Food MCB II Notes

FOOD MICROBIOLOGY II
INTRODUCTION
FOOD AS A SUBSTRATE FOR MICROORGANISMS
Microbes, plants and animals are involved in a constant interaction in the ecosystem.
Since man's food is derived from both plants and animals, it follows that his food must
contain microbes at some level of interaction. Basically, microbes use our food as a
source of nutrient for their own growth- the effect of which can be deterioration of such
foods. They can spoil food by increasing their numbers, utilizing nutrients, producing
enzymatic changes and contributing flavour through synthesis of by-products or break
down of certain compounds.
This process is just the natural course of the existence of microorganisms since their main
aim is for their healthy existence. In the course of this, they convert reduced forms of
carbon, nitrogen and sulphur in dead plant and animal tissues to the oxidized forms
required by plants. These now form the vital link in the food chain with animals.
Unfortunately, in the course of their natural existence, they frequently render our food
unfit for consumption. This can only be prevented by reducing contact between our foods
and microbes (prevent contamination), or eliminate microbes from the food, or at least
make conditions unfavourable for their existence in that food (preservation).
Preventing contact between our foods and microbes has a dual purpose of preventing
spoilage of the food and protecting consumers against pathogens. But there are areas
where the existence of microbes on our foods is desirable. Examples are found in
fermented food products such as yoghurt, beer e.t.c.
The salient questions to be answered are as follows:
(a) Why is this interaction beneficial at some times but not others?
(b) Why do some foods support microbial growth more than others?
(c) Why do some foods resist microbial spoilage?
To answer these, one must understand the characteristics of the food since it is the
substrate and its characteristics affect the presence of any microflora. To do this, one must
examine intrinsic and extrinsic parameters of food that affect microbial growth, examples
are moisture content, oxidation-reduction potential (O-R), nutrient content, presence of
Dr. Mugampoza Edriisa- Dept. of Food Processing Technology, KYU © 1

barriers, hydrogen ion (H+) concentration, temperature, relative humidity, gases in the
environment, food additives and food preservatives.
FOOD PARAMETERS (INTRINSIC FACTORS)

The parameters of plant and animal tissues that are an inherent part of the tissues are
referred to as intrinsic parameters. They are:
(1) Hydrogen ion concentration (pH).
(2) Moisture content.
(3) Oxidation-reduction (redox) potential.
(4) Nutrient content.
(5) Inhibitory/Antimicrobial constituents.
(6) Biological structures.
(1) pH and the buffering power:

All microbes have a minimal, maximal and optimum pH for growth. Yeast and moulds
are able to withstand acidic pH more than bacteria. Most foods are either acidic or neutral
in pH but the pH inherent to them normally varies with the type of food. If food is too
acidic (pH below 4.5), the spoilage organism will normally be mould and yeast. This
means foods with low pH such as fruits, soft drinks and fermented product will keep
longer compared to neutral foods. The pH of some foods could be naturally low (e.g.
lime) or acquired through accumulation of lactic acid.
The pH of the food determines the type of microbes that can be found on it. Moulds have
higher acid tolerance compared to yeast. But pH 4.0-4.5 will favour most fermentative
yeast. Most yeast would grow very well in alkaline substrate. For bacteria, pH
requirement is usually around neutrality, though the acid formers will tolerate moderate
acidity while the proteolytic ones will tolerate high (alkaline) pH.
Many foods usually contain buffers (ability to resist changes in pH). Some foods have
buffering power. This ability is usually effective over a certain pH range. Buffering helps
to maintain pH for a long enough period for appreciable yield in product over a certain
pH range. Rapid succession in microorganism is reduced. Only food rich in protein
content have high buffering ability. Low protein foods such as vegetables have low

buffering ability. pH affects both growth rate and survival of microbes in food. pH
changes as growth of organism proceeds.
pH values of some fruits:
Apples- 2.9 to 3.3;
Bananas- 4.5 to 4.7;
Limes- 1.8 to 2.0;
Oranges (juice)- 3.6 to 4.3;
Plums- 2.8 to 4.6;
Watermelons- 5.2 to 5.6;
Grapes- 3.4 to 4.5.
Most food-borne bacteria require a minimum pH value for their growth as indicated
below:
Bacillus cereus- 4.9
Clostridium botulinum- 4.6 to 5.0.
Clostridium perfringens- 5.0
Escherichia coli- 4.5.
Lactococcus lactis- 4.3.
Salmonella spp. - 4.05.
Staphylococcus aureus- 4.0.
Yersinia enterocolitica- 4.18.
Most of the meat and seafoods have a final ultimate pH of about 5.6 and above. This
makes products susceptible to bacteria as well to mould and yeast spoilage.
pH values of dairy, meat, poultry and fish products:
Dairy:
Butter- 6.4 to 6.6.
Buttermilk- 4.5.
Milk- 6.3 to 6.5.

Meat & Poultry:
Beef (ground)- 5.1 to 6.2.
Ham- 5.9 to 6.1.
Chicken- 6.2 to 6.4.
Fish & Shellfish:

Fish (most sp.)- 6.6 to 6.8.
Oysters- 4.8 to 6.3.
Crab- 7.0.
Tunafish- 5.2 to 6.1.
Shrimp- 6.8 to 7.0.
Salmon- 6.1 to 6.3.
Most vegetables have higher pH values than fruits and therefore they are subjected to
more bacterial than fungal spoilage.
pH values of some vegetables:
Broccoli- 6.5.
Cabbage- 5.4 to 6.0.
Carrots- 4.9 to 5.2; 6.0.
Cauliflower- 5.6.
Cucumbers- 3.8.
Red onions- 5.3 to 5.8.
Potatoes- 5.3 to 5.6.
Tomatoes- 4.2 to 4.3.
(2) Moisture content (concept of water activity):

One of the oldest methods of preserving foods is drying or desiccation. In this method
water is removed or available moisture, so that microorganisms cannot grow. Microbes
have absolute moisture requirement for growth. Moisture requirement is expressed as
available moisture or water activity (Aw) in the environment.

This is defined as the ratio of the vapour pressure of food substrate (or solution/solutes in
water in foods) to the vapour pressure of pure water (solvent) at the same temperature.
Aw = vapour pressure of solution/solutes in water in foods
vapour pressure of solvent (pure water)
or = P
Po
Where P= vapour pressure of solution/solutes in water in foods.
Po= vapour pressure of the solvent (usually water).
The Aw is related to relative humidity (RH) in the following way:
RH = 100 X Aw.
The Aw of pure water = 1.00.
Since vapour pressure of solution is less than that of water, Aw is always less than that of
water, Aw is always less than 1.00. In relationship to the relative humidity (RH) about a
food, the Aw is 100 times smaller. Thus, food with surrounding RH corresponding to Aw
lower than that of the food will tend to dry up while if the relative humidity corresponds
to Aw higher than that of the food, the Aw of the surface of the food will increase.
Each microbe has Aw range for growth with an optimum. These are affected by the
following factors:
(i) Kind of solutes used to reduce A w- the kind of solute does not really matter for
most microbes especially moulds.
(ii) The nutritive value of the culture medium- if the medium is rich, there will be a
lower limit of Aw.
(iii) Temperature- at the optimum temperature there will be less effect of low Aw.
(iv) Oxygen supply- at low oxygen level, anaerobes will tolerate low A w. The opposite
applies to aerobes (high oxygen level, aerobes will tolerate low Aw).
(v) pH - neutral pH favours low Aw tolerance better than acid or alkaline conditions.
(vi) Inhibitors- the presence of this narrows the range of Aw tolerance by microbes.
The Aw of most fresh foods is above 0.99. The minimum values reported for the growth
of some microorganisms in foods are presented below:

Organisms Aw
Most spoilage bacteria 0.99
Most spoilage yeasts 0.88
Most spoilage moulds 0.80
Halophilic bacteria 0.75
Xerophilic moulds 0.61
Osmophilic yeasts 0.61
The optimum Aw for most bacteria approaches 1.00(0.995-0.998), moulds will tolerate
less and 0.62 has been reported for germination of some spores. 0.70 inhibits most food
spoilage moulds. In general, bacteria require higher values of Aw for growth than fungi,
with gram negative bacteria having higher needs than gram positives. Most spoilage
bacteria do not grow below A w = 0.91, whereas spoilage moulds can grow as low as 0.80.
With respect to food poisoning bacteria, Staphylococcus aureus has been found to grow
as low as 0.86, whereas Clostridium botulinum does not grow below 0.94.
(3) Oxidation-Reduction potential(O-R):

This expresses the O-R potential (reducing and oxidizing) of the food. The O-R potential
of a substrate may be defined generally as the ease with which the substrate loses or gains
electrons. When an element or compound loses electrons, the substrate is said to be
oxidized, whereas a substrate that gains electrons becomes reduced.
oxidation
e.g. Cu ↔ Cu + e
reduction
Oxidation may also be achieved by the addition of oxygen as shown below:
2Cu + O2 → 2CuO
Therefore, a substance that readily gives up electrons is a good reducing agent, and one
that takes up electrons is a good oxidizing agent. When electrons are transferred from one
compound to another, a potential difference is created between the two compounds. The
difference can be measured as millivolts (mV). The more highly oxidized a substance is,
the more positive will be its electrical potential, the more highly reduced a substance is,

the more negative will be its electrical potential. When the concentration of oxidant and
reductant is equal, a zero electrical potential exists. The O-R potential of a system is
expressed by the symbol Eh and measured in millivolts (mV).
The O-R potential of a food is determined by:
- The characteristic O-R potential of the original food.
- The poising capacity (i.e. resistance to change in potential of the food).
- The oxygen tension of the atmosphere about the food.
- The access that the atmosphere has to the food.
Aerobic microorganisms require positive Eh values (oxidized) for growth, whereas
anaerobes require negative Eh values (reduced).
+Eh = oxidized substrate- supports aerobes.
-Eh = reduced substrate- supports anaerobes.
Among the substances in foods that help to maintain reducing conditions are sulphydryl
(-SH) groups in meats and ascorbic acid and reducing sugars in fruits and vegetables.
With respect to Eh requirements of microorganisms, some bacteria require reduced
conditions for growth initiation (Eh of about -200mV), while others require a positive Eh
for growth. In the former category are the anaerobic bacteria e.g. Clostridium, in the
latter belong the aerobic bacteria e.g. Bacillus.
Some aerobic bacteria grow better under slightly reduced condition and are called
microaerophiles e.g. Lactobacilli and Campylobacter.
Some bacteria have the capacity to grow under either aerobic or anaerobic conditions.
Such types are referred to as facultative anaerobes.
In regard to Eh of foods, plant foods (especially plant juices) tend to have Eh values of
from +300 to +400. It is not surprising to find that aerobic bacteria and moulds are the
common cause of spoilage of products of this type. Solid meats have Eh values of around
-200mV; minced meat Eh is +200mV; cheeses have Eh values from -20 to around
-200mV.
Most fresh plant and animal foods have low and well-poised O-R values in their interior.
This is due to the presence of reducing sugars and ascorbic acid in plants, and sulphydryl
group in animal tissues, so long as the food remains alive. There could be anaerobic

spoilage internally while aerobic spoilage proceeds on the surface. Processing e.g.
heating will remove this poising capacity.
(4) Nutrient content:

This is considered under 3 different aspects:
(a) Food for energy.
(b) Food for growth.
(c) Accessory food substances i.e. vitamins and related growth factors.
(a) Food for energy-

This means source of carbon and carbohydrates are mainly used. Others include esters,
alcohol, peptide, amino acid and organic acid and their fresh salts. The carbohydrates
used are largely in their simpler forms, which are soluble. Even here, the microbes differ
in their ability to use this. Many microbes are unable to use lactose and thus cannot grow
in milk whereas some yeasts are not able to use maltose. The ability of bacteria to use
some sugars is used as a classification index. Glucose is most universal for carbohydrate
users.
Those few organisms that are able to use fat for energy will only do so, if sugar is absent.
The same applies to proteolytic organism, which use some by-product of proteolysis for
energy.
Efficient utilization of the food depends on the presence of other food substances and the
osmotic pressure exerted by the kind of food energy (i.e. its concentration), e.g. 10%
glucose exerts half the osmotic pressure of 10% maltose or sucrose. The difference is
determined by their molecular weight.
(b) Food for growth-

Nitrogenous compounds mainly and, organisms differ in their ability to use these
compounds. Many organisms have no protease and depend on proteolytic organisms to
hydrolyze protein for them. The importance of any protein source depends on the types of
by-products formed such as peptides, urea, ammonia and amino acids, which may or may
not be available to other microbes. The usage is also affected by environmental

conditions. Some organisms e.g. lactic acid bacteria use polypeptides mainly and are
affected if only a limited amount of amino acid is present. If fermentable carbohydrate is
present, fermentation takes place to the suppression of the proteolytic organism. This is
known as "sparing" action on the nitrogenous compounds. It helps also to prevent the
production of unwanted nitrogenous compounds.
Many moulds are proteolytic, few genera of bacteria and fewer yeast have the ability.
Most of the bacteria will grow around pH 7 and source of carbon is usually organic,
although sometimes it is from carbon dioxide.
(c) Accessory food substance (vitamins)-

Vitamins are important as trace elements, they are usually present with few exceptions.
They are important because not all microbes are able to produce theirs. Most natural plant
and animal food have enough of different types but the amount depends totally on the
type of food while some may be totally lacking. For example, meat has high B vitamin
unlike fruits, which are also high in ascorbic acid. Processing removes some vitamins
such as thiamine, pantothenic acid, the folic group and ascorbic acid - all of which are
heat labile. Long storage especially and elevated temperatures have the same effect.
Each microbe has specific requirement, for some, this has a wide range. Example, the
coliforms that grow in a wide range of media. For others, the range is so narrow that
sometimes the vitamin has to be provided artificially. Luckily, these are mainly the
pathogens.
The presence of the necessary vitamin influences the type of food for energy or growth
the organism can use, and the level to which such food can be used. Some bacteria e.g.
Staphylococci synthesize part of their vitamins. Others, e.g. Pseudomonas, E. coli
synthesize all and others cannot, and most have them provided in media e.g. lactics and
many pathogens.
(5) Inhibitory/Antimicrobial constituents:

The stability of some foods against attack by microorganisms is due to the presence of
certain naturally occurring substances that have been shown to have antimicrobial

activity. This may be naturally present, resulting from microbial activities on the food or
be added artificially during processing. The action of the inhibitor may affect all
microbes or some specific types.
(a) Example of naturally present inhibitor includes lactenin an anticoliform factor present
in fresh milk, lysozyme in egg-white, allicin in garlic and benzoic acid in cranberries.
Lipids and essential oils, especially eugenol in cloves and cinnimic aldehyde in
cinnamon, all possess antimicrobial properties.
(b) Microbes growing on a food may produce some inhibitory by-products such as acids,
alcohols, peroxide and antibiotics, e.g. propionic acid by Propionibacterium
shermanii used in Swiss cheese manufacture inhibits moulds; alcohol produced by
yeast in wine inhibits competitors.
But some microbes are able to destroy inhibitory substances in foods, e.g. some
bacteria and moulds destroy phenolic compounds present in smoked fish and meat,
benzoic acid added to foods. Yeast resistant to sulphur dioxide can destroy SO2 and
Lactobacilli can inactivate nisin. Heating foods may destroy some of the heat labile
inhibitors or help form new ones, e.g. browning of sugar result in production of
furfural and hydromethyl furfural, which inhibits fermentative organisms.
(6) Biological structures:

Biological barriers such as skin, shells in eggs, skin on fruit and vegetables, all must have
a role in keeping the inner part of most of our fresh foods sterile. Only when these
barriers are breached, will these be contaminated. The natural covering of some foods
provides excellent protection against the entry and subsequent damage by spoilage
organisms. For example, the testa of seeds, the outer covering of fruits (e.g. fruits and
vegetables with damaged coverage undergo spoilage much faster than those not
damaged), the shell of nuts (e.g. once cracked the walnut/nutmeg are subject to spoilage),
the shells of eggs (e.g. egg when stored under proper conditions of humidity and
temperature can prevent entry of microorganisms when shells are intact) and the hide of
animals (e.g. the skin covering of fish and meats such as beef and pork prevents the

contamination and spoilage of these foods, partly because it tends to dry out faster than
freshly cut surfaces). All these are biological structures.
Artificial protection such as polyethene or plastic or wax serve some purpose- does not
prevent spoilage but determine types of contaminant and hence type of spoilage.
EXTRINSIC PARAMETERS
The extrinsic parameters of foods are those properties of the storage environment that
affect both the foods and their microorganisms. They include:
(1) Temperature of storage.
(2) Relative humidity (RH) of the environment.
(3) Gases in the environment.
(4) Presence and activities of other microorganisms.
(1) Temperature:
Microorganisms grow over a wide range of temperatures. Therefore, it would be
important to consider the different temperature growth ranges for organisms in foods as a
guide in choosing the appropriate temperature for the storage of different types of foods.
Those microorganisms that grow well at or below 7oC and have their optimum between
20oC and 30oC are said to be psychrotrophs. Those that grow well between 20oC and 45oC
and with optima between 30oC and 40oC are said to be mesophiles, while those that grow
well at and above 45oC with optima between 55oC and 65oC are said to be thermophiles.
Examples of psychrotrophic organisms (bacteria) commonly found in most foods are
Alcaligenes, Corynebacterium, Flavobacterium, Lactobacillus, Pseudomonas, Listeria,
Micrococcus and Enterococcus and others. They grow well at refrigerator temperature
and cause spoilage of meats, fish, poultry, eggs and other foods held at this temperature.
Mesophiles include Pseudomonas, Lactobacillus and Enterococcus faecalis e.t.c. They
are found in foods at refrigerator temperature, but they don't grow at this temperature.
They grow at mesophilic range, when other conditions are suitable- e.g. Enterococcus
faecalis grow over a range of 0oC to 30oC or above. Thermophiles commonly found in
most foods belong to the Bacillus and Clostridium genera. Although, a few species of

these genera are thermophilic, they are of interest to food microbiologists and food
technologists in the canning industry.
Moulds are able to grow over a wide range of temperature than bacteria. Moulds such as
Aspergillus, Cladosporium and Thamnidium can be found growing on eggs, sides of
beef and fruits. Yeasts grow over the psychrotrophic and mesophilic temperature ranges,
but generally, not within the thermophilic range.
The quality of the food product must also be taken into account in selecting a storage
temperature. While it would seem desirable to store all foods at refrigerator temperatures
or below, this is not always best for the maintenance of desirable quality in some foods.
For example, bananas keep better if stored at 13-17oC than 5-7oC. A large number of
vegetables are favoured by temperatures of about 10oC, including potatoes, cabbage and
many others.
It should be noted in every case, the success of storage temperature depends to a great
extent upon the relative humidity of the storage environment and the presence or absence
of gases such as CO2 and O3.
(2) Relative Humidity:

The relative humidity (RH) of the storage environment is important both from the
standpoint of Aw within foods and the growth of microorganisms at the surfaces. When
foods with low Aw values are placed in environments of high RH, the foods pick up
moisture until equilibrium has been established. Likewise, foods with a high Aw lose
moisture when placed in an environment of low RH. There is a relationship between RH
and temperature that should be borne in mind when choosing an appropriate storage
environment for foods. In general, the higher the temperature, the lower the RH, and vice
versa.
Foods that undergo spoilage from moulds, yeasts and certain bacteria should be stored
under conditions of low RH. Improperly wrapped meats such as whole chicken and beef-
cuts tend to suffer much surface spoilage in the refrigerator before deep spoilage occurs.
This is because the general high RH of the refrigerator and the fact that the meat spoilage
biota is essentially aerobic in nature. It is possible to lessen the chances of surface

spoilage in certain foods by storing under low conditions of RH. But the food itself will
lose moisture to the atmosphere under such conditions and thereby become undesirable.
In selecting proper environmental conditions of RH, consideration must be given to both:
(i) the possibility of surface growth and (ii) the desirable quality to be maintained in the
foods in question. By altering the gaseous atmosphere it is possible to retard surface
spoilage without lowering the RH.
The relative humidity of the atmosphere in equilibrium with the food is derived by
multiplying the Aw x 100.
Aw x 100 = Equilibrium relative humidity (ERH%).
(3) Presence and concentration of gases in the environment:

The storage of food in atmosphere containing increased amounts of CO 2 up to about 10%
is referred to as a "controlled atmosphere"(c-a) or "modified atmosphere"(MA) storage.
The effect of MA storage on plant organs was known since 1917. It was put into
commercial use in 1928. MA storage is used in a number of countries in treating fruits
such as apples and pears. Concentration of CO2 generally does not exceed 10% and is
applied either from mechanical sources or by use of dry ice (solid CO2).
CO2 has been shown to retard fungal rotting of fruits. The mechanism in retarding
spoilage is not known. It is believed that it acts as a competitive inhibitor of ethylene
action. Ethylene seems to act as a senescence factor in fruits and its inhibition would have
the effect of maintaining a fruit in a better state of natural resistance to fungal invasion.
Ozone when added to food storage environment has a preservative effect on certain
foods. It is effective against spoilage organisms and a variety of microorganisms. It is a
strong oxidizing agent and therefore it should not be used on high-lipid-content foods, as
rancidity would occur. CO2 and O3 are effective in retarding the surface spoilage of beef
under long term storage.

(4) Presence and activities of other microorganisms:
Some foodborne organisms produce substances that are either inhibitory or lethal to
others. They include antibiotics, bacteriocins, hydrogen peroxide and organic acids.
Antibiotics are secondary metabolites produced by microorganisms that inhibit or kill a
wide spectrum of other microorganisms. The useful antibiotics are produced by moulds
and bacteria of the genus Streptomyces. Some antibiotic-like substance is produced by
some Bacillus spp. and at least one antibiotic-like substance called nisin is produced by
some strains of Lactococcus lactis. Nisin contains the rare amino acids- meso-lanthionine
and 3-methyl-lanthionine. It is a bacteriocin- chemical compound produced by
microorganisms that inhibit or kill other microorganisms. For example,
Propionibacterium freudenreichii subsp. shermanii when cultured in pasteurized milk
produces a multicomponent inhibitory system that is effective against gram negative
bacteria and moulds in cottage cheese.
SUMMARY OF FOOD PARAMETERS

Each food product is characterized by a set of intrinsic parameters consisting of pH,
moisture content, O-R potential, nutrient content, antimicrobial substances, and/ or a
biological structure which is capable of preventing the entry of many microorganisms to
certain foods. When a given food is characterized with respect to these parameters, it then
becomes possible to make predictions about the perishability of that food by taking into
consideration the overall capacity of certain flora to grow within these parameters. The
microorganisms of importance in foods all have limitations relative to these parameters
and may be inhibited by one or more of them.
The extrinsic parameters of temperature of storage, relative humidity of the storage
environment, and the presence or absence of gases such as CO 2 and O3 may also be
employed to inhibit the growth of some microorganisms. The storage of dried foods in
areas of high RH. may offset the intrinsic parameter of water content and allow for
growth in an otherwise stable product. For example, citrus fruits are largely spoiled by
certain moulds; refrigerated ground beef is spoiled mainly by certain gram negative
bacteria; bread is spoiled mainly by certain moulds; etc.
MICROORGANISMS IMPORTANT IN FOOD MICROBIOLOGY

It is important to be familiar with microorganisms important in food because this will
help in their recognition, control and other uses if possible, of their various
characteristics. Moulds, yeast and bacteria are considered very important in food. Their
general characteristics will not be discussed in this lecture but the important aspects will
be highlighted.
MOULDS
Cultural characteristics:
The physical appearance of moulds on foods is often enough for identification purposes.
The shape, the size of the culture, texture and colour are all characteristics important in
identification.
Physiological characteristics:
(a) Moisture- moulds require less aw than yeast or bacteria. Total moisture content of
about 14-15% in food will greatly affect mould growth negatively, e.g. flours and
dried fruits mainly.
(b) Temperature- most moulds are mesophilic, i.e. optimum temperature is equal to 25 to
30oC. A few grow at 35 to 37oC, e.g. Aspergillus spp. Psychrotrophs exist- growth has
even been reported for some at below 5oC.
(c) Oxygen and pH- moulds are aerobic. pH range is wide: 2 to 8.5. Most prefer acidic
pH.
(d) Food requirement- they have diverse ability to use different foods, because they
possess wide range of hydrolytic enzymes.
(e) Inhibitors- they produce inhibitory substances against other microbes, e.g.
Penicillium chrysogenium produces penicillin. Some chemicals, e.g. sorbic and
propionic acid inhibits mould growth, while some kills them outrightly.
Some of the important genera known to occur in foods are:
Alternaria
Aspergillus
Aureobasidium
Botrytis
Byssochlamys
Cladosporium

Collectotrichum
Fusarium
Geotrichum
Monilia
Mucor
Penicillium
Rhizopus
Trichothecium
Wallemia
Xeromyces
Some are desirable in certain foods while some bring about spoilage or cause foodborne
illness.
YEAST
Refers generally to ascomycetes. They are single-celled and ovoid in shape. Yeast may be
useful or harmful in food. Useful aspects include fermentation in beers, wine, vinegar,
bread and cheese production. They are also grown for enzymes and for foods. Harmful
aspect includes fruit spoilage, juices, syrups, honey, wine, beer e.t.c.
Cultural characteristics:
Not very useful in identification but cause discoloured spots when growing on food. Film
yeast may form a thin film at the top of a liquid, they are mainly identified with the aid of
biochemical test.
Physiological characteristics:
(a) Moisture content- most yeast need high aw. They are able to withstand more solute
concentration than bacteria. Those that grow at high solute concentration are termed
osmophilic, while those on low solute are ordinary yeasts. Lower limits established so
far range from 0.88-0.94, though some osmophilic yeasts have been found at 0.62.
(b) Temperature- similar to those of moulds.
(c) pH- acidic (4.0-4.5) favours yeast.
(d) Food - sugars are mainly used but film yeast are able to use organic acids and alcohol.
By-products of their metabolism are important, e.g. CO2 in bread, alcohol, wine e.t.c.

Yeasts have a high capacity for mutation and can be adapted to various conditions or for
particular purposes.
Yeast groups:
Film Yeast- Pichia, Hansenula, Debaryomyces, Candida and Trichosporon grow well
on surfaces of acid foods such as sauerkraut, pickles, fruit juices, and oxidize organic
acids thereby raising pH and less tolerant organisms continue the spoilage. Hansenula
and Pichia tolerate high levels of alcohol and may be involved in its spoilage. Pichia
may impart flavours and esters on some types of wine. Debaryomyces tolerates high salt
solution and can grow on cheese brines with up to 24% salt solution. Film yeasts are poor
alcohol producers from sugars.
Apiculate or Lemon-shaped Yeast- some such as Hanseniaspora, Nadsonia and

Kloeckera are mainly spoilers of alcohols because of off-flavours and low alcohol yield.
Osmophilic Yeast- tolerates high solutes such as sugars and salts. Spoil dry fruits,
concentrated fruit juices, syrups, honey e.t.c., e.g. Saccharomyces rouxii and
Saccharomyces mellis.
Some important genera of yeast known to occur in foods are listed below:
Brettanomyces
Candida
Cryptococcus
Debaryomyces
Hanseniaspora
Issatchenkia
Kluyveromyces
Pichia
Rhodotorula
Saccharomyces
Schizosaccharomyces

Torulaspora
Trichosporon
Zygosaccharomyces
Some of them are desirable in certain foods while some are harmful.
BACTERIA
Morphological characteristics important in food microbiology:
Capsules- these are responsible for ropiness or sliminess of some foods such as bread,
milk. Also helps the organisms to resist harsh environments such as heat, chemicals e.t.c.
Spores- formed mainly by the genera Bacillus, Clostridium, Desulphotomacelum,
Sporolactobacillus and Sporosarcina. Bacillus and Clostridium are most important to
the food microbiologist. Endospores formed are very refractile, tolerant to heat, UV light
and desiccation. When cell is lysed, the spores remain dormant for years, until the
conditions are favourable then it germinates.
Cell aggregation- clumps or strings of cells are formed by some bacteria. These are more
difficult to kill than single cells.
Cultural characteristics important in food:

Bacterial growth on food makes it unattractive in appearance.
Physiology:
Growth in food produces chemical changes- hydrolysis of carbohydrate to simple sugars,
proteins to polypeptides, ammonia, amino acids, amines e.t.c., fats to glycerol and fatty
acids. Oxidation-reduction reactions for energy production. For pure knowledge of the
principles of food preservation and spoilage, it is important to understand the factors
affecting bacterial growth.
Some important genera of bacteria known to occur in food are listed below:
Acinetobacter
Aeromonas
Alcaligenes
Bacillus
Brochothrix

Campylobacter
Carnobacterium
Citrobacter
Clostridium
Corynebacterium
Enterobacter
Enterococcus
Erwinia
Escherichia
Flavobacterium
Hafnia
Kocuria
Lactococcus
Lactobacillus
Leuconostoc
Listeria
Micrococcus
Moraxella
Paenibacillus
Pantoea
Pediococcus
Proteus
Pseudomonas
Psychrobacter
Salmonella
Serratia
Shewanella
Shigella
Staphylococcus
Vagococcus
Vibrio

Weissella
Yersinia
SOURCES OF MICROBIAL CONTAMINATION OF FOOD

Microorganisms are everywhere in our environment and so can contaminate our foods.
Man's food whether from plant or animal origin harbour microorganisms on their
surfaces. Animals have microbes in their intestines too. However, the inner tissues of
healthy plants and animals contain few or no microorganisms at all. Food before or after
processing for eating are exposed to contamination with microbes from various sources:
(1) From green plants and fruits:
Plants are contaminated mostly by soil and water organisms, of these only a few persist
on the plants by virtue of their ability to adhere to the surface without being washed off
and thus able to obtain their nutritional requirement. The surfaces of these vary in
microflora but those usually found are Pseudomonas, Alcaligenes, Flavobacterium,
Micrococcus, coliforms and lactic acid bacteria. Yeast and moulds may be present. They
are transferred to the plants by raindrops, winds, insects and other crawling organisms. If
these plants are not treated to remove these bacteria and other organisms, they are
transferred into the plants. The population of microbes varies considerably. A well-
washed tomato has between 400-700 colony forming units per square centimeter
(cfu/cm2) while an unwashed tomato will have several thousand cfu/cm2. Some organisms
have been reported inside healthy fruits.
(2) From animals:

Animals are usually contaminated through their feed, their droppings and the soil. Thus,
their hides, hoofs, udder, feathers and so on are contaminated by microbes. Microbes are
usually present on the skin and in the respiratory and gastrointestinal tracts. The
important microbes are the spoilage ones, which include the ones from the intestinal tract
of the animal. Such foods can also serve as sources of infection to humans with
organisms such as Salmonella, Brucella, Mycobacterium tuberculosis, Coxiella,
Listeria betahemolytic, Streptococci, enteropathogenic Escherichia coli, parasites and
viruses being some of the transmissible ones.

(3) Sewage:
Other sources include sewage which when untreated and used for fertilizing plants serve
to contaminate raw fruits and vegetables. There is also the practice of using faecal matter
for the same purpose.
(4) Soil:
Microbes are also obtained from soil. The greatest variety of microorganisms are found in
the soil, thus, said to be the reservoir of all microorganisms. The largest repository of
microbes on earth are moulds, yeast, bacteria such as Bacillus, E. coli, Clostridium,
Alcaligenes, Proteus e.t.c.
(5) Water:
From water, there is the aspect of the natural flora coupled with contaminants from soil,
sewage, air and other sources. Contamination occurs when water is used in food
processing as (i) ingredient; (ii) washing; (iii) blanching and (iv) cooling (ice
manufacturing) e.t.c. All these must be considered especially where public health is
concerned. Escherichia coli has normally been used as an index for water purity.
(6) Air:
Organisms get into the air when wind whips up soil, when we sneeze, talk, flip up our
beddings, handkerchief and so on. Any food exposed to the air is at risk of contamination.
Microbes in air are suspended from various sources- soil, sewage, dust, sneezing,
coughing, growth of sporulating moulds e.t.c. Only very few, the spore formers survive
for long, when food is exposed they get into contact with it. They have no opportunity for
growth or multiplication but remain there. Desiccation kills off some while others
survive.
(7) Food handler:

The microflora on the hands and outer garments of food handlers reflect that of the
environment and habit of the individual. The handler of the food may serve to transfer
microbes onto it or use the surface on which the food is being handled to contaminate it
due to poor handling. The organisms may be from soil, water, air, gastrointestinal tract
(GIT), nasal and oral cavity. These usually have a build up of contaminants, thus general
hygiene is called for.
(8) Food utensils:

Food may be contaminated from utensils, surfaces and equipment used in its preparation.
Utensils (pots, cans, knives, cutting boards e.t.c.) used in the preparation of food are
hardly ever washed, and in cases where they are washed, the conditions under which they
are kept are not hygienic. This leads to a situation where subsequent use of such utensils
may contribute to the degree of contamination of food by microorganisms.
FOOD SPOILAGE
Food, which as a result of improper storage becomes unfit for human consumption is said
to be spoilt.

Fitness for food
A product is fit for 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 unfit for
food if when such an examiner refuses it as food (Thom and Hunter, 1924). This
statement solely depends on the taste of the individual e.g. most British leave their meat
to age (hang it for sometime) for the resultant flavour, while Americans will consider this
spoilt. Important to note is the fact that starving people will eat foods they will not
normally eat.
Despite individual differences, foods must meet certain criteria for assurance of fitness.
These include:
1) Reaching the desired stage of maturity e.g. fruits.
2) Freedom from pollution at any stage in production or handling.
3) Freedom from objectionable change due to microbial attack or action of enzymes
on the food. The issue of change is also dependent on certain criteria because
some changes may be desirable in certain foods but undesirable in others. Also,
the change termed spoilage may be in appearance, e.g. wilted lettuce or carrot,
which actually have neither undergone microbial spoilage nor lost any nutritive
element.
Definition and Explanation of some terms:
Food spoilage: by this we mean any organoleptic change in a food, which the consumer
considers to be an unacceptable departure from the normal.
Organoleptic change: this means any tactile, visual, olfactory and flavour change as it
relates to food. Spoilage is the common fate of all foods, though the rate of spoilage may
differ from one food type to another- for example, fruits, vegetables, fresh milk and meat
will spoil in a matter of a few days, while cereal grains, dry beans and other dry foods
may keep for almost a year or more without signs of spoilage. Food spoilage is normally
preceded by deterioration in quality.
Deterioration: this should be understood as the adverse changes in the quality of the
food, which is induced by chemical, physical and biochemical reactions taking place
within the food. A better illustration of these concepts will emerge from the following
examples; consider a mature green fruit left on the table in ambient environment of a

kitchen. With the passage of time, this fruit will pass through the following stages
sequentially:
(i) Ripening- a series of complex chemical and biochemical reactions within the
fruit tissues. Such reactions are recognizable through eventual changes in
colour (green to yellow), odour and texture (firm to soft).
(ii) Deterioration- this is the stage coming immediately after ripening and is
characterized by increased changes in colour (e.g. yellow to darkening), odour
and softening of the tissues to a point where the fruit disintegrates easily. At
this stage the fruit is said to have deteriorated in quality. Some may consider
the food spoilt at this stage.
(iii) Spoilage- next to the deterioration stage comes spoilage, and at this stage the
food is invaded and colonized by fungal mycelia and is characterized by
further softening of the inner tissues and in some cases offensive odor.
The above sequential changes ending in spoilage derived from the fact that all
biological systems are endowed with built-in mechanisms that regulate growth,
maturation, death and disintegration, followed by microbial decomposition.
Causes of Food Spoilage

Spoiled food actually implies decay or decomposition. Foods unfit for consumption due
to sanitary reasons, are not called spoilt but adulterated. Spoilage could be due to:
1) Growth and activity of microbes- sometimes several types in succession.
2) Presence of insects.
3) Action of the intrinsic enzymes of the plant or animal food.
4) Purely chemical reactions not involving the intrinsic enzyme of the food.
5) Physical changes due to freezing, burning, drying, pressure e.t.c.
Mechanisms of Food Spoilage
A. Chemical and biochemical reactions from within the food (autolysis) with or without
enzyme participation.
a) Chemically induced reactions without the participation of enzymes.
(i) Oxidative Rancidity of fats and oil, particularly those of plant origin. The
mechanism here involves the uptake of oxygen by non-conjugated unsaturated

systems in fats and oil to yield peroxides. Peroxides take up hydrogen from
the reaction environment to form hydroperoxides. Hydroperoxides, on
attaining a critical level, breakdown to release odouriferous compounds
(aldehydes, ketones and acids). These compounds are responsible for the off-
flavour characteristic of oxidized fats and oils.
Polyunsaturated fats and oils + O2 heat, light → Peroxides + H+ →Hydroperoxides
Dintegration
→Aldehydes, ketones, acids e.t.c.
Oxidative rancidity is encouraged by light, high temperature, certain divalent and
polyvalent metals. Lipoxidase enzyme found in some oil seeds has been shown to
catalyze the oxidation of fats and oils too. Nevertheless, oxidative rancidity is more
widespread and typical for fats and oils.
(ii) Caramelization Reaction
Heat Polymerization
Sugars → Anhydrous sugars → Brown caramel
Sugars when heated under controlled conditions in the absence of water, form
anhydrous-sugars, which readily polymerize to produce brown pigments called
caramels. Caramelization is desirable in sweets, toffees and syrups but is not
appreciated in a number of products where brown colour is undesirable. Such
products are considered spoilt.
(iii) Maillard Reaction
Polymerization
Reducing sugar + Primary or Secondary amine → Amine sugar →
Melanoidins (brown)
Reducing sugars such as glucose and lactose (not sucrose) when in contact with
amino acids, proteins and other primary and secondary amines will form amino
sugars. These polymerize to form brown coloured melanoidins. The sequence of
reactions leading to melanoidin formation is called Maillard reaction.
(iv) Off-colour development in red meats:
Myoglobin + O2 → Oxymyoglobin → Metamyoglobin
(pinkish) (bright red) (brownish)

Freshly cut meat surface in contact with oxygen of the air becomes bright red due to
oxymyoglobin. Further exposure to oxygen leads to the formation of metamyoglobin
(brownish). Although, these changes in colour have no serious negative effect on the
nutritive value of the meat, consumers invariably will regard such meat as spoilt and
reject it.
b) Reaction within the food, due to uncontrolled activities of enzymes:

Enzymes are biological catalysts, which accelerate the rate of specific chemical
reactions in biological systems without undergoing any change at the end of the
reaction. Uncontrolled activities of certain food enzymes result in the development of
spoilage symptoms as exemplified below:
(i) Off-flavour development (rancidity) in fats and oils- lipolysis
Lipase
Fat + H2O → Free fatty acids + Glycerol
In the presence of water, lipases hydrolyze fats or glycerides to yield fatty acids and
glycerol in a process often referred to as hydrolytic rancidity. Hydrolytic rancidity is a
common phenomenon in palm oil extracted from palm fruits with water if the oil has
high residual water content. Off-odour perse is not a regular characteristic of
hydrolytically degraded fat. This becomes the case when the released fatty acids are
of low molecular weight and therefore volatile at ambient temperature. A good
example is milk fat that gives out off-odour on hydrolysis due to the resultant low
molecular weight fatty acids (mainly butyric acid). This is not the case with
groundnut and palm oils, which contain higher molecular weight fatty acids. Taste
will however change.
(ii) Food browning mediated by enzymes

Phenolase Autopolymerization
Polyphenol + O2 → Orthoquinone → Brown pigment
i.e. Melanin

Enzymes that promote food browning are often collectively referred to as phenolases.
They catalyze the oxidation of polyphenols particularly the orthophenols to the highly
reactive intermediate known as quinines in the presence of oxygen and phenolase.
The quinines polymerize spontaneously to brown coloured polymers (melanin).
Enzyme-mediated browning is a common event in peeled or injured Irish potatoes,
yams, cocoyams, apples and many other fruits and vegetables which are rich in
polyphenols. The enzyme phenolase and oxygen are readily available in the food
environment. A cut or injury to the plant material (cell) is needed to allow the enzyme
and O2 make contact.
To prevent enzymic browning, freshly peeled yams or potatoes should be kept under
water or cut surfaces of such foods should be rubbed with oil to keep-off oxygen.
(iii) Trimethylamine formation in fish
Enzymes released in a dead fish by the gut flora mediate the spoilage of the fish
through the production of trimethylamine (with offensive NH3-like smell) from
nitrogenous compounds in the fish muscle.
B. Spoilage due to the activities of microorganisms:

Microbial spoilage of foods is brought about by the development and biochemical
activities of microorganisms (bacteria, yeasts and moulds). The spoilage process
starts with the invasion of the surface and later the interior of the foods by the specific
spoilage organisms. Not all microorganisms have the potential to cause food spoilage.
Only those microorganisms with the structural and physiological properties which
cause the breakdown of (a) the natural defenses of the fresh food (e.g. the shell of
eggs, testa of seeds, cuticle of vegetables), (b) the physical and chemical selective
factors operating in the food environment, can colonize, invade and spoil a given
food. Such physio-chemical selective factors include temperature, pH, oxidation-
reduction potential, water activity, antimicrobial constituents of the food environment
e.t.c. The way these factors operate, individually and in combination cannot be
discussed here. Virtually, all cases of microbiologically induced spoilages are enzyme
mediated. The type of spoilage encountered depends on a number of factors, one that

is chemical composition of food. This will determine the nature and composition of
the degradation products produced. For example,
a) Carbohydrates are degraded to various organic acids and CO2 by bacteria, yeasts
and fungi.
Glucose Lactobacilli
Fructose } → Lactic acid
Lactose
b) Lipids in butter and cheese are hydrolyzed by numerous bacteria and fungi to free
fatty acids and glycerol.
c) Proteins are degraded to mixtures of polypeptides, peptides and amino acids and
their degradation products such as NH3, H2S and indole by proteolytic anaerobic
bacteria.
All these metabolic degradation products of carbohydrates, proteins and fats impart
undesirable odours and flavours to foods, in some cases the products may be toxic.
Classification of Food According to the Ease of Spoilage-
Foods can be classified into three main groups on the basis of their shelf-life or
perishability:
1) Stable or Non-perishable foods- these foods do not spoil unless they are handled
carelessly. They should be stored in a cool, dry place. They can be stored for one
year. They should be picked and cleaned before storage. For instance, grains can
be washed with water to remove any dust and dirt sticking to them. These should
then be dried in the sun, allowed to cool and stored in containers with tight fitting
lids. Examples include sugar, flour, whole grains, whole nuts, dry salted meat and
fish, hydrogenated fat, vegetable oil, canned foods, jams e.t.c.
2) Semi-perishable foods- will remain stable for fairly long period if properly
stored. They are less likely to decay due to microbiological contamination than
other perishable foods. Natural chemical breakdown is also slower in such foods.
If they are stored in a cool place with adequate ventilation they have a moderately
long shelf-life. Foods of this group can be stored for a week to a couple of months
at room temperature without the development of any undesirable changes in

flavour and texture. Examples include potatoes, onions, some types of apples,
dates, nuts, processed cereals, frozen foods (kept at 0 to -180C) e.t.c.
3) Perishable foods- most of the foods eaten daily belong here. These foods contain
high amounts of protein, moisture and other nutrients; they are an ideal medium
for microbial growth. They also spoil easily by natural enzymatic changes. They
have a very short shelf-life of a few hours to a few days, after which they spoil
rapidly. Examples include most fruits and vegetables (such as bananas, pineapple,
papaya, green leafy vegetables, tomatoes e.t.c.), meat, eggs, poultry, meat, fish,
milk and milk products e.t.c.
Factors Affecting Kind and Number of Microbes in Foods

Plant and animal foods usually contain some microbes along with their intrinsic enzymes.
Type of organisms present will depend on those that are able to strike an equilibrium
between other types of organisms present and their enzymes. Only one or a few groups
may be able to reach population that can cause spoilage. Sometimes there is a succession
of spoilage organisms. The following factors greatly influence the kinds of microbes
present:
1) Contamination- this may increase or add new microbes to the food including
spoilage organisms. They may come from handlers, wash water, processing
surfaces e.t.c. These factors increase risk of spoilage and decreases ease of
preservation.
2) Growth- of microbes in or on the food increases the microbial load and this
increases the chances of spoilage of the food.
3) Pretreatments- could change the whole course of the microbial changes in the
food. Microbes may be removed or added by this process, these include washing
with antiseptics, treatment with UV light, ozone, sulphur dioxide or germicidal
vapours, high temperature and storage conditions.
Factors Influencing Microbial Growth in Foods

1) Associative Growth- since there are various kinds of microbes present on a fresh
food, different levels of competition exist, with all conditions provided. Bacteria grows
faster than yeasts and yeast grows faster than moulds. Some of the microbes may be
antibiotic to each other, symbiotic or synergistic.

Most important is metabolic effect, one microbe makes conditions favourable for
another’s growth. This is what happens in succession of microbes in spoiled foods e.g. in
raw milk, Streptococcus lactis grows first, reducing the pH until the acidity kills them,
then Lactobacilli grows and reduce the pH further. They are followed by yeast and
moulds, which now reduce the acidity and proteolytic bacteria grows.
2) Effect of environmental conditions- these conditions are interrelated and determine

which organisms will outgrow each other in a food. The factors include physical and
chemical state of the food, oxygen tension(availability of oxygen) and temperature.
i) Physical state and structure of the food, whether it is has been frozen, heated,
moistened or dried, together with its biological structure may determine whether the food
will spoil and the type of spoilage.
The water content of the food and whether it is available to the microbes (Aw) is very
important in determining the growth of organisms. This is of course related to the relative
humidity of the environment about the food. If relative humidity is lower than Aw of food,
it loses water and from the inner parts, more water is drawn out. Spoilage of solid foods
is restricted or begins at the surface mainly. The Aw of the surface influences the type of
microbe e.g. bread will favour moulds more; syrups and honey will have osmophilic
yeast; meat; fish and eggs will have bacteria. Aw of 0.70 may not favour most spoilage
organisms at room temperature. Bacillus subtilis in bread causes ropiness by hydrolyzing
starch.
Freezing prevents microbial growth but also damages tissues releasing moisture, which
can be used for growth by organism when the food thaws. Likewise, heat treatment may
kill microbes but it will change the physical state of the food.
ii) Chemical properties of the food- this determines the suitability of the food as a
substrate for microbial activities. They include pH, nutrient content, moisture content,
oxidation-reduction potential and inhibitory substances.
iii) Temperature- if unsterile and moist, any food can spoil at temperatures from –5 to
70oC. The holding temperature of the food will determine the type of microbes present,
35 to 37oC and above do not favour moulds and yeast. But they grow very well at room
temperature.
Chemical Changes caused by Microbes

Since there are numerous kinds of organic compounds in foods, the changes in food will
depend on what chemical compound is being metabolized.
(1) Changes in nitrogenous organic compounds- nitrogen in food is largely protein. This
is hydrolyzed to peptides, polypeptides, and amino acids using proteases. Anaerobic
breakdown of protein, peptides or amino acids results in offensive odours- putrefaction.
(2) Changes in non-nitrogenous organic compounds-
(a) Carbohydrate- broken down to give energy. Aerobically, water and carbon dioxide are
the end products. Anaerobically, we get one of 6 different fermentation steps viz:
(i) Alcoholic fermentation e.g. yeast- ethanol and carbon dioxide produced.
(ii) Simple lactic fermentation- homofermentative organisms e.g. lactic acid
bacteria.
(iii) Mixed lactic fermentation e.g. heterofermentative lactic acid bacteria.

(iv) Coliform type of fermentation- lactic, acetic and formic acids, ethanol, carbon
dioxide, hydrogen produced.
(v) Propionic fermentation- Propionibacterium producing propionic acid, acetic
acid.
(vi) Butyric- butyl-isopropyl fermentation by anaerobic bacteria.
(b) Organic acids- usually present as salts are oxidized to carbonates, causing the medium
to become more alkaline.
(c) Lipids- lipids are hydrolyzed to fatty acids and glycerol. Phospholipids are broken
down to their phosphate, fatty acids and nitrogenous base e.g. choline.
(d) Pectic substances- in plants, the water-insoluble parent pectic substance (protopectin)
is converted to pectin (a water-soluble polymer of galactoronic acid, which contains
methyl ester linkages). Pectinesterase hydrolyzes methyl ester linkage of pectin to
yield pectic acid and methanol.
FOOD PRESERVATION
There are 8 groups of foods. Four each of plant and animal origin.
Foods of Plant origin:
(i) Cereals and their products.

(ii) Sugar and their products.
(iii) Vegetables and their products.
(iv) Fruits and their products.
Foods of Animal origin:
(i) Meat and their products.
(ii) Poultry and eggs.
(iii) Fish and sea food.
(iv) Milk and their products.
Most of these foods are readily decomposed by microbes while others are fairly stable.
The perishable ones can fairly be kept for long period if special preservative methods are
used.
METHODS OF FOOD PRESERVATION:
(i) Asepsis- means prevention of contamination (keeping out microbes).
(ii) Removal of microorganisms in case where contamination has already
occurred.
(iii) Maintenance of anaerobic condition especially in sealed containers.
(iv) High temperature treatment denatures enzymes and kills off microbes.
(v) Low temperature slows down microbial growth and therefore preserves food.
(vi) Drying reduces aw by tieing up water, using solids or hydrophilic colloids.
(vii) Chemical preservation could be natural (as produced by microbes) or
synthetic.
(viii) Irradiation- UV light kills off bacteria.
(ix) Mechanical destruction e.g. grinding, high pressure treatment e.t.c.
More often than not, a combination of two or more of those methods is used. The net
effect is more efficient and not the full dose of any of the methods is required.
PRINCIPLES OF FOOD PRESERVATION

There are 3 general principles underlying food preservation:
(a) Prevention or delay of microbial decomposition-
(i) asepsis, (ii) removal of microorganisms-filtration, (iii) preventing growth and
activities of microbes e.g. low temperature, drying, chemical preservatives e.t.c., (iv)
killing the microbes- heating and irradiation.
(b) Prevention or delay of self decomposition-
(i) destruction or inactivation of enzymes-blanching, (ii) prevention or delay of purely
chemical reactions, e.g. oxidation, using anti-oxidants.
(c) Prevention of physical damage, insects, animal e.t.c.
DELAY OF MICROBIAL DECOMPOSITION

The principle here is to delay or prevent the growth of microbes present in the food. This
does not attempt to remove the microbes already present but rather tries to prevent their
increase in population. The microbial growth curve is usually taken into consideration.
The main aim being to lengthen the lag phase, as long as possible. The ways to achieve
this are as follows:
(a) Preventing or limiting contamination with spoilage micro-organisms.
(b) Avoid contamination with organisms already in the log phase.

(c) Make the environmental conditions unfavourable for the growth of the
organisms e.g. low moisture, pH, oxidation-reduction potential, presence
of the inhibitor or unfavourable foods.
(d) Damage the organism using heat, irradiation e.t.c.
The more unfavourable the condition, the longer the generation time, e.g. a drop in
temperature will increase generation time. If one cell has 30 minutes generation,
1,000,000 cells will be available in 10 hours, but only 1,000 if the generation time is 60
minutes.
PREVENTION OF MICROBIAL DECOMPOSITION OF FOODS

If microbial growth is prevented, those present could still cause spoilage, although at a
slower rate. They have to be completely removed and recontamination prevented, if
spoilage is to be prevented. In this, care must be taken to avoid build up of strains
resistant and the following aspects must receive special attention:
(A) Asepsis-
The best way to avoid microbial spoilage is to avoid contamination of the food. In nature,
the asepsis is demonstrated by the inner tissues of the healthy plants and animals, which
are free of microbes. If microbes are present, they are unlikely to cause spoilage.
Protective coverings such as shell of nuts, skin of fruits, which provide good protection.
The ‘load’ of the microbes on or in the food and its kinds and numbers are important. The
kinds may include spoilage organisms, those desirable in fermentation or even pathogens.
The number determines the ease of preservation or spoilage of the food. To achieve
asepsis in industries, the following practices have been employed:
(a) Packaging of foods- wraps, cans e.t.c.
(b) Avoid simple contamination during production and handling of foods, e.g.
dairy industries.
(c) Heating of sealed evacuated cans.
(d) Sanitary methods in meat-packing industry, e.g. abattoirs during slaughter,
handling and processing.
(B) Removal of Microbes-

May not achieve much but helps. It can be done through filtration, centrifugation,
washing, trimming e.t.c. Filtration is the most successful and depends on the pore size of
the filter, e.g. asbestos pad, sintered glass, diatomaceous earth, unglazed porcelain,
membrane pads e.t.c. The liquid is filtered through a previously sterilized ‘bacteria-proof’
filter made of sintered glass, diatomaceous earth, unglazed porcelain, membrane pads, or
similar material, and the liquid is forced through by positive or negative pressure.
Example of food sterilized by filtration are fruit juices, beer, soft drinks e.t.c.
Centrifugation or sedimentation used in drinking water, sometimes is not generally
effective, because some but not all of the microbes are removed. When centrifugation is
applied to milk, it is used to take out other suspended materials and not to remove
bacteria, although centrifugation at high speeds removes most of the spores.
Washing only removes some but not all microbes, but may be dangerous if the water adds
spoilage organisms or increases the moisture so that growth of spoilage organisms is
encouraged.

Trimming is removal of spoiled portions of a food or discarding spoiled samples.
(C) Maintenance of Anaerobic Conditions

This helps to keep foods in sealed containers. ‘Complete fill’ or evacuation of headspace
or replacement with carbon dioxide or nitrogen will make conditions anaerobic. Spores of
some aerobic spore-forming microbes are resistant to heat and may survive in canned
food but may be unable to germinate or grow in the absence oxygen. Production of
carbon dioxide during fermentation and its accumulation at the surface will help to make
conditions anaerobic there and prevent the growth of aerobic microbes.
(D) Application of Heat (High Temperature) in Food Preservation

Heating kills microbes by denaturing proteins and inactivating enzymes. The type and
degree of heat treatment depends on the types and number or population of microbes to
be killed, and whether they are vegetative or spores. The degree of heat treatment on the
foods. To fully understand the effect of heat treatment on microbes, it is important to
examine the factors that affect the heat resistance of cells and spores.
Factors Affecting Heat Resistance
Heat resistance by microbes is measured by the time it takes for the heat at given
temperature to cause the death of those microbes. This resistance is not equal between
cells and spores.
The factors which control this include the following:

1) Temperature-time relationship: the time it takes to kill cells or spores under a
given set of condition reduces as the temperature increases. For example, Bigelow
and Esty (1920), showed that for 115,000 spores/ml of Bacillus
stearothermophilus (flat sour bacteria) the following was obtained:
Temperature (oC) Time to destroy all spores (minutes)
100 1,200
110 190
120 19
130 3
135 1
2) Initial concentration of spores or cells: the higher the number of cells or spores,
the greater the heat treatment required to kill them off. Again, according to
Bigelow and Esty (1920), at 120oC and pH 6.0:
Initial conc. of spores, number/ml Time required to kill all spores, min. at 120oC
50,000 spores 14 minutes
5,000 10 minutes
500 9 minutes
50 8 minutes
3) Previous history of the cells/spores: this mainly refers to the condition under,
which the cells/spores are formed. The more favourable, the condition, the higher
resistance of the cells/spores.

(a) Culture medium- the richer the medium in which the cells/spores grow, the
more resistant they will be.
(b) Incubation temperature- resistance is promoted by optimum growth
temperature and further enhanced if the temperature approaches the
maximum for growth.
(c) Phase of growth or age- vegetative cells are most resistant during late lag
phase and maximum stationary phase but least resistant in logarithmic
phases, spores are more resistant in the first few weeks and therefore
decline.
(d) Desiccation- some spores are more resistant when dried.
4) Composition of substrate in which cells/spores are heated:
(a) Moisture- the type of heat applied depends on the type of food material.
Moist heat is more effective than dry heat. For moist heat, heating at
120oC/15minutes achieves sterilization. The same effect is achieved for
dry heat at 170oC/2 hours or 180oC/1 hour.
(b) pH- neutral pH favours heat resistance but acid pH is more effective than
alkaline. This factor is especially important in canned foods and have been
classified as follows by Cameron (1940):
(i) Low acid foods- pH > 5.3 e.g. meat, fish, poultry, milk, corn, beans e.t.c.
(ii) Medium acid foods- pH 5.3 to 4.5 e.g. spinach, asparagus, pumpkin.
(iii) Acid foods- pH 4.5 to 3.7 e.g. tomatoes, pears, pineapples e.t.c.
(iv) High acid foods- pH 3.7 and below e.g. berries, sauerkraut, those food
with the least pH require the least treatment.
The heat process required in the canning of foods will increase with their pH
(for the listed acid foods).
(c) Other constituents of the foods- NaCl in low concentration (0.5 – 3.0%)
protect some spores i.e. increase the heat resistance of microorganisms. At
higher concentration however, the effect is reversed. So, also does sugar
and colloid substances (especially proteins and fats, are protective against
heat) because they tie up moisture to protect the organisms. Antiseptics
and germicidal compounds reduce the resistance of organisms. The
presence of these in a given food increases the effect of heat on
microorganisms. This explains the use of hydrogen peroxide (H 2O2) in
combination with heat to reduce the bacterial content of sugar and milk.
Concept used in Heat Treatment of Foods- Thermal Death Time (TDT) and
Thermal Death Point (TDP)
All modern thermal processing is based on two concepts- TDT and TDP.
The heat resistance of microbes is expressed in terms of thermal death time (TDT).
TDT is the time needed to kill a stated number of cells or spores under specific condition
at a particular temperature.
Majority thermal death time (MTDT) - is time needed to kill most of the cells or
spores. Thermal death rate (TDR) - expressed as the rate of killing.
Thermal death point (TDP) - temperature required to completely destroy cells or
spores, within a stated time or temperature needed to kill all cells in 10 minutes.

The Thermal Death Time (TDT) Curve
Using the growth-no growth procedure, a series of tubes in specific replicates (e.g. 6) can
be used to determine the TDT curve. At different heating temperatures of 110 oC, 115oC
and 120oC the experiment can be performed as follows. At specific intervals, replicates of
tubes can be removed and plated to see if any cell survived. The point at which no growth
occurred is the destruction point, and the point at which the last assay showed growth is
the survivor point. Example,
Temperature Time (min.) Number of tubes Number positive
o
110 C 25 6 6
40 6 6
80 6 5
110 6 0
o
115 C 10 6 6
14 6 6
22 6 2
28 6 0
o
120 C 3 6 6
4 6 6
7.5 6 0
TDT decreases logarithmically as temperature increases. Bigelow and Esty demonstrated

that strains of thermophiles differ in their heat resistance. Bacillus stearothermophilus
strain No. 1518 is the standard test organism for studies in heat processing of low-acid
foods.
Thermal death times are plotted as logarithms on semi-logarithmic graph paper and
temperature as arithmetic values to give a thermal death time curve. The curve is a
straight line indicating that the order of death by heat is logarithmic, i.e. that the death
rate is constant.
Z-value: this is the interval in temperature (oF) required for the line to pass through one
logarithmic cycle (the slope of the line). It also represents the degrees Fahrenheit ( oF)
required to reduce the thermal death time ten-fold.

F-value: time in minutes required to destroy the organism in a specified medium at 250oF
(121.2oC). Both the Z- and F-values will vary with the heat resistance and concentration
of the test organism and with the medium in which it is heated.
From the Z and F values, the process time (t) can be calculated as follows,
tmin = F antilog 250 – T
Z
The above formula is used to calculate the Process time (t) required to destroy or kill
microorganisms at temperature T in canned foods.
Decimal Reduction Time (D-VALUE)

Formerly it was common to express heat resistance in terms of lethal combinations of
time and temperature (TDT, TDP, and F-value). Later the D-value (the D symbol is used
for the decimal reduction time) was introduced, i.e., that time in minutes of heating at a
specified temperature to cause a 90% reduction in the count of viable spores (or cells).
This is the time for the survivor curve to traverse one logarithmic cycle. D-value is not
completely adequate either. Prof. Mossel of the University of Utrecht has proposed the
use of an integrated parameter- MPED n (Most Probable Effective Dose of thermal energy
to achieve n (overall decimal reductions in cell numbers under given set of conditions of
temperature, pH, water activity e.t.c).
Note that over the range of temperatures usually used in food sterilization, the
relationship between D and temperature is essentially exponential. Log D plotted against
Temperature therefore gives a straight line, the slope gives a quantitative measure of the
sensitivity to heat and the graph can be used to calculate process time for food
sterilization.
Food Sterilization- Autoclaving

The aim is usually to kill all microorganisms including bacterial endospores. Usual
procedure is to heat at 1.1kg/cm2 (15 lb/in2) steam pressure, which yields a temperature of
121oC. Heating is usually done in an autoclave, although pressure cookers may be used
for small batches of materials. At 121oC, the time of processing is 10 – 15 minutes
(exposure time only), because for bulky batches, heat transfer to the interior will be slow.
Longer exposure times will also be required when large volumes of liquids are being
autoclaved because large volumes take longer time to attain sterilization temperature.
Vegetative Cell, Spores and Heat Resistance
Vegetative cells and bacterial endospores vary considerably in heat resistance. For
example, in the autoclave a temperature of 121oC is normally reached at which condition
endospores may require 4 to 5 minutes for a decimal reduction, whereas vegetative cells
may require only 0.1 to 0.5 minute at 65oC. Thus, the practice of heat sterilization
revolves around procedures for killing bacterial endospores.
Process time/Temperature Survivors
65oC/30min. Byssochlamys, Penicillium, Aspergillus,
Alcaligenes tolerans, Streptococcus
faecalis.
80oC/1min. Corynebacterium, Microbacterium,

Bacillaceae.
o
*120 C/3min. Clostridium botulinum A&B, some
Bacillus spores.
o
120 C/4min. Bacillus stearothermophilus, Clostridium
nigrificans, Cl. sporogenes.
o
120 C/5min. Clostridium thermosaccharolyticum
*Recommended heat process for Clostridium botulinum in foods.
Sporing microorganisms with thermal resistance greater than that of Clostridium

botulinum often involved in spoilage of canned foods.
Type of spoilage Trivial or common Scientific name Optimum
name temperature
Flat sour Flat sour bacteria Bacillus 50oC
stearothermophilus
Putrid swell Putrefactive Clostridium 37oC
anaerobe sporogenes 3679
Sulphide spoilage Sulphur stinker Clostridium 55oC
nigrificans
Hard swell Thermophilc Clostridium 75oC
anaerobe thermosaccharolyticu
m
Heat Penetration
In heating food for preservation, the part of it that heats slowest is the critical point. The
part is usually near the center of the container. The heating process could be by
conduction and convection. From adequate knowledge on the rate of penetration, the
thermal process needed to treat the food can be calculated.
The factors that determine the time required to bring the center of the food to the
sterilizing temperature are as follows:
1) The material composition of the container, glass heats slowly. Metal faster.
2) Size and shape of the container- larger container heat longer because of longer
distance to center and less surface per volume or weight. Shape is important, long
slim container heats faster than compact ones. Even if volume is the same.
3) Initial temperature of the food- the higher the starting temperature, the longer the
food is maintained at the lethal range of microbes.
4) Retort temperature- the higher the temperature, the faster the food will reach
sterilization temperature and remain for longer period.
5) Consistency of can contents and their shape/size:
(a) Pieces that retain identity e.g. peas, beans- can heat faster. If they are small,
heating is rapid. If large, time must be given for heat to penetrate center.

(b) Pieces that becomes mashy or viscous- heat slowly e.g. Irish potatoes, pumpkin
e.t.c.
(c) Pieces that form layers- these as they settle due to heat form layers. Layering is
principally determined by the degree of fill of the container. Horizontal layers
interfere with convectional current. Vertical layers allow convectional current.
6) Rotation and Agitation- rotation increases rate of penetration. Processed food
must be cooled rapidly to remove chances of over condensing.
Heat Treatments used for Processing Foods

In heating food, note must be made on the potential effect of heat on the food and if
two or more preservation methods are to be employed. The desire to remove all or
most spoilage organisms, to prevent recontamination and reduce chances of growth of
survivors later. Three categories of heating can be recognized:
1) Pasteurization 2) Heating at 100oC and 3) Heating above 100oC.
1) Pasteurization
Heat treatment that kills most but not all of the microorganisms present. Temperature
usually below 100oC. Any method could be used, steam, hot water, dry heat, electric
current e.t.c. But products are rapidly cooled thereafter. Pasteurization is used for the
following reasons:
(i) More heat will change the food quality e.g. milk.
(ii) The main aim is to kill pathogens as with market milk.
(iii) The spoilage agent is not very resistant to heat e.g. yeast in fruit juices.
(iv) Any spoilage organism is handled by additional preservative method
e.g. refrigeration.
(v) Competing organisms are killed to aid growth of desired ones e.g.
fermentation.
After treatment, additional preservation methods include:
(a) Keeping out microbes e.g. milk.
(b) Keeping out microbes e.g. milk processing.
(c) Maintenance of anaerobic conditions.
(d) High sugar concentration e.g. condensed milk.
(e) Use of chemical preservatives.
There are two main methods of pasteurization:
1) High Temperature Short Time (HTST) method e.g. 71.7oC for 15 seconds in
market milk. This is the minimal treatment given to market milk; it is more
satisfactory than other treatments in terms of preserving the flavour of the milk. It
is used in almost all modern dairies. It is used mainly because Coxiella burnetti
(which causes Q-fever) is a bit resistant to heat.
2) A low Temperature Long Time Holding (LTH) method employs lower
temperature for longer time or period e.g. 62.8oC for 30 minutes in market milk.
E.g.
Food Pasteurization Pasteurization Time (min.)
o
Tempearature ( C)
Ice cream mix 71.1 30
82 – 85 16 – 20seconds

Grape wine (bulk) 82 – 85 1
Fruit wine 62.8 or over (bottled hot) 1
Beers 60 or over 20 or time varying with
temperature.
Dried fruits (depending on 65 – 85 30 – 90
kind of fruit and size of
package)
Bottled grape juice 76 – 77 30
Bulk grape juice 80 – 85(flash treatment) 1
Carbonated juices 66 30
2) Heating at about 100oC

Sufficient to kill all vegetative cells but not spores. Boiling, immersion in boiling
water or steam is usually employed. Also used for blanching vegetables by immersing
for few seconds. In baking, inner temperature of bread never reaches 100 oC even
though the oven is much hotter. Unsealed canned foods heat at temperature of liquid
in it. So, also when food is left to simmer. All are at 100 oC in roasting, frying, inner
temperature of most of the foods rarely up to 100oC.
3) Heating above 100oC

Achieved by means of steam under pressure. Mainly used for canning.
Canning
Preservation of food in sealed containers. Mostly in tin, cans (coated with steel) or
glass container perfected by Nicholas Appert (French) in 1809. Now in modern times,
most cans are steel plate coated with tin to achieve greater thinner and more even
coating of tin.
Canned food is heated in special containers called retorts at about 110oC to 121oC for
intervals ranging from 25 to over 100 minutes. The precise time and temperature
depend on the nature of the food. Sometimes canning does not kill all the
microorganisms, but only those that will spoil the food (any remaining bacteria are
unable to grow). After heat treatment, the cans are cooled as rapidly as possible,
usually with cold water.
(E) Application of Low Temperatures in Food Preservation

Low temperatures are effective to varying degrees in protecting foods from microbial
spoilage. Only psychrotrophs (minimum –5oC; optimum 20 – 30oC; maximum 35 –
40oC). Psychrophiles (minimum –15oC; optimum 10 – 15oC; maximum 18 – 20oC)
are important in the spoilage of chilled, refrigerated or frozen foods.
Despite the ability of some microorganisms to grow at low temperatures, there is a
lower limit below which reproduction is impossible. At –30 oC, cellular growth ceases
but enzymatic reactions can occur, albeit slowly. The lower limit for biochemical
reactions in aqueous systems is around –140oC. From the foregoing, freezing may
stop microbial growth but may not cause its death. Freezing is in fact one of the best
ways of preserving microbial cultures. Under the proper conditions such as:

a) presence in the suspending medium of glycerol at
10% final concentration (act by reducing the
freezing point of the cell content and thus reducing
the severity of dehydration effect and preventing ice
crystal formation, after penetrating the cells);
b) presence of serum albumin, dextran and polyvinyl-
pyrrolidone at 10-5 to 10-3M final concentration.
These do not penetrate the cells but protect by
preventing freeze damage to the cell membrane.
Explanation of Death of microorganisms at Low Temperatures

1. Decrease in reaction rate of enzymatic
processes: the rate of reaction rule on
biological reactions stipulates that a
temperature decrease of 10oC leads to a 2-
fold reduction in the reaction rate. This
slowed down reaction rate could lead to
cell starvation in the long run.
2. Ice formation: this leads to the
concentration of solutes resulting in a
reduction in water activity, which
eventually leads to death of the cells.
3. Freezing injury: intracellular ice formation
makes the ice crystals participate in a
mechanical way in the killing of cells
through disruption of structures e.g.
plasma membrane and the cell wall.
Storage Life of Frozen Foods

Foods such as fruits and vegetables may be blanched to inactivate enzymes before frozen
for storage. The total period for which a food can be safely kept in a frozen state without
appreciable loss in quality will depend upon the nature of the food, its processing,
packaging and storage temperature.
Orange juice will keep for 27 months at –18oC; 10 months at –12oC; 4 months at –7oC.
Green beans and peas will keep for 11 – 12 months at –18 oC; 3 months at –12oC; 1 month
at –7oC.
Raw chicken will keep for 27 months at –18oC; 15.5 months at –12oC; 8 months at –7oC.
Effects of Freezing and Thawing on Frozen Foods

Fluctuations (freezing and thawing) result in greater damage to the food than either the
freezing or storage. A fluctuation of 3 oC in freezing storage temperature below –18 oC can
cause considerable damage to the food. During thawing, the ice crystals melt but on
freezing they once again turn into ice crystals, but this time much larger in size, and
capable of causing greater physical damage to the product.
Those microorganisms that survived the frozen storage become active when the food is
thawed. Thawing of foods prior to culinary preparation takes a long time leading

eventually to damage due to proliferation of microorganisms that have survived the
freezing and storage, e.g. when frozen whole egg is thawed in running water at 21oC, 12
hours will be needed for thawing during which time the microbial population increases to
about 300% of the original count. If air at 21 oC is used, the duration will be 36 hours and
the increase in microbial load will be 750%. Thus, it is recommended that thawing should
be as rapid as possible followed by cooking.
Preservation by Cooling or Chilling

Cold storage is a short-term preservation technique. The foods are washed to remove
contaminants and sorted to remove over-ripe, damaged materials before cooling or
chilling is applied. Prior to loading with the foods, the cold store rooms must be properly
cleaned, disinfected with either chemicals or radiation.
Storage Life of Plant and Animal Foods at various Temperatures in Days

Food 0oC 22oC 38oC
Animal flesh 6 – 10 1 1
Fish 2–7 1 1
Poultry 5 – 18 1 1
3
Dry meat and fish 10 and more 350 and more 100 and more
Fruits “ “ “
Leafy vegetables 3 – 20 1–7 1–3
Root crops 90 – 300 7 – 50 2 – 20
Dry seeds 103 and more 350 and more 100 and more
(F) Application of Radiation in Food Preservation

Certain forms of electromagnetic radiation are used in food preservation. These
include gamma rays from cobalt 60 and x-rays and highly accelerated electrons from
special machines.
The electromagnetic radiation energy absorbed by food (or any object for that matter)
can be measured in Radiation Absorbed Dose (RAD), which is equivalent to 10 -5
Joules of energy absorbed per gram of material. The International Standards
Organization (ISO) has now replaced the RAD with the Gray (Gy), which
corresponds to 100RAD. Heavy doses are reported as Kilo Gray (KGy) equivalent to
100,000RAD. Gamma rays have very short wave length and therefore possess great
penetrating powers. The absorption of radiation by living cells causes changes in the
cells, which result in death (through lethal mutation and damage to the nuclear
material).
Sensitivity of Organisms to Gamma Radiation

Different types of cells differ in their sensitivity to radiation:

Dose Effect
0.1Gy No effect on living organisms
1 – 10Gy Lethal man
0.01 – 1KGy Lethal to insects; parasites
1KGy Inhibits sprouting of tubers; delay fruit
ripening
1 – 10KGy Strong reduction in microbial load
10 – 50KGy Commercial sterilization of foods,
elimination of viruses.
Acceptability of use of Ionizing Radiation in Foods

The use of ionizing radiation in food processing has failed to gain the desired wide
spread acceptability among consumers and governments for fear of possible toxicity.
This is in spite of several decades of research on Radiolysis and toxicity of radiolytic
products have failed to justify such fears. Based on overwhelming evidence of safety
of irradiated foods the Joint Expert Committee on Wholesomeness of irradiated foods
along with the WHO has recommended that any food irradiated to an average dose of
10KGy or less is wholesome for humans and should be approved without testing.
Among the food items currently processed for safety or long-term storage are
potatoes, onions, spices, garlic, wheat flour, strawberries. Top on the list of countries
that accept irradiated foods are the Netherlands, Chile, Bangladesh, South Africa and
Belgium.
Advantages: the advantages of food irradiation are:
1) Possibility of irradiating foods in their final packaging due to the strong
penetrating power of ionizing radiation. The risk of recontamination is thus ruled
out.
2) Irradiation treatment does not heat up the food and thus results in shelf-stable
products that are close to the fresh state in texture, flavour and colour. Loss of
vital nutrients like vitamins and essential amino acids is minimal.
3) Food irradiation is of particular advantage in developing countries where the
standards of hygiene are low during food processing.
Practical Irradiation of Foods:

Radurization: a process designed to kill or inactivate food spoilage organisms. This
is applied to disinfect the surfaces of fruits, spices, meat, fish and dry ingredients for
the enhancement of wholesomeness during cold storage. The operative dose is usually
4 – 5 KGy. Radurization is superior to ethylene oxide fumigation, which leaves toxic
residue on the food.
Radicidation: a process designed to kill or render harmless all disease-causing

organisms (except viruses and spore-formers). The operative dose does not exceed
10KGy and the aim is to destroy such organisms as Trichinella spiralis (0.3KGy);
Taenia saginata (0.3KGy); Salmonella, Vibrio parahaemolyticus and
Staphylococcus aureus (5KGy).

Foods subjected to radicidation include meat, eggs and others liable to contamination
with pathogens. Radicidation must be accompanied by refrigeration.
Radappertization: the use of radiation to achieve commercial sterilization of food. This

treatment will ensure the destruction of pathogenic organisms including spores of
Clostridium botulinum and food spoilage organisms. Radappertized food can be stored at
room temperature like thermally processed foods. Examples are meat, poultry, fish and
vegetables. Dairy products are unsuitable due to flavour changes. Astronauts and organ
transplant patients are maintained on radappertized foods.
(G) Application of Drying in Food Preservation

Drying usually is accomplished by the removal of water, but any method that reduces the
amount of available moisture, i.e., lowers the A w in a food is a form of drying. For
example, dried fish may be heavily salted so that moisture is drawn from the flesh and
bound (or tied up) by the solute and is thus, unavailable to microbes. Sugar may be added
to sweetened condensed milk to reduce the amount of available moisture.
Moisture may be removed from foods by a number of methods mentioned briefly below:
i) Natural Dehydration (Solar drying)-
Natural dehydration is a low-cost method in which water is removed by the heat of the
sun. It is used to dry grains as well as to dry certain fruits (e.g. raisins, apricots, figs,
pears, prunes and peaches), vegetables, fish, meat, milk and curd, in countries with hot
climates. The process is slow and depending upon the conditions used, spoilage and
pathogenic bacteria as well as fungi can grow during drying.
ii) Mechanical Drying-

This is a controlled process and drying is achieved in a few seconds to a few hours. Some
of the methods used are tunnel drying (in which a food passes through a tunnel against
the flow of hot air and the water is removed); roller drying (in which a liquid is dried by
applying a thin layer on the surface of a roller drum heated from inside); spray drying (in
which a liquid is sprayed in small droplets, which then come in contact with hot air that
dries the droplets instantly). Vegetables, fruits, fruit juices, milk, coffee, tea, and meat and
soups are examples of foods that are dried by mechanical means.
iii) Freeze-Drying-
This method is relatively costly to use. It can be used for solid and liquid foods. It
involves freezing the food initially at low temperature, and then exposing the frozen food
to a relatively high vacuum environment plus heat applied at the drying shelf. The water
molecules are removed from the food by sublimation (from solid state to vapour state)
without affecting its shape or size. This method has been used to produce freeze-dried
vegetables, fruits, fruit juices, coffee, tea, fish and mea products.
iv) Foam Drying-

This consists of whipping a product to produce a stable foam to increase the surface area.
The foam is then dried by means of warm air. The method itself has little lethal effect on

microbial cells and spores. Liquid products, such as egg white, fruit purees, and tomato
paste, are dried in this way.
v) Smoking-
Many meat and fish products are exposed to low heat and smoke for cooking and
depositing smoke on the surface at the same time. The heating process removes water
from the products, thereby reducing their A w. Heat kills many microbes. The growth of
the survivors is controlled by low Aw as well as the many types of antimicrobial
substances present in the smoke.
vi) Intermediate Moisture Foods (IMF)-

These are foods that contain approximately 10-40% moisture content (Aw value of about
0.70 to 0.90) and have non-refrigerated shelf stability. They can be eaten without
rehydration, but they are shelf-stable for a fairly long period of time without refrigeration
and considered microbiologically safe. The low Aw value and relatively high moisture is
obtained by adding water-binding solutes and hydrophilic colloids. Microorganisms can
survive in the products; but due to low A w, bacteria cannot grow. However, yeasts and
moulds can grow in some. To inhibit their growth, specific preservatives, such as sorbate
and propionate are added. Examples include various soft candies, jams, jellies, honey,
many dried fruits, some bakery products, some meat products (such as pepperoni, ham,
dry sausages) and some dried fish.
(H) Application of Chemical Preservatives/Food Additives in Food Preservation

Various chemical agents can be used to preserve foods, and these substances are closely
regulated by the Food and Drug Administration(F.D.A). They include simple organic
acids (propionates, benzoates, sorbates, acetates e.t.c.), sulfites/sulphur dioxide, ethylene
oxide as a gas sterilant, sodium nitrite, ethyl formate, sugar and salt, wood smoke, spices
and other condiments. The effectiveness of many of these chemical preservatives depends
on the food pH. As an example, sodium propionate is most effective at lower pH values,
where it is primarily undissociated and able to be taken up by lipids of microorganisms.
Breads, with their low pH values, often contain sodium propionate as a preservative.
These compounds, ‘generally recognized as safe’ (GRAS), are used with grain, dairy,
vegetable, and fruit products.
Foods can have antimicrobial components through the following ways: occurring
naturally in the food; formed during processing of the food; added as ingredients in the
food. Those that are added have to be GRAS-listed and approved by regulatory agencies.
Some of the chemical preservatives/food additives added to foods directly or indirectly
are mentioned briefly below:
i) Nitrites and Nitrates-
These are employed as salts in curing solutions and curing mixtures of meats. Nitrites
decompose to nitric acid, which forms nitrosomyoglobin when it reacts with the haem
pigments in meats and thereby forms a stable red colour. Nitrates act as reservoir for
nitrite, and their use is being restricted.
It has been shown that nitrites have inhibitory effect on Clostridium botulinum in meat
products such as bacon and canned or processed hams. Nitrates have a limited

antimicrobial effect on organisms, and are not considered as a good chemical
preservative.
ii) Sulphur Dioxide and Sulphites-

Sulphur dioxide and various sulphites, including sodium sulphite, sodium bisulphate,
potassium sulphite, potassium bisulphate, sodium metabisulphite and potassium
metabisulphite are used to control microbes (and insects) in soft fruits, fruit juices, lemon
juices, beverages, wines, sausages, pickles, and fresh shrimp.
In addition to the antimicrobial action of sulphur dioxide and sulphites, they are also used
as antioxidants (to prevent enzymatic and nonenzymatic changes) in fresh and dried fruits
and vegetables (salads) to prevent browning or discolouration in some foods.
iii) Epoxides (Ethylene Oxide, Propylene Oxide)-

Ethylene oxide and propylene oxide are used as fumigants to destroy microbes (and
insects) in grains, cocoa powder, gums, nuts, dried fruits, spices and packaging materials.
They have germicidal effect and destroy cells, spores and viruses. Ethylene oxide is more
effective. They are alkylating agents and react with various groups (e.g. sulphydryl,
amino and hydroxide groups) in cellular macromolecules, particularly structural proteins
and enzymes, and adversely affect their functions. They react with some food
components, such as chlorides, and form toxic compounds that can remain as residue in
treated foods. Propylene oxide is permitted only as packaging fumigant for some dried
foods.
iv) Organic Acids and their salts-

Propionates- sodium or calcium propionate is used widely in the prevention of mould
growth and ropiness in baked foods, cheese foods and spreads.
Propionic acid- a short chain fatty acid affects cell membrane permeability. It is found
naturally in Swiss cheese as a built up preservative.
Benzoates- sodium benzoate has been incorporated into jams, jellies, margarine,
carbonated beverages, fruit salads, pickles, fruit juices, e.t.c. It is relatively ineffective at
pH values near neutrality and effective at acidic pH.
Sorbates- sorbic acid, as calcium, sodium or potassium salt, used directly in foods as an
antimicrobial additive and as a spray, dip or coating on packaged materials. It is used in
cheeses, cheese products, baked foods, beverages, syrups, fruit juices, jams, fruit
cocktails, dried fruits, pickles and margarine. Sorbic acid and its salts inhibit yeast and
moulds but are less effective against bacteria. They are effective at low pH values.
Acetates- acetic acid and its derivatives, e.g. monochloroacetic acid, peracetic acid,
dehydroacetic acid and sodium diacetate, have been recommended as preservatives, but
not all are approved by the F.D.A.
Acetic acid in the form of vinegar is used in mayonnaise, pickles, e.t.c. It is more
effective against yeasts and bacteria than moulds.
v) Antibiotics-
Several antibiotics have been used tested on raw foods, mainly the proteinaceous ones
like meats, fish, and poultry in order prolong their storage time at chilling temperatures.
Aureomycin (chlortetracycline) has been found superior to other antibiotics because of its

broad spectrum of activity. Streptomycin, neomycin, polymyxin, nisin, subtilin,
bacitracin have been used, but have not been successful. Nisin has been used in Europe to
suppress anaerobes in cheese and cheese products. Natamycin is effective against yeasts
and moulds, it is used in orange juice, fresh fruits, sausage and cheese, its usage has also
been banned in some countries.
vi) Sugar and salt-

These compounds lower the Aw and therefore, have an adverse effect on microbes.
Sodium chloride is used as brines and curing solutions or is directly applied to foods.
Sugars, like glucose or sucrose can be used to make water unavailable to microbes and
the osmotic pressure they exert on them. Examples of such foods are sweetened
condensed milk, fruits in syrups, jellies and candies.
vii) Wood Smoke-

Many processed meat products and fishes are processed with smoke generated by burning
hardwood. Smoking of foods usually has two main purposes: to impart desirable flavour,
texture and colour to the products; other benefit is to prolong the shelf-life of smoked
food products. The smoke contains several different types of chemicals that deposit on
the surface of the food, which have antibacterial properties, e.g. formaldehyde, phenols
and cresols.
viii) Spices-
Many spices, condiments, and plant extracts are known to contain antimicrobial
compounds, e.g. cinnamic aldehyde in cinnamon, eugenol in cloves, paramene in oregano
and thymol in thyme. They have both bacteriostatic and fungistatic properties depending
on the active ingredient.
MICROBIAL CONTAMINATION, PRESERVATION AND SPOILAGE OF

DIFFERENT KINDS OF FOODS
CEREALS AND CEREAL PRODUCTS e.g soybeans, sorghum, millet, barley, maize,
flours, breads, cakes and other bakery products.

Sources of Contamination: harvested grains retain some of the microorganisms plus
contamination from soil, insects, and other sources. During slicing of baked products,
contamination may take place from microorganisms in the air, on the knives, or on the
wrapper
Storage Conditions:
- Use of low temperature- storage temperature of 4 to 7 oC is recommended for dry
products.
- Use of heat.
- Use of chemical preservatives, e.g. acetic acid in dough, sodium and calcium
propionate, sodium diacetate and sorbates have been widely used.
Spoilage: cereal grains and meals contain moulds, yeasts, and bacteria, which are ready
to grow if enough moisture is provided. Different moulds are involved, but most common
species are Aspergillus, Penicillium, Mucor, Rhizopus and Fusarium. These moulds
produce mycotoxins, which are hazardous to human health. Moulds involved in the
spoilage of bread are the so-called ‘bread mould’, Rhizopus stolonifer, Penicillium
expansum, Aspergillus niger, Neurospora sitophila and also species of Mucor or
Geotrichum. Ropiness in bread is caused by Bacillus subtilis or Bacillus licheniformis.
SUGAR AND SUGAR PRODUCTS e.g. sucrose (cane and beet sugar), molasses,
syrups, honey and candy.
Sources of Contamination: from sugarcane and the soil contaminating it; plastic tubing,
buckets or other collection vessels in syrups; nectar of flowers and honeybees in honey;
ingredients, air, dust and handling in candies. Bagging of raw sugar also may add some
microorganisms. During the refining process of the raw sugar, contamination may come
from equipment, and organisms are added during bagging.
Storage Conditions:
- Use of controlled atmosphere.
- Use of chemical preservatives during sugar refining.
- Boiling and complete-fill of the container in syrups.
- Pasteurization of commercially distributed honey.
Spoilage: spoilage is caused by osmophilic or xerotolerant microorganisms. Certain
yeasts, especially those of the genus Saccharomyces, and certain moulds. Species of
Bacillus and Leuconostoc have also been implicated as possible spoilage problems.
Osmophilic yeasts are chief cause of honey spoilage, species of Zygosaccharomyces
mellis, Z. richteri or Torula mellis. Candies are not subject to microbial spoilage because
of high sugar content and low moisture content, except for chocolates with soft centers,
which under certain conditions burst or explode.
FRUITS AND VEGETABLES

Sources of Contamination: soil, during storage and transportation, boxes, lugs, trucks
and during harvesting. The processing plant, mechanical damage and wash water.
Storage Conditions:
- Use of heat.
- Use of low temperature- chilling and freezing.
- Drying.
- Use of preservatives.

Spoilage: fruits and vegetables are capable of supporting the growth of bacteria, moulds
and yeasts, consequently one or all can spoil them. The higher water content of
vegetables favour the growth of spoilage bacteria, and the relatively low carbohydrate
and fat contents in soluble form, favourable pH and oxidation-reduction potential. The
bacteria isolated from vegetables include Erwinia spp., Pseudomonas spp., Bacillus and
Clostridium spp. Most fruits contain on the average water- 85%, carbohydrate- 13%,
protein- 0.9%, fat- 0.5%, while ash is 0.5% and vitamins. The fruits differ from
vegetables in having less water but more carbohydrate. On the basis of this nutrient
content, fruits are capable of supporting the growth of bacteria e.g. Erwinia and
Pseudomonas spp.
MEAT AND MEAT PRODUCTS

Sources of Contamination: include soil, water, feed, manure as well as its natural
surface flora and the intestinal contents containing the intestinal organisms. However,
knives, clothes, air, hands and clothing of workers are intermediate sources of
contaminants. Special equipment such as grinders, sausage stuffers and casings, and
ingredients in special products e.g. fillers and spices may be add undesirable organisms in
appreciable numbers.
Storage Conditions:
- Use of heat. The canning of meat is a specialized technique.
- Use of low temperature- chilling meat promptly and rapidly to temperature near
freezing and chilling storage at only slightly above the freezing temperature.
- Freezing temperature. Halves or quarters can be cut off and promptly frozen.
- Use of irradiation. Use of U.V. rays in conjunction with chilling storage.
- Drying.
Spoilage: meats are the most perishable of all important foods. The reason is simply that
they contain sufficient nutrient materials for the growth of microorganisms. The
microorganisms are as follows: Bacillus, Micrococcus, Salmonella, Staphylococcus.
However, Clostridium, Lactobacillus, Leuconostoc, Pediococcus, Streptococcus and
Brevibacterium are present in most meats, fresh or cured and can grow even at
refrigerator temperature.
FISH AND SEA FOODS e.g. fish, shell fish (shrimps, lobsters, crabs and cray fish) and
mollusk (oysters, clams, squid e.t.c.)
Sources of Contamination:
Storage Conditions:
- Use of heat.
- Use of low temperatures- chilling, freezing.
- Preservation by drying.
- Use of preservatives.
Spoilage: both salt water and fresh water fish contain comparatively high levels of
protein and other nitrogenous constituents. The carbohydrate content is nil while fat
content varies from very low to rather high values depending on species. The bacteria
responsible for spoilage include gram-negative rods e.g. Pseudomonas, Acinetobacter
and Moraxella types. The implicated spoilage bacteria include Pseudomonas,

Acinetobacter, Moraxella in crustacean shell-fish while molluscan shell-fish include the
following genera: Pseudomonas, Proteus, Clostridium, Bacillus, Escherichia,
Enterobacter, Lactobacillus, Flavobacterium, Shewanella and Micrococcus.
EGGS AND POULTRY

Sources of Contamination: the sources of contamination as applied to meat as well as
the skin of live birds, lining of the body cavity occurs during washing, plucking and
avisceration.
Storage Conditions: same as those of meat except the plucking and dressing of the fowl.
- Use of heat, dressed chickens and other fowls may be canned, whole or dissected in
their own juices or in jelly.
Spoilage: isolates from poultry and poultry products could include members of the
following genera: Enterobacter, Escherichia, Alcaligenes, Bacillus, Flavobacterium,
Salmonella and Staphylococcus.
MILK AND MILK PRODUCTS e.g. market milk and cream, butter, cheese, yoghurt,
condensed and dried milk products.
Sources of Contamination: external of the udder during milking, milking machines,
cans or pipelines, milking by hand, clothes, personnel and the external environment of the
cow.
Storage Conditions:
- Use of heat- pasteurization and ultra pasteurization.
- Use of low temperature, with the exception of canned milk and dry milk, most dairy
products require the use of low temperature- refrigeration and freezing.
- Drying and use of preservatives- sorbic or propionic acid or one of their salts to a
limited extent.
Spoilage: Milk and dairy products e.g. butter, cream, cheese are all susceptible to
microbial spoilage because of their chemical composition. Milk is an excellent medium
for all of the common spoilage organisms, including yeasts and moulds. Bacteria
implicated in the spoilage include the following genera: Enterobacter, Staphylococcus,
Streptococcus, Leuconostoc, Lactobacillus, Microbacterium, Propionibacterium,
Micrococcus, Proteus, Pseudomonas, Bacillus, coliforms and others. There are the
psychrophiles- those that survive low temperature (below 20 oC); the mesophiles- grow in
temperature of 25oC – 40oC; thermophiles- grow in temperature of 45oC – 60oC.
CANNED FOODS
Sources of contamination:
Storage Conditions:
Spoilage: the objective of canning of foods is to destroy absolutely all the
microorganisms present. Nevertheless, canned foods undergo microbial spoilage under
certain conditions. The main reasons are under-processing, inadequate cooling,
contamination of the can resulting from leakage through seams and pre-process spoilage.
Canned food spoilage organisms may be characterized as follows:
1. Mesophilic Organisms:
- Putrefactive anaerobes.
- Butyric anaerobes.

- Aciduric flat sours.
- Lactobacillus.
2. Thermophilic Organisms:
- Flat-sour spores.
- Thermophilic anaerobes producing sulphides.
- Thermophilic anaerobes not producing sulphides.
The common types are Bacillus spp., Bacillus coagulans, B. stearothermophilus are
thermophilic spoilers, while mesophilic spoilers are B. polymyxa, B. macerans,
Clostridium spp. e.g. C. pasteurianum, C. butyricum, lactobacilli and others.
BAKED FOODS AND CONFECTIONARY PRODUCTS

Sources of contamination: grains, soil, milling process, blending, conditioning and
insects.
Storage Conditions:
- Use of heat.
- Use of low temperature- refrigeration and freezing.
- Use of chemical preservatives e.g. sodium and calcium propionate.
- Use of irradiation.
Spoilage: baked foods e.g. bread may undergo a type of spoilage known as ropiness,
which is caused by the growth of certain strains of Bacillus subtilis. B. cereus and B.
licheniformis have been isolated from baked products. In addition, Staphylococcus
aureus has been found in bread.
FERMENTED FOODS
Sources of contamination: raw materials e.g. soybeans, sorghum, millet, barley, maize,
sugar and sugar syrups.
- Equipment and vessels- if not properly washed or cleaned.
- Pitching yeasts recovered from a previous batch of fermentation, ordinarily it may
contaminated with bacteria or wild yeasts.
Storage Conditions: Bottled and pasteurized.
Spoilage: there are a good number of fermented foods that are susceptible to the growth
of spoilage microorganisms e.g. beers can be spoiled by Acetobacter, Lactobacillus,
Pediococcus, and Zymomonas anaerobium. Wines can be spoiled by genus Acetobacter.
Sauerkraut is the product of lactic acid fermentation of fresh cabbage. The spoilage
bacteria include Lactobacillus brevis causes soft kraut, slimy kraut is caused by
Lactobacillus plantarum and L. brevis, while pink kraut is caused by Torula spp. Pickles
result from lactic acid fermentation of cucumbers. The following bacteria are implicated
in the spoilage: Bacillus nigrificans, Enterobacter spp., Lactobacilli and Pediococci.
MISCELLANEOUS FOODS
DEHYDRATED FOODS: typical examples of dehydrated foods are macaroni, spaghetti
and tapioca. Enterobacter cloacae is the commonest isolate from macaroni.

FOOD-BORNE DISEASES OF MICROBIAL ORIGIN
Etiological and Epidemiological Concepts:
Food-borne diseases of microbial origin are of two types:
a) Food Infection: refers to food-borne illnesses caused by the entrance of bacteria or
fungi or viruses or protozoa or helminthes(parasitic worms) or rickettsia into the body

through ingestion of contaminated food and the reaction of the body to their presence or
to their metabolites.
Therefore, food infection could be bacterial or non-bacterial.
b) Food Intoxication: this refers to food-borne illnesses caused by the presence of a

toxin formed in the food. It could also be bacterial or non-bacterial.
Food Poisoning: is often called food intoxication and refers to food-borne illnesses
caused by the presence of a bacterial, fungal or algal toxin formed in the food.
Concept of Microbial Pathogenicity

Microbial pathogenicity- is the ability of a parasite or pathogenic microorganism to gain
entrance to a body to produce physiological or anatomical changes, that is disease.
Virulence- the degree of pathogenicity or ability to cause infection by a pathogenic
organism is referred to as virulence.
Food infections can be divided into two types:
(1) Those in which the food does not ordinarily support
growth of the pathogens but merely carries them,
i.e., pathogens such as those causing tuberculosis,
diphtheria, the dysenteries, typhoid fever,
brucellosis, cholera, infectious hepatitis, Q-fever
e.t.c.
(2) Those in which the food can serve as a culture
medium for growth of the pathogens to numbers
that will increase the likelihood of infection of the
consumer of the food. These include Salmonella
spp., Vibrio parahaemolyticus and
enteropathogenic Escherichia coli.
The controversial status of Clostridium perfringens food-borne illness and Bacillus
cereus gastroenteritis is recognized. Both should have been classified as food
intoxications rather than food infections since toxins might be released by B. cereus as a
result of autolysis of the cells in the food or by Cl. perfringens during sporulation in the
food. A large number of viable cells must be consumed in both cases, which implies the
release of toxin invivo, rather than in the food.
Viral pathogenicity- numerous viral infections may result in the death of animal or the
destruction of plants. This consequence could be considered a reduction of potential
foods available for man. In addition, many of the animal viruses can result in diseases of
man e.g. encephalitis, hepatitis, measles, mumps, rabies, small pox and yellow fever.
Many of the enteroviruses including the coxsackie viruses, ECHO viruses and Norwalk
virus may result in human gastroenteritis.
Susceptibility/Resistance to Infection
The resistance of a consumer at risk and hence the minimum infection dose (MID)
depends on the following: (a) diet; (b) general physical condition; (c) immune status; (d)
acidity of gastric juice; (e) character of the intestinal flora; (f) neutralizing effect of the
food.

In addition to these determining factors, the mode in which the organism is ingested will
determine the number of cells required to initiate infection. Normally, more than 10 6 cells
per gram of food of enteric pathogen is needed to initiate an enteric infection. However, 1
– 10 cells/gram may cause infection if ingested with small volume of water or food
between meals. Under this circumstance, the cells pass quickly from the stomach to the
duodenum thus avoiding exposure to the bactericidal effect of the gastric fluid.
Morbidity by Food-borne Disease of Microbial Origin

Information on this aspect is poor due to severe under reporting to Public Health
Authorities. Only about 1 – 10% of actual cases are generally recorded. Food-borne
diseases result from the so-called dual failure i.e. contamination of food followed by
temperature abuse leading to proliferation.
MICROBIAL FOOD-BORNE INFECTIONS

SALMONELLOSIS: virtually all Salmonella serotypes when present in foods in
sufficiently high numbers can cause febrile gastroenteritis within roughly ten hours. A
striking aspect of salmonellosis is the almost universal and permanent predominance of
about a dozen serotypes in human food-borne diseases. These include S. typhimurium;
S. newport; S. stanleyville; S. panama; S. heidelberg; S. brandenburg; S. oranenburg
and S. derby. Others serotypes such as S. raus; S. good; S. carro and S. veile do often
appear but disappear again rather fast.
The predominance of these serotypes is due to their high degree of persistence in water
and sewage and certain foods. Their identification is based on agglutination tests for the
O- and H-antigens.
Foods involved: all foods of animal origin and a few vegetable products. This is due to
the high carrier rate of healthy food-producing animals, particularly pigs, calves and
poultry. Salmonellosis is therefore an important zoonosis(disease of animal that affect
man).
Prevention: (a) Consumption of insufficiently heated foods of animal origin must be
avoided.
(b) Foods processed for safety should be protected against recontamination by soiled
equipment.
(c) Proliferation of occasional contaminant should be prevented through adequate
refrigeration.
SHIGELLOSIS:
Caused by Shigella dysenteriae, S. sonnei S. boydii and S. flexneri. They cause bacillary
dysentery. Whereas the great majority of Salmonellae is spread primarily by healthy
carrier animals so that humans are mostly secondarily infected, the spread of Shigellae is
virtually limited to man. Food therefore has significance only as a non-specific vector and
no particularly dangerous food items are known.

Foods involved: all foods that are not too acidic or too dry to allow growth or at least
survival of Shigellae introduced by extensive handling can play an aetiological role. E.g.,
milk, beans, potato, turkey, salads.
Absorption of Shigella species with food takes about 10 hours to produce febrile, often
blood stained diarrhoea. When absorbed with food, the infective dose (ID 50) is generally
high but very low with water (10ml). They can easily survive the gastric environment. Of
the four species of Shigella, outbreaks due to S. sonnei are the most frequent. This is
thought to be due to higher resistance to adverse environmental conditions. Shigella
pathogenicity is determined by: (a) presence of smooth lipopolysaccharide-somatic
(LPS)-O-antigen; (b) possession of genes for the invasion of epithelial cells followed by
proliferation therein; (c) ability to produce a toxin complex in the epithelial cells.
Epithelial cell invasion and action of the toxin complex (neuro-cyto-enterotoxins) are
responsible for the clinical manifestations of shigellosis.
Prevention:
1) Chill foods rapidly in small quantities.
2) Foods should be prepared under good sanitary conditions.
3) Protect and treat water for consumption.
4) Sewage should be disposed off properly.
GASTROENTERITIS DUE TO E. coli

Majority of E. coli are harmless enteric commensals (normal flora). Some strains
however can cause febrile gastroenteritis. Outbreaks have been reported in adults and
children- travelers diarrhoea and infantile diarrhoea. These E. coli strains may be as
important as Salmonella and Shigella in diseases of young people. Apart from
Rotaviruses, E. coli constitutes the commonest enteric pathogen in hospitalized children.
Three distinct pathogenic E. coli groups have been recognized namely: (1)
Enterotoxigenic E. coli; (2) Enteroinvasive E. coli; Enteropathogenic E. coli.
Sources: the sources of food infection are human and animal due to dual failure.
Prevention: (1) Prevention of contamination of fresh food and of recontamination of
foods already processed for safety..
(2) Control of microbial proliferation through refrigeration.
CAMPYLOBACTERIOSIS
Caused by Campylobacter fetus subsp. jejuni. Common disease in man. Incubation
period 2 – 10 days. Acts through invasiveness with low ID 50. Grows under
microaerophilic condition. Foods involved are similar to those of Salmonella.
Symptoms: severe abdominal pains and explosive diarrhoea.
Foods involved: many animal and animal by-products used for human and animal feeds
e.g. chicken carcasses and faeces, swine carcasses and faeces, sheep carcasses and faeces,
turkeys, pork sausages, various red meats and ground beef.
Prevention: controlled by avoiding the dual failure.
YERSINIOSIS
Caused by Yersinia enterocolitica. Y. enterocolitica causes febrile gastroenteritis often
with rheumatoid sequalae. Enteropathogenic strains include 0:3; 0:5; 0:8; 0:9. Survives
and proliferates under cold storage- a great problem therefore for food safety and quality

assurance. Produces ST-toxin invitro (foods) but not at 37oC. ST-toxin therefore may be
of no significance in pathogenicity during food infection.
Foods involved: are same as Salmonella.
Prevention: control of the dual failure.
Clostridium perfringens FOOD-BORNE DISEASE

That Cl. perfringens causes food-borne gastroenteritis has been known since about 1930.
Based on ability to produce α, β, ε and ί (iota) exotoxins. Five types of Cl. perfringens
are distinguishable- Types A, B, C, D and E. When ingested with foods in numbers
exceeding 106/gram, Types A and C cause gastroenteritis in man. The illness is less severe
with Type A. Type C causes severe necrotic enteritis. The foods mostly involved are meat
and meat products; milk and milk products; fish and fish products; in which some
occasional spores had survived and which under inadequate refrigeration germinates. The
mechanism of gastroenteritis is extracellular production and subsequent release of an
enterotoxin by cells having colonized the lower gastrointestinal tract (i.e. a food infection
case). Another view of the mechanism of gastroenteritis claims that the spores germinate
in the food, grow quickly, producing the required dose of the enterotoxin in a short time.
This is thought to explain the clinical manifestation within 8 – 22 hours of food
consumption. Recovery takes place within 24 hours- no fatalities.
Prevention: temperature is a single most important factor in the control of Cl.
perfringens. Growth is severely restricted below 15 to 20 oC and thus cooling below 10oC
is usually an effective means of control. Heating above 75oC destroys the organism.
FOOD-BORNE GASTROENTERITIS FROM Vibro parahaemolyticus

The earliest recorded incidence of food-borne illness by this organism occurred in Osaka,
Japan. Incidents are invariably associated with the consumption of raw or improperly
cooked oceanic foods- e.g. shellfish, crustacean foods and fishery products.
Clinical Symptoms: include febrile gastroenteritis often with bloody diarrhoea. High
numbers of the order of more than 105 cells/gram food are required for food infection.
V. parahaemolyticus food-borne illness is not restricted to the Far Eastern Pacific as
earlier thought. Therefore, all oceanic or sea foods should be carefully monitored.
Prevention:
1) Foods should be cooked thoroughly and chilled rapidly in small quantities.
2) Avoid cross contamination from saltwater fish.
3) Sanitize equipment properly.
4) Avoid using seawater for rinsing foods to be eaten raw or for cleaning.
MICROBIAL FOOD-BORNE INTOXICATIONS

BOTULISM: this is caused by toxins produced in the food prior to ingestion by
Clostridium botulinum, an obligately anaerobic gram positive bacterium.
Botulism is the most serious food-borne disease of microbial origin. The toxin botulin is
a neurotoxin and is a protein. Seven types of this toxin A to G are presently known. Types
A, B, E are the most toxic to man, with Type A as the most potent. Recently, Type G toxin
has been implicated as the cause of sudden death in man.

Pathogenesis: following the ingestion of these toxins with food, the toxins are absorbed
from the gastrointestinal tract and reach susceptible neurons by way of the blood stream.
Between 18 – 30 hours early symptoms of weakness, dizziness and severe dryness of the
mouth and pharynx appear. With Type E toxin, nausea and vomiting are manifested too.
Later, neurological symptoms- blurring of vision, dilation of the pupil of the eye, inability
to swallow, difficulty in speech, urinary retention, generalized weakness of skeletal
muscles and respiratory paralysis which finally results in death.
Foods involved: these include low and medium acid canned foods e.g. string beans,
sweet corn, beef, asparagus, spinach, peas, meat, fish, poultry and milk and rarely, low
acid foods like tomatoes, apricots, pears and peaches. The low acid canned food should
be subjected to a heat treatment of 120oC for 4 minutes (equivalent to 12D-processing),
referred to as ‘botulinum cook’ to prevent botulism. For canned cured meats- increasing
the salt content will in combination with the sodium nitrite used for curing, increase the
efficacy of heat treatment below the 12D treatment.
Under normal circumstances, foods with pH less than 4.5 should be botulinum safe for as
long as:
1) the acid is homogeneously distributed.
2) There are no other contaminants in the food that would dissimilate the acid thus
increasing the pH to a hazardous level.
Prevention:
1) Boil any canned food for at least 15 minutes at 100oC.
2) All swollen canned foods should be rejected (Note the case of Clostridium botulinum
e.g. Type E.
3) Reject foods that have been cooked, held and not reheated.
4) In smoking of fish, the internal temperature of the fish should not be less than
82oC for 30 minutes.
5) In an outbreak, individuals who consumed the same food as the patient should be
treated with polyvalent antitoxin.
6) Laboratory high risk workers should be immunized with a pentavalent toxoid.
STAPHYLOENTEROTOXICOSIS
A microbial food intoxication caused by Staphylococcus aureus. It is more common than
other forms of microbial food-borne intoxications. 80% of strains of human origin are
enterotoxigenic. Enterotoxigenic strains are coagulase positive, although occasional
coagulase strains may be enterotoxigenic. All coagulase positive strains are not
enterotoxigenic. Enterotoxigenic strains are salt-tolerant, and nitrite tolerant and
consequently can grow on cured meat under favourable environmental conditions. 8
serological types of enterotoxin are produced- A, B, C 1, C2, D, E, F, G, all are
polypeptides. Type A is usually involved in most food-poisoning incidents.
The enterotoxins are produced under conditions which favour cell multiplication.
Minimum temperature = 7 – 10oC.
Minimum pH = 4.8 – 5.5.
Foods involved: are usually those with relatively low water activity (a w) e.g. hams,
sausages, pastry and ice cream. In those with high aw growth and toxin production by
Staph. aureus is either inhibited by competitive microorganism or destroyed by

metabolic activities of other food bacteria whose development is either inhibited or
retarded by reduced aw.
Staph. enterotoxin is resistant to heat- can withstand boiling for 60 minutes. Heating can
thus not detoxify already formed toxin in foods. The D-value ranges between 1 – 3 hours
at 100oC or 10 – 40 minutes at 120oC.
Clinical Manifestation: period between food ingestion and the onset of symptoms is 2 –
3 hours. In this respect it could be confused with Salmonella infection. The major
symptoms include salivation, nausea, abdominal cramp, profuse vomiting and diarrhoea-
often bloody. No fever. Duration- 1 – 2 days. Usually no treatment is given except in
extreme cases when ORT can be administered to restore electrolyte balance.
Prevention: prevention of staphyloenterotoxicosis is achieved through the exclusion of
food handlers with Staph. infections- colds, boils e.t.c. from food processing and
handling.
Bacillus cereus INTOXICATION

Bacillus cereus, a gram positive spore-forming bacterium produces two types of food-
borne intoxications. One is characterized by irritation of the intestinal mucosa resulting in
diarrhoea, while the other is characterized by acute nausea and vomiting with diarrhoea in
some patients. The first has an incubation time of 10 hours and the latter 1 – 5 hours.
About 108 cells/gram of food are required for disease production. Smaller numbers are
required in compromised hosts. Recovery comes within 12 hours.
Foods involved: include cooked and fried rice, pudding, vegetable soup, cooked meat,
poultry.
Prevention:
1) Chill foods rapidly in small quantities.
2) Hot foods should be held at 65oC or above.
3) Personal hygiene should be practiced, proper sanitary conditions observed during
food processing.
4) Leftover foods should be reheated to 71.1oC.
TOXOFLAVIN AND BONGKREKIC ACID INTOXICATION

This brand of intoxication is caused by Pseudomonas cocovenans. Outbreaks have been
reported from Java in Indonesia as a result of the consumption of ‘bongkrek klappa’, a
mould fermented coconut product. Pseudomonas cocovenans produces two orally active
toxins- toxoflavin and bongkrekic acid. The toxicity of the former(toxoflavin) is due to
formation of intracellular hydrogen peroxide. The latter (bongkrekic acid) is an
unsaturated pentacyclic, tricarboxylic acid that acts by interfering with carbohydrate
metabolism.
FOOD-BORNE PRESSOR AMINE SYNDROME

This presents generally as facial flushing (redness of face and neck), dizziness and a
bright red rash, once in a while respiratory distress too. The syndrome is caused by
pressor amines, which are pharmalogically active compounds e.g., histamines, tyramines
and phenylethylamines absorbed with food. Several bacteria of common occurrence in
foods would decarboxylate the constituent amino acids leading to the formation of the

pressor amines. A food rich in peptides containing large amounts of the relevant parent
amino acid will result in high enough levels of pressor amine as to be of clinical
significance.
Like staphyloenterotoxins, pressor amines are heat stable and have been found in e.g.
canned fish products.
Individual sensitivity to orally administered pressor amines varies widely. This is due to
great variations in levels of monoamine oxidase (MAO) in the gut of different individuals
and in persons under medication. The higher the gut level of MAO, the higher the
tolerance for pressor amine absorbed with food. Dose of 100mg/100g food will generally
lead to clinical syndrome.
MYCOTOXINS
These are fungal metabolites and some are highly toxic to many animals and potentially
toxic to human beings. Interest in the problem of food-borne mycotoxins came to the fore
when it became apparent that mould growth on certain relatively dry agricultural products
led to highly destructive diseases of man and animals. These included alimentary toxic
aleukia (ATA) caused by ingestion of overwintered cereals, mouldy rice toxicosis and
aflatoxicosis in poultry. Today, more than 220 different types of moulds produce
mycotoxins active against man and animals when growing on foods. These
mycotoxigenic moulds are found mainly within the genera Aspergillus (43 species),
Penicillium (52 species), Fusarium (20 species) and Cladosporium (8 species). The
mycotoxins cause serious diseases of the liver, kidney, the circulatory system, the blood-
forming system and even the nervous system. There is epidemiological evidence that
chronic absorption through food of certain mycotoxins may result in primary hepatoma in
man, some cause tremors- an alarming pharmalogical activity in man. Other diseases
linked to mycotoxins are encephalopathy seen in Thailand, and endemic nephropathy
observed in the Balkans. Hence, such mould prone foods like cereals, flours, bread, nuts,
fruits and juices, jams, meat products, cheese, spices, dried soups and smoked fish should
be monitored for mycotoxin content.
Consumption of soft cheese ripened by mould e.g. Roquefort and Camembert cheese
should be with caution. Other foods worthy of cautious consumption are mould
fermented foods and fungal enzymes used in food preparation.
A common characteristic of mycotoxin in spite of their chemical diversity is their small
molecular weight. This accounts for their high thermostability making it virtually
impossible to inactivate clinically significant mycotoxins to safe level by any heat
treatment currently applied in food processing.
By virtue of their low molecular weights they can sometimes diffuse away from mould
colonies on food. Removal of mouldy portions of affected food therefore may not entirely
eliminate mycotoxins.
Mould prone foods should, as a preventive measure be stored under condition of
temperature and water activity that would not permit mould growth and metabolism.
Some Fungal Food-borne Disease Mycotoxins
1. Aflatoxin: a toxin produced by Aspergillus flavus. The resulting A. flavus toxin was
called aflatoxin. They are found in cereal grains- sorghum, maize, millet. Aspergillus
parasiticus also produces aflatoxins B1 and B2, G1 – G2 and M1 – M2. They are found in
milk, urine of infected humans.

2. Patulin: patulin(clavacin, expansin, clavatin e.t.c.) is produced by a large number of
Penicillium species, including Penicillium claviforme, P. expansum, P. patulum. This
mycotoxin has been found in mouldy bread, sausage, fruits(including pears, pineapples,
grapes, bananas), apple juice and cider.
3. Ochratoxin: Aspergillus ochraceus, A. alliaceus, A. ostianus, A. melleus and other

species of Aspergillus. Among Penicillium that produce ochratoxin A are Penicillium
viridicatum and P. palitans. The mycotoxin has been found in cocoa, dried beans, corn,
soybeans and fruits.
4. Citrinin: Penicillium citrinum and P. viridicatum and other fungi. It has been
recovered from polished rice, mouldy wheat bread.
There are other mycotoxins such as alimentary toxic aleukia(ATA), penicillic acid,
sterigmatocystin, luteoskyrin, roquefortine e.t.c.
VIRAL FOOD-BORNE DISEASES

Not much is known about the incidence of viruses in foods than about bacteria and fungi
for several reasons. Firstly, being an obligate parasite. They do not grow on culture media
as bacteria and fungi do. Secondly, they do not replicate in foods. Thirdly, laboratory
virological techniques are not practicable in many food microbiology laboratories.
Finally, not all viruses of potential interest to food microbiologists can be cultured by
existing methods.
The following viruses have been implicated in food borne diseases:
1. Poliomyelitis caused by Polioviruses types I, II, III, have
been found in milk (pasteurized and raw), possibly other
beverages and prepared foods.
2. Infectious hepatitis caused by infectious hepatitis virus A &
E, found in milk, other beverages, shellfish and oysters.
3. Acute non-bacterial gastroenteritis caused by Coxsackie
virus, ECHOviruses and Norwalk virus.
INFECTIONS OCCASIONALLY TRANSMITTED BY FOOD

These include bacterial, viral and helminthes diseases. They do not give rise to acute
gastroenteritis normally associated with food-borne diseases discussed earlier, yet may be
transmitted by foods.
These diseases have very low MID (minimum infective dose, i.e., the lowest number of
microorganisms required to cause disease) in contrast with the classical food-borne
diseases.
Examples of Occasional Food-borne Pathogens:
1. Corynebacterium diphtheriae causes diphtheria. Lancefield Group A Streptococci
causes septic sore throat. Both are properly controllable by proper thermal
treatment of food and protecting foods from recontamination by sanitary
handling.

2. Mycobacterium bovis from raw milk and Brucella melitensis also from raw milk
were responsible for much of the outbreaks of tuberculosis and undulant (Malta)
fever in the past. Today, due to their heat lability, proper pasteurization of milk
has eliminated incidents of these diseases arising from milk, in the advanced
countries.
3. Note Coxiella burnetti (Rickettsiae) agent of Q-fever (Queensland or query fever)
transmitted by dust and milk. Susceptible to pasteurization.
FOOD-BORNE PARASITIC INFECTIONS

Some parasitic infections are caused by the following parasites:
1. Anisakiasis caused by Anisakis spp. (a nematode) found in raw or insufficiently
cooked fish.
2. Amebic dysentery (Amebiasis) caused by Entamoeba histolytica found in water
contaminated with sewage and moist food contaminated with human faeces.
3. Beef and Pork tapeworms caused by tapeworms (Taenia saginata and Taenia
solium) respectively found in raw beef and pork or insufficiently cooked beef or
pork.
4. Trichinosis caused by Trichinella spiralis found in raw or insufficiently cooked
pork and pork products.
MICROBIAL FOOD QUALITY CONTROL

Objectives of Microbiology test in Quality Control
Microbiology tests in quality control are tests established with a lot of objectives amongst
which are the following:
1. To ensure the supply of safe, nutritious and decently presented food.
2. To protect consumers from contaminated food, which can be dangerous to their
health.

3. To prevent adulteration of consumers goods because adulteration/imitation can
cause consumer to eat decomposed, injurious or poisoned food. Adulteration can
also cause producers loss of customers to unauthorized sellers as well as mar the
image of producers by using similar packages and false labels.
4. To promote better quality control of foods by food processors and distributors and
hence encourage development of the food industry on sound scientific lines.
5. To ensure production of standard food and hence improve export potentials.
6. To check quality of imported foods.
The essence of this quality control system and all quality control systems in general is
that it helps in building confidence in consumers. The consumers will develop confidence
in the product they buy, they can trust that, the food is safe for consumption.
As a result of increased awareness of food-borne disease and problems associated with
the sanitary and microbiological quality of food, together with increased international
trade in food products, microbiological bodies for quality control were established to
ensure the practice of the above listed points on behalf of consumers.
On behalf of producers, quality control settings were designed to help in the following
ways:
1. Production of wholesome food ensures the confidence of consumers and hence
can increase sales of producers.
2. Microbiological food quality control will also lead to general improvement in the
quality of food by retarding or eliminating microbial growth, which could easily
cause spoilage and this ensures better shelf life than unmonitored food, which is
handled carelessly. Increased shelf life helps manufacturers to be able to increase
productivity without fear of their spoilage prematurely.
3. Identification of contaminated food before packaging and distribution to
consumers is of great advantage to manufacturers because quick solution can be
applied. For instance, if microbial count is not high, growth of pathogen can be
further prevented by one or more properties of the food- increase in acidity or
sodium chloride content helps other processes of preservation like pasteurization.
Set up of Food Microbiology Quality Control Laboratory

A typical food microbiology laboratory for quality control does necessarily look special
from any other microbiology laboratory. However, since it has to handle a lot of samples
from raw materials, materials in process at various stages and finished products, it needs
to have a lot of room and storage facilities. Some basic equipment for the quality control
laboratory added to those normally seen in general microbiological laboratory should
include: a functional centrifuge; water bath with a functional thermostat; as many
sinks as possible; a medium preparation room and various media for culturing
microorganisms usually found in foods; analytical balance for dispensing and
quantification of media; deep freezer for storage of food samples; hot air oven;
colony counters; pipettes of various sizes; membrane filter apparatus and
membrane filter; incubators; microscopes and slides; homogenizer; sharp knife for
cutting.

The set up however is not of any particular design but should contain such equipment and
chemicals that are relevant to food microbiology. Useful equipment for sampling are very
important for aseptical handling of materials. The most important precaution to be taken
is the hygienic handling of the food materials. This will ensure that no handler
contaminates or introduces his own microbial load into the samples. A good food
standard has to be set so as to give the assurance that the food received by the consumers
are pure, safe and of the quality claimed. This food standard has to conform with that set
in the area of jurisdiction where the food has to be sold. These standards of various
regulating bodies have to be available in the laboratory. These standards differ from one
regulatory body to the other and even from one country to the other. But the joint
FAO/WHO food standards commission is a forum for the cooperation among nations to
develop and agree upon various international standards.
It is equally of importance to mention that the laboratory should have a good water
supply. This should of course be good quality water normally graded as class 1. Thus, it is
necessary that most industries should have their own treatment plant to ensure this grade
of purity.
ENUMERATION AND DETECTION OF MICROORGANISMS

SAMPLING METHOD
Sampling, put simply, is the taking of a small quantity to represent a whole or larger
population. Unfortunately, the sample can never be exact as the population it represents.
It is even less exact in microbiological quantifications. But effort is made to achieve as
reasonable a result as possible. Samples are taken from foods with sterile implements
such as knives, spoons or spatulas, weighing pans or even specimen plastic
jars/containers.
These samples are taken from different areas of the food. The top, bottom and center. If
the food is in the liquid or semi-liquid form, a mix is made before the sample is taken. If
the sample is taken in the field, it has to be transported to the laboratory as fast as
possible for analysis. In some cases, it may be necessary to put the sample into transport
media, if the organisms are fastidious or the time taken to the laboratory lasts more than
six hours.
Sample Collection and Handling in Microbiology Quality Control
Sample collection and handling in microbiological assay is a very important aspect
because its handling can make all the difference in the types of microbial load present.
(1) In sample collection, to avoid false result, clean (sterile) containers must be used.
If dirty or unsterile containers are used, the microbial flora of the container might
be what is being enumerated rather than what is contained in the sample itself.
(2) Proper and immediate labeling must be done to prevent samples from different
sources being mingled and confused. Avoid the use of water-soluble markers,
other markers (water resistant) or adhesive tape can be used.
(3) The samples must be properly capped to avoid contamination from external
sources due to improper handling.
(4) Samples should be kept at an appropriate temperature to avoid alteration of
characteristics of the microflora either positively or negatively. High temperature
sometimes might render some microflora unviable. Warm environment might lead
to conducive environment for multiplication. Adequate temperature for

preservation must be present. For example, sufficient refrigeration must be
provided to prevent destruction or growth of microflora in the sample. Perishable
samples collected in non-frozen state must be refrigerated preferably at 0 to 4.4oC
from time of collection. Samples collected while frozen should be kept solidly
frozen. Samples should be examined within 36 hours after collection.
Sampling devices and Instruments
Methodology used in sampling as well as the type of equipment adopted for sampling are
two important things in sampling. Instruments and devices are as follows:
1. Stirrer, drills, spoons, scoops, spatula, pipettes, swabs e.t.c. usually used in taking
samples from bulk materials.
2. Sterile scissors, knives, can openers (used for opening of packaged and canned
food).
3. Non-water soluble labels and markers.
4. Refrigerators capable of chilling samples to 0-5oC in a few hours.
5. Insulated containers for transporting frozen or chilled samples.
6. Sterilizing equipment such as autoclave and oven.
7. Sterilizing agents such as 70% alcohol, methylated spirit e.t.c.
8. Sample containers -usually sterilizable e.g. glasswares, bottles or that comes
sterile from manufacturer (disposables).
Note: Sampling instruments may be presterile and kept in protective covers. Presterile
multiple use containers must be cleaned and sterilized after each sampling.
Sample containers must be cleaned, dried sterile, leak proof, wide mouth and be of
suitable size to hold samples of a particular product under examination.
Multiple use of containers must be avoided to prevent contamination of samples. Broken
glass containers or leaking cover should be avoided in order to reduce contamination rate.
The sampling operation should be planned in advance and all needed equipment and
containers at hand. Sampling instruments should be handled as little as possible before
wrapping and must be protected from contamination until after sampling is completed.
Above all, experienced and qualified individual or technicians should be available to
carry out sampling and enumeration of samples in any quality control laboratory.
Methodology
The number of samples taken should be representative of the batch e.g. 10 samples per
batch, 5 – 10 batches from each establishment. In bulk materials, both deep and
superficial samples are taken. Each bulk container must be sampled. In factory sampling,
it is more important to take a small number of samples at different times during the day,
than to take a lot of sample at any given time. Where resources are limited and GMPs
(good manufacturing practices) instituted, concentrate on finished products, but if level of
microorganisms begin to increase, work back taking on-line sample to defeat the faulty
critical step.
The containers used for the samples are usually screw capped disposable plastic or
aluminium containers, large mono or polythene bags. It is not good to use glass jars since
these are prone to breakages, which could contaminate the product or cause injury to
personnel. Instruments for removing portions of bulk material vary according to the
nature of the substance. For frozen food, a wrapped and sterilized brace and bit or a clean
chopper, washed in methylated spirit and flamed is used.

Individually wrapped and sterilized spoons and spatulas or wooden tongue depressors are
used for soft materials. For prepackaged materials, it is better to examine several
complete packs as offered for sale.
Foods like whole meat carcases, where the organisms are on the surface, swabs of the
surface are made with sterile cotton wool. For vegetables, the surfaces are washed out.
Sample Preparation and Enumeration
Samples taken are usually in the liquid, powdery and solid forms. For the liquid samples
like milk and juices, sterile pipettes or measuring cylinders are used to transfer aliquots to
sterile diluents, serial dilution of the sample is made to reduce the number of
microorganisms before enumeration takes place.
For powdery foods, 10g of the food is weighed using a sterile weigh pan or grease-proof
paper and transferred into 100ml of sterile diluent (which may be distilled water, ringers
solution, normal saline, peptone water and so on). If the sample is suspected of heavy
contamination. This initial 1:10 will further be diluted. If the powdery food substance
does not dissolve in water but forms a suspension. It is then centrifuged to throw down
the suspension and the supernatant is used. This is then diluted as wished using the serial
dilution technique.
For solid foods, like fish, meat, salads and so on, an aliquot is weighed into a sterile
diluent aseptically. This is transferred into a sterile homogeniser, blended, then
centrifuged and the supernatant used. Serial dilution is carried out at will and then
enumeration can then take place.
Enumeration
It is often necessary to report on the size of the bacterial or yeast population in a sample.
Unfortunately, accuracy is demanded where it is not needed. It is noted that errors of
90% in counts of about 10 to 100,000 per milliliter are not usual even with the best
possible technique. It is therefore necessary to combine maximum care in technique with
a liberal interpretation of results. Thus, a single test is valueless and figures obtained from
it can only be interpreted if the product is regularly tested and the normal range known.
So, replicates of a test is normally done.
There is no completely satisfactory method of examination and enumeration of microbes
in food stuffs. The method selected and the examination performed will depend upon
local conditions such as availability of staff, space and materials, number of samples to
be examined and the period of time allowed to obtain a result. Most laboratories or
quality control microbiologist have developed their own sub-optimum methods of
standards according to experience, to give a ‘pass’ or ‘fail’ mark to a batch of products.
Some enumeration methods often used in food quality control include total counts, using
the direct microscopic slide method or the counting chamber or Breeds method (BMC).
Another is the viable count using the pour plate or streak plate method, roll tube method,
and the membrane filter method, then the Dye reduction tests. There is another called the
most probable number (MPN) based on the assumption that bacteria are normally
distributed in liquid media.
Sampling Procedure for the following:
1. Liquid Products (e.g. milk, fruit juices, beer and water at source) may contain only few
bacteria but are subsequently contaminated, and the degree may be heavy or light
depending on the sanitary precautions and careful handling procedures employed. These
samples are usually mixed properly to enable homogenization before drawing is done

using scooper or tap device. The sample containers should not be filled more than ¾ way
to enable shaking during enumeration. Other hygienic procedures are adopted to prevent
contamination e.g. flaming. For instance, milk on leaving the udder of a healthy cow
contains few microorganisms. Method of milking- either hand or machine adds more to it
and the utensils used, e.g. milk pails e.t.c. Then on bulking in tanks still increases the
microbial load as shown below:
Point of Sampling Usual Range
Aseptically drawn milk 500 – 1000TPC/ml
Milk Machine/Pail 1000 – 10000TPC/ml
Bulk Milk Tank (farm) 5000 – 20000TPC/ml
To test a sample of milk for microbial quality, a given quantity is taken, diluted and
plated out. The result in the table above shows the appropriate acceptable range for fresh
milk. Other methods that can be used are Breeds count and dye reduction method, which
is also used in milk classification. If faecal contamination is suspected, coliform plate
count can be carried out.
2. Dehydrated Products (milk powder, cassava flour, yam flour, corn flour, garri e.t.c.).
The recommended microbiological methods for dry powdery products include standard
plate count, direct microscopic counts, coliform plate count and yeast and mould count.
Occasionally, thermoduric, thermophilic and psychrophilic bacterial counts are carried
out when it is expected that the presence of such organisms may affect the keeping
quality or future use of the product.
Taking of samples should be done with sterile spoon, spatulas, excess humidity should be
prevented and several samples should be taken to ensure representative samples.
3. Frozen Products (e.g. frozen dessert, ice cream e.t.c.). The keeping of food products in
the frozen state for a long time is capable of inhibiting microbial growth with reduction in
the viable counts of organisms present. A high bacterial count of frozen product may
indicate poor sanitary quality or poor manufacturing practice, but a low to moderate
count does not always prove the opposite. The methods useful here are standard plate
count (SPC) and coliform plate count (CPC).
Frozen bulky foods may be sampled with sharp sterile object. Frozen samples should be
kept frozen until arrival in the laboratory, i.e. it must be kept in heat conserving container
to prevent thawing. The sample to be assayed is thawed to room temperature. The
packages are crushed and 50g weighed out into 450ml of sterile diluent and
homogenized. It may be necessary to centrifuge to eliminate debris of food. Serial
dilutions are made to 10-5 dilution for standard plate count on plate count agar (PCA).
Frozen food products of acceptable sanitary quality should not produce counts exceeding
50,000 per gram.
For coliform counts, 1:5 and 1:10 dilutions are made and plated out on coliform media
like MacConkey agar, Eosin-methylene blue (EMB) agar or violet red-bile (VRB) agar.
But the most common is VRBA, the colonies here appear dark-red and of 0.5mm
diameter, showing evidence of precipitation of bile salts. Coliform counts of 2,000 or
more per gram of new products for freezing is indicative of poor manufacturing practice

and 50 coliforms of frozen material is equally indicative of possible contamination from
soil, equipment or personnel.
4. Canned Products (e.g. canned beef, fruit juices, vegetables e.t.c.). Before products are
canned, they have undergone one or more processing operations inimical to most
microorganisms, and even after canning, they undergo pasteurization to ascertain their
stability or keeping quality. Thus, the assaying of these products, need only the sterility
test to ascertain their sanitary quality. The medium used is the sterility medium or
thioglycolate medium. The food suspension is prepared aseptically and inoculated. 2ml
sample each into 8 tubes of sterility medium. The tubes are mixed thoroughly and
incubated in duplicates i.e. 2 at 35oC and 2 at 55oC aerobically, 2 at 35oC and 55oC
anaerobically, for 48 – 72 hours. Look for evidence of cloudiness indicating growth and
non-sterility of tubes. If a tube appears doubtful, gram-stain and observe under the
microscope.
Examination of many samples helps in getting accurate results, because the number of
spoilt cans might be small in a batch. Physical examination of can is usually necessary to
detect swollen cans, rotten cans and batch numbers noted.
The surface of the can should be swabbed before opening with sterile flamed opener.
Aseptic condition must be maintained throughout the sampling period. The sampling
procedure of normal and swollen cans is the same, extra caution is only taken to prevent
spilling over of swollen can e.g. use of funnel.
Bacteriological Examination of Water

Sample Collection: when collecting water, for examination, care must be taken so that the
depth is about 1feet(i.e. 30cm) below the water surface so that not only the surface
organisms are collected. Also, this has to be done at three different feet levels at three
different positions away from the water bank. Sterile bottles of least 100ml capacity are
used. A clamp should be used to hold the bottles vertically, until the depth is reached
before tilting the bottle to collect. Samples so collected should reach the laboratory in an
hour’s time, this is because (i) organisms with very short generation time e.g. E. coli will
populate the entire flora of the water, (ii) the corked bottle creates anaerobic environment,
which could kill off the aerobes. On getting to the laboratory depending on the bacterial
load, from experience and/or expectations, serial dilutions are made of the water sample.
Analysis of Sample: the methods employed in water analysis microbiologically are
general viable count and coliform test using E. coli as indicator.
1. Estimation of total numbers using the spread plate technique or the pour plate
technique. The medium of choice is nutrient agar, because aquatic bacteria favour poor
medium. Quarter (¼) strength nutrient agar is preferred. Better still, environmental
stimulative medium (ESM) can be used.
2. E. coli an indicator organism for faecal contamination. The detection of coliforms in

water sample is by the most probable number (MPN) method. This gives an index and
not the actual number in a given water sample. The test is in three parts:
(a) Presumptive Test: the principle underlying this test is that the coliforms are known to
ferment lactose with production of acid and gas. When samples are added to the tryptone
lactose broth and incubated at 35 – 37 oC for 48 hours, the presence of gas within 48±

hours in the fermentation tube constitutes a positive reaction. Then the MPN table is
consulted.
To confirm the presence of E. coli and differentiate it from other coliforms, the following
test is carried out. Subculture positive tubes into brilliant green lactose bile broth
(BGLBB) in duplicates and then into one tube of peptone water. One tube of BGLBB is
incubated at 35oC for coliforms while the other and the peptone tube are incubated at
44oC and inspect after 6 hours and 24 hours. The peptone water is tested for indole
production after 24 hours. The positive BGLBB at both temperatures are recorded and
read off the MPN tables of positively confirmed coliforms at 35 – 37oC and E. coli at
44oC. Only E. coli produces gas and indole at 44oC.
(b) Confirmatory test: positive tubes are subcultured into eosin methylene blue agar
(EMB), these are incubated at 35 – 37 oC for 24 hours. Typical colonies are reddish,
nucleated with or without metallic sheen. Atypical colonies appear opaque, non-nucleated
and mucoid after 24 hours. Other colonies are negative. With typical colonies confirmed,
there is no need to continue to the completed test.
(c) Completed test: pick one or two typical colonies or if there is no typical colony
present, 2 more or more colonies considered most likely are transferred, each to a lactose
broth in fermentation tube and nutrient agar slant and incubated at 35± 0.5 oC for 24
hours. Only those slopes whose corresponding fermentation tube are positive are gram
stained and results recorded. Confirmation of gas in the secondary tube and
demonstration of gram negative non-spore forming rods may be considered as
satisfactory test.
Grade of Potable Water:
Grade A: has no coliform in 100ml sample of water.
Grade B: has only 1 coliform in 100ml sample of water.
Grade C: has 1 - 3 coliforms in 100ml sample of water.
Grade D: has 3 –10 coliforms in 100ml sample of water.
Above 10 coliforms per 100ml, the water is no longer potable.
Food Sampling
Samples of foods for investigation are collected in sterile containers. These samples could
either be in solid or liquid forms depending on the nature of the particular food item.
After collection, care should be taken so that the samples collected are transported
without delay to the laboratory for analysis. In the laboratory, the samples are analysed by
culturing them on selected media that will permit the growth of the microorganism that
may be present in the samples. The solid samples could be plated out directly on nutrient
media or in most cases, are diluted serially before plating out. The choice of serial
dilution before plating out, was informed by the fact that if there are too many microbes
in the sample, direct plating out of the sample might give growth of microorganisms too
numerous to count (confluent growth). Similarly, liquid samples could be plated out
directly or plated out after serial dilution. The choice of media for cultivation of the
microbes depends to a large extent on the type of food item under investigation and the
type of microbes that are being investigated. In view of these and other factors, the
following culture media are often used in studying food samples for the presence of

microbes: nutrient agar, MacConkey agar, malt extract agar, nutrient broth, MacConkey
broth e.t.c.
The samples cultured on any of the above media should be incubated at a chosen
temperature, which will equally depend to a large extent on the nature of the suspected
microorganisms. However, most of the food samples are incubated at temperatures
ranging from 25oC – 50oC. The period of incubation is between 24 hours – 72 hours.
However, in some cases, incubation could be extended up to 5 days or even 7 days. After
the period of incubation, the plates are examined for the growth of bacteria that may be
present in the food sample under analysis.
The gross appearance of bacterial culture in various media provides clues to the identity
of the organism and should always be noted. Description of the colonies on agar plates
should include size, shape, colour, appearance by reflected/transmitted light and
appearance when examined under the microscope. Growth in broth may consist of
uniform turbidity or cloudiness, a sediment at the bottom of the culture or a membrane on
the surface. It should be noted however, that these growth characteristics are frequently
perculiar to certain species, although, as in the case of morphology, temporary variations
occur under different cultural conditions.
The following colonial forms/shapes have been observed in bacteria on solid media, they
are circular, irregular, filamentous and spindle. The colours of the colonies range from
white, cream, yellow, orange, grey, milk, pink e.t.c.
Cell morphology- the shape and arrangement of the cells are determined by microscopic
examination of wet mounts or hanging drop preparations and stained smears.
Examination of the microorganisms also reveals motility and gives evidence of presence
of flagella. Non motile bacteria frequently display Brownian movement, a vibratory
motion caused by molecular activity in the fluid. The cell arrangements and shape are
better observed when the cells are stained especially with gram stain. Gram stain is a kind
of staining procedure that is used to study the morphology of bacteria.
INDICES OF FOOD SANITATION AND MICROBIOLOGICAL STANDARDS

AND CRITERIA
The aim of examining foods for microorganisms are two folds:
(1) For spoilage organisms which grow in/on foods to produce
off-odours, off-flavours and generally reduce its palatability.
(2) For pathogens which cause several human diseases. These
examinations and their limitations are discussed.

Direct Examination of Foods for possible Pathogens
This involves direct microscopic count (DMC), Gram stain, spore stain, flagella and
capsule stains are made of the food sample and likely pathogens are identified and
counted. This is easier for fluids and semi-solids. For solid foods, a known quantity (10g)
is homogenized in a known quantity of sterile distilled water (100ml), it is then
centrifuged and the supernatant used. For enumeration, a graduated slide (like
haemocytometer) is used. The result is expressed as number of organisms per gram of
food.
Limitations:
(i) The sample of food taken may not be representative of all.
(ii) Both pathogens and non-pathogens are enumerated.
(iii) Both dead and living cells are enumerated.
(iv) The homogenizing action may destroy some organisms.
Total Plate Count(TPC) for Mesophilic Aerobes

This method is more reliable than DMC but the result cannot be obtained until after
incubation period of 24 – 48 hours. The food is mixed very well and measured out (mls
for liquids and grams for solids). Homogenization and centrifugation is done for solids
and supernatant is taken. Depending on suspected microbial load, the sample is diluted
serially 1:10, 1:100, 1:1000 and so on. Two methods can be then used for plating out viz
pour plate or spread plate technique.
In pour plate method, sterile molten agar in water bath at about 45oC is used. 0.1ml of
sample from each dilution is placed on the petri dish and 10ml of agar is poured onto it. It
is then swirled round to mix and left to solidify.
In spread plate method, the agar is solidified on plates and 0.1ml of sample is placed on it
and spread with a glass spreader. The plates are then incubated at room or between 25o –
30oC for 48 hours. Plates with discrete colonies are counted. Only plates with 30 – 300
colonies are accepted. Those with less than 30 are unreliable and above 300 are too
numerous to count (TNTC).
Limitations:
(i) The result has to wait for 24 – 48 hours.
(ii) It does not isolate spoilage from pathogens.
(iii) Some organisms may not grow at all due to,
- Non-specialized or enriched medium.
- Temperature of incubation and
- Anaerobes.
(iv) With low levels of contamination, results may be mis-leading.
Coliform Count
Coliforms are of faecal origin and their presence in foods is an indication of faecal
contamination. They are gram-negative, non-sporing rods, producing acid and gas
from lactose within 48 hours.
The method used is the MPN technique since it is most suitable where low level of
contamination is expected as in milk, water and foods. For solid foods, measure out
aliquots as appropriate and homogenize, centrifuge and collect supernatant. Dilutions
are made where necessary. Using 9 or 15 tubes carry out as described in water
sampling. The MPN table gives the most probable number of organisms per gram of

solids or per ml of liquid foods. If foods like milk where turbidity is masked, dyes or
coloured indicators are added. Note that only tubes that develop turbidity and gas
within 48 hours are positive for coliforms. If presumptive tubes are all negative, no
further test is carried out. Where the confirmatory plates show atypical colonies, do
completed test, but where they are typical, completed test is not necessary.
Limitations:
(i) It takes 3 or 4 days to get results.
(ii) The number given is simply a probability.
(iii) Sampling errors are rife.
(iv) Organisms that have suffered metabolic stress in foods are not resuscitated.
Total Enterobacteriacea Count

Enterobacteriacea is a group of gram-negative non-spore forming rods, which inhabit
the gastrointestinal tract (GIT) of man and animals. They all ferment dextrose but few
ferment lactose. The lactose fermenters are called coliforms, these include
Escherichia, Klebsiella, Citrobacter and Enterobacter. Others are non-coliforms. All
ferment carbohydrates producing gas and/or acid.
Plate count of enterobacteriacea is made using differential media like EMB(Eosin
Methylene Blue), MacConkey agar and others. These media inhibit the growth of
gram-positive organisms and also differentiates the lactose fermenters (appear reddish
on EMB) from lactose non-fermenters (appear colourless). From a suitable dilution of
the food sample, 0.1ml is inoculated on the medium and spread with a spreader and
incubated at 35 – 37oC for 24 – 48 hours, 30 – 300 cfu are taken. Lactose fermenters
and lactose non-fermenters ratio are also taken.
Pathogens as an Index of Food Sanitation

Pathogens in foods indicate that the food is unsafe for human consumption and grossly
contaminated during processing and storage. Most of these pathogens are of intestinal
origin and so, two coliform organisms Escherichia coli and Enterobacter aerogenes are
used as indices. Where they occur in large quantities in food, it is an indication of/that:
(i) the food may be a source of food poisoning/infection and therefore unacceptable.
(ii) unwholesome raw material source unduely contaminated during processing,
packaging, storage and handling.
(iii) introduction of filth such as faecal matter, pus cells, mould mycelium e.t.c.
(iv) the food will not have the keeping quality expected of it.
Microbiological Criteria for Foods

The chief purposes of microbiological criteria for foods are to give assurance that,
- the food is acceptable from the public health standpoint.
- the food is of satisfactory quality.
- the food is of aesthetic value, free from the introduction of filth e.g. faecal material,
pus cells, parts of vermin, mould mycelium e.t.c.
- the food will have keeping qualities that is expected of the food product.

Many difficulties have been encountered in establishing and applying microbiological
standards for foods. Microbiological standards usually have been based on total numbers
of organisms, number of indicator organism, or number (or total absence) of pathogens;
although there have been disagreements as to which, counts should be considered as
significant, what the indicator organism should be, and whether pathogens can be
demonstrated.
The National Academy for Science, Food Protection Committee, has suggested the
following definitions of microbiological criteria:
(a) A Microbiological Specification- is the maximum acceptable number of
microorganisms or of specific types of microorganisms, as determined by prescribed
methods, in a food being purchased by a firm or agency for its own use.
(b) A Recommended Microbiological Limit/Guideline- is the suggested maximum

acceptable number of microorganisms as determined by prescribed methods in a food.
(c) A Microbiological Standard- is that part of a law or administrative regulation

designating the maximum acceptable number of microorganisms or of specific types of
microorganisms, as determined by prescribed method(s), in a food produced, packed or
stored, or imported into the area of jurisdiction of the enforcing agency.
The National Academy of Science, Food Protection Committee, 1985, came out with
certain recommendations:
(1) Testing procedure and standards should be adapted to a particular kind of food.
(2) A numerical relationship should be demonstrated between the standard and
hazard.
(3) Tolerances should be allowed for admitted inaccuracies of sampling and analysis.
(4) Any suggested criteria should be tried out first on a voluntary basis.
Some Coliform Standards of Foods

1. Milk: they should not exceed 10 coliforms/ml of grade A and certified pasteurized
milk, or yoghurt, cultured milk or milk products, and in pasteurized dried milk not over 1
coliform/gram (MPN).
2. Precooked frozen foods: not over 10 coliforms/gram (MPN).
3. Crab: not over 100 coliforms/gram of crab meat (MPN).
4. Shell fishes: not more than 100 coliforms/gram (MPN) and not over 500,000/gram at
35oC(Plate Count).
5. Meat and poultry products: (TPC) raw-surface mesophilic flora 10 – 10 2/cm2.

Psychrotrophs- 3 – 4/cm2. Coliforms- 10 – 102/gram of tissue. Cooked meat- heat
resistant bacterial spores, not more than 100/gram, fermented meat products e.g. sausages
should not exceed 106/gram. Staphylococcus 103/gram and less tolerated. On dried meat,
10 – 100/gram.

6. Eggs and egg products: freshly broken eggs 1/gram or less of Salmonella, egg
products up to 100/gram tolerated (TPC).
7. Fruits and vegetables: mostly commensals are encountered on the surfaces of fruits and
vegetables, 102 – 104/gram acceptable.E. coli should be less than 10/gram and same for
Staphylococcus. Yeast and moulds dominate fruits and vegetables and lactic acid
bacteria.

Food MCB II Notes

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FOOD MICROBIOLOGY II

Dr. Mugampoza Edriisa- Dept. of Food Processing Technology, KYU © 1

FOOD PARAMETERS (INTRINSIC FACTORS)

(1) pH and the buffering power:

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Fish & Shellfish:

(2) Moisture content (concept of water activity):

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(3) Oxidation-Reduction potential(O-R):

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(4) Nutrient content:

(a) Food for energy-

(b) Food for growth-

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(c) Accessory food substance (vitamins)-

(5) Inhibitory/Antimicrobial constituents:

Dr. Mugampoza Edriisa- Dept. of Food Processing Technology, KYU © 9

(6) Biological structures:

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(2) Relative Humidity:

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(3) Presence and concentration of gases in the environment:

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SUMMARY OF FOOD PARAMETERS

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Apiculate or Lemon-shaped Yeast- some such as Hanseniaspora, Nadsonia and

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Cultural characteristics important in food:

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SOURCES OF MICROBIAL CONTAMINATION OF FOOD

(2) From animals:

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(7) Food handler:

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(8) Food utensils:

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Causes of Food Spoilage

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b) Reaction within the food, due to uncontrolled activities of enzymes:

(ii) Food browning mediated by enzymes

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B. Spoilage due to the activities of microorganisms:

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Factors Affecting Kind and Number of Microbes in Foods

Factors Influencing Microbial Growth in Foods

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2) Effect of environmental conditions- these conditions are interrelated and determine

Chemical Changes caused by Microbes

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PRINCIPLES OF FOOD PRESERVATION

DELAY OF MICROBIAL DECOMPOSITION

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