INTRODUCTION TO MICROBES IN
FOODS
Chapter 3: Source of Microorganisms in Foods
Microorganisms get into food from both natural sources
and from external sources to which a food comes into
contact from the time of production until the time of
consumption.
 Natural sources for foods of animal origin include
skin, hairs, feathers, gastrointestinal tract, urogenital
tract, respiratory tract, milk ducts(teat canal) in
udders of milk animals.

Natural micro-flora exist in ecological balance with their
hosts, and their types and levels vary greatly with the
types of plants and animals as well as their geographical
locations and environmental conditions.
 Besides natural microorganisms, foods can be
contaminated with different types of microorganisms
coming from outside sources such as: air- soil- sewagewater- feeds- human- food ingredients- equipmentpackages- insects


Microbial types and their levels from these
sources getting into foods vary widely and
depend on the degree of sanitation used
during the handling of foods.
PREDOMINANT MICROORGANISMS IN
DIFFERENT SOURCES
Plants( Fruits and Vegetables)
 The inside tissue of foods from plant sources are
essentially sterile except for few porous vegetables(
radishes, onion, cabbage).
 Some plants produce natural antimicrobial metabolites
that can limit the presence of microorganisms.
 Fruits and vegetables harbor microorganisms on the
surface ; their types and level vary with:
soil condition, type of fertilizer, water used,
air quality.

EXAMPLES OF MICROORGANISMS IN PLANTS
Molds, Yeasts, lactic acid bacteria,Pseudomonas,
Alcaligenes, Micrococcus, Erwinia, Bacillus,
Clostridium, Enterobacter.
 Pathogens like enteric types can be present if the soil is
contaminated with untreated sewage.
 Factors increasing the number of
microorganisms in plants:
- Diseases of the plant, damage of the surface, long delay
between harvesting and washing, unfavorable storage
and transport before processing and improper storage
after processing

REDUCTION OF MICROBIAL LOAD IN FOODS
OF PLANT ORIGIN
Use
of treated sewage for fertilizers
Damage reduction during harvesting
Quick washing with good quality
water to remove soil and dirt.
Storage at low temperature before
and after processing.
ANIMALS, BIRDS, FISH AND SHELLFISH
Food animals and birds normally carry many types of
indigenous microorganisms in gastrointestinal tract,
respiratory tract, urogenital tract and teat canal in the
udder as well as in skin, hooves, hair and feathers.
 The number of these microorganisms depends on the
specific organ e.g. in large intestine >1010/g.
 Many animals act as carriers, they can harbor
pathogens like S. aureus, E. coli, Campylobacter, and
Listeria without showing symptoms.
 Laying birds may carry S. enteritidis in the ovaries.

Fish and shellfish carry normal flora in the scales,
skin and digestive tracts. They can also carry
pathogenic bacteria e.g. V. parahaemolyticus .
 Many spoilage and pathogenic microorganisms can get
into foods of animal origin (milk, egg, meat, fishery
products) during production and processing.
 Milk can be contaminated with fecal materials on the
udder surface.
 Meat can be contaminated with intestinal contents
during slaughtering.


Meat from food animals and birds can be
contaminated with several spoilage and pathogenic
microorganisms from skin, hair and feathers e.g. S.
aureus, Micrococcus, Propionibacterium,
Corynebacterium, Molds and Yeasts.
PREVENTION OF FOOD CONTAMINATION
FROM ANIMAL SOURCES






Good housing and supply of uncontaminated feed and
water.
Testing animals and birds for pathogens and culling the
carriers.
Using good quality water for washing the carcases.
Hair removal.
Removal of Contaminated parts
Proper cleaning of the udder before milking



Cooling milk immediately after milking and
processing .
Collection of eggs quickely after laying.
Fish and marine products should be harvested
from unpolluted water, and stored properly.
AIR
Microorganisms are present in dust and moisture
droplets in the air.
 Microorganisms do not grow in dust.
 Microorganisms in air are transient and variable
depending on the environment.
 Level of Microorganisms in air is controlled by:
 The degree of humidity, Size, Level of dust particles,
Temperature, Air velocity and Resistance to drying .

 Dry
air with low dust content and higher
temperature has a low microbial level.
EXAMPLES OF MICROORGANISMS PRESENT IN AIR







Spores of Bacillus Spp.
Clostridium Spp.
Molds and Yeasts
Cells of Gram Positive Bacteria e.g. Micrococcus and
Sarcina
If the surroundings contain a source of pathogens e.g.
animal and poultry farms or a sewage treatment plant,
different types of bacteria including pathogens and viruses
can be transmitted via air.
Control Measures:
Removing potential sources, Air filtration, Reducing
humidity, Installing ultra violet lamps.
SOIL
Soil used to grow agricultural produce and raise
animals and birds contains several varieties of
microorganisms.
 Microorganisms can multiply in soil and their numbers
can be very high(billions/g).
 Microorganisms Found In Soil:
 Molds and Yeasts, Enterococcus, Bacillus, Clostridium,
Enterobacter, pseudomonas, Proteus, Micrococcus and
Parasites.
 Soil contaminated with fecal materials can be the in
foods. source of enteric pathogens and viruses

SEWAGE
Sewage can contaminate crops with microorganisms
when it is used as fertilizer e.g. enteropathogenic
bacteria, viruses and parasites.
 Sewage should be treated before using as fertilizer to
kill pathogens.
 Effective washing of foods following harvesting is
important.

WATER
Contamination of foods with pathogenic bacteria ,
viruses and parasites has been recorded.
 Waste water can be recycled for irrigation.
 Potable water may contain bacteria capable of
causing food spoilage such as Pseudomonas,
Alcaligenes, flavobacterium.
 Improperly treated water can contain pathogenic and
spoilage microorganisms.
 Water used in food processing should be of higher
microbial quality than that of potable water.

HUMANS
Between production and consumption, foods come in
contact with different people handling the foods, they
include:
 People working in farms
 People working in processing plants.
 People handling food in restaurants
 People in retail stores and at home.
 Human carriers have been a source of pathogenic
microorganisms in food.

MAJOR SOURCES OF MICROBIAL
CONTAMINATION OF FOODS BY HUMANS
Improperly cleaned hands.
 Lack of aesthetic sense and personal hygiene.
 Dirty clothes and hair.
 Presence of minor cuts and infection in hands and
face.
 Diseases(Influenza, Throat infection, Hepatitis A
and others).

Pathogens such as S. aureus, Salmonella, Shigella,
Pathogenic E. coli, Hepatitis A can be introduced into
food from human sources.
 Control Measures:
 Proper training of personnel in personal hygiene.
 Regular checking of health
 Maintaining efficient sanitary and aesthetic
standards.

FOOD INGREDIENTS
Many ingredients can be the source of spoilage and
pathogenic microorganisms
 e.g. Spices have very high populations of mold and
bacterial spores
 The ingredients should be produced under sanitary
conditions.
 Setting up acceptable microbial specifications for the
ingredients.

EQUIPMENT
Equipment can be a source of contamination
 Many microorganisms can get in food from equipment
e.g Salmonella, Listeria, Enteric bacteria,
Micrococcus, Escherichia, Pseudomonas,
Lactobacillus, Leuconostoc, Clostridia, Bacillus, Yeast
and Molds.
 Proper cleaning and sanitation of equipment should
be emphasized.

MISCELLANEOUS
Food might be contaminated from other sources;
 Packaging and Wrapping materials
 Containers
 Flies
 Vermins
 Birds
 House pets
 Rodents
 Proper care should be taken not to contaminate food
from these sources.

Section One: Basics of Food
Microbiology
Chapter 6: Factors that influence microbes in foods
Dr. Ali Salman Bin Thani
Food ecosystems, homeostasis, and
hurdle technology
•
•
1.
2.
•
The food ecosystems composed of two
components: the environment and the organisms
that live in it
The food environment is composed of two factors:
Intrinsic factors (e.g., pH, water activity [aw], and
nutrients)
Extrinsic factors (e.g., temperature, gaseous
environment)
Food constitutes multiple micro-environments,
were small parts might exhibit different extrinsic
factors
Dr. Ali Salman Bin Thani
Detection and enumeration of
microbes in food
Plate count:
Defining bacterial colony:
Could refer to a single bacterial cell, however
exceptions include, e.g., Spirochetes were multiple
cells might associate to give single colony
Therefore, ideally microbiologists would use CFU
(colony forming unit) which might refer to chain
of 10 or clamp of 100 cells
•
Dr. Ali Salman Bin Thani
Total plate count
Plate count is performed to count
bacterial cells between 25-250 colonies.
 The average number is then multiplied by
the dilution factor to get the bacterial cell
number in the original sample
 The above procedure is known as the
total plate count

Dr. Ali Salman Bin Thani
Total plate count
However, the total plate count is limited by the
following factors:
 It counts only those bacteria that can grow at
the selected agar medium and specified
temperature
 If you did this experiment in aerobic conditions
then you most likely missed the anaerobic
bacteria in the same medium
 You will miss bacteria growing at different
temperatures (e.g., psychrophile will prefer
temperatures below 15C)

Dr. Ali Salman Bin Thani
Total plate count
Total plate count involves serial dilution to get the
desired number of colonies per plate
Dr. Ali Salman Bin Thani
Selective media
Selective media: Some ingredients of the
medium favor the growth of target bacteria ,,
e.g., Staphylococcus aureus…medium is BairedParker agar
 Some drawbacks for selective media:
1. If the target cell is injured then it will be killed
by selective media
2. Some selective media are not entirely
selective…making the need for further
identification steps

Dr. Ali Salman Bin Thani
Differential media
S. aureus forms black colonies when grown on BairdParker agar
Dr. Ali Salman Bin Thani
The Most Probable Number (MPN)
method
Used to estimate low numbers of
organisms< 30 CFU/ml
 At least three 10-fold serial dilutions are
inoculated
 The pattern of turbid tubes with
microbial growth is recorded
 The pattern is used to derive the most
probable number of bacteria from a
statistical chart

Dr. Ali Salman Bin Thani
The Most Probable Number (MPN)
method
Dr. Ali Salman Bin Thani
The Most Probable Number (MPN)
method
Combination of
positives
MPN index/100 ml
95% confidence limits
Low
er
Upper
4-2-0
22
6.8
50
4-2-1
26
9.8
70
4-3-0
27
9.9
70
Dr. Ali Salman Bin Thani
Enrichment media and injury
Enrichment media is used to recover
injured pathogens
 Injured cells and cells that are “viable but
non-culturable” (VNC) pose additional
threats to food microbiologists
 Sub-lethal levels of heat, radiation, acid or
sanitizers may injure rather than kill cells
 However, under appropriate conditions
pathogens/injured cells can recover

Dr. Ali Salman Bin Thani
Normal microbiological quality of
foods and its significance

Raw and ready to eat meat products:
Carcasses normally contain average of 10-3 bacterial
cells/in2
 Carcasses of birds have higher incidences of Salmonella
contamination (fecal sources) than to those of animals
 The type of the predominant microbes during storage
of meat depend on: pH of the meat (which is low in
beef 5.4 and high in birds 6.0), high protein content, and
low carbohydrates and the environment

Dr. Ali Salman Bin Thani
Normal microbiological quality of
foods and its significance



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

Raw and pasteurized milk:
Rich in proteins and carbohydrates (lactose)
Because of its high carbohydrate content, the shelf life of raw and
pasteurized milk is limited
During refrigeration of raw milk psychrotrophs grow e.g.,
Pseudomonas, coliforms and Bacillus. They affect the acceptance
quality of raw milk, making the flavor and texture undesirable by
producing proteinases and lipases
Grade A pasteurized milk can have standard plate counts of
20,000/ml and less than 10 coliforms/ml
Thermodurics microbes are those escaping/surviving the
pasteurization step
Dr. Ali Salman Bin Thani
Normal microbiological quality of
foods and its significance
Other food materials:
 Several antimicrobial factors present in
egg albumin, such as lysozyme, conalbumin
(binds iron), avidin (binds biotin), all
control bacterial growth
 However, at favorable growth
temperatures microbes can enumerate in
the yolk which is rich in nutrients and has
a pH of 7

Dr. Ali Salman Bin Thani
Normal microbiological quality of
foods and its significance
Fruits have high carbohydrate contents
and low pH thus favoring the growth of
molds, yeast and lactic acid bacteria
causing food spoilage
 Canned food are either:
 heat treated to obtain commercial
sterility if their pH was 4.6 or above
 Or those with a pH below 4.6 were a
heat treatment of 100⁰C is enough

Dr. Ali Salman Bin Thani
Normal microbiological quality of
foods and its significance
Soft drinks with pHs between 2.5-4.0, allow only
aciduric microorganisms e.g., molds, yeast, lactic acid
bacteria to grow
 Bottled water should contain less than 10-100
bacteria/100 ml and less than 10 coliforms/100ml
 Mayonnaise and salad dressings pH between 3.5-4.0,
allow only aciduric microorganisms e.g., molds, yeast,
lactic acid bacteria to grow, however pathogens are
expected to grow in low calorie mayonnaise as the pH
for it is normally above 4.5

Dr. Ali Salman Bin Thani
Bacterial cells and injury
When cells are subjected to mild stress and
were plated on both rich nonselective and
selective media
 The difference between the population of cells
able to form colonies on each medium
represents the number of injured cells
 For example, 107 CFU of a population / ml are
found on the nonselective medium and 104 CFU
/ ml grow on the selective medium
 Then 9.9 x 106 CFU / ml were injured

Dr. Ali Salman Bin Thani
Bacterial cells’ injury and repair
Dr. Ali Salman Bin Thani
Importance of cell injury to food
safety
Overestimation of heat effect and injured
cells may be misleading as cells might
repair before food is eaten and cause
illness
 Recovery of injured and pathogen
bacteria may come from one of the food
ingredients (e.g., salt, organic acid)

Dr. Ali Salman Bin Thani
Viable but non-culturable
Salmonella, Campylobacter, Escherichia,
Shigella and Vibrio are able to form
dormant VNC
 These are non-sporulating bacteria able
to shrink and become small spherical
bodies
 Detection usually maintained through
microscopic examinations: Fluorescent
probes and stains

Dr. Ali Salman Bin Thani
Viable but non-culturable

VNC is always induced by nutrient
limitations, or other external factors e.g.,
when Vibrio vulnificus is shifted to
refrigeration temperature it becomes
VNC but still lethal to mice
Dr. Ali Salman Bin Thani
Intrinsic factors that influence
microbial growth





pH: Yeast & molds can tolerate acidic conditions more
than bacteria
Some food materials might have inherited low pH
values (acidic).
Examples include: Fermented products of lactic acid
bacteria
Vegetable juices with low buffering power, permit
decrease in the pH of food
Milk is known for being high in protein content
permitting the growth and acid production during
fermentation of milk
Dr. Ali Salman Bin Thani
Intrinsic factors that influence
microbial growth
pH: Most foodborne pathogens cannot
grow below pH 4.4, and most bacterial
cells will require an optimum pH value
between 7 to 9.
 However, most foods are not that alkaline
 Therefore, most bacteria will try to
maintain a neutral pH for their growth

Dr. Ali Salman Bin Thani
Mechanisms applied by bacteria to
sense changes in acidity:
Organic acids enter the cell only in the
protonated (undissociated) form. Once inside
the cell they dissociate releasing H+:
 Cells may sense the released ions
 The change in pH may affect the
transmembrane proton gradient
 Changes in the acidity inside the cell may result
in the protonation or deprotonation of amino
acids, changing the structure and function of
proteins and signal the cell to change the pH

Dr. Ali Salman Bin Thani
Maintaining the pH of the microbial
cell:
Changing the pH of cells will denature the
proteins present in the cytoplasm and restrain
the metabolic activities leading to the death of
microorganism
 Salmonella enterica serovar Typhimurium has
three mechansims to maintain the intracellular
pH (pHi):
1. Homeostatic response
2. The acid tolerance response
3. Synthesis of acid shock proteins

Dr. Ali Salman Bin Thani
Maintaining the pH of the microbial
cell:
These different mechanisms depend upon
different changes in the external pH (pHo):
1. Homeostatic response: At pHo > 6.0,
Salmonella will adjust their pHi through the
homeostatic response: would maintain the pHi
by increasing the activity of proton pumps to
expel more protons from the cytoplasm. This
process is not sensitive to protein synthesis
inhibitors

Dr. Ali Salman Bin Thani
Maintaining the pH of the microbial
cell:
The acid tolerance response (ATR) is
triggered by pHo of 5.5 to 6.0:
 There are 18 of these ATR
 Involves the membrane-bond ATPase
proton pump and maintains pHi of > 5.0
at pHo values as low as 4.0
 This process is sensitive to protein
synthesis inhibitors
2.
Dr. Ali Salman Bin Thani
Maintaining the pH of the microbial
cell:
Synthesis of acid shock proteins:
 Triggered by pHo from 3.0 to 5.0 and
constitute a set of regulatory proteins
distinct from the ATR proteins
3.
Dr. Ali Salman Bin Thani
Intrinsic factors that influence
microbial growth
Water activity (aw):
 All microorganisms require water in an
available form to grow and metabolize
 Availability of water is measured by water
activity (aw)
 aw= Vapor pressure of water in food (P) /
Vapor pressure of pure water at the same
temperature (Po)

Dr. Ali Salman Bin Thani
Water activity (aw):
Movement of water vapor from food to the air depends
upon many factors:
 Moisture content of the food
 The food composition
 The temperature and humidity of air e.g. potato chips
get soggy in a humid day
 At constant temperature, water in the food equilibrates
with water vapor in the air
 This is called equilibrium moisture content
 At this point the food neither gains nor loses water to
the air

Dr. Ali Salman Bin Thani
Water activity (aw):





The water activity can also be measured using
the relative humidity of the air surrounding the
food, called equilibrium relative humidity (ERH)
aw = ERH (%)/100
The third way to express aw is on a mole
fraction bases:
aw = moles of solvent/(moles of solvent + moles
of solute)
Or aw = n1/(n1 + n2),,,,,,, aw = 55.5/ (55.5 + n2)
Dr. Ali Salman Bin Thani
Water activity (aw):

Foods at the same (aw) may have different
moisture contents due to their chemical
composition and water binding capacity
Moisture content of various dry or
dehydrated foods (aw 0.7 at 20°C)
Food
% Moisture
content
Grains
4-9
Milk powder
7-10
Dehydrated
vegetables
12-22
Dried soups
13-21
Dried fruits
18-25
Dr. Ali Salman Bin Thani
Reducing the (aw) of food
(preservation):
Dehydration (removing available water)
 Hot air removes water by evaporation
 Freeze drying removes water by
sublimation (conversion of ice to vapor
without passing through the liquid stage)

Dr. Ali Salman Bin Thani
Effect of aw on shelf life and
microbial growth

Decreasing the aw increases the lag phase of growth leading to
decrease in growth rate
Organisms
inhibited
aw
Foods
Shelf life
Clositridium botulinum
Salmonella, most
bacteria
1.00
Fresh foods, meat, fish, Days
poultry, fruits, and
vegatables
Staphylococcus aureus
(anaerobic)
Most yeasts
Staphylococcus aureus
(aerobic)
0.90
Cured meat
Weeks
Most molds
0.8
Syrups, salted foods
Months
0.85
Dr. Ali Salman Bin Thani
Extrinsic factors that influence
microbial growth
These are external factors to the food
 Temperature and gas composition are the
main extrinsic factors influencing
microbial growth
 Slow cooling: hot food in a deep or
large container takes more time to cool
than in a shallow tray, thus allowing
microbes to grow

Dr. Ali Salman Bin Thani
Extrinsic factors that influence
microbial growth
Hazard is the potential to cause harm or
be a source of damage
 Risk is the probability that the hazard
will lead to injury
 For example, beef might be considered
hazardous due the potential presence of
pathogenic bacteria, however, if cooked
well, then there is no risk

Dr. Ali Salman Bin Thani
Homoeostasis and hurdle
technology
Homeostasis: An attempt by a
microorganism to maintain a constant
intracellular state, e.g., maintenance of pH
 Hurdle technology de-optimizes
several factors to inhibit or stop growth
of food-borne pathogens, e.g., pH and aw
 Thus expenditure of microbial energy is
driven towards cell maintenance rather
than growth

Dr. Ali Salman Bin Thani
Introduction
Chapter 6: Food Microbiology
Factors Influencing Growth of
Microorganisms in Food

Understanding factors that influence microbial growth
essential to maintaining food quality
◦ In production and preservation
Conditions naturally present in food termed intrinsic
factors
 Environmental conditions are termed extrinsic factors
 Factors combine to determine which microbes grow
in particular food and at what rate

Factors Influencing Growth of
Microorganisms in Food

Intrinsic factors
◦ Multiplication of food greatly influenced by
inherent characteristics of food
 Microbes multiply most rapidly in moist, nutritionally
rich, pH neutral foods
◦ Intrinsic factors include





Water availability
pH
Nutrients
Biological barriers
Antimicrobial chemicals
Factors Influencing Growth of
Microorganisms in Food

Intrinsic factors
◦ Water availability
 Foods vary dramatically in terms of water availability
 Fresh meats and milk have high water content
 Supports microbial growth
 Breads, nuts and dried foods have low water availability
 Defined populations can grow in these specific
environments
 Water activity (aw) used to designate amount of
water available in foods
 Pure water has aw of 1.0
 Most bacteria require aw of above 0.90
 Most fungi require aw of above 0.80
Factors Influencing Growth of
Microorganisms in Food

Intrinsic factors
◦ pH
 Important in determining which organisms can survive and thrive
on specific foods
 Many microorganisms inhibited by acid conditions
 Exception include lactic acid bacteria
 Lactic acid bacteria used in fermentation process of food
production
 Also prime cause of spoilage of unpasteurized milk and other foods
 Fungi able to survive at relatively low pH
 Most acid foods spoil from fungal contamination as opposed to bacteria
 pH can determine bacteria’s ability to produce toxin
 Toxin production of many organisms is inhibited by acid pH
Factors Influencing Growth of
Microorganisms in Food
 Intrinsic factors
◦ Nutrients
 Nutrients present in food determine organisms that
can grow in foods
◦ Biological barriers
 Rinds, shells and other outer coverings help protect
foods from microbial invasion
 Microorganisms will eventually breakdown coverings and
cause spoilage
◦ Antimicrobial chemicals
 Some foods contain natural antimicrobial chemicals
that inhibit growth of organisms responsible for
spoilage
Factors Influencing Growth of
Microorganisms in Food

Extrinsic factors
◦ Extent of microbial growth largely dependent on
storage of food
◦ Microbes multiply rapidly in warm, oxygen-rich
environments
◦ Extrinsic factors include
 Storage temperature
 Atmosphere
Factors Influencing Growth of
Microorganisms in Food

Extrinsic factors
◦ Storage temperature
 Storage temperature affects rate of microbial growth
 Below freezing water availability is significantly decreased
 Water crystallizes and is unavailable halting microbial
growth
 At low temperature (above freezing) enzymatic action is very
slow or non-existent
 Results in inability of microbe to grow
Factors Influencing Growth of
Microorganisms in Food

Extrinsic factors
◦ Atmosphere
 Presence or absence of oxygen affects type of
microbial population
 Obligate aerobes cannot grow under anaerobic conditions
 Obligate anaerobes will grow in anaerobic conditions
 Including certain foodborne pathogens
Microorganisms in Food and Beverage
Production
Acid produced in yogurt, cheese and pickled
vegetables inhibit growth of many spoilage
organisms and foodborne pathogens
 Fermentation historically important method of
food preservation

Microorganisms in Food and Beverage
Production

Lactic acid fermentations by lactic acid bacteria
◦ Tastes of yogurt, pickles, sharp cheeses and some
sausages due to production of lactic acid by lactic
acid bacteria

Cheese, yogurt and other fermented milk products
◦ Milk is sterile in cow’s udder
 Rapidly becomes contaminated during milking and handling
 Lactic acid bacteria generally reside ON the udder
◦ Aesthetic features of milk change due to
production of acid
 Causes milk proteins to coagulate or curdle
 Sours flavor
Microorganisms in Food and Beverage
Production


Production of fermented milk products do
not rely on naturally occurring lactic acid
bacteria
Starter cultures added to milk
◦ Strains carefully selected to produce desirable
flavors and textures

Starter cultures must be carefully maintained
and protected against contamination
Microorganisms in Food and Beverage
Production

Cheese production
◦ Can be made from milk of wide variety of
animals
 Cow’s milk most common
◦ Cheeses classified as very hard, hard, semi-soft
and soft
 Classification passed on percentage of water content
Microorganisms in Food and Beverage
Production

Cheese production
◦ Cottage cheese easiest
cheese to make
 Pasteurized milk inoculated with
starter culture
 Culture causes milk proteins to
coagulate
 Coagulated proteins called
curd
 Curd heated and cut into small
pieces to facilitate drainage of
liquid waste
 Waste termed whey
Microorganisms in Food and Beverage
Production

Cheese production
◦ Most other cheeses undergo further microbial
processing termed ripening or curing
 Cottage cheese is unripened
◦ Enzyme rennin is added to fermenting milk to
hasten protein coagulation
◦ Curds salted after whey is separated and pressed
and ripened to encourage changes in texture and
flavor
 Ripening can take weeks to years
 Longer ripening produces more acidic sharper cheese
 Certain organisms produce certain characteristics
» Propionibacterium shermanii  Swiss cheese
» Penicillium roquefortii  Roquefort, and gorgonzola
Microorganisms in Food and Beverage
Production

Yogurt
◦ Pasteurized milk is concentrated slightly then
inoculated with starter culture
◦ Mixture is incubated for several hours at 40° C 45° C for several hours
 Thermophilic bacteria grow rapidly at higher
temperatures
 Produce lactic acid and other end products
 Contribute to flavor
◦ Controlled incubation ensures proper levels of acid
and flavor compounds
Microorganisms in Food and Beverage
Production

Acidophilus milk
◦ Sweet acidophilus milk retains flavor of fresh
milk because it is not fermented
 Culture is added immediately before packaging
 Bacteria are added for purported health benefits
 Prevent and reduce severity of some diarrheal diseases
Microorganisms in Food and Beverage
Production

Pickled vegetables
◦ Pickling originated as way to preserve
vegetables
 Particularly cucumbers and cabbage
◦ Pickling uses naturally occurring lactic acid
bacteria residing on vegetables
 Unlike fermentation of milk products which relies on
starter culture
Microorganisms in Food and Beverage
Production

Fermented meat products
◦ Traditionally were produced by letting small
numbers of lactic acid bacteria to multiply to
dominance
 Natural fermentation of meat inherently risky
 Incubation that initiates fermentation can support growth
and toxin production of pathogens
» Clostridium botulinum and Staphylococcus
aureus
Microorganisms in Food and Beverage
Production

Alcoholic fermentations by yeast
◦ Some yeasts ferment sugars to produce ethanol
and carbon dioxide
◦ Yeasts are used to make variety of alcoholic
beverages as well as vinegar and bread
 Alcoholic beverages include
 Wine
 Beer
 Distilled spirits
Microorganisms in Food and Beverage
Production

Wine
◦ Product of alcoholic fermentation of naturally
occurring sugars in juices of fruit
 Most commonly grapes
◦ Commercially made wine produced by crushing
selected grapes
 Stems are removed and solids collected
 Entire grape used in red wines
 Juice only used in white wines
 Solids removed after one day and juice fermented to produce
rose wines
Microorganisms in Food and Beverage
Production

Wine
◦ Fermentation must be carefully
controlled to ensure proper reactions
◦ Sulfur dioxide is added to inhibit growth
of natural microbial population
 These convert alcohol to acetic acid
(vinegar) and most responsible for
spoilage
◦ Fermentation process is initiated by
addition of selected strains of yeast
◦ At completion of fermentation wine
siphoned several times to separate juice
from sediment
◦ Wines then aged in oak barrels
◦ Wine is filtered for clarification then
bottled
Microorganisms in Food and Beverage
Production

Beer
◦ Production of beer is multistep
process
 Designed to breakdown starches in
grain to produce simple sugars
 Sugars are fermented
◦ Yeast lack enzymes to convert grains
to alcohol
 Malted barley (malt) contains enzymes
◦ Malt and starch, sugars and other
adjuncts soaked in warm water
 Termed mashing
 Enzymes in malt act on starches
converting to fermentable starches
◦ Spent grains removed
 Remaining liquid called wort
Microorganisms in Food and Beverage
Production

Beer
◦ Hops are added to wort
 Gives beer distinct bitter taste
 Also has natural antimicrobial
substances
◦ Hops/wort mixture boiled
 Extract flavor of hops
 Concentrate wort
 Inactivates enzymes and precipitates
proteins
◦ Wort centrifuged to remove solids
and cooled
◦ Brewer’s yeast added to initiate
fermentation
 Bottom fermenters clump and sink to
bottom of fermentation tank
 Produces lager beers
 Top fermenters distributed throughout
 Produces porter and stout beers
Microorganisms in Food and Beverage
Production

Distilled spirits
◦ Fermentation process nearly same as beer
 Wort is not boiled
 Degradation of starch continues through fermentation
◦ When fermentation is complete ethanol is purified
and distilled
◦ Different types of spirits made with different
substrates
 Rum  fermentation of molasses
 Scotch whiskey  fermentation of barley the aged
 Tequila  fermentation of agave plant
Microorganisms in Food and Beverage
Production

Vinegar
◦ Aqueous solution of at least 4% acetic acid
◦ Product of oxidation of ethanol
◦ Strictly aerobic process
 Fermenting bacteria are obligate aerobes
◦ Organisms can tolerate high concentration of acid
◦ Vinegar generator produces available oxygen to
hasten oxidation
 Sprays alcohol on biofilm of acid bacteria on wood chips
 Alcohol trickles down and is oxidized by bacteria
Microorganisms in Food and Beverage
Production

Bread
◦ Bread rises due to carbon
dioxide produced through
fermentation of sugars by
baker’s yeast
 Any alcohol produced evaporates
during baking
◦ Characteristic flavor of sour
dough bread due to the
addition of lactic acid bacteria
to bread making ingredients
Food Spoilage

Food spoilage encompasses any
undesirable change in food
◦ Spoiled food is generally not harmful

Spoiled food considered unsafe because
high numbers of spoilage organisms
indicate foodborne pathogen may be
present
Food Spoilage

Common spoilage bacteria
◦ Wide range of bacteria important in food spoilage
• Pseudomonas can metabolize a wide variety of compounds
◦ Psychrophilic organisms can multiply in refrigerator
 Most common genera include
– Erwinia
– Acetobacter
– Alcaligenes
◦ Endospore forming organisms can survive cooking
and in some cases canning processes
 Prevalent spore formers include
– Clostridium species
– Bacillus species
Food Spoilage

Common spoilage fungi
◦ Wide variety of fungi spoil foods
 Some of the most common include
– Rhizopus
– Alternaria
– Penicillium
– Aspergillus
– Botrytis
◦ Fungi grow readily in acidic low-moisture
environments
Foodborne Illness

Commonly referred to as food poisoning
◦ Occurs when a pathogen or its toxin is
consumed
◦ Consumers must employ sound preserving,
preparation and cooking techniques to avoid
hazards of food products
◦ Estimated millions of cases of food poisoning
occur each year
 Vast majority could have been prevented
Foodborne Illness

Food intoxication
◦ Illness resulting from consumption of an exotoxin
produced by organisms growing in food product
 When food is ingested it is the toxin responsible for illness not
organism
◦ Common causes of foodborne intoxication are
• Staphylococcus aureus
• Clostridium botulinum
Foodborne Illness

Staphylococcus aureus
◦ Produces toxin that causes nausea and vomiting
◦ Thrives in moist, rich foods in which other
organisms have been killed or inhibited
 Survives well in unrefrigerated foods with high salt
content
◦ Source of S. aureus generally human carrier
 Organism is inoculated into food during preparation
 Food left at room temperature allows organism to
grow and produce toxin
 Toxin is heat stable and not inactivated by cooking
Foodborne Illness

Botulism
◦ Paralytic disease caused by ingestion of a neurotoxin
 Produced by Clostridium botulinum
◦ Growth of organism or production of toxin may not
result in changes in taste or appearance of food
◦ Canning process designed to destroy endospores
 Processing errors can allow germination of endospores
 Errors extremely rare in commercial canning
 Home canned foods should be boiled for 10 to 15 minutes
immediately before consumption
 Heat destroys toxin
Foodborne Infection
Foodborne infection requires consumption of
living organisms
 Symptoms do not appear for at least one day
after ingestion
◦ Major symptom usually diarrhea
◦ Thorough cooking of food immediately before
consumption will kill organisms

 Prevent infection
◦ Foodborne illness commonly caused by
• Salmonella
• Campylobacter
• Escherichia coil O157:H7
Foodborne Infection
• Salmonella and
Campylobacter
◦ Commonly associated
with poultry products
◦ Inadequate cooking can
result in foodborne
infection
◦ Cross-contamination
can result in transfer of
pathogens to other
foods
 Cutting boards and knives
often become contaminated
Foodborne Infection
• Escherichia coil
O157:H7
◦ Causes bloody diarrhea
◦ Sometimes develops into
hemolytic uremic syndrome
(HUS)
 Life threatening
– E. coli O157:H7 responsible
for several large food
poisoning outbreaks
◦ Ground meats are
troublesome source of
foodborne infection
 ground meat should be
cooked thoroughly through
Food Preservation

Preventing growth and metabolic activities of
organisms that cause spoilage and foodborne illness
preserves food quality

Preservation methods include
◦ Canning
◦ Pasteurization
◦ Cooking
◦ Refrigeration
◦ Freezing
◦ drying,/reducing water availability
Food Preservation

Canning
◦ Destroys all spoilage and pathogenic organisms
◦ Low acid foods use steam under pressure to
destroy endospores
 Acidic food methods not as stringent
 Spore forming bacteria can’t grow or produce toxin in high acid
environment

Pasteurization
◦ Heating foods under controlled conditions at high
temperatures for short periods
 Reduces number of spoilage organisms
 Does not alter taste of food significantly
Food Preservation

Cooking
◦ Can destroy non-spore forming organisms
◦ Alters characteristics of food
◦ If heat is uneven some organisms may survive in
undercooked portion of food

Refrigeration
◦ Preserves food by slowing growth rate of
spoilage organisms
 Many organisms unable to multiply in low
temperatures
Food Preservation

Freezing
◦ Stops microbial growth
 Water unavailable due to ice formation
◦ Portion of organisms remaining can grow when
food is thawed

Drying/reducing water availability
◦ Inhibits microbial growth by decreasing
available moisture
 Molds may grow eventually
Section One: Basics of Food
Microbiology
Chapter 9: Spores and their significance
Dr. Ali Salman Bin Thani
Spores in the food industry
Diseases and spoilage caused by spore formers are
usually associated with thermally processed foods
 Three species of spore formers are producing toxins:
1. Clostridium botulinum
2. Clostridium perfringens
3. Bacillus cereus
 Other spore formers are involved in food spoilage
 Both groups are important in food industry and
contribute to food borne illness and spoilage of low
acid foods (equilibrium pH ≥ 4.6)

Dr. Ali Salman Bin Thani
Spores in the food industry
low acid foods (equilibrium pH ≥ 4.6):
 Packaged in can, bottles, or other hermetically
(vacuum) sealed containers
 Other spore formers include:
1. Cause spoilage of high acid foods, equilibrium
pH < 4.6
2. Pschrotrophic spore formers cause spoilage of
refrigerated foods
3. Fungi producing heat resistant ascospores
and cause spoilage acidic foods

Dr. Ali Salman Bin Thani
Low acid canned foods
Low acid canned food is defined by FDA and
USDA as food items with final pH > 4.6 and
water activity (aw) of > 0.85
 Low acid foods are packaged in hermetically
sealed containers and are heat processed to
achieve commercial sterility
 Commercial sterility: uses heat to inactivate
food borne pathogens as well as any spoilage
microorganisms that can grow in the food
under normal non-refrigerated conditions

Dr. Ali Salman Bin Thani
Low acid canned foods
Other factors could be used to achieve
commercial sterility:
 Preservation e.g., acidification or lowering aw
 These are usually combined to reduce heat
treatments
 All of these methods are applied to achieve one
goal, inactivation of the Clostridium botulinum
spores

Dr. Ali Salman Bin Thani
Low acid canned foods
In thermal processing, two values are used
to describe thermal inactivation
1. The D value is the time required for a 1log reduction in viability of a microbial
population (a 1-log reduction equates to
killing 90% of the population
❖ The D value represents the resistance of
an organism to a specific temperature

Dr. Ali Salman Bin Thani
Low acid canned foods
2.
The z value is the temperature change
required to change the D value by a factor of
10
❖ The
z represents the relative resistance of an
organism to inactivation at different
temperatures
Dr. Ali Salman Bin Thani
Low acid canned foods
Generally, low acid foods in hermetically sealed
containers are heated to achieve 3 to 6 min at a
temperature of 121°C (250°F) or equivalent at
the coldest spot of the food
 This ensures the inactivation of most heat
resistant Clostridium botulinum spores with a
D121°C of 0.21 min and a z of 10°C (18°F)

Dr. Ali Salman Bin Thani
Bacteriology of sporeformers of
public health significance
Three species of sporeformers are
involved in foodborne illness:
1. C. botulinum
2. C. perfringens
3. B. cereus

Dr. Ali Salman Bin Thani
Bacteriology of sporeformers of
public health significance
C. botulinum:
 Considered the principal microbial hazard
in heat processed vacuum packed foods
and in minimally processed refrigerated
foods
 The bacterium is a gram positive,
anaerobic, spore forming bacilli that
obtain energy by fermentation
1.
Dr. Ali Salman Bin Thani
Bacteriology of sporeformers of
public health significance
C. botulinum:
 The species of C. botulinum are heterogeneous
and produce antigenically distinct toxins A
through G
 Type I: mesophiles, produce heat resistant
spores and make proteases that cause overt
spoilage
 Type II: low temperature growers, with spores
less heat resistant and are nonproteolytic
1.
Dr. Ali Salman Bin Thani
Bacteriology of sporeformers of
public health significance
C. perfringens:
 It is widespread in soils and in the
intestine of human
 Grows well in high protein foods such as
meat
 Two classes of spores are produced, heat
resistance and heat sensitive
2.
Dr. Ali Salman Bin Thani
Bacteriology of sporeformers of
public health significance
B. cereus
 Can produce heat-labile enterotoxins
causing diarrhea
 Or produce heat stable toxins causing
emesis (vomiting)
3.
Dr. Ali Salman Bin Thani
Spoilage of acid and low acid canned and
vacuum packaged foods by sporeformers






Heat resistance fungi:
Cause spoilage of acidic foods e.g., fruits
Thick wall ascospores of heat resistance fungi
can survive heating at ≥ 85C for 5 minutes
Known heat resistance fungi include:
Neosartorya and Talaromyces
These are involved in producing mycotoxins
Control of foodborne pathogenic fungi uses
heat, aw and oxygen manipulation, plus
application of antimycotic agents
Dr. Ali Salman Bin Thani
Spore biology and structure
Seven layers: exosporangium, coat, outer
membrane, cortex, germ cell wall, inner
membrane, and core
 Underlying the exoporium is the spore
coat, which protects the spore cortex
from attack by lytic enzymes
 The outer spore membrane helps
keep the spore impermeable to small
molecules

Dr. Ali Salman Bin Thani
Spore biology and structure
The cortex is made of peptidoglycan which
differs from the peptidoglycan of vegetative cells
by the presence of dipicolinic acid DPA
 DPA in spore cortex is involved in dehydration
of the spore core and much of spore resistance
 The germ cell wall is between the cortex and
the inner membrane
 The structure of the germ cell wall is identical
to its counterpart in vegetative cells

Dr. Ali Salman Bin Thani
Spore biology and structure





The inner spore membrane structure is
similar to that found in vegetative cells
“phospholipids”
The spore core contains: DNA, ribosomes,
enzymes, DPA and divalent cations
The spore core contains unique molecules
required for formation of the spore; large pool
of small acid soluble proteins (SASP)
These comprises 10 to 20% of spore proteins
SASPs are found bonded to spore DNA
Dr. Ali Salman Bin Thani
Spore biology and structure
Water content of the spore compared to
vegetative cells:
 Vegetative cells have 4 grams of water per
gram of dry weight
 Spore core has only 0.4 to 1 grams of
water per gram of dry weight
 The core’s low water content is
responsible for spore dormancy and
spore resistance

Dr. Ali Salman Bin Thani
The ASAP molecules
These play important role in spore
resistance
 The binding of these proteins to DNA
render it resistant to chemical and
enzymatic cleavage, e.g., the DNA
backbone is saved from ultraviolet (UV)
light by altering the DNA’s
photochemistry

Dr. Ali Salman Bin Thani
Molecules present in spores
Many macromolecules present in the vegetative
cells are absent in spores: amino acids
nucleotide biosynthetic enzymes
 Small molecules such as high energy compounds
e.g., deoxynucleoside triphosphates, and
coenzyme A (acyl-CoA) are present in very low
numbers
 Enzyme-substrate pairs are stable for months to
years in the spore core, however, they degrade
in 15 to 30 min after germination

Dr. Ali Salman Bin Thani
Spore resistance
Spore resistance is due to three factrs:
1. Spore core dehydration
2. SASP
3. impermeability

Dr. Ali Salman Bin Thani
Spore resistance




Both core dehydration and ASAP are important to
overcome the following stress conditions:
Freezing and desiccation: this process can cause
DNA damage and mutagenesis. However, the DNA is
protected by the ASAP
Pressure resistance: spores are killed more rapidly at
lower pressures than at higher pressures. This is
because spore germination is promoted by lower
pressures but not by higher pressures
Thus the spores are allowed to germinate by lower
pressures and then are killed
Spore resistance
Radiations resistance: spore uses the core
dehydration to prevent DNA damage
 Chemical resistance: chemical resistance is
maintained by the impermeability of the spore to
different chemical agents
 Spore heat resistance: spores of thermophiles are
more heat resistant than spores of mesophiles, and
spores of the same strain prepared at various
temperatures are most heat resistant when prepared at
the highest temperature
 The spore resistance to heat is due to two factors;
alpha/beta type SASP and core dehydration

Dr. Ali Salman Bin Thani
The cycle of sporulation and
germination





Three steps:
Sporulation: unequal cell division producing the
forespore and the sporangium, the later will engulf the
spore forming the endospore
Activation: induction usually by heat and involves
addition of nutrients “germinant”
Germination: lose of the DPA and SASP
Outgrowth: marked by cell ability to start synthesis of
macromolecules and commence of cell division
Dr. Ali Salman Bin Thani