2. Detailed Content
1. Introduction- Definition, historical development in food microbiology
and its significance
2. Microbiology of milk and milk products like cheese, butter, ice-
cream, milk powder
3. Microbiology of meat, fish, poultry and egg products
4. Microbiology of fruits and vegetable products like jam, jelly, sauce,
5. Microbiology of cereal and cereal products like bread
6. Microbial spoilage of foods – food borne pathogens, food poisoning,
food infection and intoxication
7. Concept, determination and importance of TDT, F, Z and D value;
factors affecting the heat resistance of microorganisms
8. Anti-microbial agents–physical and chemical agents-Their mechanism
3. Chapter 1
Introduction- Definition, historical development in food microbiology
and its significance
1.1) Introduction: The objective of the food microbiology is to develop ability to:
• describe the nature of micro-organisms, their classification, identification, growth
• describe the importance of micro-organisms for food processing, fermentations,
food spoilage and food poisoning;
• describe and evaluate both qualitatively and quantitatively the action of anti-
microbial agents, chemical and physical;
• apply basic microbiological principles to the production, handling, processing and
marketing of foods;
• perform basic laboratory techniques for studying micro-organisms;
• describe routine microbiological analyses for milk and other raw materials and for
finished food products;
• select and evaluate analytical techniques for specific purposes and materials;
• identify appropriate techniques for use in quality control procedures;
• perform practical analyses of raw materials, food products, environmental
samples and packaging materials; and
• understand the principles of test design.
The content includes microbiological diversity; fungi: yeasts and moulds; bacterial
structure; bacterial growth and nutrient requirements; bacterial classification and
identification; viruses: structure, replication and identification; methods for cultivation
and identification of micro-organisms; control of micro-organisms; organisms of
importance to dairy/food industry; microbiological quality of raw milk; rationale for
microbiological testing; principles of test design; sample preparation; and
microbiological testing of milk and dairy products and food.
4. 1.2) Definition: Food microbiology is the study of the microorganisms which inhabit,
create or contaminate food. Of major importance is the study of microorganisms causing
However "good" bacteria such as probiotics are becoming increasingly important in
food science. In addition, microorganisms are essential for the production of foods such
as cheese, yoghurt, other fermented foods, bread, beer and wine.
1.3) Historical development in food microbiology:
It is not known exactly when our ancestors recognized the invisible creatures, now
designated as microorganisms, in food.
From the time of Greeks until the discovery of biogenesis, spoilage of food,
especially of meat and fish, was thought to be due to spontaneous generation, such as the
development of maggots. When the presence of different types of bacteria in many food
was discovered, their appearance through spontaneous generation was explained to be the
cause of food spoilage. Schawnn (1837) and Helmholtz (1843) associated the presence of
microorganisms (bacteria) in food with both putrifactive and fermentative changes of
foods. However, they did not believe in spontaneous generation, but they could not
explain how microorganisms could bring about those changes. Finally, Pasteur resolved
the mystery by explaining that contamination of food with microorganisms from the
environment and their subsequent metabolic activities and growth were the causes of
fermentation of grapes, souring of milk, and putrefaction of meat.
Pasteur, in the 1860s, recognized yhe role of yeasts in alcohal fermentation. He also
showed that souring of wine was due to growth of acetic acid-producing bacteria
(Acetobacter aceti), and developed the pasteurization process to selectively eliminate
these undesirable bacteria from wine. Like fermentation, cheese ripening was suggested
by Martin in 1867 to be of microbial origin. John Lister, in 1873, was able to isolate
milk-souring bacteria (Lactococcus lactis) by serial dilution procedure. Cienkowskii, in
1878, isolated the bacteria (Leuconostoc mesenteroides) associated with slime formation
in sugar. In 1895, microbial enumeration of milk was developed by Von Geuns.
5. 1.4) Significance: An individual who has completed courses in food technology (both
lecture and laboratory) should gain knowledge in the following areas:
• determination of microbial quality of foods and food ingredients using appropriate
• determination of microbial type(s)involved in spoilage, health hazards and the
identification of the sources;
• design corrective procedures to control the spoilage and pathogenic
microorganisms in food;
• identify how new technologies in food processing can have specific
microbiological problems and design methods to overcome the problem;
• design effective sanitation procedure to control spoilage and pathogen problems
in food processing facilities;
• effective use of desirable micsoorganisms to produce fermented food;
• design method to produce better starter cultures for use in fermented foods and
• food regulation(state, federal,international)
To be effective, in addition to the knowledge gained, one has to able to communicate
with different groups of people about the subject (food microbiology and its relation to
food science). An individual with good common sence is always in a better position to
sense a problem and correct it quickly.
1.5) Importance of microorganisms in foods:
The importance of microorganisms in food has increased greatly. Their role in food can
be either desirable or undesirable.
1.5. A) Undesirable role of microorganisms in foods:
i. Food borne diseases: Many pathogenic microorganisms (bacteria, molds and viruses)
can contaminate food during various stages of their handling between production and
consumption. Consumption of these foods can cause food borne diseases. Food borne
diseases not only can be fatal, but they can cause large economic losses. Foods of animal
origin are associated more with foodborne diseases than foods of plant origin.
6. Mass production of foods, introduction of new technologies in the processing and storage
of foods, changes in food consumption pattern and the increase in imparts of food from
other countries have increased the chances of large outbreaks as well as the introduction
of new pathogens.
ii. Food spoilage: Except for sterile foods, all foods harbor microorganisms. Food
spoilage stems from the growth of these microorganisms in food or is due to the action of
microbial heat-stable enzymes. New marketing trends, the consumer desire for foods that
are overly processed and preserved, extented shelf life and changed of temperature abuse
between production and consumption of foods have greatly increased the chances of food
spoilage and in some instances, with new type of microorganisms.
1.5. B) Desirable role of microorganisms in foods:
i. Food bioprocessing: Many food grade microorganisms are used to produce different
types of fermented foods using raw material from animal and plant sources. Consumption
of these foods has increased greatly over the last 10 to 15 years and is expected to
increase still more in future. There have been great changes in the production and
availability of these microorganisms to meet the large demand.
ii. Food biopreservation: Antimicrobial metabolites of desirable microorganisms are
being used in food in place of nonfood preservatives to control pathogenic and spoilage
microorganisms in foods.
iii. Probiotics: Consumption of foods containing the live cells of bacteria that have
apparent health benefits has generated interest among consumers. The efficiency of these
bacteria to produce these benefits is being investigated.
7. Chapter 2
Microbiology of milk and milk products like cheese, butter, ice-cream,
2.1) Microbiology of Milk
Milk drawn from a healthy milk animal already contains some bacteria. Most of the
changes which take place in the flavor and appearance of milk, after it is drawn from
udder are the results of the activities of microbes. These microbes are of two types i.e.
favourable–which bring favourable changes in flavour & appearance while pathogenic-
which may cause diseases. The favourable are carefully propagated while pathogenic
(unfavourable) are destroyed to make the milk & its products safe for human
2.1.1) Following are the important microbes found in milk:
A).Bacteria: Bacteria are microscopic, unicellular, occurs in the form of spherical,
cylindrical or spiral cells. Size of bacteria is the range of 1-5µ. Spore forming bacteria
produce trouble in dairy industry because of their resistance to pasteurization &
B).Moulds: Multi-cellular, at maturity are as Mycelium. Useful in cheese making which
is responsible for defects in butter and other milk products. Most spores of moulds are
destroyed by pasteurization.
C).Yeast: Unicellular, larger than bacteria. Destroyed during pasteurization.
D).Viruses: viruses are ultra-microscopic forms of like can be destroyed by
pasteurization or higher heat treatment.
2.1.2) Growth of micro-organisms
Bacteria multiply during production and holding of milk, depending on storage time and
conditions. The changes take place in the physico-chemical properties of milk are result
of the activities of the individual microbial cells during their period of growth and
reproduction or of substances produced during such activity.
8. 2.1.2. A) Stages of growth:
i. Initial stationary phase
ii. Lag phase (Phase of adjustment)
iii. Accelerated growth phase (log phase)
iv. Maximum stationary phase
v. Phase of accelerated death.
2.1.2. B) Factors Influencing Growth:
i. Food supply – Milk and its products are good food source, provides all food
ii. Moisture – Milk contains adequate moisture to development.
iii. Air – Supplies O2 to aerobic bacteria and moulds.
iv. Acidity or pH – Preferably range 5.6 to 7.5.
v. Preservatives – Check growth depending upon concentration.
vi. Light – More or less harmful.
vii. Concentration – High sucrose or salt content check growth.
viii. Temperature – Important means for controlling growth.
According to their optimum growth temperature, bacteria can be classified into:
Psychotropic – can grow at refrigeration temperature 5-70
Mesophilic – can grow at temperature 20-400
Thermophilic – can grow at temperature above 500
2.1.2 C) Products of Microbial Growth:
i. Decomposition products (fats, proteins, sugars).
2.1.2 D) Results of Microbial Growth in Milk:
i. Souring: Most common, due to transformation of lactose into lactic acid & other
volatile acids & compounds, principally by lactic acid bacteria.
ii. Souring & gassiness: Caused by coil group, indicates contamination of milk and its
9. iii. Aroma production: Due to production of desirable flavour compounds.
iv. Proteolysis: Protein decomposition leading to unpleasant odour.
v. Ropiness: Long threads of milk are formed while pouring.
vi. Sweet curdling: Due to production of a renin like enzyme curdles milk without
2.1.2 E) Destruction of Micro-organisms:
May be done by following means:
i. Heat – Most widely Pasteurization & sterilization used.
ii. Ionizing radiation – Such as ultraviolet rays etc.
iii. High frequency sound waves – Supersonic and ultrasonic.
iv. Electricity – Microbes are destroyed actually by heat generated.
v. Pressure – Should be about 600 times greater than atmospheric pressure.
vi. Chemicals – Includes acids, alkalis, hydrogen peroxide, halogens etc.
2.1.3) Action of Microbes on Milk:
Microbes Action Result
Souring Lactose-lactic acid
4. E coil Souring &
Lactic acid & gases
Affects cheese ripening.
Proteolysis off flavors.
Ropiness Ropi milk
Bitter flavour to cream
8. B. substallis Sweet
9. St. Attacks Flavors curd.
10. paracitrovorus citric acid
2.2) Microbiology of Butter
Butter is made as a means of extracting and preserving milk fat. It can be made directly
from milk or by separation of milk and subsequent churning of the cream. Butter contains
around 15% water, 81%fat and generally less than 0.5% carbohydrate and protein.
2.2.1) Sources of contamination
In addition to bacteria present in the milk other sources of bacteria in butter are (1)
equipment, (2) wash water, (3) air contamination, (4) packing materials, and (5)
2.2.1. A) Equipment
In smallholder butter-making, bacterial contamination can come from unclean surfaces,
the butter maker and wash water. Packaging materials, cups and leaves are also sources
of contaminants. Washing and smoking the churn reduces bacterial numbers. But
traditional equipment is often porous and is therefore a reservoir for many organisms.
When butter is made on a larger processing scale, bacterial contamination can come from
holding-tank surfaces, the churn and butter-handling equipment.
A wooden churn can be a source of serious bacterial, yeast and mould contamination
since these organisms can penetrate the wood, where they can be destroyed only by
extreme heat. However, if care is taken in cleaning a wooden churn this source of
contamination can be controlled.
2.2.1. B) Wash water
Wash water can be a source of contamination with both coliform bacteria and bacteria
associated with defects in butter. Polluted water supplies can also be a source of
2.2.1. C) Air
Contamination from the air can introduce spoilage organisms: mould spores, bacteria and
yeasts can fall on the butter if it is left exposed to the air. Moulds grow rapidly on butter
exposed to air.
11. 2.2.1. D) Packaging
Care is required in the storage and preparation of packaging material. Careless handling
of packaging material can be a source of mould contamination.
2.2.1. E) Personnel
Personnel pass organisms to butter via the hands, mouth, nasal passage and clothing.
Suitable arrangements for disinfecting hands should be provided, and clean working
garments should not have contact with other clothes.
2.2.2) Types of spoilage in butter:
i. Surface taint or Putridity: This condition is caused by “Pseudomonas putrefaciens”
as a result of its growth on the surface of finished butter. It develops at temperature with
in the range 4 to 7o
C and may become apparent within 7 to 10 days.
ii. Rancidity: This condition is caused by the hydrolysis of butterfat with the liberation
of free fatty acids. The causative organism is “Pseudomonas fragi”, although
“Pseudomonas fluorescens” is sometimes found.
iii. Malty flavour: This flavour reported to be due to the growth of “Lactococcus lactis”.
iv. Skunklike: odor is reported to be caused by “Pseudomonas mephitica”.
v. Discoloration: Black discolorations of butter have been reported to be caused by
vi. Fungal spoilage: Butter undergoes fungal spoilage commonly by species of
Cladosporium, Alternaria, Aspergillus, Mucor, Rhizopus, Penicillium and Geotrichum.
These organisms can be seen growing on the surface, where they produce coloration
referable to their particular spore colors.
2.2.3) Control of micro-organisms in butter
Salting effectively controls bacterial growth in butter. The salt must be evenly dispersed
and worked in well. Salt concentration of 2% adequately dispersed in butter of 16%
moisture will result in a 12.5% salt solution throughout the water-in-oil emulsion.
Washing butter does little to reduce microbiological counts. It may be desirable not to
wash butter, since washing reduces yield. The acid pH of serum in butter made from
ripened cream or sour milk may control the growth of acid-sensitive organisms.
12. Microbiological analysis of butter usually includes some of the following tests: total
bacterial count, yeasts and moulds, coliform estimation and estimation of lipolytic
Yeast, mould and coliform estimations are useful for evaluating sanitary practices. The
presence of defect producing types can be indicated by estimating the presence of
All butter contains some micro-organisms. However, proper control at every stage of the
process can minimize the harmful effects of these organisms.
2.3) Microbiology of Cheese:
A) Cottage cheese: This is a type of cheese and undergoes spoilage by bacteria, yeast
and molds. The most common spoilage pattern displayed by bacteria is a condition
known as “slimy curd”. Alcaligenes spp. have been reported to be among the most
frequent causative organisms, although Pseudomonas, Proteus, Enterobactor, and
Acinetobactor spp have been implicated. Penicillium, Mucor, Alternaria, and
Geotrichum all grows well on cottage cheese, to which they impart stale, musty, moldy
and yeasty flavors.
B). Ripened cheese: This is also type of cheese and has low moisture content make them
insusceptible to spoilage by most organisms, although molds can and do grow on these
products. Some ripened cheese has sufficiently low oxidation reduction potential to
support the growth of anaerobes. Anaerobic bacteria sometimes cause the spoilage of
cheese when water activity permits growth to occur. Clostridium species especially C.
pasteurianum, C. butyricum and C. sporogenes have been reported to cause gassiness of
One aerobic spore former, Bacillus polymyxa, has been reported to cause gassiness.
All the organisms utilize lactic acid with the production of CO2, which is responsible for
gassy condition in these milk products.
2.4) Microbiology of Ice-cream:
In frozen desserts include ice-cream, the ingredients may be
i. Various combination of milk,
13. ii. Cream,
iii. Evaporated milk,
iv. Condensed milk,
v. Dried milk,
vi. Coloring material,
x. Sweetening agents,
xi. Egg and egg products and
Any of these may contribute microorganisms to the product and affect the quality of
dessert as judged by its bacterial content or its content of specific kind of bacteria such as
coliforms. The desserts are not ordinarily subject to spoilage, however, as long as they
kept frozen. The only important type of spoilage takes place in the ingredients before
they are mixed or in the mix before it is frozen. So, mix is pasteurized before frozen.
14. Chapter 3
Microbiology of meat, fish, poultry and egg products
3.1) Microbiology of meat:
Meat is an ideal culture medium for many organisms because it is high in moisture, rich
in nitrogeous foods of various degrees of complexity, and plentifully supplied with
minerals and accessory growth factors. Also, it is usually has some fermentable
carbohydrate (glycogen) and is at a favorable pH for most microorganisms.
The factors that influence the growth of microorganisms are as follows:
a. the kind and amount of contaminmation with microorganisms and the spread of these
organisms in the meat;
b. physical properties of the meat;
c. chemical properties of the meat;
d. availability of oxygen;
3.1.1) General types of spoilage of meat: The common type of spoilage of meats can be
classified on the basis of whether they occur under aerobic or anaerobic conditions and
whether they are caused by bacteria, yeast and molds.
3.1.1.A) Spoilage under aerobic conditions by bacteria: Under aerobic conditions
bacteria may cause the following:
i. Surface slime: which may be caused by species of pseudomonas, Acinetobacter,
Moraxella, Alcaligenes, Streptococcus, Leuconostoc, Bacillus and Micrococcus. Some
species of Lactobacillus can produce slime. The temperature and the availability of
moisture influence the kind of microorganisms causing surface slime.
15. ii. Changes in color of meat pigments: The red color of meat, called its “bloom”, may
be changed to shades of green, brown or gray as a result of the production of oxidizing
compounds, e.g., peroxides, or of hydrogen sulfide, by bacteria.
iii. Changes in fats: The oxidation of unsaturated fats in meats takes place chemically in
air and may be catalysed by light and copper. Lipolytic bacteria may cause some lipolysis
and also may accelerate the oxidation of the fats.
iv. Phosphorescence: This rather uncommon defect is caused by phosphorecent or
luminous bacteria. e.g., photobacterium spp., growing on the surface of meat.
v. Varoius surfaces colors due to pigmented bacteria: Thus “red spot” may be caused
by Serratia marcescens or other bacteria with red pigments. Pseudomonas syncyanea can
impart a blue color to the surface. Yellow discoloration may caused by bacteria with
yellow pigments, usually species of Micrococcus. Chromobacterium lividum and other
bacteria give greenish-blue to brownish-black spots on stored beef.
vi. Off-odrs and off-tastes: Undesirable odors or tastes, that appear in meat as a result of
the growth of bacteria on the surface are give other signs of spoilage.
3.1.1.B) Spoilage under aerobic conditions by Yeast: Under aerobic conditions yeasts
may grow on the surface of meats, causing:
iii. Off odors and tastes
iv. Discoloration- white, cream, pink or brown-due to pigments in the yeasts.
3.1.1. C) Spoilage under aerobic conditions by Molds: Aerobic growth of molds may
cause the following:
i. Stickiness: Incipent growth of molds makes the surface of the meat sticky to the touch.
ii. Whiskers: When meat is stored at temperature near freezing, a limited amount of
mycelial growth may take place without sporulation. Such white fuzzy growth may be
caused by a number of moulds, inclubing Thamnidium chaetocladiodes, Mucor mucsdo
iii. Black spot: This usually is caused by Cladosporium herbarum, but other molds with
dark pigments may be responsible.
16. iv. White spot: Sporotrichum carnis is the most common cause of white spot, although
any mold with wet, yeastlike colonies, e.g.,Geotrichum can cause white spot.
v. Green patches: These are caused for the most part by the green spores of species of
Penicillium such as P. expansum, P. asperulum.
vi. Decomposition of fats: Many molds have lipases and hence cause hydrolysis of fats.
Molds also aid in the oxidation of fats.
3.1.2) Spoilage under anaerobic conditions: Facultative and anaerobic bacteria are able
to grow within the meat under anaerobic condition and cause spoilage.
i. Souring: The term implies a sour odor and perhaps taste. This could be caused by
formic, acetic, butyric, propionic and higher fatty acids or other organic acids such as
lactic or succinic. Souring can result from (a) action of the meat’s own enzymes during
aging or ripening, (b) anaerobic production of fatty acids or lactic acid by bacterial
action, or (c) proteolysis without putrifaction, caused by facultative or anaerobic bacteria.
Acid and gas formation accompany the action of “butyric” Clostridium species and
coliform bacteria on carbohydrates.
ii. Putrefaction: The putrefaction is the anaerobic decomposition of the protein with the
production of foul-smelling compounds such as hydrogen sulfide, ammonia and amines.
It usually is caused by Clostridium, but facultative bacteria may cause putrifaction or
assist in its production.
iii. Taint: This word is also applied to any off-odor or off-taste.
Not only air but temperature has an important influence on the type of spoilage to
be expected in meat. When meat held at temperatures near 0o
C, as recommended,
microbial growth is limited to that of molds, yeast and bacteria able to grow at low
temperatures. These includes many of the types that produce sliminess, discoloration, and
spots of the growth on the surface and many that can cause souring, such as Psedomonas,
Acinetobactor Moraxella, Alcaligenes, Lactobacillus, Leuconostoc, Streptococcus, and
3.2) Microbiology of fish:
Like meat, fish may be spoiled by autolysis, oxidation or bacterial activity or most
commonly by combination of these. Most fish flesh is considered more perishable than
17. meat because of more rapid autolysis by the fish enzymes and because of less acid
reaction of the fish flesh that favors microbial growth. Also many of unsaturated fish oils
seem to be more susceptible to oxidative deterioration than are most animal fats.
All species of fish, when properly chilled, will stay fresh for longer periods than those
that are not preserved in any way. The micro flora of living fish depends on the microbial
contents of the water in which the fish live. Fish micro flora includes bacteria such as
species of Pseudomonas, Alcaligenes, Vibrio, Serratia, Micrococcus etc. The level of
contamination can increase with the type of processing and subsequent handling.
Fish is also susceptible to autolysis, oxidation and lipolysis in addition to microbial
spoilage. Its preservation, therefore, should involve a combination of treatments.
3.2.1) Factors influence kind and rate of spoilage: The kind and rate of fish spoilage
varies with a number of factors, such as
i).The kind of fish: The various kinds of fish differ considerably in their perishability.
Thus some flat fish spoil more readily than round fish because they pass through rigor
mortis more rapidly, but the flat fish keeps longer because of low pH (5.5) of its flesh.
Certain fatty fish deteriorate rapidly because of oxidation of the unsaturated fats of their
ii).The condition of the fish when caught: Fish that are exhausted as the result of
struggling, lack of oxygen, and excessive handling spoil more rapidly than those brought
in with less ado, probably because of the exhaustion of glycogen and hence smaller drop
in pH of the flesh.
iii).The kind and extent of contamination of the fish flesh with bacteria: These may
come from mud, water, handlers and the exterior slime and intestinal content of the fish
and are supposed to enter the gills of the fish, from which they pass through the vascular
system and thus invade the flesh, or to penetrate the intestinal tract and thus enter the
body cavity. In general, the greater the load of bacteria on fish, the more rapid the
iv).The storage temperature: Chilling the fish is the most commonly used method for
preventing or delaying bacterial growth and hence spoilage until the fish is used or is
otherwise processed. The cooling should rapid as possible 0 to -1C, and this low
18. temperature should be maintained. Obviously, the warmer the temperature, the shorter the
storage life of the fish.
v).Use of an antibiotic ice or dip
3.2.2) Bacteria causing spoilage: The bacteria most often involved in the spoilage of
fish are part of the natural flora of the external slime of fishes and their intestinal
a) The predominant kinds of bacteria causing spoilage vary with the temperatures at
which the fish are held, but at the chilling temperatures usually employed, Pseudomonas
are most likely to predominate with Acinetobacter, Moraxella, and Flavobacterium
b) At high temperature, bacteria of the genera Micrococcus and Bacillus.
c) Other genera as having species involved in fish spoilage, such as Escherichia, Proteus,
Serratia, Sarcina and Clostridium. Most of these would grow at ordinary atmospheric
temperatures and probably would do little at chilling temperatures.
d) Normally Pseudomonads increase in numbers on chilled fish during holding.
The bacteria grow first on the surfaces and later penetrate the flesh. Fish have
a high content of nonprotein nitrogen and autolytic caused by their own enzymes increase
the supply of nitrogenous foods (e.g. amino acids and amines) and glucose for bacterial
e) A musty or muddy odor and taste of fish has been attributed to the growth of
Streptomyces species in the mud at the bottom of the body of water and absorption of the
flavor by the fish.
f) Discolorations of fish flash may occur during spoilage like yellow to greenish-yellow
colors caused by Pseudomonas fluorescens, red or pink colors from growth of Sarcina,
Micrococcus, Bacillus species or by molds or yeasts and a chocolate-brown color by an
Pathogens parasitizing the fish may produce discoloration or lesions.
19. 3.3) Microbiology of poultry:
a) The enzymes of the fowl contribute to deterioration of the dressed bird; bacteria are the
chief cause of spoilage, with the intestines a primary source of these organisms.
b) The work done on the bacterial spoilage of poultry has indicated that most bacterial
growth takes place on the surfaces, i.e., the skin, the lining of the body cavity, and any
cut surfaces, and the decomposition products diffuse slowly in to the meat.
c) A surface odor has been noted when the bacterial count on the skin was about 2.5
million per square centimeter. This took about 4 weeks at 0o
C and 5 weeks at 1.1o
one set of experiments.
d) Eviscerated poultry held at 10o
C or below is spoiled mostly by Pseudomonas and to a
lesser degree by yeasts, e.g., Torulopsis and Rhodotorula.
e) About 10o
C, micrococci usually predominated, and their also is growth of alcaligenes
and flavobacterium. In time the surface of meat usually becomes slimy. Small amounts
(1to5ppm) of iron in the wash water may favor bacterial growth on the surface and
production of the fluorescent pigment pyoverdine by pseudomonades; more iron will
reduce pigmentation. About 100 ppm of magnesium is optimal for pigment production by
f) Iced, cut-up poultry often develops a slime that is accompanied by an odor described as
tainted, acid, sour or dishraggy. This defect is caused chiefly by species of Pseudomonas,
although. Alcaligenes also may be concerned.
g) Similar bacteria grow weather the temperature is as low as 0o
C or as high as 10o
enormous numbers must be present, about 108
per square centimeter, before the odor
20. h) It should be kept in mind that chemical changes in poultry meat other than those
caused by microorganisms occur during refrigerated storage and will in time reduce the
3.4) Microbiology of egg:
To cause spoilage of an undamaged shell egg, the causal organisms must do the
a) Contaminate the shell,
b) Penetrate the pores of the shell to the shell membranes (usually the shell must be moist
for this to occur),
c) Grow through the shell membrane to reach the white (or to reach the yolk if the
touches the membrane),
d) Grow in the egg white, despite the previously mentioned unfavorable conditions there,
to reach the yolk, where they can grow readily and complete spoilage of the egg. Bacteria
unable to grow in the white can reach the yolk and flourish there only when the yolk
touches the inner cell membrane.
The time required for bacteria to penetrate the shell membranes varies with the
organisms and the temperature but may take as long as several weeks at refrigerator
temperatures. The special set of environmental conditions, the selective egg-white
medium, and the low storage temperature of about 0o
C combine to limit the number of
kinds of bacteria and molds that can cause spoilage chiefly to those to be mentioned.
In general, more spoilage of eggs is caused by bacteria than by molds. The
Types of bacterial spoilage, or “rots,” of eggs go by different names.
i) Among the three chief ones are the green rots, caused chiefly by Pseudomonas
fluorescens, a bacterium that grows at 0o
C; the rot is so named because of the bright-
green color of the white during early stages of development. This stage is noted with
difficulty in candling but shows up clearly when the egg is broken. Later the yolk may
21. disintegrate and blend with the white so as to mask the green color. Odor is lacking or is
fruity or “sweetish,” The contents of eggs so rotted fluoresce strongly under ultraviolet
ii) A second important group of rots are the colorless rots, which may be caused by
Pseudomonas, Acinetobacter, Alcaligenes, certain coliform bacteria, or other type of
bacteria. These rots are detected readily by candling, for the yolk usually is involved,
except in very early stages, and disintegrates or at least shows a white incrustation. The
odor varies from a scarcely detectable one to fruity to “highly offensive.”
iii) The third important group of rots are the black rots, where the eggs are almost opaque
to the candling lamp because the yolks become blackened and then break down to give
the whole-egg contents a muddy-brown color. The odor is putrid, with hydrogen sulfide
evident, and gas pressure may develop in the egg. Species of Proteus most commonly
cause these rots, although some species of Pseudomonas and Aeromomnas can cause
black rots. Proteus melanovogenes* causes an especially black coloration in the yolk and
a dark color in the white. The development of black rot and of red rot usually means that
used for storage.
iv) Pink rots occur less often, and red rots are still more infrequent. Pink rots are caused
by strains of Pseudomonas and may at times be a late stage of some of the green rots.
They resemble the colorless rots, except for a pinkish precipitate on the yolk and a pink
color in the white. Red rots, caused by species of Serratia, are mild in odor and are not
v) Different rots by ten different species of the genera Pseudomonas, Alcaligenes,
Proteus, Flavobacterium, and Paracolobactrum. These rots have been characterized as
fluorescent, green and yellow, custard, black, red, rustly red, colorless, and mixed. The
list of secondary invaders in the genera Enterobacter, Alcaligemnes, Escherishia,
Flavobacterium, and Paracolobactum. These bacteria can grow in the egg but cannot
Spoilage by fungi: The spoilage of eggs by fungi goes through stages of mold growth
that give the defects their names. Very early mold growth is termed pin-spot molding
because of the small, compact colonies of molds appearing on the shell and usually just
inside it. The color of these pin spots varies with the kind of mold; Penicillium species
22. cause yellow or blue or green spots inside the shell, Cladosporium species give dark-
green or black spots, and species of Sporotrichum produce pink spots.
In storage atmospheres of high humidity a variety of molds may cause superficial
fungal spoilage, first in the form of fuzz or “whiskers” covering the shell and later as
more luxuriant growth. When the eggs are stored at near-freexzing temperatures as they
usually are, the temperatures are high enough for slow mycelial growth of some molds
but too low for sporulation, while other molds may produce asexual spores.
Microbiology of fruits and vegetable products
Like jam, jelly, sauce, juice
4.1) Introduction: The deterioration of raw vegetables and fruits result from physical
factors, action of their own enzymes, microbial action, or combination of these agencies.
Mechanical damage resulting from action of animal, birds, or insects or from bruising,
wounding, bursting, cutting, freezing, desiccation, or other mishandling may predispose
towards increased enzymatic action or the entrance and growth of microorganisms.
Previous damage by plant pathogens may make the part of the plant used as food unfit for
consumption or may open the way for growth of saprophytes and spoilage by them.
Contact with spoiling fruits and vegetables may bring about transfer of organisms causing
spoilage and increasing the wastage. Improper environment condition during harvesting,
transit, storage, and marketing may favor spoilage. Most of the discussion to follow will
be concerned with microbial spoilage, but it always should be kept in mind that the plant
enzymes continue their activity in raw plant foods. If oxygen is available, the plant cell
will respire as long as they are alive, and hydrolytic enzymes can continue their action
after death of the cells.
4.2). Microbiology of fruits and vegetable:
The most common and predominant type of spoilage varies not only with the kind of fruit
and vegetable but also to some extent with the variety. Microbial spoilage may be due to
(1) plant pathogens acting on the stems, leaves, flowers, or roots of the plant, on the fruits
23. or other special parts used as foods, e.g., roots or tubers, or on several of these locations,
(2) saprophytic organisms, which may be secondary invaders after action of a plant
pathogen or may enter a healthy fruit and vegetable, as in case of various “rots”, or grow
in its surface, as when bacteria multiply on moist, piled vegetables.
At times a saprophyte may succeed a pathogen or a succession of saprophytes may be
involved in the spoilage. Thus, for example, coliform bacteria may grow as secondary
invaders and be present in appreciable numbers in fruit and vegetable juices if rotten
products have been included.
Although each fruit and vegetable has certain types of decomposition and kinds of
microorganisms predominant in its spoilage, some general types of microbial spoilage are
found more often than the rest in vegetables and fruits. The most commonly occurring
types of spoilage are as follows:
1. Bacterial soft rot, caused by Erwinia carotovora and related species, which are
fermenters of pectins. Pseudomonas marginalis and clostridium and Bacillus spp.
have also been also isolated from these rots. It results in a water-soaked
appearance, a soft, mushy consistency and often a bad odor.
2. Gray mold rot, caused by species of Botrytis, e.g., B. cinerea, a named derived
from the gray mycelium of the mold. It is favored by high humidity and a warm
3. Rhizopus soft rot, caused by species of Rhizopus, e.g., R. stolonifer. A rot results
that often is soft and mushy. The cottony growth of the mold with small, black
dots of sporangia often covers masses of the foods.
4. Anthracnose, usually caused by Colletotrichum lindemuthianum, C. coccodes,
and other species. The defect is a spotting of leaves and fruit or seedpods.
5. Alternaria rot, caused by Alternaria tenuis and other species. Areas become
greenish-brown early in the growth of the mold and later turn to brown or black
6. Blue mold rot, caused by species of Penicillium digitatum and other species. The
bluish-green color that gives the rot its name results from the masses of spores of
24. 7. Downy mildew, caused by species of Phytophthora, Bremia and other genera.
The molds grow in white, woolly masses.
8. Watery soft rot, caused chiefly by Sclerotinia sclerotiorum, is found mostly in
9. Stem-end rot, caused by species of molds of several genera, e.g., Diplodia,
Alternaria, Phomopsis, Fusarium, and others, involve the stem ends of fruits.
10. Black mold rot, caused by Aspergillus niger. The rot gets its name from the
dark-brown to black masses of spores of the mold, termed “smut” by the
11. Black rot, often caused by species of Alternaria but sometimes of
Ceratostomella, Physalospora, and other genera.
12. Pink mold rot, caused by pink-spored Trichothecium roseum.
13. Fusarium rots, a variety of types of rots caused by species of Fusarium.
14. Green mold rot, caused usually by species of Cladosporium but sometimes by
other green-spored molds, e.g., Trichoderma.
15. Brown rot, caused chiefly by Sclerotinia species.
16. Sliminess or souring, caused by saprophytic bacteria in piled, wet, heating
Fungal spoilage of vegetables often results in water-soaked, mushy areas, while fungal
rots of fleshy fruits such as apples and peaches frequently show brown or cream-colored
areas in which mold mycelia are growing in the tissue below the skin and aerial hyphae
and spores may appear later. Some types of fungal spoilage appear as “dry rots”, where
the infected area is dry and hard and often discolored. Rots of juicy fruits may result in
The character of the spoilage will depend on the product attacked and the attacking
organism. When the food is soft and juicy, the rot is apt to be soft and mushy and some
leakage may result. There are, however, some kinds of spoilage organisms that have a
drying effect so that dry or leathery rots or discolored surface areas may result. In some
instances most of the mycelial growth of the mold is subsurface and only a rotten spots
25. shows, as in most rotting of apples. In other types of spoilage the growth of the mold
mycelium on the outside is apparent and may be colored by spores.
4.3) Microbiology of fruits and vegetable products like jam, jelly, sauce:
A). Spoilage by yeast: Since yeasts and their spores are killed by most pasteurization
treatments, their presence in canned food is the result of either gross underprocessing or
leakage. Canned fruits, jams, jellies, fruit juices, syrups and sweetened condensed milk
have been spoiled by fermentative yeasts, with swelling of the cans by the CO2 produced.
Film yeasts may grow on the surface of jellied products, but their presence indicates
recontamination or lack of heat processing, plus poor evacuation.
B). Spoilage by molds: Molds probably are the most common cause of the spoilage of
home-canned foods, which they enter through a leak in the seal of the container, jam,
jellies, marmalades, and fruit butters permit mold growth when sugar concentration are as
high as 70 percent and in the acidity usually present in these products. It has been
claimed that adjustment of the soluble extract of jam to 70 to 72 percent sugar in the
presence of a normal 0.8 to 1.0 percent acid will practically remove the risk of mold
spoilage. Strains of Aspergillus, Penicillium, and Citremyces, found growing in jellies,
and candied fruits are able to grow in sugar concentration up to 67.5 percent.
Acidification to pH 3 prevented growth of the first two molds, and heating the foods at
C for 1 min killed all strains. Some molds are fairly resistance to heat, especially those
forming the tightly packed masses of mycelium called sclerotia. The high sugar content
of syrups about fruits helps protect microorganisms against heat, and this protective
effect is increased if the sugar and fruit are added to the can separately, localizing the
sugar during processing.
4.4). Microbiology of fruits and vegetable juices:
Juices squeezed or extracted from fruits are more or less acid, depending on the product,
the pH ranging from about 2.4 for lemon juice, up to 4.2 for tomato juice, and all contain
sugars, the amount varying from about 2 percent in lemon juice, up to almost 17 percent
in some sample of grape juice. Although molds can and do grow on the surface of such
26. juices if the juices are exposed to air, the high moisture content favors the faster-growing
yeasts and bacteria. Which of the latter will predominate in juices low in sugar and acid
will depend more on the temperature than on the composition. The removal of solids
from the juices by extraction and sieving raises the oxidation-reduction potential and
favors the growth of yeasts. Most fruit juices are acid enough and have sufficient sugar to
favor the growth of yeasts within the range of temperature that favors them, namely, from
15.6 to 35o
Therefore, the normal change to be expected in raw fruit juices at room temperatures is
an alcoholic fermentation by yeasts, followed by the oxidation of alcohol and fruit acids
by film yeasts or molds growing on the surface if it is exposed to air or the oxidation of
the alcohol to acetic acid if acetic acid bacteria are present.
In addition to the usual alcoholic fermentation, fruit juices may undergo other changes
caused by microorganisms:
1). the lactic acid fermentation of sugars, mostly by heterofermentative lactic acid
bacteria such as Lactobacillus pastorianus, L. brevis, and Leuconostoc mesenteroides in
apple or pear juice and by homofermentative lactic acid bacteria such as lactobacillus
arabinosus L.leichmanii , and Microbacterium,
(2). the fermentation of organic acids of the juice by lactic acid bacteria, e.g.,
Lactobacillus pastorianus, malic acid to lactic and succinic acids, quinic acid to
dehydroshikimic acid, and citric acid to lactic and acetic acids,
(3). slime production by Leuconostos mesenteroldes, Lactobacillus brevis, and L.
plantarum in apple juice and by L. plantarum and streptococci in grape juice.
27. Chapter 5
Microbiology of cereal and cereal products like bread
5.1) Microbiology of cereal: Cereal grains and meals and flours made from them should
not be subject to microbial spoilage if they are prepared and stored properly because their
moisture content is too low to support even the growth of molds. If, however, these
products become moistened above the minimum for microbial growth, growth will
follow. A little moistening will permit only growth of molds, but more moisture will
allow the growth of yeasts and bacteria.
Although the microbial load of cereal grains, meals and flours may not constitute
a spoilage problem by itself, the number and the types of microorganisms in such
products are of concern since these products are used in formulation of many other foods.
The microbial contribution of cereal grains and flours to convenience foods is an
important consideration from a public health aspect and as a source of possible spoilage
Since cereal grains and meals ordinarily are not processed to reduce their natural
flora of microorganisms greatly, they are likely to contain molds, yeasts, and bacteria,
which are ready to grow if enough moisture is added. In addition to starch, which is
unavailable to many organisms, these grains contain some sugar and available nitrogen
compounds, minerals, and accessory growth substances; and the amylases will release
28. more sugar and proteinases will yield more available nitrogenous foods if the grains are
moistened. A little added moisture will result in growth of molds at the surfaces, where
air is available. A wet mash of the grains or a mash of the meals will undergo an acid
fermentation, chiefly by the lactic acid and coliform bacteria normally present on plant
surfaces. This may be followed by an alcoholic fermentation by yeasts as soon as the
acidity has increased enough to favor them. Finally, molds (and perhaps film yeasts) will
grow on the top surface, although acetic acid bacteria, if present, may oxidize the alcohol
to acetic acid and inhibit the molds.
Factors: The major factors involved in the spoilage of stored grain by molds include:
A. Microbial content,
B. Moisture levels above 12 to 13 percent,
C. Physical damage, and
Numerous different molds can be involved, but the most common are species of
Aspergillus, Penicillium, Mucor, Rhizopus and Fusarium. As previously mentioned,
many of these molds can produce mycotoxins. Grains spoiled by molds represent a
potential animal or human health hazard as well as a great economic loss.
5.2) Microbiology of cereal product like bread:
The fermentation takes place in the doughs for various kinds of bread, where it
noted that some changes caused by microorganisms are desirable and even necessary in
making certain kinds of bread. The acid fermentation by lactics and coliform bacteria that
is normal in flour pastes or doughs may be too extensive if too much time is permitted,
with the result that the dough and bread made from it may be too “sour”. Excessive
growth of proteolytic bacteria during this period may destroy some of the gas holding
capacity so essential during the rising of the dough and produce sticky dough. Sticky
doughs, however, are usually the result of overmixing or gluten breakdown by reducing
agent, e.g., glutathione. There also is the possibility of the production by microorganisms
of undersirable flavors other than the sourness.
Historically, the chief types of microbial spoilage of baked bread have been
moldiness and ropiness, usually termed
29. (1) “Mold” and
Molds are the most common and hence the most important cause of the spoilage of bread
and most bakery products. The temperatures attained in the banking procedure usually
are high enough to kill all mold spores in and on the loaf , so that molds must reach the
outer surface or penetrate after baking. They can come from the air during cooling or
thereafter, from handing, or from wrappers and usually initiate growth in the crease of the
loaf and between the slices of sliced bread.
Chief molds involved in the spoilage of bread are the so-called bread mold,
Rhizopus stolonifer (syn., R nigricans), with its white cottony mycelium and black dots of
sporangia; the green-spored Penicillium expansum or P. Stoloniferum; Aspergillus niger
with its greenish- or purplish-brown to black conidial heads and yellow pigment diffusing
into the bread; and Monialla (Neurospora) sitophila, whose pink conidia give a pink or
reddish color to its growth. Species of Mucor or Geotrichum or any of a large number of
species of other genera of molds may develop.
Mold spoilage is favored by:
(1). heavy contamination after baking, due, for example, to air heavily laden with mold
spores, a long cooling time, considerable air circulation, or a contaminated slicing
(2). slicing, in that more air is introduced into the loaf,
(3). wrapping, especially if the bread is warm when wrapped, and
(4). storage in a warm, humid place. There is little growth of commercial importance on
bread crust in a relative humidity below 90 percent.
Ropiness of bread is fairly common in home-baked bread, especially during hot weather,
but it is rare in commercially baked bread because of the preventive measures now
employed. Ropiness is caused by a mucoid variant of Bacillus subtilis or B. licheniformis,
formerly called B. mesentericus, B. panis, and other species names. The spores of these
species can withstand the temperature of the bread during baking, which does not exceed
30. 100°C, and can germinate and grow in the loaf if conditions are favorable. The ropy
condition apparently is the result of capsulation of the bacillus, together with hydrolysis
of the flour proteins (gluten) by proteinases of the organism and of starch by amylase to
give sugars that encourage rope formation. The area of ropiness is yellow to brown in
color and is soft and silky to touch. In one stage the slimy material can be drawn out into
long threads when the bread is broken and pulled apart. The unpleasant odor is difficult to
characterize, although it has been described as that of decompose or overripe melons.
First the odor is evident, then discoloration, and finally softening of the crumb, with
strictness and stringiness.
Microbial spoilage of foods – food borne pathogens, food poisoning,
food infection and intoxication
6.1). Food borne diseases: Safe, nutritious foods are essential to human health and well-
being. However, food-borne diseases pose a significant problem worldwide. Food-borne
diseases are any illness resulting from the consumption of food contaminated with one or
more disease-producing agents. These include bacteria, parasite, viruses, fungi, and their
products as well as toxic substances not of microbial origin. These different diseases have
many different symptoms, so there is no one “syndrome” that is food borne illness.
However, the microbe or toxin enters the body through the gastrointestinal tract, and
often causes the first symptoms there, so nausea, vomiting, abdominal cramps and
diarrhea is common symptoms in many food borne diseases.
Types of microbial food borne diseases:
On the basis of mode of illness, Food borne diseases can be divided into three groups.
A. Intoxication: Illness in this case occurs as a consequence of ingestion of a pre formed
bacterial or a mold toxin due to its growth in a food. A toxin has to be present in the
contaminated food. Once the microorganism have grown and produced toxin in a food,
31. there is no need of viable cells during the consumption of the food for illness to occur.
e.g., Staph food poisoning
B. Infection: Illness occurs as a result of the consumption of food and water
contaminated with entero pathogenic bacteria. It is necessary for the cells of
enteropathogenic bacteria to remain alive in the food or water during consumption. The
viable cells even if present in small numbers have the potential too establish and multiply
in the digestive tract to cause the illness. e.g., Salmonellosis.
C. Toxicoinfection: Illness occurs from the ingestion of a large number of viable cells of
some pathogenic bacteria through contaminated food and water. Generally the bacterial
cells either sporulate or die and release toxin(s) to produce symptoms. e.g., Bacillus
Factors: The main factors responsible for the food borne illness include:
a) Improper holding temperature during processing
b) Inadequate cooling during storage
c) Contaminated equipments and utensils
d) Food from unsafe source
e) Poor personal hygiene
f) Adding contaminated ingredients to cooked foods
6.2) Food borne pathogens:
A detailed account of major food borne pathogens:
A). Staphylococcal food poisoning
Caused by bacterium Staphylococcus aureus, Staphylococcal food poisoning is one of the
most common types of food poisoning.
Biology of Staphylococcus aureus:
Staphylococcus aureus is a spherical bacterium (coccus) which on microscopic
examination appears in pairs, short chains, or bunched, grape like clusters. They are
Gram-positive, facultative anaerobes, but grow rapidly under aerobic conditions. They
are mesophiles with a growth temperature range of 7 to 48o
C and have the ability to grow
at low Aw (0.86), low pH (4.8), and high salt and sugar concentration of 15% and in the
32. presence of NO2. Staphylococcus aureus are naturally present in the nose, throat, skin and
hair of healthy humans, animals and birds. They can be present in infections such as cuts,
wounds, abscesses and facial acne etc. The contamination generally occurs through these
sources because of improper handling by infected persons.
Disease and symptoms:
About 30gm or ml of food containing toxins produced by 106
cells per gram (ml)
for a normal healthy individual is sufficient to cause the symptoms. The primary
symptoms are salivation, nausea and vomiting, abdominal cramps & diarrhea. Secondary
symptoms are sweating, chills, headache and dehydration.
Sensitive immunological tests viz., ELISA etc has been developed for this purpose.
Although the disease is very rare, the disease has a high fatality rate. Botulism is caused
by the bacterium Clostridium botulinum.
Biology of Clostridium botulinum:
Clostridium botulinum is an anaerobic, Gram-positive, spore-forming rod that produces a
potent neurotoxin. Spores of Clostridium botulinum are widely distributed in soil,
sewage, mud, sediments of marshes, lakes and coastal waters, plant-fruits and vegetables
and intestinal contents of animals and fishes. Cells are sensitive to low pH (<4.6), lower
Aw (0.93), and moderately high salt (5.5%). The spore are heat resistant (killed at 115o
and survive in foods that are incorrectly or minimally processed. Spores do not germinate
in the presence of nitrate (250ppm). Foods most commonly associated are low acid
vegetables (green beans, corn, spinach, asparagus, pepper and mushrooms) and fruits
(figs and peaches) and also fermented, improperly cooked and smoked fish and fish eggs.
Disease and symptoms:
At initial stage (generally 12h to 36h, but can be 2h), some gastrointestinal symptoms
(nausea, vomiting, diarrhea and constipation) are evident. Neurological symptoms appear
in a short time particularly when the amount of toxin consumed is more, which includes
blurred or double vision, difficulty in swallowing, breathing and speaking, dryness of the
33. mouth, and paralysis of different involuntary muscles that spreads to the lung and heart.
Death usually results from respiratory failure.
The presence of C. botulinum can be determined by enumeration techniques using
selective agar media and followed by anaerobic incubation. The presence of toxins is
more often tested by immunological methods like ELISA etc.
C). Salmonella Gastroenteritis:
S. enteritids contamination and food production in centralized facilities, leading to large
and widespread outbreaks incase contamination occurs.
Biology of Salmonella:
Salmonella is a rod shaped, Gram-negative, non-sporulating, facultative anaerobic motile
bacterium. They are mesophiles with a growth temperature range of 5 to 46o
optimum growth temperature of 35 to 37o
C. They are sensitive to low water activity
(<0.94) and low pH (4.5 or below). There are over 2000 serovars of Salmonella based on
flagellar, somatic and capsular antigens, which are capable of causing salmonellosis in
humans. Salmonella are natural inhabitants of gastrointestinal tracts of animals, birds,
pets, frogs, turtle and insects. They are also present in soil, water and sewage
contaminated with fecal matter from where they can contaminate foods directly or
indirectly and cause Salmonellosis. Foods generally associated with outbreaks of
Salmonella includes raw meats, poultry, eggs, milk and dairy products, fish, shrimp, frog
legs, yeast, coconut, sauces and salad dressing, cake mixes, cream-filled desserts and
toppings, dried gelatin, peanut butter, cocoa, and chocolate. Various Salmonella species
have long been isolated from the outside of egg shells.
Disease and symptoms:
The disease starts with the penetration and passage of Salmonella organisms from gut
lumen into epithelium of small intestine where inflammation occurs; there is evidence
that an enterotoxin may be produced, perhaps within the enterocyte. S. typhi and the
paratyphoid bacteria normally cause septicemia and produce typhoid or typhoid-like
fever in humans. Other forms of salmonellosis generally produce milder symptoms which
34. includes nausea, vomiting, abdominal cramps, diarrhea, chills, fever, headache and
Immunological methods which are used to detect Salmonella include ELISA, enrichment
serology and fluorescent antibody technique.
D). Pathogenic E.coli
E.coli was considered a harmless, Gram negative, motile, non-sporulating; rod shaped,
facultative anaerobic bacterium, a normal inhabitant of GIT of humans and warm
blooded animals and birds. As it is present high number in intestine, it has been used as
an index organism of possible fecal contamination of enteric pathogens in foods and
water. Since in mid 1940s, evidence has accumulated that certain E.coli strains cause
diarrhea, particularly in infants, and they were then designated as pathogen. The four
groups of pathogenic E.coli are:
a. Enterotoxigenic Escherichia coli (ETEC)
b. Enteropathogenic Escherichia coli (EPEC)
c. Enterohemorrhagic Escherichia coli (EHEC)
d. Enteroinvasive Escherichia coli (EIEC)
Enterotoxigenic Escherichia coli (ETEC):
Disease: Gastroenteritis/travelers diarrhea is the common name of the illness caused by
Infective dose: A relative large dose (100 millions to 10 billions bacteria) of ETEC is
probably necessary to establish colonization of the small intestine, where these organisms
proliferate and produce toxins which induce fluid secretion.
Symptoms: The most frequent clinical syndrome of infections includes watery diarrhea
abdominal cramps, low-grade fever, nausea and malaise.
Associated food: Contamination of water with human sewage may lead to contamination
of foods. Infected foods handlers may also contaminate foods. These organisms are
infrequently isolated from dairy products such as semi-soft chesses.
Identification methods: with the availability of a gene probe method, foods can be
analyzed directly for the presence of ETEC, and the analysis can be completed in about 3
35. days. Alternative methods which involve enrichment and plating of samples for isolation
of E.coli and their subsequent confirmation as toxigenic strains by conventional toxin
assays take at least 7 days.
E). Bacillus cereus Gastroenteritis
Biology of Bacillus cereus:
The causal organism is a bacterium Bacillus cereus, which is a Gram-positive, facultative
aerobic spore former whose cells are large rods and whose spores do not swell the
sporangium. A wide variety of foods including meats, milk, vegetables, and fish have
been associated with diarrheal type food poisoning. The vomiting-type outbreaks have
generally been associated with rice products; however, other starchy foods such as potato,
pasta and cheese products have also been implicated.
Disease and symptoms:
The symptoms of Bacillus cereus diarrheal type food poisoning are the onset of watery
diarrhea, abdominal cramps and pain after 6-15 hours of consumption of contaminated
food. Nausea may accompany diarrhea, but vomiting (emesis) rarely occurs. The emetic
type of food poisoning is characterized by nausea and vomiting within 0.5 to 6 h after
consumption of contaminated food. Occasionally, abdominal cramps and/or diarrhea may
Bacillus cereus can be enumerated by surface plating.
Control measures: General control measures for prevention of food borne diseases
a. By employing proper sanitary measures.
b. By the use of chemical agents in preventing the growth of microorganisms.
c. Removing left over food promptly from work surfaces and utensils in food
d. By cooking or applying proper and desired processing treatment to the food
36. e. By avoiding chances of re-contamination of food, hence storing in it proper
f. Maintaining good personal hygiene, like food handlers should not have any open
cuts and wounds.
g. Maintaining proper sanitation and preventing contamination of water to be used.
Concept, determination and importance of TDT, F, Z and D value;
factors affecting the heat resistance of microorganisms
7.1) Thermal death time: Bacteria are killed by heat at a rate that is very nearly
proportional to the number present in the system being heated. This is referred as
logarithmic order of death, which means that under constant thermal conditions the same
percentage of the bacterial population will be destroyed in a given time interval,
regardless of the size of the surviving population. In other words, if a temperature kills
90% of the population in the first minute of heating, 90% of the remaining population
will be killed in the second minute, 90% of what is left will be killed in the third minute,
and so on. The logarithmic order of death also applies to bacterial spores, but the slope of
the death curve will differ from that of vegetative cells, reflecting the greater heat
resistance of spores.
Thermal death time curve can be described in two ways:
37. a. Thermal death rate curve: It provides data on the rate of destruction of a
specific organism in a specific medium or food at a specific temperature.
b. Thermal death time curve: A thermal death time curve for a specific organism
in a specific medium or food provides data on the destruction times for a defined
population of that organism at different temperatures.
7.2) D value: The concept of “D value” which is defined as the time in minutes at a
specified temperature required to destroy 90% of the organisms in a population. Thus, the
D value or decimal reduction time, decreases the surviving population by one log cycle.
e.g. If the quantity of food in a can contained one million organisms and it received heat
for a time equal to four D values, then it would contain 100 surviving organisms.
7.3) Z value: The Z value is the number of degrees required for a specific thermal death
time curve to pass through one log cycle.
Different organisms in a given food will have different Z values, which characterize
resistance of the populations to changing temperature.
7.4) F value:
The F value is defined as the number of minutes at a specific temperature required to
destroy a specified number of organisms having a specific Z value. Thus, the F value is a
measure of the capacity of a heat treatment to sterilize.
→ Since F values represent the number of minutes to diminish a population with a
specific Z value at a specific temperature, and Z values as well as temperature vary, it is
convenient to designate a reference F value. Such a reference value is Fo value, which
equals the number of minutes at 121o
C required to destroy a specified number of
organisms whose z values is 10 o
C. If such a population is destroyed in 6 min at 121o
then the heat treatment was equal to an Fo of 6. Other temperatures for different times can
have same lethality as this heat treatment. If they do, then they also can be described as
having an Fo value of 6. If they have less lethality, they have an Fo value of less than 6 and
vice-versa. The Fo value of heat treatment is thus a measure of its lethality and the Fo
value is also known as the “sterilization value” of the heat treatment.
38. 7.5) Factors affecting the heat resistance of microorganisms:
The killing of microorganisms by heat is supposed to be caused by denaturation of the
proteins and especially by the inactivation of enzymes required for metabolism. The heat
treatment necessary to kill organisms or their spores varies with the kind of organism, its
state and the environment during heating. Depending on the heat treatment employed,
only some of the vegetative cells, most or all of the cells, part of the bacterial spores, or
all of them may be killed. The heat treatment selected will depend on the kind of
organisms to be killed.
Cells and spores of microorganisms differ widely in their resistance to high temperatures.
Some of these differences are the result of factors that can be controlled, but other are
characteristic of the organisms and cannot always be explained. There are differences in
heat resistance within a population of cells or spores. A small number of cells have low
resistance, most of the cells have a medium resistance, and a small number have high
resistance. Conditions of growth may favor one or other of these groups, and, by
selection, cultures that are more or less heat-resistance than usual can be produced.
Certain factors are known to affect the heat resistance of cells or spores and must be
keep in mind when microorganisms are compared and when heat treatments for the
destruction of an organism are considered. The chief known factors are as follows:
1. The temperature-time relationship: The time for killing cells or spores under a given
set of conditions decreases as the temperature is increased. This is illustrated in table:
Table: Effect of temperature of heating on time needed to kill spores or cells
C Thermal death time, or time to destroy all
39. 135 1
2. Initial concentration of spores: The more spores or cells present, the greater the heat
treatment necessary to kill all of them. This is illustrated in table:
Table: Effect of initial numbers of spores on time required to kill them
Initial concentration of spores,
Thermal death time, or time required to
kill all spores, min at 120oC
3. Previous history of the vegetative cells or spores: The condition under which the
cells have been grown and spores have been produced and their treatment thereafter will
influence their resistance to heat.
a. Culture medium: The medium in which growth takes place is especially important.
The effect of the nutrients in the medium, their kind, and the amount vary with the
organism, but in general the better the medium for growth, the more resistant the cells or
spores. The presence of an adequate supply of accessory growth factors usually favors the
production of heat-resistant cells or spores.
Carbohydrates, amino acids, and organic acid radicals have an effect, but it is difficult to
predict. A small amount of glucose in a medium may lead to increased heat resistance,
but more sugar may result in the formation of enough acid to cause decreased heat
b. Temperature of incubation: The temperature of growth of cells and the temperature
of sporulation influence heat resistance. In general, resistance increases as the incubation
temperature is raised toward the optimum for the organism and for many organism
increases further as the temperature approaches the maximum for growth.
Escherichia coli, for example, is considerably more heat-resistant when grow at 38.5o
which is near its optimum temperature, than at 28o
c. Phase of growth or age: The heat resistance of the vegetative cells varies with the
stage of growth and of spores with their age. Bacterial cells show their greatest resistance
during the late lag phase but almost as great resistance during their maximum stationary
40. phase. The cells are least resistance during their phase of logarithmic growth. Very young
(immature) spores are less resistant than are mature ones. Some spores increase in
resistance during the first weeks of storage but later begin to decrease in resistance.
d. Dessication: Dried spores of some bacteria are harder to kill by heat than are those
kept moist, but this apparently does not hold for all bacterial spores.
4. Composition of the substrate in which cells or spores are heated: The material in
which the spores or cells are heated is so important that it must be stated if a thermal
death time is to have meaning.
a. Moisture: Moist heat is a much more effective killing agent than dry heat, and as a
corollary dry materials require more heat for sterilization than moist one. In the
bacteriological laboratory about 15 to 30 min at 121o
C in the moist heat of an autoclave
will effect sterilization of ordinary materials, but 3 to 4 hr at 160 to 180o
C is necessary
when the dry heat of an autoclave is employed.
b. Hydrogen-ion concentration (pH): In general, cells or spores are more heat
resistance in a substrate that is at or near neutrality. An increase in acidity or alkalinity
hasten killing by heat, but a change toward the acid side is more effective than a
corresponding increase in alkalinity.
c. Other constituent of the substrate: The only salt present in appreciable amounts in
most foods is sodium chloride, which in low concentration has a protective effect on
Sugar seems to protect some organisms or spores but not others. The optimal
concentration for protection varies with the organism: it is high for some osmophillic
organisms and low for others, high for spores and low for non osmophillic cells. The
protective effect of sugar may be related to a resulting decrease in aw. A reduced aw does
result in an increase in observed heat resistance.
Solutes differ in their effect on bacteria. Glucose, for example, protects Escherichia coli
and Pseudomonas fluorescens against heat better than sodium chloride at aw levels near
the minimum for growth. On the other hand, glucose affords practically no protection or
is even harmful to Staphylococcus aureus, whereas sodium chloride is very protective.
Colloidal materials, especially proteins and fats, are protective against heat.
41. Chapter 8
Anti-microbial agents – physical and chemical agents -
Their mechanism of action
8.1) Anti-microbial agents:
Many chemical compounds, either present naturally or formed during processing or
legally added as ingredients, are capable of killing microorganisms or controlling their
growth in foods. They are, as a group, designated as antimicrobial inhibitors or
Examples: 1. some of the naturally occurring preservatives can be present in sufficient
amount in foods to produce antimicrobial action, such as lysozyme in egg white, and
organic acids in citrus fruits.
2. Similarly, some of the antimicrobials can be formed in enough quantities during food
processing to control undesirable microbial growth, such as lactic acid in yogurt
3. Some agents are added to improve the functional properties of food such as
Objective: Antimicrobial chemicals are used in food in relatively small doses, either to
kill undesirable microorganisms or to prevent or retard their growth in food.
42. These antimicrobial agents should be suitable for application in food.
• This compound should not only have the antimicrobial properties but it also
should not affect the normal quality of a food i.e. texture, flavor or color.
• It should not interact with food constituent and become inactive.
• It should have a high antimicrobial property at pH, water activity and storage
temperature of the food.
• It should be stable during the storage life of the food.
• Finally, it should be economical and easily available.
• The regulatory requirements include the expected effectiveness of an
antimicrobial agent in a food system.
• It should be effective in small concentration and should not hide any fault of a
food i.e. poor quality and spoilage.
• The most important, it should be safe for human consumption.
8.2) Types of antimicrobial agents:
1. Chemical agents
2. Physical agents
8.2.1). Chemical agents:
A). Nitrite (NaNO2 and KNO2):
Curing agents that contain nitrite and together with salt, sugar, spices, ascorbate and
erythorbate are permitted for used in heat processed meat, poultry and fish products to
control growth and toxin produced by clostridium botulinum. Nitrate and nitrite are also
used in cheese to prevent gas flowing by Clostridium butyricum and Clostridium
Mechanism of action:
The mechanism of antimicrobial action of nitrite is not properly understood, but there is
indication that the inhibitory effect is produced in several ways. These include reaction
with some enzymes in the vegetative cells and germinating spores, restriction of bacterial
use of iron, and interference with membrane permeability limiting transport. In addition
43. to clostridial species, nitrite is inhibitory to some extent to Staphylococcus aureus,
Escherichia, Pseudomonas, and Enterobacter spp. at 200ppm. The antimicrobial effect of
NO2 is enhanced at lower pH (5.0 to 6.0), in the presence of reducing agent (e.g.
ascorbate, erythorbate, and cystiene) and with sorbate.
B). Sulfur dioxide (SO2) and sulfites (SO3):
Sulfur dioxide, sodium sulfite (Na2SO3), sodium bisulfide (NaHSO3) and sodium
metabisulfite (Na2S2O5) are used to control microorganisms (and insects) in soft fruits,
fruit juices, lemon juices, beverages, wines, sausages and pickles. They are most effective
against molds and yeasts than bacteria.
Mechanism of action:
The antimicrobial action is produced by the undissociated sulfurous acid that rapidly
enters inside the cell and reacts with thiol groups in structural proteins, enzymes and
cofactors, as well as with other cellular components.
The concentrations used ion foods vary greatly in different countries. Mostly, 200 to 300
ppm is generally permitted for antimicrobial use. Sulfur dioxide and sulfites are also used
as antioxidants in fresh and dried fruits and vegetables to prevent browning. However,
people with respiratory problems can be mildly to severely allergic. The products need to
be labeled to show the presence of sulfites.
Some lactic acid bacteria produced H2O2 under aerobic condition of growth and due to
the lack of cellular catalase, or peroxidase, they release it into the environment to protect
themselves from its antimicrobial action. It is strong oxidizing agent and can be
antimicrobial against bacteria, fungi and viruses.
Mechanism of action:
Raw milk contains lactoperxidase enzyme and thiocyanate (SCN-
). In the presence of
H2O2, lactoperoxidase generates hypothiocyanate anion (OSCN-
), which at milk pH can
be in equilibrium with hypothiocyanous acid (HOSCN). Both OSCN-
and HOSCN are
strong oxidizing agents and can oxidize the –SH group of proteins, such as membrane
proteins of gram-negative bacteria that are especially susceptible. This system is
inactivated by pasteurization.
44. Hydrogen peroxide is permitted in refrigerated raw milk and raw liquid eggs (about
25ppm) to control spoilage and pathogenic bacteria. Prior to pasteurization catalase (0.1
to 0.5 g/1000 lb) is added to remove the residual H2O2. It produces antimicrobial action
by its strong oxidizing property and damage to cellular components, especially the
membrane. Due to its oxidizing property, it can produce undesirable effects in food
quality such as discoloration in processed meat, and thus has limited use in food
D). Epoxides (ethylene oxide and propylene oxide):
Ethylene oxide and propylene oxide are used as fumigants to destroy microorganisms
(and insects) in grains, cocoa powder, gums, nuts, dried fruits, spices and packaging
materials. They are germicidal and effective against cells, spores and viruses.
Mechanism of action:
Ethylene oxide is more effective. They are alkylating agents and react with various
groups (e.g., -SH, -NH2 and -OH) in cellular macromolecules, particularly structural
proteins and enzymes, and adversely affect their functions.
They can react with some food components, such as chlorides and form toxic compounds
that can remain as residue in treated foods. They can be toxic at higher concentrations (as
residue), particularly to people who are sensitive to them.
E). Butylated Hydroxyanisol (BHA), Butylated Hydroxytoluene (BHT), and t-Butyl
BHA, BHT and TBHQ are primarily used at 200 ppm or less as antioxidants to delay
oxidation of unsaturated lipids. Additionally, they have antimicrobial properties and thus
can be regarded as indirect antimicrobials.
Mechanism of action:
In concentration ranging from 50 to 400 ppm, BHA inhibits growth of many gram-
positive and gram-negative bacteria; however some species may be resistant to it. They
can also effectively prevent growth and toxin production by molds and growth of yeasts,
but BHA seems to be more effective. The antimicrobial action is most likely produced by
45. their adverse effect on the cell membrane and enzymes. Their antimicrobial effectiveness
increases in the presence of sorbate, but decreases in foods with high lipids and at low
Chitosan, a polycationic polymer, is obtained by alkaline hydrolysis of chitin from the
shells of Crustaceae. It has many applications in foods, including food preservation due
to its antimicrobial capability.
Mechanism of action:
It causes destabilization of the cell wall and cell membrane functions and is effective
against bacteria, yeasts and molds.
G). Ethylenediaminetetraacetate (EDTA):
The sodium and calcium salts of EDTA at 100 ppm are approved for use in foods to
chelate trace metals in order to prevent their adverse effect on food quality. At low doses
(5000 ppm), EDTA appears to have no toxic effect and mostly passes through the GI tract
Mechanism of action:
By itself, EDTA may not have much antimicrobial effect, but due to its ability to chelate
divalent cations, it can destabilize the barrier functions of the outer membrane of the
Gram-negative bacteria and, to some extent, the cell wall of Gram-positive bacteria. In
this way, it enhances the antimicrobial action of other chemicals, especially those that are
membrane acting, such as surface active compounds, antioxidants, lysozymes, and
EDTA is also inhibitory for germination and outgrowth of spores of Clostridium
botulinum. In the presence of divalent cations in the environment (e.g., dairy products),
the effectiveness of EDTA is greatly reduced.
46. The enzyme lysozyme is present in large quantities in some foods such as egg white and
shellfish (oysters and clams) and in small amount in milk and some plant tissues.
Mechanism of action:
It hydrolyzes the mucopeptide layer present in the cell wall of Gram-positive bacteria and
in the middle membrane of Gram-negative bacteria. However, Gram-negative bacteria
become sensitive to the lysozyme effect only after the barrier function of the outer
membrane is destabilized by chemical (i.e., EDTA) and physical (e.g., freezing or
heating) stresses. The antimicrobial effect is manifested by the lysis of cells. Lysozyme is
more effective at pH 6.0 to 7.0 and at concentration of about 0.01 to 0.1%. It can be used
in wine (sake) to prevent the growth of undesirable lactic acid bacteria.
Many spices, condiments, and plant extracts are known to contain antimicrobial
compounds. Some of these include cinnamic aldehyde in cinnamon; eugenol in cloves,
allspice and cinnamon.
Mechanism of action:
Depending upon an active agent, they have bacteriostatic and fungistatic properties. Due
to the small amounts used as spice in foods, they probably do not produce any
antimicrobial effects. However, the antimicrobial components can be used in higher
concentrations as essential oils. It is expected that in future the antimicrobial compounds
from plants, especially food plants, will be studied more effectively and thoroughly.
8.2.2). Physical agents:
Microorganisms can be physically removed from solid and liquid foods by several
methods. In general, these methods can partially remove microorganisms from foods and
by doing so they reduce the microbial level and help other antimicrobial step(s) that
follow them become more effective. They are generally used with raw foods before
Centrifugation is used in some liquid foods, such as milk, fruit juices, and syrups, to
remove suspended undesirable particles.
47. Mechanism of action:
The process consists of exposing the food in thin layers to a high centrifugal force. The
heavier particles move outwards and separated from the lighter liquid mass. Although
this is not intended to remove microorganisms, spores, large bacterial rods, yeasts and
molds can be removed, due to their heavier mass. Under high force, as much as 90% of
the microbial population can be removed.
Filtration is used in some liquid foods, such as soft drinks, beer, wine and water, to
remove undesirable solids and microorganisms and to give a sparkling clear appearance.
As heating is avoided or given only at minimum levels, the natural flavor of the products
and heat-sensitive nutrients (e.g., vitamin C in citrus juices) are retained to give the
products natural characteristics. The filtration process can also be used as step in the
production of concentrated juice with better flavor and higher vitamins.
Mechanism of action:
Many types of filtration system are available. In many filtration processes, coarse filters
are initially used to remove the large components; this is followed by ultrafiltration.
Ultrafiltration methods, depending on the pore size of the filter materials (0.45 to 0.7
µm), are effective in removing yeasts, molds and most bacterial cells and spores from the
Filtration of air is also used in some food processing operations, such as spray drying of
milk, to remove dust from air used for drying. The process also removes some
microorganisms with dust, and they reduce the microbial level in food from this air.
Fruits and vegetables showing damage and spoilage are generally trimmed. In this
manner, areas heavily contaminated with microorganisms are removed.
Mechanism of action:
Trimming of outside leaves in cabbage used for sauerkraut production also helps reduce
microorganisms coming from soil. Trimming is also practiced for the same reason to
remove visible mold growth from hard cheeses, fermented sausages, bread and some low
48. pH products. Although this method helps remove highly contaminated areas, it does not
ensure complete removal of the causative microorganisms.
Fruits and vegetables are washed regularly to reduce temperature (that helps to reduce
metabolic rate of a produce and microbial growth) and remove soil. Washing also helps
remove the microorganisms present, especially from the soil. It is also used for shell eggs
to remove fecal materials and dirt.
Mechanism of action:
With high pressure, the effectiveness of hot water, steam, ozonated water and water
containing chlorine, acetic and propionic acids, lactic acid have been studied to determine
their effectiveness in removing microorganisms, particularly enteric pathogens such as
salmonella spp., E.coli. Some of these agents also have bactericidal properties. However,
the studies showed that all of these agents are capable of reducing bacterial
contamination to a certain level and that a combination of two or more components may
be better. However, the suitability of the combinations as well as their concentrations and
duration of application must be determined.