2Chapter 2 (2).pdf

Food Microbiology
 is the study of the specific microorganisms in food and their
beneficial and harmful effects on the quality and safety of
raw and processed food.
 Food microbes can be beneficial, neutral, or harmful to
humans
 Foods are not only nutritious to consumers, but are also
excellent source of nutrients for microbial growth
January 8, 2024 Applied Microbiology 2

Lab.
Management
Food borne
disease
Water Quality
Food
fermentation Food Spoilage
Quality Control
Food
Preservation
Food Hygiene
Food
Microbiologist

Some important genera known to occur in food
Bacteria Yeast Mold Protozoa
• Two important questions that must be answered to tell whether a certain food is
safe or not:
– What is the total number of MO’s present per g or per ml
– What types of MO’s present
Aspergillus
Penicillium
Fusarium
Botrytis
Alternaria
Mucor
Rhizopus
Cladosporium
Cryptococcus
Candida
Saccharomyce
Zygosaccharomyce
Trichosporon
Rhodotorula
Torula
Cryptosporidium parvum
Entamoeba histolytica
Giardia lamblia
Toxoplasma gondii
Erwinia
Pseudomonas
Alcaligenes
Acinetobacter
Moraxella
Aeromonas
Proteus
Serratia
Salmonella
Shigella
Staphylococcus

Primary Sources of microorganisms in food
 Soil and water
 Air and dust
 Plant and plant products
 Food utensils and
equipment
 Animal feeds
 Animal hides
 Intestinal tracts of humans
and animals
 Food handlers
In order to manage microbial contamination and growth
Hazard Analysis Critical Control Point (HACCP) approach is widely used.
 This approach emphasizes monitoring the quality of food ingredients at critical
process handling steps.
A safe product will result if the individual steps are carefully controlled.

FACTORS AFFECTING MICROBIAL GROWTH IN FOOD
 classified in two types.
A/ Intrinsic Factors (parameters)
B/ Extrinsic factors
 Intrinsic parameters
– food related parameters
– inherent part of the food
These parameters include:
1. pH
2. Moisture content
3. Nutrient content Factors related to the food itself.
4. Antimicrobial constituents
5. Biological structures
6. oxidation reduction potential

1. pH
 Microbes are sensitive to the pH in their environment.
 Most microorganisms grow best at pH values around 7.0 (6.6–7.5)
 Some exception
 Lactobacillus requires acidic env.
 Vibrio requires alkaline env.
 Pathogenic bacteria more fastidious (pH) than molds and yeasts
 Microorganisms
 Acidophiles: grow at an optimum PH well below neutrality
 Neutrophiles: grow best at neutral PH
 Alkalophiles: grow best under alkaline conditions

 The natural or inherent acidity of foods by nature way of protecting the
respective plant or animal tissues from destruction by microorganisms.
 For example,
 vegetables generally have a moderately acidic PH and thus are spoiled by soft-rot
producing bacteria such as Erwinia carotovora and Pseudomonads,
 fruits, a lower pH prevents bacterial growth and spoilage is caused by yeasts and
molds.
 The pH sensitive genus Shewanella plays an important role in fish spoilage but not in
normal meat
Table . Approximate pH ranges of different microbial groups.
Microorganisms Minimum Optimum Maximum
Most Bacteria 4.5 6.5 – 7.5 9.0
Yeasts 1.5 – 3.5 4.0 – 6.5 4.0 – 6.5+
Molds 1.5 – 3.5 4.5 – 6.8 8.0 – 11.0

2. Moisture Content
 Water is essential for the growth and metabolism of all cells. Its reduction or total
removal could affect the cellular activities. The form in which water exists within the
food is important as far as microbial activity is concerned.
 There are two types of water - free and bound.
Bound water is present within the tissue and is vital to all the physiological processes
within the cell.
Free water exists in and around the tissues and can be removed from cells without
seriously interfering with the vital processes.
 Free water is essential for the survival and activity of micro-organisms. Therefore, by
removing free water, the level of microbial activity can be controlled.
 Bacteria require more water than yeasts, which require more water than moulds to
carry out their metabolic activities.

 The amount of water available for micro-organisms is referred to as the
water activity (aw).
 Pure water has a aw 1.0.
 Almost all microbial activity is inhibited below aw of 0.6.
 fungi are inhibited below aw of 0.7
 yeasts are inhibited below aw of 0.8
 bacteria below aw 0.9.
 Lower aw inhibits microbial growth
 The aw of foods lowered by;
 drying
 freezing to change the state of water from liquid to solid
 increasing or decreasing the conc. of solutes by adding salt or sugar
 Simply by drying a food, one can control or eliminate spoilage processes

 A dry food like bread is generally spoiled by molds but not by
bacteria.
 Decreasing aw order:
Bacteria > Yeast > Mold
 Water activity is an important property that can be used to predict
food safety, stability and quality.
 Below 0.60, no microbiological growth is possible. Thus the dried
foods like milk powder, cookies, biscuits etc are more shelf-stable
and safe as compared to moist or semi-moist foods.

3. Physical (biological) structure of the food
 Vegetables and fruits have outer skins (peels and rinds) , shell of eggs and
nuts, outer covering of testa of seeds, hide of animals that provide
protection against entry and subsequent spoilage by microorganisms
4. Availability of nutrients
 In order to grow and function normal
 water
 source of carbon, energy, nitrogen
 minerals
 vitamins and growth factors
 In general, molds have the lowest requirement, followed by yeasts, gram-
negative bacteria, and gram-positive bacteria.

5. Presence of antimicrobial agents
 Many foods contain natural antimicrobial substances
 essential oils in spices (eugenol in cloves and cinnamon,)
 lactaferrin, lactoperoxidase and lysozyme in milk
 ovatransferrin, avidin, lysozyme and ovoflavoprotein in hen‟s egg albumin.
 The hydroxycinnamic acid derivatives (p-coumaric, feluric, caffeic and
chlorogenic acids) found in fruits, vegetables, tea and other plants
possess antibacterial and antifungal activity
 generally fungi are more sensitive than most bacteria

B/ Extrinsic Parameters (Factors)
 storage environment that affect both the foods and their mo’s
 The extrinsic parameters of foods are not substrate dependent.
These are:
 Temperature of storage
 Relative humidity of environment Environmental related factors
 Presence and concentration of gases
Time
1. Temperature of Storage:
 lowest reported for growth is -34oc
 Highest reported for growth is 90oc

Temperature classes of microorganisms
 Microorganisms grouped into four based on cardinal points for growth.
A/ Psychrophiles (cold lover): Those organisms grow well at or below 7oC
and have their optimum between 10oC and 150C.
 Cause spoilage at 5–7◦C of meats, fish, poultry, eggs, and other foods normally
held at this temperature.
B/ Mesophiles: Those grow well between 20◦C and 45◦C with optima b/n
30◦C & 40◦C.
C/ Termophiles: Those grow well at and above 45◦C with optima b/n 55◦C
& 65◦C.
D/ Hyperthermophiles: have very high temperature optimum (hot lover,
up to 115 ◦C).

Cont’d…
• Alcaligenes and Pseudomonas grow well at refrigerator temp
and cause spoilage of meat, fish, poultry and eggs
• Mesophiles found on foods held at refrigerator temp but do not
grow at this temp are known as psychrotrophs and those that
survive at high temp but cant grow are thermoduric
• Important thermophilic bacteria in foods belong to the genera
Bacillus and Clostridium
• Molds grow over wider ranges of temp than bacteria
• Many molds are able to grow at refrigerator temp
– e.g. Aspergillus, Cladosporium and Thamnidium
• Storing foods at refrigerator temp is not always the best method
– e.g. banana(13-17oc), various vegetable (10oc)
If temperature is too hot or too cold, microorganisms will not grow.

2. Relative Humidity of Environment (RH)
 food having a low water activity are stored in an atmosphere of high
relative humidity water will transfer from the gas phase to the food.
 Once micro-organisms have started to grow and become physiologically
active they usually produce water as an end product of respiration.
 Thus they increase the water activity of their own immediate environment
 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.
 Foods that undergo surface spoilage from molds, yeasts and certain
bacteria should be stored under conditions of low RH.

3. Presence and Concentration of Gases in the Environment
 Carbon dioxide (CO2) is the single most important atmospheric gas that is used to
control microorganisms in foods. It along with O2 are the two most important gases
in modified atmosphere packaged (MAP) foods.
 The storage of foods in atmosphere containing 10% of CO2 is referred to as
“Controlled Atmosphere”.
 Inhibitory effect of Co2 increases with decreasing with temp due to
 Increased solubility of Co2 at lower temp
 Formation of carbonic acid(pH reduction )
 Gram negative are more sensitive to Co2 than gram positive
 Pseudomonas is the most sensitive but LAB are the most resistant
 Ozone (O3) is the other atmospheric gas that has antimicrobial properties, but
because it is a strong oxidizing agent, it should not be used on high-lipid-content
foods since it would cause an increase in rancidity.
 protozoan was about three times more sensitive to O3 at 25◦C than at 5◦C

Time
• Most microbes need time to
multiply to high numbers that
can cause food spoilage or
illness.
• Leaving foods in the "danger
zone" of 4-60°C for extended
periods allows growth.
• Time is also needed for
chemical hazards to develop,
such as putrescine formation
during decomposition
(spoilage).

Food Spoilage
 Food spoilage: is the undesirable deterioration of food quality that
may result in changes in odour, taste, and appearance of food or
objectionable chemical & physical changes in food.
 It is the most serious economic problem in food processing industry
 Spoilage can occur through both microbial and non-microbial routes.
Autolysis – self destruction, caused by enzymes present in the food.
 Microbial spoilage – caused by the growth of bacteria, yeasts and
mould

Causes of Food Spoilage
• Physical injury (light, oxygen, heat, humidity)
 Atmospheric oxygen can react with some food components which may cause
rancidity or color changes.
• Autolysis – self destruction, caused by enzymes present in the food
• Microorganisms (yeasts, molds and bacteria)
• Insect or rodent infestation
• Time
Therefore in general every kind of food should be stored in a
dark dry place and at a proper temperature.

Cont’d…
• The main purpose of adopting principles of food
spoilage is to minimize food contamination via:
– Good management process
– Acceptable sanitary process
– Rapid movement of food through processing plant
– Well-tested preservation procedures, and so on.

Microbial deterioration of food is evidenced by alteration:
 in the appearance (color changes, pockets of gas/ swelling)
 texture (soft & mushy), color, odor
 flavor or slime formation
Microorganisms manifest their presence in the food in one
of several ways:
 they can cause spoilage
 they can cause food borne illness
 they can transform a food’s properties in a beneficial way, food
fermentation.

 The Causes of Food Spoilage Classify foods in terms of potential
for spoilage.
 Three categories based on likelihood of spoilage
 Perishable
 Nutrient rich, moist and unprotected by coverings.
 Semi-perishable
 Can store sealed for months without spoiling.
 Many fermented foods are semi-perishable.
 Nonperishable
 Dry or canned foods that can be stored indefinitely
 Often nutrient poor, dried, fermented, or preserved

SPOILAGE OF FISH
• Fish contain high levels of proteins and other nitrogenous
constituents
• The carbohydrate content of these fish is nil
• Fresh iced fish are spoiled by bacteria(Pseudomonas and
Acinetobacter, Moraxella)
• Many fish-spoilage bacteria are capable of good growth
between 0◦C and 1◦C. (Pseudomonas spp.3◦C)

The microorganisms known to cause fish spoilage

SPOILAGE OF MEATS
• Meats are the most perishable of all major foods
• With respect to fungal spoilage of fresh meats,
• Thamnidium, Mucor, and Rhizopus, all of which produce “whiskers” on
beef;
• Cladosporium, a common cause of “black spot”;
• Penicillium, which produces green patches;
• Sporotrichum and Chrysosporium, which produce “white spot.”
• Unlike the spoilage of fresh beef, ground beef or hamburger
meat is spoiled exclusively by bacteria
• Pseudomonas, Alcaligenes, Acinetobacter, Moraxella, and Aeromonas

• During spoilage process, first simple carbohydrate are exhausted; at
this time off odor may or may not be evident , then Pseudomonas,
Alcaligenes, serratia, Moraxella, and Aeromonas utilize free amino
acids and related simple nitrogenous compound as a source of
energy. Foul odor then appears from H2S, NH3 and indole.
Food Types of Spoilage Spoilage Microorganisms
MEAT
Fresh
Putrefaction
Clostridium, Pseudomonas, Proteus,
Alcaligenes, Chromobacterium
Souring
Chromobacterium, Lactobacillus,
Pseudomonas
Cured
Mouldy Penicillium, Aspergillus, Rhizopus
Souring Pseudomonas, Micrococcus, Bacillus
Slimy Leuconostoc
Vacuum
Packed
Souring
Greening
Lactobacillus, Carnobacterium,
Leuconostoc

Poultry- Egg
• The most common form of bacteria spoilage of egg is
know as rotting
• Green rot by pseudomonas fluerescens
• Colorless rot by Pseudomonas, Acinetobacter
• Black rots by Proteus, Pseudomonas and Aeromonas
• Pink rots by Pseudomonas
• Red rot by Serratia
• Custard rot by Proteus vulgaris and P. intermedium
• Mold spoilage of egg is generally called pinspots because of the
appearance of mycelial growth on the side up on candling
• Caused by penicillium and cladosporium

Dairy products
• Milk is an excellent growth medium for all of the common spoilage organism
• Raw milk held in refrigerator temp for several day shows the presence of
Enterococcus, Lactococcus, streptococcus, Lactobacillus, Micrococcus, Proteus,
Pseudomonas, Leuconostoc…
• Spoilage of pasteurized milk is caused by the growth of heat resistant Streptococci
utilizing lactose to produce lactic acid
• Ropiness is caused by the growth of Alcaligenes viscolactis in raw milk
Food Types of Spoilage Spoilage Microorganisms
DAIRY
MILK
Bitterness Pseudomonas spp.
Souring Lactobacillus thermophilus
Sweet curdling Bacillus cereus
CHEESE
Green discoloration Penicillium
Green to black discoloration Cladosporium
Black discoloration Candida
Sliminess (high pH) Pseudomonas spp.
“Gassy” cheese Coliforms, LAB, Clostridia

Beer
• Industrial spoilage of beers is known as beer infection. This condition is
caused by yeast and bacteria.
• The spoilage pattern of beer is classified in to 4 groups
– Ropiness – the liquid becomes viscous and pours as an oily stream
• Caused by Acetobacter, Lactobacillus, Pediococcus cerevisie and Gluconobacter
cerevisie
– Sarcinae sickness – caused by Pediococcus cervisiae which produces honey
like odor
– Sourness- caused by Acetobacter spp. The bacteria oxidized ethanol to acetic
acid
– Turbidity and off odors are caused by Zymonas anaerobia and several
yeasts such as saccharomyces spp

Foodborne Diseases
• Definition: Any disease of an infectious or toxic nature caused
by, or thought to be caused by, the consumption of food or
water.’ WHO
• Food borne illnesses can be fatal as well as cause suffering and
discomfort.
• Foodborne illness could be:
1. Food Intoxication (Food poisoning)
2. Food Infection

1. Food Intoxication (Food poisoning)
- is caused by eating food containing a toxin.
• Toxins are damaging biochemical substances
synthesized by microorganisms.
• Toxins are virulence factors of many pathogens.
• Based on their chemical composition and way of
action, toxins are classified into:
A. Exotoxins
B. Endotoxins

A. Exotoxins
 are protein toxins & released from the cell into the
surrounding
 extracellular diffusible toxins
 highly potent, toxic & often fatal
 is heat stable and not inactivated by cooking, Example:
– Botulinum toxin &Tetanus toxin produced by C. botulinum C. tetani
– Staphylococcus aureus

B. Endotoxins
 are lipopolysaccharide toxins
 found in the cell wall of G-ve bacteria
 released upon cell death
 less toxic & rarely fatal
• Example:
– Shiga toxin causing bacillary dysentry, produced by
Shigella dysentriae

Toxins
• ergotism
– toxic condition caused by growth of a fungus in grains
• aflatoxins
– carcinogens produced in fungus-infected grains and
nut products
• fumonisins
– carcinogens produced in fungus-infected corn

2. Food Infection
 is caused by eating food containing a living pathogenic microbes.
 Symptoms do not appear for at least one day after ingestion
 is transmitted by milk, water, or solid food contaminated
 Foodborne illness commonly caused by
• Campylobacter
• Salmonella (causes salmonellosis and typhoid fever)
• Shigella (causes bacillary dysentery or shigellosis)
• E.coli (causes diarrhea, urinary tract infections…etc)
• V.cholerae (causes cholera) and so on.

Methods of food preservation
• Preservative is a substance or a chemical that is added to
products such as food, beverages, pharmaceutical drugs, etc to
prevent decomposition by microbial growth or by undesirable
chemical changes
• Food preservatives: Any substance added to a processed food
in order to extend its shelf life(GRAS)
• Major Approaches
– Asepsis – prevention of contamination
– Micro-biostatic – Inhibition
– Microbicidal – killing microbes
– Canning – sealing prevents contamination
- anaerobic condition inhibits aerobes
- heat treatment kills microbes

• Microorganisms of greatest concern in food
preservation are “extremists”
Thermophiles
Halophiles
Psychrophiles
Obligate anaerobes
Acidophiles

Food Preservation Methods
1. High Temperatures
- safest and most reliable
- used to kill microbes
A. Pasteurization
• The process of heating a liquid to destroy harmful bacteria without
materially changing the composition, flavor, or nutritive value of the
liquid.
• Used to reduce microbial spoilage of alcoholic drinks, milk, diary
products, etc
B. Canning NB: Clostridium botulinum

• Used to preserve highly perishable foods (milk, meat,
vegetables, etc)
A. Refrigeration (4oC)
B. Freezing – below 0oC
3. Others
– Dehydration
– Smoking
– Radiation – lethal
- applied on surfaces only
– Chemicals
• High concentration of salt
• High concentration of sugar
• Using chemical preservatives (acetic, lactic, benzoic acids)

Fermentation and Traditionally Fermented
Foods
Fermentation – is the conversion of sugars to ethanol
production by yeasts or organic acids by lactic acid bacteria.
Fermented foods - foods which have been subjected to the
action of microorganisms or enzymes so that desirable
biochemical changes caused significant modification to the
food.

Benefits of Fermentation
• Increases the vitamins, minerals, enzymes and
protein content of the food
• Increases nutritional value
• Increases flavor, texture and aroma
• Improves digestion
• Prevents spoilage, growth of pathogens
• Increases shelf life

• Microorganisms undergoing fermentation are
– naturally occur on the food
– prepared as starter culture
 The type of microbial flora that develops in each
fermented food depends on
 intrinsic factors like water activity, pH, salt concentration,
availability of O2, composition of the food matrix, and
 extrinsic factors such as temperature, relative humidity and other
parameters.

Fermented Diary Products
• The fermentation of milk is primarily carried out
by lactic acid bacteria.
A.Cultured Buttermilk – microbial fermentation of
skim milk to produce a flavor and aroma of true
milk
Buttermilk – the liquid after the coagulated fat is
separated as butter

B. Yogurt – is a sour, creamy food made by fermenting milk with a
mixture of Streptococcus thermophilus and Lactobacillus
acidophilus.
•Yogurt is rich in protein, calcium, Vit B6, Vit B12
C. Cheese – milk curds separated from the liquid portion of the milk
(whey).
•The curdling of milk is achieved by using the enzyme rennin (casein
coagulase) and lactic acid bacteria starter culture (Lactobacillus,
Lactococcus, Streptococcus)

D. Fermented Meat
• Various lactic acid bacteria are involved in
fermentation of meat eg. Sausage
E. Fermented Beverages
• Alcoholic fermentation of sugars by yeasts
(Saccharomyces cerevisiae)
• The d/ce in d/t alcoholic beverages is due to:
- different sources/substrates
- the production methods

• Beer – fermentation of grain sugars
• Wine - fermentation of fruit sugars (grapes)
• Vinegar - fermentation of fruit or grain sugars followed
by oxidation of ethanol
 Sauerkraut - fermentation of shredded cabbage
 Pickles - fermentation of cucumbers
F. Fermented Vegetables

MAKE A GROUP OF 3 AND DISCUSS THE ON ETHIOPIAN
TRADITIONALLY FERMENTED FOOD
1. Borde
2. Shameta
3. Enjera
4. Cheka
5. Teji
6. Tela
7. Areke
8. Kotcho
9. Ergo,
10. Ayib,
11. yetenetere qibe,
12. arrera,
13. aguwat etc
Discuss with Substrate and
the processes shortly used

Table 1 Some Traditional fermented foods of Ethiopia and other parts of the world
S.N
o
Product Geographical
location
Substrate Microorganisms
involved
Product
Nature
Product use
1 Injera Ethiopia Teff, maize wheat,
barley, sorghum
Enerobacteriaceae,
lactic acid bacteria
Bread-like.
Moist
Bread substitute
2 Kotcho Ethiopia Enset LAB Bread-like
3 Shamita Ethiopia Barley Bacillus, LAB Liquid Meal replacement
4 Borde Ethiopia Maize, Barley,.. Bacillus, LAB Liquid Meal replacement
5 Siljo Ethiopia Safflower LAB Liquid
6 Natto Northern
Japan
Soyabean Bacillus natto Solid Cake, as a meat
substitute
7 Pozol S. Mexico Maize Molds, yeasts,
bacteria
Dough,
spongy
Diluted with H2O ,
drunk as basic food

Health benefit of fermented foods
• Fermented foods were historically used for preservation and flavor
enhancement, but have gained attention for their potential health
benefits.
• Microorganisms involved in fermentation have been associated with
synthesizing vitamins, producing bioactive peptides, and removing
non-nutrients.
• These bioactive peptides have various health benefits
– blood pressure reduction and anti-microbial effects.
– as antioxidant, reduce cancer, anti-inflammatory, and anti-diabetic
properties,

PROBIOTICS
• Probiotics is a concentrated supplements of beneficial live
bacteria culture taken orally intended to improve our
health.
• It is a friendly bacteria which play vital role in keeping us
fit and healthy.
• Probiotics means “for life”
• The good friendly bacteria are good because they promote
the body’s natural to keep our body fit and to help our
digestion

The example of probiotics in food
• Milk- baby milk nowadays is added with Lactobacillus
acidophilus and Bifidus bacteria
• Yogurt- rich with live bacteria culture such as Lactobacillus
bulgaricus and Streptococcus thermophillus
• Cheese- friendly bacteria that is added in cheese is
Lactobacillus
• Buttermilk- Lactobacillus bulgaris

Prebiotics
• Prebiotics are non-digestible fibers that promote the growth
and activity of probiotic-type bacteria in the gut.
• They serve as food for these beneficial bacteria, helping them
to thrive and maintain a healthy balance in the gut
microbiome.
• Prebiotics can be found in certain fruits, vegetables, and
whole grains, as well as in some dietary supplements.
• Consuming prebiotics can help support digestive health and
overall well-being.

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