condition (frozen, fresh), time, date, mode of delivery and write
description of the specimen immediately.
• Examine seals for faults or damage and describe and note the details on the
label.
• Weigh/ Measure the specimen as received and prior to opening.
27
Practical Manual  Odour and taste
Food Microbiology
• Smell the food. The odour of a food can give clues to the nature of the
complaint (volatile substances, deterioration, chemical taints etc.) Food
may be required to be tasted CAREFULLY at this point.
Spoilage
• Visually observe spoilage by turbidity, gas production, bubbling etc.
Mouldy food
• Document a full description of the affected areas recording the types of
colonies present, their colours and their textures. Measure the area(s) of
suspect mould as soon as possible and in three dimensions if applicable.
Ensure that the dimensions and numbers of individual colonies are noted.
Microscopic Examination of Foods
Principle
Microscopes are instruments that are capable of producing a magnified image
of a small objects including microorganisms. In a food microbiology
laboratory Compound Microscopes are most commonly used. These
microscopes are light illuminated. They are used in observation and description
of the microscopic morphology of bacteria, fungi, parasites and host cells in
various stained and unstained preparations.
Figure 5.1: Labelled diagram of a compound microscope

28
Procedure to Focus the Specimen in Microscope Visual and
Microscopic
1. Obtain a prepared slide of contaminated food/ isolated microorganism. Examination of Raw
and Processed
Mount the slide onto the stage of the microscope. Product
2. Start with the lowest power objective in place. Using the course
adjustment knob, move the objective lens to its lowest point. Look through
the ocular and focus upward with the coarse adjustment until an image
comes into view. Use the fine adjustment to obtain maximum clarity.
From this point on, do not use the coarse adjustment; doing so can result in
damage to the lens, slide or both. Adjust the iris to allow enough light for
maximum visibility and contrast. Usually, this will be about half the
maximum iris opening. Too much light can wash out the details of the
image.
3. Move the slide to a point of interest. Move the next objective lens into
place and adjust the fine focusing knob, and adjust the iris as necessary.
Repeat this step with the highest power, non-oil lens.
4. Note that as the power of the objective lens increases, the distance between
the objective and the specimen (working distance) decreases. Also, as
magnification increases, the field of view (visible area) and depth of
field/focus (visible thickness) decrease. Moving the fine adjustment up and
down allows viewing of other areas along the depth of thickness of the
specimen).
5. To use the oil-immersion lens, move the turret halfway between the high-
power air (non-oil) lens and the oil lens. Place a drop of immersion oil
directly on the slide. Move the oil-immersion lens into place and adjust the
fine focusing knob. Adjust the iris as necessary. Make sure that the
immersion oil does not get on the air lenses. Make note of the differences
and similarities between the organisms.
6. After using the oil lens for a specimen, wipe the lens with a piece of lens
paper. Do not use anything but lens paper to clean microscope lenses.
Usually, lens-cleaning fluids are not necessary unless the lens is
exceptionally dirty.
For Getting the Best Possible Image
1. Use lens tissue, to clean the ocular and objective lenses; do not use any
other kind of paper. You may also need to clean the slide.
2. Always begin to focus the microscope with the low power, coarse focusing
knob.
3. For best viewing at high power, white light is essential. The higher the
power of the objective lens, the less will be the depth of field.
Microorganism’s Morphology Using the Microscope
Moulds
Mould mycelium and spores can be observed in unstained wet mounts at
magnifications of x100 although direct observations of “mouldy” material
through the lid of a Petri dish or specimen jar at lower magnifications with the
plate microscope are also informative (but keep the lid on!). Routine
29
Practical Manual  identification of moulds is based entirely on the appearance of colonies to the
Food Microbiology
naked eye and of the mycelium and spores in microscopical preparations.
Figure 5.2: Aspergillus sp. under microscope
Yeast
Yeast can be seen in unstained wet mounts at magnifications ×100.
Figure 5.3: Yeast under microscope
Bacteria
Bacteria are much smaller and can be seen unstained at ×400 but only if the
microscope is properly set up and all that is of interest is whether or not they
are motile. A magnification of ×1000 and the use of an oil immersion objective
lens for observing stained preparations are necessary for seeing their
characteristic shapes and arrangements. If there is doubt that a food has caused
food poisoning or has undergone microbial spoilage, the original product or a
low serial dilution of it should be used to prepare a slide for direct microscopic
examination. The Gram stain reaction and cellular morphology of the bacteria
on the slide may indicate the need for other types of examination. A
microscopic examination must be made, even though the food may have
undergone heat treatment and the microorganisms involved may no longer be
viable.
30
5.2.2 Requirements Visual and
Microscopic
1. Glass slides, 25 x 75 mm, with etched portion for labelling; 1 slide for each Examination of Raw
and Processed
blended food sample (10-1 dilution) Product
2. Wire loop, 3-4 mm, platinum-iridium or nichrome, gauge No. 24 or 26

3. Gram stain reagents
Hucker's crystal violet
Solution A
Crystal violet (90% dye content) 2 g
Ethanol, 95% 20 ml
Solution B
Ammonium oxalate 0.8 g
Distilled water 80 ml
Mix solutions A and B. Store 24 h and filter through coarse filter paper.
Gram's iodine
Iodine 1g
Potassium iodide (KI) 2g
Distilled water 300 ml
Hucker's counterstain (stock solution)
Safranine O (certified) 2.5
Ethanol, 95% 100 ml
Working solution: Add 10 ml stock solution to 90 ml distilled water.
1. Compound Microscope, with oil immersion objective lens (95-100X) and
10X ocular
2. Immersion oil
3. Methanol
4. Xylene
5.2.3 Procedure
1. Prepare film of blended food sample (10−1 dilution).
2. Air-dry films and fix with moderate heat by passing films rapidly over
Bunsen or Fisher burner flame 3 or 4 times. Alternatively, air-dry films and
fix with methanol 1-2 min, drain excess methanol and flame or air-dry (this
is particularly helpful for foods with a high sugar content).
3. Cool to room temperature before staining.
4. De-fat films of food with high fat content by immersing films in xylene 1-2
min; then drain, wash in methanol, drain, and dry.
5. Stain film by Gram-staining procedure.
31
Practical Manual  Procedure for gram staining
Food Microbiology
1. Fix air-dried films of food sample in moderate heat. Stain films 1 min with
crystal violet-ammonium oxalate solution.
2. Wash briefly in tap water and drain. Apply Gram's iodine for 1 min. Wash
in tap water and drain.
3. Decolorize with 95% ethanol until blue color is no longer released (about
30 s). Alternatively, flood slides with ethanol, pour off immediately, and
reflood with ethanol for 10 s.
4. Wash briefly with water, drain, and apply Hucker's counterstain (safranine
solution) for 10-30 sec. Wash briefly with water, drain, blot or air-dry, and
examine.
5. Examine under oil immersion and 10X ocular; adjust lighting systems to
Koehlor illumination.
6. Examine at least 10 fields of each film, noting predominant types of
organisms, especially clostridial forms, Gram-positive cocci, and Gram-
negative bacilli.
5.2.4 Observations
• Observe the bacteria under the microscope.
Gram positive bacterium Gram negative bacterium

• Record the Results in table given below.
Table 1: Gram characteristic, size, shape of two bacterial as determined
following Gram staining and observation using a compound
microscope
Bacterium Gram Reaction Cell Size Cell

(µm) Shape
Unknown 1
Unknown 2
5.2.5 Results
• Large numbers of Gram-positive cocci on the slide may indicate the
presence of staphylococcal enterotoxin, which is not destroyed by the heat
treatments that destroy enterotoxigenic Staphylococcus aureus strains.
32
• Large numbers of sporeforming, Gram-positive rods in a frozen food Visual and
specimen may indicate the presence of Clostridium perfringens, an Microscopic
Examination of Raw
organism that is sensitive to low temperatures. Other Gram-positive, and Processed
sporeforming rods such as Clostridium botulinum or Bacillus cereus may Product
also be present in the food.
• When the microscopic examination of suspect food discloses the presence
of many Gram-negative rods, consider the symptoms and incubation
periods reported for the illness under investigation and select the specific
examination method for isolating one or more of the following genera:
Salmonella, Shigella, Escherichia, Yersinia, Vibrio, or Campylobacter.
33
Food Microbiology
EXPERIMENT 6 ENUMERATION OF
BACTERIA BY DILUTION
AND PLATING
Structure
6.1 Introduction
Objectives
6.2 Experiment
Principle
Requirements
Procedure
Observations
Calculations
Results
6.1 INTRODUCTION
The ability of microorganisms to grow and reproduce in food products is well
known. Microorganisms may cause spoilage of the food product. Because of
their very small size, counting the number of bacteria in a food sample can be
difficult. Although direct counts are possible with a microscope, they require a
lot of time and expertise. An easier method is to spread bacteria over a wide
area (i.e. nutrient agar plate) and count the number of colonies that grow. If
the bacteria are spread out enough, each bacterial cell in the original sample
should produce a single colony. Usually, bacterial samples must be diluted
considerably to obtain reasonable counts.
Objectives
• enumerate bacteria in food samples; and
• isolate pure colonies of bacteria.
6.2 EXPERIMENT
6.2.1 Principle
Since bacterial cell numbers are usually very high in your original sample,
plating out this sample in an undiluted fashion would just lead to the creation
of a bacterial lawn (a smear of many, many individual bacteria colonies that
are all growing next to or on top of one another). Bacterial cell numbers need
to be reduced, which is done by repeatedly diluting the amount of bacteria you
have in your sample. A small amount of bacteria sample is mixed with a
diluent solution (such sterile water or nutrient broth), and then successive
dilutions are made. A small amount of each of the diluted bacteria samples is
then spread onto an agar plate. The numbers of bacteria colonies that grow on
each plate are counted. By working backwards using multiplication with the
“dilution factor” (the number of times that you have diluted the bacteria
sample with the diluent solution), you will be able to make a determination of
the numbers of bacteria in your original sample.
34
Enumeration of
Bacteria by Dilution
For example, 10,000,000 cell per ml diluted to 100 cells per ml. It is virtually and Plating
impossible to count 10,000,000 cells on the surface of the agar. However, it is
much easier if we dilute the sample and only have to count 300 cells. Diluting
is performed in increments because we must obtain a plate with between 30
and 300 colonies (for statistical purposes). Figure 6.1 shows how isolated
colonies should look like.
Figure 6.1: Bacterial colonies separated by spread plate method
6.2.2 Requirements
• Nutrient agar plates (6)
• Large sterile tubes (2)
• Tubes with 9 ml of sterile nutrient broth (11)
• Sterile transfer pipettes
• Sterile sticks (2)
• Micropipettor
• Bacteria spreader
• 70% alcohol
• Food samples ( say Sample A has been stored in a refrigerator for 4 days;
• Sample B has been stored frozen)
6.2.3 Procedure
DAY 1
1. Label 9 dilution broth tubes as follows:
A 10−2, A 10−3, A 10−4, A 10−5, A 10−6, A 10−7,
B 10−2, B 10−3, B 10−4
2. Label 6 agar plates as follows:
A 10−6, A 10−7, A 10−8
B 10−3, B 10−4, B 10−5
3. Label one large sterile tube A and the other large sterile tube B. Weigh
aseptically 1 gram of sample A, and place it in sterile tube A. Add the
contents of one tube of dilution broth to the food sample, and shake the
sample until the suspension appears fairly uniform. Repeat this with
sample B and sterile tube B.
4. Serial Dilutions
Precaution: You must use a new sterile pipette for each of the dilution
steps.
35
Food Microbiology
Use a sterile pipette to transfer 1 ml of the suspension from large tube A to
the culture tube labeled A 10−2. Mix the contents thoroughly by pipeting
up and down several times. Use a new pipette to transfer 1 ml from tube A
10−2 to tube A 10−3 and mix thoroughly as before. Continue this series of
dilutions into tubes A 10−4, A 10−5, A 10−6 and A 10−7.
Repeat this series of dilution using sample B. Transfer 1 ml of suspension
from large tube B into tube B 10−2 and mix thoroughly. Serially transfer,
as before, into tubes B 10−3 and B 10−4.
5. Plating bacteria
Use a micropipettor to withdraw 0.1 ml of liquid from tube A 10−5 and
place it onto the surface of the agar plate labeled A 10−6. (NOTE: Plating
0.1 ml of a 10−5 dilution will give you the same number of colonies as
plating 1 ml of a 10−6 dilution; the agar plate cannot absorb 1 ml of liquid,
so the smaller volume is used.)
Sterilize the bacterial spreader by dipping it into a beaker of alcohol.
Remove and shake off the excess. Carefully run the spreader through the
flame of a Bunsen burner and allow the alcohol to burn off. Cool the
spreader by holding it against the condensation on the inside of the petri
dish lid. Gently spread the liquid culture onto the surface of the agar by
moving the spreader in a circular manner while rotating the plate. This will
ensure an even distribution of bacteria.
6. Repeat step 5 with the remainder of the A cultures:
Spread 0.1 ml from culture tube A 10−6 onto plate A 10−7
Spread 0.1 ml from culture tube A 10−7 onto plate A 10−8
7. Repeat step 5 with the B cultures:
Spread 0.1 ml from culture tube B 10−2 onto plate B 10−3
8. Allow plates to absorb the cultures, then turn plates upside-down and
incubate overnight at 37° C.
Precautions
1. Before plating, be sure to label each plate with its dilution, date and
"food".
2. Mix the samples thoroughly before plating.
3. After pipetting the correct amount of sample in each plate, spread the
sample with a bactispreader evenly over the entire surface of the agar.
4. Remember to use aseptic technique. Invert plates. Incubate at 37°C.
6.2.4 Observations
On DAY 2, after incubating the plates, count the colonies on the plate. Each
colony represents one cell initially plated. For statistical purposes, pick a
plate with between 30 and 300 colonies.
36
Enumeration of
Bacteria by Dilution
and Plating
PLATE # COLONIES PLATE # COLONIES

A 10−6 B 10−3
A 10−7 B 10−4
A 10−8 B 10−5
Bacterial count in sample A Bacterial count in sample B (food):
(refrigerated food):
6.2.5 Calculations
Determine the number of cells/gram in the original sample of food by
multiplying the number of colonies on a plate by the dilution factor of that
plate.
Calculations dilutions X amount = "plated dilution"

made inoculated
dilution factor (simply X # colonies = # CFUs/ml (or gram)
the inverse of the plated of the original
dilution) undiluted sample
Example
If a plate labelled 10−7 has 87 colonies, then the sample has 87×107 = 8.7×108
colonies per gram.
6.2.6 Result
Find the average number of cells/g by adding the results from all of your
plates and dividing by the number of plates.
37
Food Microbiology
ANNEXURE-1
Composition of different media used for microbial study
1. Standard Plate Count Agar (SPCA)

Tryptone - 5.0g
Yeast extract - 2.5g
D-glucose - 5.0g
Agar - 15.0g
Distilled water - 1000ml
pH - 7.0
2. Nutrient Agar for Bacterial count
Beef extract - 3.0g

Peptone - 5.0g
Agar - 15.0g
Distilled water - 1000 ml
pH - 7.0
3. Potato Dextrose Agar for Fungal count
Potato, peeled and diced - 200g

D-glucose - 20g
Agar - 15.0g
Boil 200g of peeled and diced potato for 1 hr in a

litre of water. Filter and make up the volume to 1litre
and add rest of the constituents.
4. Violet Red Bile Agar (VRBA) for coliform count
Yeast Extract - 3.0g

Peptone - 7.0g
Sodium Chloride - 5.0g
Bile Salts - 1.5g
Lactose - 10.0g
Neutral red - 0.03g
Crystal violet - 0.002g
Agar - 13.0g
Distilled water - 1000ml
pH - 7.4
38

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Classification of

UNIT 1 CLASSIFICATION OF Microorganisms

1.2 VARIOUS TYPES OF MICROORGANISMS

d) Aerobes: require atmospheric oxygen for growth, e.g., Acetobacter aceti.

e) Facultative anaerobes: can grow with or without atmospheric oxygen.

f) Obligate anaerobes: do not grow in atmospheric oxygen.

Some useful bacteria

Yeast Product Spoilage

Group of microorganism Minimal aw value

Halophillic bacteria 0.75

Xerophillic fungi 0.65

Osmophillic Yeasts 0.60

− Halophillic bacteria: Bacteria which grow in high salt solutions

1. Classify bacteria according to their morphology.

1.3 CHARACTERISTICS (MORPHOLOGICAL,

1.4 LET US SUM UP

1.5 KEY WORDS

Yeasts : Unicellular fungi, larger than bacteria, which

Molds : Multicellular, filamentous fungi devoid of

Endospore : Heat resistant structures formed by bacteria under

Capsule : Slimy material composed of polysaccharides that

Mycostatic : Substances which are inhibitory to mold growth.

Fungicidal : Substances capable of killing the fungi.

# 1.6 ANSWERS TO CHECK YOUR PROGRESS

Check Your Progress Exercise 2

2.2 HYDROGEN-ION CONCENTRATION (pH)

2.2.1 Effect on Microbial Growth

Food pH range Organism pH range

pH minima and maxima of microorganisms also varies due to other important

2.2.2 Effect on Microbial Ecology and Food Spoilage

2.2.3 Inhibition of Microbes by Weak Acids

Neutral pH Plasma membrane

Low pH external environment

2.2.4 Buffers in Foods

2.3 MOISTURE REQUIREMENT/WATER ACTIVITY

2.3.1 Effect on Microbial Growth and Activity

xerophilic (dry-loving) molds and osmophilic (preferring high osmotic

Organisms Water Organisms Water

Molds differ considerably in optimal aw and range of aw for the germination of

2.3.2 Ways of Reducing Water Activity

Graph 2.1: Effect of sodium chloride concentration on water activity

2. Hydrophilic colloids (gels) make water unavailable. As little as 3 to 4

Graph 2.2: Effect of temperature on water activity

4. Oxygen supply: Growth of aerobes takes place at a lower aw in the

2.4 OXIDATION REDUCTION POTENTIAL

2.4.1 Redox Couples in Food

1. Redox couples present

2.4.3 Effect on Microbial Growth and Ecology

at an Eh of +100 to +500 mv, and other oxidative Gram-negative rods. These

In the presence of limited amounts of oxygen the same aerobic or facultative

2. Enlist the physiological groups of microorganisms based on their oxygen

4. How is the poising capacity of food destroyed?

Microorganisms use foods as a source of nutrients and energy. Each kind of

2.5.1 Foods for Energy

The carbohydrates, especially the sugars, are most commonly used as an

Bacteria differ in their ability to utilize different foods as a source of energy.

2.5.2 Foods for Growth

Microorganisms differ in their ability to use various nitrogenous compounds

2.5.3 Accessory Food Substances or Vitamins