Envh 3171: FOOD

MICROBIOLOGY

Dante R. Santiago, PhD

 CHAPTER 1. BASIC MICROBIOLOGY

Microorganism or simply microbes are living entities that cannot be

seen by the naked eye, that is, these are microscopic in size.

Microbes:
 ubiquitous; present everywhere
 in humans, animals, plants, other living things, soil, water and air
 can multiply everywhere except in the air
 the unseen majority on earth
Types of microbes
1. Procaryotes (pro = before; karyote = nucleus)
• nucleus of cell not bounded by nuclear membrane
• location of nucleus in cell is called nucleoid
• include all bacteria
• cell size range: 1 to 10 µm
Two types of bacteria:
• Gram-positive (G+) and Gram-negative (G–)
• Two types of Gram-positive bacteria: spore-formers (e.g.,
Bacillus, Clostridium) and nonspore-formers (Lactobacillus,
Corynebacterium)
2. Eucaryotes (eu = true); cells with nuclei.
• include fungi (molds and yeasts) and protozoa
• cell size range: 20 to 100 µm
Yeast – unicellular
Molds – multicellular
Protozoa – unicellular
3. Viruses
• no cellular structure
• molecular in nature consisting of protein capsules enclosing
nucleic acids (either RNA or DNA, never both).
• sometimes, a lipid envelope surrounds the virus particle.

4. Prions
• proteinaceous infection particles
• cause transmissible spongiform encephalopathies (TSE); mad
cow disease
• mode of transmission is poorly understood
Bacterial vegetative cells
and spores
Role of microbes in nature
 Microbes are the ultimate decomposers
• degrade dead organic matter to simpler products
• mineralize such degradation products

Mineralization: process of converting organic matter to its inorganic

components resulting in the production of carbon dioxide, inorganic
nitrogen compounds (e.g., ammonia) and other inorganic
components such as sulphur (from amino acids) and phosphorus
(from nucleic acids)

 Microbes are involved in making the soil fertile, purifying water,

animal and plant diseases, fermentation (e.g., making wine,
vinegar, some foods such yogurt, etc.), food spoilage and food-
borne diseases.
MICROBIAL GROWTH
Growth is the increase in number or mass of cells as the result of cell
multiplication.

Microbes, as any living organism, require at least the following for

growth and reproduction:
• Source of energy (electron) for ATP production, e.g., carbohydrates
• Source of carbon for enzyme and biomass production, e.g.,
carbohydrates
• Source of nitrogen for enzyme and biomass production, e.g., proteins
and amino acids
Proteins can serve as alternate carbon and energy source
• Electron acceptor for termination of energy conversion process, e.g.,
oxygen or nitrate
Other growth nutrients:
• Phosphorus for nucleic acid and membrane production
• Sulfur for protein production
• Vitamins as co-enzymes (many microbes can produce their own
vitamins)
• Trace elements (Fe2+, Mg2+, Zn2+) as enzyme co-factors, from
proteins
Foods contain the nutrients required for cell development and
multiplication and can serve as media for microbial growth.
Amount (%) of Nutrients
Type of Carbo- Proteins Fats and Oils Non-protein
Food hydrates Nitrogen

Milk 48 3.4 37
Ox meat 1.2 19.5 11≥ 1.6
Lamb meat ≈1 17.1 14.8 ≈1
Fish meat ≈1 15 to 20 25 <1
Chicken ≈1 20.2 7.2 ≈1
meat
Bread 52.3 8.2 3.3
Orange 11.2 0.9 0.5
Peas 17.7 6.7 0.4
Cell division:
Bacteria – binary fission
Eucaryotes – mitosis

Cellwall
Cell membrane
P arent genome
R eplicated genome
R ibosome

Binary fission
Terms used in the study of microbial growth:
Growth rate – change in cell number or cell mass per unit time
Generation – interval for the formation of two cells from one
Generation time – time required for doubling of the population

Doubling of cell:
• two cells become four expressed as 21 → 22
• four cells become eight, as 22 → 23 and so on
• geometric increase in cell number

Relationship between the number of cells from the initial time and
after a certain period is: N = No2n
Where:
• N – the final cell number
• No – the initial cell number
• n – number of generations
The generation time is calculated as g = t/n, where t is the time
expressed in hour or minute.
To compute for n:
N = No2n
logN = logNo + n log2
n = log N – log No ÷ log 2
If No = 5 x 107 and N = 1 x 108, then n = 8 – 7.69 ÷ 0.301 = 1
generation.
If the population doubles in 2 h, then g = t/n = 2/1 = 2 h.
Problem: If a culture with 10,000 cells has a generation time of 2 h,
how many cell will be in the culture after 8 h?

Solution: n = 8 h ÷ 2 h per generation = 4 generations.

Therefore, N = 24 x 104 = 1.6 x 105 cells
The microbial growth curve
Physical factors affecting microbial growth:
1. Temperature
For every microbe, there is a minimum, optimum and maximum
temperature (the three cardinal temperatures)
Optimum temperature is always nearer the maximum than the
minimum temperature.
Types of microbes according to temperature tolerance:
• Psychrophiles – those with low temperature optima
• Mesophiles – with midrange temperature optima
• Thermophiles – with high temperature optima

2. pH
Each organism has a pH range within which growth is possible
• Acidophiles – grow best at low pH
• Neutrophiles – grow best at pH 6 – 8
• Alkalinophiles – grow best at high pH
3. Osmotic pressure
Effect of solute and water on cells
• Halophiles – tolerate high salt concentration
• Osmophiles – tolerate high sugar content
• Xerophiles – tolerate very dry environment

4. Oxygen
Some organism may live in presence or total absence of molecular
oxygen
Aerobes – capable of growth at full oxygen tension
Microaerophiles – require O2 at reduced level
Facultative anaerobes – under appropriate nutrient conditions, can
grow with either presence or absence of O2

Anaerobes – cannot tolerate molecular oxygen

Obligate anaerobes – inhibited or killed by O2
 CHAPTER 2. MICROBES IMPORTANT IN FOOD
MICROBIOLOGY
Characteristics of microbes in relation to growth on foods:
Bacteria:
• Short generation time (in minutes)
• Grow at near neutral to higher pH, high moisture level, moderate osmotic
pressure
• Grow in meats, milk, fish, egg, vegetables, fruits, nuts, cereals, sugar, bottled
water, mayonnaise
Yeasts:
• Moderate generation time
• Grow at low pH, high moisture level, high osmotic pressure
• Grow in fruits, sugar, softdrinks
Molds:
• Longest generation time among microbes (in hours)
• Grow at low pH, low moisture level, high osmotic pressure
• Grow in vegetables, fruits, grains, flour, bread, sugar, softdrinks, mayonnaise,
hard cheese
Important genera of bacteria found in foods:

 Thermotolerant – able to grow at 50OC and above: Bacillus, Clostridium,

Pediococcus, Streptococcus, Lactobacillus
 Thermoduric – able to survive pasteurization treatment: Micrococcus,
Enterococcus, Lactobacillus, Pediococcus, Bacillus and Clostridium spores
 Psychrotrophic – able to grow at refrigeration temperature (≤ 5OC: Pseudomonas,
Altermonas, Alcaligenes, Flavobacterium, Serratia, Bacillus, Clostridium,
Lactobacillus, Leuconostoc, Carnobacterium, Brochothrix, Listeria, Yersinia,
Aeromonas
 Halophilic – grow at ≥ 10% sodium chloride: Bacillus, Micrococcus, Staphylococcus,
Pediococcus, Vibrio, Corynebacterium
 Acidophilic – grow at ≤ pH 4: Lactobacillus, Pediococcus, Lactococcus,
Enterococcus, Streptococcus
 Osmophilic – grow at high sugar content: Staphylococcus, Leuconostoc,
Lactobacillus
 Lipolytic – hydrolyse triglycerides (lipases): Micrococcus, Staphylococcus,
Peudomonas, Altermonas, Flavobacterium
Important mold species found in food

 Aspergillus – Asp. flavus (rotting of grains, beans, produces aflatoxin),

Asp. oryzae (ferments starch to alcohol), Asp. niger (ferments
sucrose to acid)
 Alternaria tenuis – rotting of tomatoes, rancidity of dairy products,
may produce mycotoxin
 Fusarium solani – rotting of citrus fruits, potatoes and grains
 Geotricum candidum – spoilage of dairy products
 Mucor rouxii – spoilage of vegetables
 Penicillium spp – rotting of fruits, vegetables, grains, bread and meat
 Rhizopus stolonifer – spoilage of fruits, vegetables, and bread
Important yeast species found in foods:

 Saccharomyces cerevisiae – leavening of bread, ferments sugar to

alcohol
 Pichia membranaefaciens – spoilage of beer, wine and brine
 Rhodotorula glutinis – produce pigment and discolors meat and fish
 Candida lipolyticum – spoilage of food high in acid, salt and sugar,
rancidity of dairy products
 Zygosaccharomyces bailii – spoilage of high-acid food such as
ketsups, pickles, mayonnaise, mustard and salad dressings
Viruses and protozoa do not grow in food but are introduced by
contamination from infected food handlers

Important viruses found in foods

 Hepatitis A virus – causes infectious hepatitis

 Poliovirus – causes poliomyelitis

Important protozoa found in foods

 Entamoeba histolytica – causes amoebic dysentery

 Giardia lamblia (= G. duodenalis) – cause of giardiasis
Microbes used in food preparations:
Food Raw Material Organism
Bread Flour Saccharomyces cerevisiae
Cheese, cottage Milk Lactococcus lactis
Cheese, cheddar Milk Lactococcus lactis, L.
cremoris, Lactobacillus
casei
Pickled cucumber Fresh cucumber Pediococcus cerevisiae,
Lactobacillus plantarum
Miso Soybean Aspergillus oryzae,
Zygosaccharomyces rouxii
Soy sauce Soybean Asp. oryzae, Zy. rouxii, Lac.
delbroeckii
Wine Grape juice Saccharomyces pombe
 CHAPTER 3. MICROBIAL CONTAMINATION AND
SPOILAGE OF FOODS
SOURCES OF MICROBES FOUND IN FOOD
Raw foods always contaminated by microbes because of contact with
humans, machineries, the soil, the added water and the air during
handling.
 Airborne bacteria – predominantly Gram-positive rods and cocci
unless there is a recent contamination of air with Gram-negative
bacteria by aerosol from animal or human source
If plate count agar in Petri dish exposed to air:
• Pigmented colonies: micrococci or corynebacteria; pigment protect microbes from
damage due to visible and UV radiation
• Large white colonies to cream-colored colonies: aerobic spore-forming rods of
genus Bacillus
• Small raised, tough colonies: filamentous bacteria e.g., Streptomyces or related
actinomycetes genera
 Airborne fungi – predominantly molds
Penicillium, Aspergillus – frequent cause of food spoilage
Fusarium
Cladosporium – C. herbarium grow well at refrigeration temperature
on surface of chilled meats
 Animal hides, feathers – major source of contaminant in meat and
poultry
Spore-formers: Bacillus, Clostridium
Gram-positive cooci: Staphylococcus, Enterococcus
Gram-positive rods: Lactobacillus, Corynebacterium
Gram-negative rods: Alcaligenes, Enterobacter, Pseudomonas
Psychrotrophs: Psychrobacter, Shewanella
 Soil – very competitive environment because its physico-chemical
properties can change very rapidly
Bacillus and Clostridium: produce resistant endospores and are
common in soil
Fungi: those that produce conidia (spores) and sclerocia (small fruiting
bodies)

 Water – major source of contaminants in food

Halophilic bacteria: in marine fishes
Enterobacteriaceae and Staphylococcus: human-associated bacteria
Vibrio parahaemolyticus: in shellfishes
Cyanobacteria: photosynthetic bacteria, produce toxins
 Plants – have complex microflora including soil microbes
Yeasts – Sporobolomyces, Bullera on leaf surfaces
Molds – Cladosporium, Penicillium, Aspergilus, Fusarium
Aspergillus flavus – aflatoxin-producing, in stored grains
Bacteria, G(-): Pectobacterium (formerly Erwinia), Erwinia,
Pseudomonas, Xanthomonas
Bacteria, G(+): Lactobacillus, Leuconostoc in fermented vegetables

 Human skin normal flora: Staphylococcus, Propionibacterium

Note: Spores of spore-forming bacteria may survive heating and

remain in the food; under favorable conditions these may germinate
and multiply using the food as nutrient source.
Other microbes may come in as contaminants from the air, machines,
food-handlers, some added ingredients especially spices.
FOOD SPOILAGE
Food spoilage – change in the character of food due to
microorganisms so that it is no longer acceptable

Manifestations of spoilage:
• Visible microbial growth in the form of surface slime or colonies
• Degradation of structural components of food that cause loss of
texture
• Presence of gas (bubbles) and/or pigments
• Off-odors and flavors

Microbial spoilage is relatively sudden onset, not gradual but

unexpected due to exponential nature of microbial growth
 Microbes cause spoilage of food in two ways:
• Through growth and active metabolism of food components by
living microbial cells
• Through extracellular and intracellular enzymes (after cell
lysis) that react with food components even in absence of living
microbial cells, e.g., meat spoilage.

Types of food in relation to ease of spoilage:

 Stable/non-perishable food – sugar, flour, pasta; not spoiled
unless improperly handled, e.g., moistened
 Semi-perishable food – bread, potatoes, fruits; if properly
handled and stored may remain unspoiled for some time
 Perishable – meats, fish, poultry, milk; readily spoiled unless
preservation methods are used
Factors influencing microbial survival and growth in food

INTRINSIC PARAMETERS
Characteristics or parameters inherent to plant and animal tissues.

 pH
• Most microbes grow best at pH near neutral (neutrophiles) and few
grow at pH below 4
• pH of fruits, vegetables and juices fall below neutral and usually
undergo yeast and mold rather than bacterial spoilage
• Yeasts and molds can grow at pH values ≤ 3.5
• Some foods have buffering activity; meats are highly buffered
because of much protein while vegetables have less proteins and
thus have weak buffering capacity
Table 3. pH ranges of some food spoilage microbes
Microbe Minima Maxima
Molds 0.4 11

Yeasts 1.4 8.5

Salmonella spp 3.6 9.5
Listeria 4.2 9.2
monocytogenes
Escherichia coli 4.4 9.8
Costridium 4.4 8.4
botulinum
Bacillus cereus 5.0 9.5
Shigella spp 5.0 9.2
Table 4. pH range of some food products
Food pH Range Food Product pH Range
Product
Lime 1.8 – 2.2 Orange 3.6 – 4.3
Sugar beet 4.2 – 4.4 Banana 4.5 – 4.7
Beans 4.6 – 6.5 Carrot 4.9 – 6.0
Pumpkin 4.8 – 5.2 Potato 5.3 – 5.6
Beef 5.2 – 6.2 Cabbage 5.4 – 6.0
Onion 5.3 – 5.8 Butter 6.1 – 6.4
Milk 6.3 – 6.5 Chicken 6.2 – 6.4
 Water activity or moisture
Water activity (AW) – measure of the availability of water in a food in
free form and available for biological functions.
Water may be bound to hydrophilic molecules or may dissolve
solutes in the food.
This bound water is not available for biological functions and does
not contribute to water activity.

where: P is the vapour pressure of the food (< 1) and PO, the vapour
pressure of pure water (=1).

The approximate minimum water activity values for growth of

microbes are as follows:
Most bacteria – 0.9 Halophilic bacteria – 0.75
Most yeasts – 0.88 Xerophilic molds – 0.61
Most molds – 0.80 Osmophilic yeasts – 0.61
 Reduction-oxidation potential
Redox potential (Eh) – ease in which substrate losses or gains electron.
A substance that readily losses electron is good reducing agent and a
substance that readily gains electron, good oxidizing agent.
When electron is transferred from one compound to another, an
electrical potential difference is created between the two compounds.

Eh measured in mV
Oxidized compound, Eh = + mV
Reduced compounds, Eh = – mV
Aerobic microbes require positive Eh values for growth while
anaerobic microbes require negative Eh values.
Eh of foods:
• Plants foods (juices: 300 to 400 mV) • Minced meat: 200 mV
• Solid meats: – 200 • Cheese: –20 to –200 mV
 Composition (Nutrient content)
• Water – must always be available
• Energy source – sugars, alcohols, amino acids for most microbes;
starches, cellulose, fats for some microbes
• Nitrogen source – amino acids for most microbes; nucleotides for some
microbes
• Growth factors:
B vitamins – as co-enzymes; required in low quantities;
available in all foods
Minerals – as co-factors; also required in low quantities
Gram-positive bacteria – least capacity to synthesize B vitamins; require
external source
Gram-negative bacteria, molds – able to synthesize B vitamins

Gram-negative bacteria and molds are able to grow in fruits which are low in
B vitamins. Meats have higher B vitamins so that Gram-positive bacteria able
to grow. Low B vitamins content plus low pH and positive Eh of fruits explain spoilage
caused by mold rather than by bacteria.
 Antimicrobial constituents
Plant essential oils have antimicrobial activities:
• Eugenol in cloves
• Allicin in garlic
• Cinnamic aldehyde and eugenol in cinnamon
• Allylisothiocyanate in mustard

Cow’s milk has antimicrobial constituents such as lactoferrin, an iron-

binding glycoprotein inhibitory to some food-borne bacteria and
lactoperoxidase.
EXTRINSIC PARAMETERS
These are properties of storage environment that affect both the food
and their microorganisms
 Storage temperature:

Table 5. Cardinal temperatures (OC) for microbial growth

Group Minimum Optimum Maximum
Thermophiles 40 to 45 55 to 75 60 to 90
Mesophiles 5 to 15 30 to 40 40 to 47
Psychrophiles -5 to 5 12 to 15 15 to 20
Pschrotrophs -5 to 5 25 to 30 30 to 35
Note: Pscychrophiles are obligate cold-loving; psychrotrophs are
facultative
Refrigerated (5 to 10OC) meat, fish, poultry, eggs and others are
commonly spoiled by Pseudomonas and Enterococcus, especially
Enterococcus faecalis which grow at 0 to 30OC.
Molds such as Aspergillus, Cladosporium, Thamnidium grow at
refrigeration temperature on eggs, beef and fruits. Yeasts also grow at
psychrotrophic and mesophilic range.
 Relative humidity of environment
Relative humidity is important for microbes growing on surfaces of
foods.
• Foods with low Aw but stored at high RH, absorb water which leads
to increased surface and subsurface Aw which results in growth of
microbes
• Foods with high Aw lose moisture when stored in environment with
low RH. Whole chicken and beef cuts should be stored at low RH
since meat spoilage biota essentially are aerobic in nature.
 Presence and concentration of gases in environment
Carbon dioxide – used to control molds and Gram-negative bacteria
(sensitive to CO2) in food.
It has bacteriostatic effect because it increases acidity of the food
surface:
CO2 + H2O  H2CO2  HCO2 + H+

Other effects of CO2:

• physiological changes in cytoplasmic membrane,
• inhibition of key enzymes especially those involving
carboxylation/decarboxylation reactions in which CO2
is a reactant
• reactions with protein amino groups causing changes in enzyme
properties and activities.
 Activity of other microbes
General microbial interference – nonspecific inhibition or destruction of
one microbe by another in the same habitat.
• Bacterial interference – bacteria present in food prevent entry of
spoilage bacteria
E.g., fresh ground beef normal biota of 105 cells/g prevents growth of
Listeria monocytogenes
E.g., aerobic bacteria in fresh meats prevent growth of Clostridium
botulinum
• Lactic antagonism – lactic acid bacteria inhibit or kill closely related
and food-poisoning or food-spoiling organisms when in mixed
population due to:
(a) production of antimicrobial substances: antibiotics, bacteriocins
against Gram-negative bacteria, diacetyl, hydrogen peroxide
against anaerobic bacteria,
(b) depletion of nutrients and competition for adhesion sites
(c) depression of pH, and (d) combination of these mechanisms.
Changes in food due to spoilage
Spoilage of meat is signaled by off-odor when microbial load reached
107 cfu/g or cm2
• Microbes then switch from diminishing glucose levels to amino acids
as growth substrate.
• Bacterial metabolism produce complex mixtures of volatiles such as
esters, alcohols, ketones and sulfur-containing compounds which
collectively comprise the decay odor
• Odor production
 First stage spoilage: Buttery or cheesy odor due to metabolites of
lactic acid bacteria, Brochothix and Enterobcteriaceae such as
diacetyl (2,3-butanone), acetoin (3-hydroxy-2-butanone),3-
methylbutanol, 2-methylpropanol
  Second stage spoilage: pseudomonads begin to increase and
produce sweet or fruity odor from esters derived from amino
acids, esterification of acids and alcohols generated by first stage
spoilage
 Putrid odor due to amino acid metabolism and production of
methane thiol, dimethyl sulfide and dimethyl disulfide
 Later stages: meat increases in pH due to ammonia and amines
from amino acid metabolism
• At microbial number of 108 cfu/g or cm2, slimy growth becomes
visible on meat.
Spoilage detection level:
• Bacteria and yeasts multiply and reach approximately 107 cells/g or
ml of a food from a level normally found in a food (e.g., meat
normally contains up to 103 cells/6.5 cm2 (1 inch2)
• Spoilage detection level – level at which microbes produce
detectable changes in color, odor and tecture of food accompanied
by slime formation
 It varies from 107 to 108cells/g or ml depending on food type.
 Slime formation generally detectable at ≥ 108cells/g or ml or sq
cm
Shift in predominant microbe during food spoilage:

Most foods initially have various microbial contaminants.

When spoiled, the food contains only one or two predominant microbial
types.
• Beef sample (pH 6) contains 1% Pseudomonas spp., 11%
Acinetobacter and Morexella, 13% Brochothrix thermosphacta and
75% others (Micrococcus, Staphylococcus, Pseudomonas spp., lactic
acid bacteria and Enterobacteriaceae).
• After storage at 2OC for 12 days, microbial count reaches 107 with
99% Pseudomonas spp. and 1 % other bacterial groups.
 CHAPTER 4. SPOILAGE OF SPECIFIC FOODS

1. MILK
Milk – high water activity and near neutral (6.4 to 6.6) pH so a good
microbial growth medium
• Raw milk naturally contains 102 – 103 cfu/ml (cfu – colony forming
unit). Milk from some quarters may be sterile.
• Microbial population consist of micrococci, streptococci and
Corynebacterium bovis.
• Pathogens such as Salmonella, Campylobacter, Mycobacterium bovis
and M. tuberculosis come in as contaminants.
 First two bacteria form the human handlers
 Two others from the infected cow itself.
Chilled (below 7OC) milk may contain psychrotrophic bacteria such as
Acinetobacter, Aerobacter, Alcaligenes, Flavobacterium, Pseudomonas
and Bacillus spp.
These bacteria are post-pasteurization contaminants and the causes of
spoilage of stored milk.

Spoiled milk exhibits the following:

• Bitty cream character, i.e., small proteinaceous fat globules float
and adhere to the sides of containers, including cups and crockery.
• Due to the hydrolysis of phospholipids by lecithinase of Bacillus
cereus.
• Psychrotrophs, particularly lactobacilli, denature the proteins which
then form the sediment in milk.
• Denaturation – due to the production of acids which lowers the pH
and destroys the native structure of the proteins.
Raw milk contains thermodurics (microbes that survive pasteurization)
• These include: Microbacterium, Micrococcus, Enterococcu,
Lactobacillus and Alcaligenes.
• The common food-poisoning bacterium, Bacillus cereus, is a major
cause of spoilage in milk.

2. BEEF
Beef – has high water content and abundant nutrients so also good
growth medium
Main source of microbes:
• Skinning of animal spread the contaminating bacteria to the surface
of meat.
• Removal of the visceral organs transfers of microbes from the gut to
the meat.
Change of pH in beef
• Oxygen is cut off from muscle tissues after death.
• Metabolism of glycogen to glucose continues for some time
resulting in accumulation of lactic acid.
• pH of the muscle tissues right after slaughtering is about 7.4 and
lactic accumulation lowers the pH to about 5.4 to 5.5.
• This condition favors the growth of acid-loving bacteria and fungi.
• Psychrotrophic bacteria represent only a small percentage of
microbes in the beginning but predominate later when the meat is
kept at constant chilling temperature.
Spoilage of meat is mainly caused by Acinetobacter, Brochothrix,
Enterobacter, Pseudomonas, Psychrobacter and Serratia.

Deterioration of meat occurs in the following phases:

• First indication of spoilage is production of off odor as microbial count
increases to 107 cfu/cm2.
 Glucose metabolism by Enterobacteriaceae, lactic acid bacteria
and Brochothrix results in the production of diacetyl, butanedione,
acetoin, butanol and methylpropanol which give buttery or cheesy
odor.
• When glucose is exhausted, amino acids are used as energy sources
by Pseudomonas and Moraxella resulting in the formation esters.
 These bacteria cause the esterification of acids and alcohols
produced during the previous spoilage phase.
 These esters have a sweet or fruity smell
• Putrid odor is emitted due to production of sulfur volatiles such as
methane thiol, dimethyl sulfide and dimethyldisulfide by
Pseudomonas and Acinetobacter from metabolism of amino acids.
• Later stages of decomposition is characterized by increase in meat
pH as ammonia and amines (putrecine and cadaverine) which,
however, do not contribute to odor are produced
• At this phase, surface slime becomes visible due to increase in
microbial number to 108 cfu/cm2.
FISH
Fish is an aquatic animal so its normal flora consists of psychotrophs
such as Pseudomonas, Shewanella, Psychrobacter, Vibrio,
Flavobacterium and Cytophaga.
• Skin of fish may contain bacteria from 102 to 107 cfu/cm2
• Fish carcass contains less glucose than mammalian tissues, amino
acids are immediately used as energy and carbon sources producing
hydrogen sulfide , methylmercaptan, dimethylsulfide and esters.
• Putrefaction of fish meat is similar to that of mammalian meat.
PLANT PRODUCTS
Plants have:
• physical barriers such as cuticle, bark and fruit skin that prevent
microbial invasion.
• also have chemical barrier such as lignin, a polymer of polyphenol
 Phenol has anti-microbial activity
Invasion of the internal plant tissues, post-harvest, is mainly due to
contaminants that entered through wounds
 Cereals
• Stored dry grains can be attacked primarily by xerophilic molds such
as Cladosporium, Alternaria, Epicoccum and Fusarium.
• Such molds produce biological water as products of metabolism and
also generate heat (self-heating)
• These conditions allow the growth of Penicillium and Aspergillus,
initially, and bacteria, subsequently.
• Grain spoilage:
 first marked by mold growth and production of musty odor
 then caking of the grains due to mycelia and slime formation
 complete decay proceeds when other organisms grow on the
grains because of increased moisture and favorable temperature.
 Pulses
Pulses – leguminous plants such as peas, beans, ground nut (peanut)
and lentils
• These contains relatively high amounts of oils are thus are
attacked by lipolytic molds such as Aspergillus, Penicillium,
Paecilomyces and Rhizopus.
• Spoilage is similar to that of cereals.

 Fruits
Fruits have high water activity and low pH
• Spoiled by molds and yeasts
• Citrus fruits, e.g., oranges, are decomposed by Penicillium italicum
and P. digitatum. Apples are spoiled by Penicillium expansum,
Venturia inequalis and Monilinia fructigena
• The fruits exhibit mycelia growth on the skin and putrefaction in the
interior tissues.
 Leafy vegetables
Leafy vegetables have high water activity and relatively high pH
• Attacked by bacteria and also molds
• First there is softening of the tissues due to hydrolysis of pectin (the
major component of lamella between cells) thus weakening the
integrity of the leaf tissues
• Later, bacterial or fungal growth is seen on the surface of leaves
• Causal microbes are the bacteria Pectobacterium, Pseudomonas,
Xanthomonas and Corynebacterium as well as the fungi Botrytis
cinerea, Fusarium and Aspergillus.
 CHAPTER 5. PRINCIPLES AND METHODS OF FOOD
PRESERVATION

CONTROL BY HEAT
Purpose of control by heat:
• To destroy the microbial vegetative cells and spores in order to retain
the acceptability and nutritional qualities of a food.
 Control of growth of surviving microbes is accomplished by other
control methods following heat treatment.
• To destroy undesirable enzymes that would otherwise adversely
affect acceptance quality of food.
 Heating helps destroy or reduce the activity of heat-stable
proteinases and lipases produced by some microbes.
• To destroy undesirable microbes before adding starter culture for
fermentation of food, e.g., fermentation of milk to produce yoghurt
Mechanism of action
 On vegetative cells:
• Disrupts the cell membrane and cell wall
• Denatures, breaks and degrades DNA, ribosomal RNA and
proteins
• Damages vital functions and structural components of cells
 On spores
• Damages structural component of spore coat and structures
destined to become cell membrane and cell wall
• Causes inability to use water foe hydration during germination
• Causes death due to inability to germinate or to grow out of
spore
Factors affecting heat control method
 Composition (amount of carbohydrates, proteins, lipids and solutes)
that provide protection to microbes against heat
• Aw – high Aw increases resistance to heat inactivation
• pH – low pH increases susceptibility to heat
 Nature of organism
• Spores more resistant than vegetative cells of yeasts, molds and
bacteria
• Thermophilic and thermoduric cells destroyed at 75 – 80OC in 5 – 10
min
• Molds, yeasts, many bacteria and viruses destroyed at 65 OC in 10
min
• Yeast and mold spores destroyed at 65 – 70OC in few minutes but
spores of some molds survive at as high as 90OC for 4 – 5 h
• Bacterial spores killed at 80 – 100OC for few minutes but some
survive after boiling for 24 h
• All organisms destroyed at 121OC in 15 min
Methods of heat treatment of foods
 Pasteurization or low heat processing to destroy relatively heat
sensitive microbes
Purpose of pasteurization:
• To destroy all vegetative cells of pathogens and large numbers of
associated and spoilage microbes such as yeast, molds, bacteria and
viruses
• To destroy natural enzymes such as phosphatase in milk

Thermoduric microbes survive pasteurization temperatures, so additional

treatment of product is needed such as:
(1)refrigeration
(2) modification of the atmosphere
(3) addition of chemical preservatives
(4) reduction of Aw.
Pasteurization methods for milk:
• Low temperature long time (LTLT) – 62.8OC for 30 min
• High temperature short time (HTST) – 71.7OC for 15 sec
• Ultra high temperature – 135OC for 1 sec
• “Sterilized” – > 100OC for 20 – 40 min
Immediately milk is cooled to 40 F, packaged and maintained at that
temperature until consumed.
 High heat treatment
• For high acid or low pH (pH < 4.6) products such as tomato, fruit
products and acidified foods (pickles), use100OC heat
 Clostridium botulinum spores cannot germinate at low pH
 Bacillus coagulans vegetative cells, Lactobacillus, Leuconostoc,
yeats and molds can grow at low pH but are heat-sensitive
• For milk, use ultrahigh temperature processing at 150OC for 2 – 3 min
then store at < 30OC
 Milk will have 3-months shelf-life
LOW TEMPERATURE CONTROL
Objectives:
• To prevent or reduce growth of microbes; 90% of vegetative cells can
die if food is frozen
• To prevent catalytic activity of microbial enzymes especially heat-
stable proteinases and lipases
• To reduce spore germination
Mechanism of action
• Microbial activities associated with growth will slow down.
 Usually doubling time doubles for every 10OC reduction in
temperature, e.g., if cells divide every 60 min at 22OC, cells will
divide in 120 min at 12OC and in 240 min at 2OC.
• Lag and exponential phases of growth and germination time of
spores of psychrotrophs become increasingly longer at 0 – 1OC.
• Crystalization of water to ice causes cell injury by damaging cell
membrane, breaking DNA, degrades RNA and inactivate enzymes
causing cell death.
Factors influencing low temperature preservation of foods
• Nature of food – foods with high solid content (protein,
carbohydrate, and lipid, e.g., meats), pH near neutral, higher A w and
absence of microbial inhibitors facilitate survival and growth of
microbes at refrigeration temperatures (5OC)
• Nature of microbes
 Psychrotrophs can continue growing at refrigeration
temperatures
 Spores of Clostridium can germinate at 2OC; Bacillus spores
germinate at slightly higher temperature
Methods
• Ice chilling between 0 - 1OC for fish, meat, cut fruits and vegetable
salads
• Refrigeration at 4 - 5OC for many foods
• Freezing: home freezers at – 20OC; dry ice (solid carbon dioxide) at –
78OC
Storage at frozen state:
• Red meat: 6 – 12 months
• Poultry: 3 months
• Fruits and vegetables: 3 – 6 months
CONTROL BY REDUCTION OF WATER ACTIVITY
Objectives:
• To prevent or reduce growth of cells and germination of spores
• To cause injury and death of cells

Mechanism of action
• Removal of water or addition of solutes and hydrophilic colloids (which
cannot enter cells) cause the free water to flow out of the cells
(osmosis) to establish equilibrium
• Loss of water causes osmotic shock and plasmolysis during which cell
do not grow (in dormancy) or later die
• A 0.005 reduction in Aw from 0.955 to 0.950 reduces intracellular water
content by 50% in Staphylococcus and by 40% in Salmonella
thyphimurium
Methods
• Sun drying – grains, fruits, vegetables, fish, meat
• Mechanical means
 Tunnel drying – food travels through tunnel against flow of hot air
 Roller drying – liquid food applied thinly on heated roller
 Spray drying – liquid food sprayed as small droplets and dried by
hot air
 Smoking – for meat and fish; low heat (63OC) and smoke
CONTROL BY LOW pH AND ORGANIC ACIDS
Objective:
To control of microbial growth by reducing pH with addition of weak
acids
Mechanism of action:
Microbes maintain internal cytoplasmic pH of 6.5 – 7.0 in acidophiles
and 7.5 – 8.0 in neutrophiles
• For each 1.0 unit change in environmental pH, the internal pH drops
by 0.1 unit
• Addition of acids to food increases the environmental proton gradient
which affecting cell’s transmembrane proton gradient
 Organic acids are lipophilic (except citric acid) and readily enter
microbial cells by osmosis
 These acids dissociate inside cells, releasing protons and anions
(removed from or metabolized by cells)
 Protons reduce internal pH, disrupt proton gradient and affect
protein functions and structural integrity
Acids used in food:
• Acetic acid – 5 to 10% as acid or sodium/calcium acetate for pickles,
salad dressings and sauces
Inhibitory concentration of acetic acid against microbes:
Salmonella: 0.02%
Staphylococcus aureus: 0-.01%
Bacillus cereus: 0.02%
Aspergillus spp: 0.01%
Saccharomyces spp: 0.5%

• Propionic acid as acid or salts of calcium or sodium at 0.1 – 0.2% in

bakery products, cheese, jam, jellies and tomato puree
• Lactic acid as acid or salts of sodium or potassium up to 2% in
carbonated drinks, salad dressings, pickled vegetables, low-heat
processed meat products and sauces
• Citric acid at 1% in nonalcoholic drinks, , jams and jellies, baking
products, cheese, canned vegetables and sauces
• Sorbic acid at 0.05 – 0.2% in nonalcoholic and alcoholic drinks,
processed fruits and vegetables, dairy desserts, mayonnaise, salad
dressings, sandwitch spreads, mustard
• Benzoic acid at 0.05 – 0.2% in nonalcoholic and alcoholic drinks,
beverages, pickles, mayonnaise, salad dressings, mustard and
cottage cheese
• p-Hydroxybenzoic acid (paraben) as methyl, ethyl, butyl and propyl
parabens in nonalcoholic and alcoholic drinks, beverages, fruit
fillings, jams and jellies, pickles, salad dressings, spreads and
mustard
CONTROL BY MODIFIED ATMOSPHERE
Objectives:
• Control or reduce growth of undesirable microbes in food
• Retard enzymatic and respiratory activities in fresh food
• Prevent of growth of yeast, molds and aerobic bacteria

Mechanism of action:
• Presence of CO2 or N2 or both extends the lag and exponential
phases of microbes
• Rapid penetration of CO2 into cells alter cell membrane permeability
• Dissolution of CO2 to carbonic acid in cells reduces pH and interferes
with enzymatic functions
Methods:
• Controlled atmosphere packaging (CAP) – atmosphere in storage
facility altered and levels of gasses continually monitored
 Expensive to operate
 For long term storage of fruits and vegetables
• Modified atmosphere packaging (MAP) – food is enclosed in high gas-
barrier packing material; air removed from package and flushed with
100% CO2 or 100% N2 or both and sealed hermetically
 For fresh pasta, bakery products, fresh and cooked fish and sea
foods, sandwiches, raw meat, some vegetables
• Vacuum packaging – removal of air from package and sealing
hermetically
 For meat and meat products
 If Aw reduced and pH lowered, product shelf-life extended beyond 4
weeks
CONTROL BY ANTIMICROBIAL PRESERVATIVES
Objective:
To kill or prevent or retard growth of bacteria and fungi with use of
small doses

Chemicals:
• Nitrite (Na or K salts) – 200 ppm for heat-processed meat, poultry and fish
 Reacts with some enzymes in vegetative cell and germinating spores
 Restrict bacterial use of iron (co-factor of enzymes)
 Interfere with membrane permeability and limiting water and nutrient
transport
• Sulfur dioxide (SO2) and sulfite (SO32-) – 200 to 300 ppm for soft fruits, fruit juices,
beverages, wined, sausages, pickles fresh sea foods against molds and yeasts
 Sulfite as sodium sulfite (Na2SO3), sodium bisulfite (NaHSO3) or sodium
metabisulfite (Na2S2O3)
• Glycerol monolaurate (monolaurin) – up to 500 ppm against anaerobic bacteria
and to enhance thermal inactivation of Bacillus spores
 For meat, chicken sausages, minced fresh meat, other foods
CONTROL BY IRRADIATION
In the electromagnetic spectrum, energy exists as waves and the
intensity of energy increases as the waves get shorter.
• Waves with < 300 nm length are UV, X-rays, beta rays, gamma rays
and cosmic rays
• Exposure to ionizing radiation does not result in radioactivity
• Gamma rays have high penetration power (~ 40 cm) and used for
food at dose level of 10 kGy
• Cobalt-60 and cesium-137 are good sources of gamma rays

Objectives:
• Complete or partial destruction of molds, yeast and bacterial cells,
spores and viruses
• Killing of worms and insects in food
• But could not destroy toxins and undesirable enzymes
Mechanism of action:
• High energy gamma rays (10-1 to 10-2 nm) strip electrons from atoms
and molecules that absorbed them in a fraction of a second
 Removal of electrons from outer shells of atoms results in
formation of negatively and positively charged ions pairs
 Ionization adversely affects normal biological systems
• Direct effects:
• Damage DNA by removal of electrons
• Ionization of water into hydrogen and hydroxyl radicals (highly
reactive) cause oxidation, reduction and breakdown of C-C bonds of
molecules including DNA
• Ionization damages membranes and other structures causing lethal
injury to cell
Advantages:
• Effective pasteurization of food
• Large pieces of food can be processed
Disadvantages:
• Some loss of vitamins and flavor from food
• Potential production of carcinogens in food
Dose:
• 1 rad – quantity of ionizing radiation that results in absorption of
100 ergs of energy per gram of irradiated material
• 1 Gray (Gy) = 100 rad
• 1 Gy is the dose equivalent to 1 joule (= 107 ergs) absorbed by 1 kg
of food
Application:
For fruits, grains, meat, fish and sprouting vegetables
Lethal doses of gamma rays to microbes:
• Molds, yeasts, bacterial cells = 0.5 – 10 kGy
• Bacterial spores = 10 – 50 kGy
• C. botulinum = 30 -60 kGy
• Viruses = 10 – 200 kGy
 CHAPTER 6. FOOD IN RELATION TO DISEASE

Definitions:
• Toxin – protein, glycoprotein or lipopolysaccharide poison
elaborated by a living organism
• Intoxication – ingestion of food containing active preformed toxin,
produced by microbial growth in food, causing disease
 Ingested viable cells are not necessary for disease development
 Symptoms develop quickly as early as 30 min after ingestion
• Infection – ingestion of an infective dose of viable cells that
establish, multiply and produce toxin in digestive tract
• Toxico-infection – ingestion of live cells that produce toxins
 Ingested cells of nonspore-formers multiply in digestive tract and
release toxin when cells die
FOOD-BORNE INTOXICATION
Feature of Organism Staphylococcus aureus Clostridium botulinum
Cell morphology Round Rod
Gram reaction Positive Negative
Spore formation Absent Present
Natural habitat Nose, throat, skin Soil, GIT
Growth conditions:
Oxygen Aerobe Obligate anaerobe
Temperature (OC) 7 to 48 3 to 45
Aw ≤ 0.86 > 0.93
Ph 4.8 > 4.6
NaCl (%) 15 > 5.5
Toxin heat stability Thermostable enterotoxin Thermolabile neurotoxin
(affects involuntary
muscles)
Food association Protein-rich foods Canned, chilled foods
Staphylococcus intoxication:
• Most frequently occurring foodborne disease worldwide
• Cells survive in food with high sugar content and 15% nitrite
• Cells killed at pasteurization temperatures
• Dose – 30 g or ml of food with 100-200 ng toxin (7 types)
• Toxin cause salivation, nausea, vomiting, abdominal cramps and diarrhoea
• Prevention – people with acne not allowed to handle food, warming of cooked
food for more than 2 h not advisable
• Identification – cells by selective media, toxin by serology
Botulism:
• Neurotoxin types A, B, E and F blocks nerve signals, cause irreversible paralysis of
involuntary muscles
• Dose – 1 ng/kg body weight (extremely potent)
• Symptoms – blurred or double vision, difficulty in swallowing, breathing and
speaking, dryness of mouth, paralysis of different organs, then death by
respiratory failure
• Infant botulism – ingestion of spores through food
• Prevention – 12D concept in canning, especially home canning, heat chilled food
to destroy toxin; 1D is time of exposure to certain temperature to reduce microbial
number by 1 log cycle, i.e., 90%
 TOXICO-INFECTION
Feature of Organism Bacillus cereus Clostridium perfringens
Cell morphology Rod Rod
Gram reaction Positive Positive
Spore formation Present Present
Natural habitat Soil, GIT Soil, GIT
Growth conditions:
Oxygen Facultative anaerobe Obligate anaerobe
Temperature (OC) 4 to 50 10 to 52
Aw > 0.95 < 0.93
pH 4.9 to 9.3 <5
NaCl (%) 10 > 5.0
Dose (cells/g) 106 to 108 ≥ 5x105
Toxin heat stability Thermostable emetic Thermolabile enterotoxin
enterotoxin (formed during sporulation
Thermolabile enterotoxin in intestines)
Food association Meats, vegetables, soups Chilled or roasted foods,
meat stew
Bacillus cereus gastroenteritis:
• Outbreaks are not frequent
• Toxins – emetic (vomiting) and enteric (gastroenteritis) formed during growth and
released when cell lyse
• Symptoms appear 6-12 h after eating contaminated food; abdominal pain, profuse
watery diarrhoea, nausea, vomiting if with emetic toxin
• Resolved in 24 h
• Prevention – do not store food chilled for long time (cells are psychrotrophs),
holding temperature of heated food at > 60 OC
• Detection – cells by selective media
Clostridium perfringens gastroenteritis:
• In most cases, large numbers of cases are involved
• Outbreaks generally due to keeping warm cooked food for several hours before
serving, e.g., restaurant, cafeteria, school canteens and parties
• Disease has mild symptoms, many incidence probably not reported
• Symptoms appear 8-24 h after eating contaminated food; abdominal pain,
diarrhoea, nausea, vomiting
• Resolved in 24 h
• Prevention – thorough cooking of food, improper holding temperature
• Detection – selective media in anaerobic condition
 FOOD-BORNE BACTERIAL INFECTION

Feature of Organism Salmonella enterica Listeria monocytogenes

Cell morphology Rod Rod
Gram reaction Negative Positive
Spore formation Absent Absent
Natural habitat GIT Soil, GIT
Growth conditions:
Oxygen Facultative anaerobe Facultative anaerobe
Temperature (OC) 5 to 46 1 to 44
Aw > 0.95 < 0.93
Ph ≥ 4.6 ≥ 4.6
Dose (cells/g) 105 to 106 100 to 1000
Toxin heat stability Thermostable cytotoxic Hemolysin (formed during
factor exponential growth in
Thermolabile enterotoxin intestines)
Food association Many foods, chicken, raw Chilled foods, milk, dairy
foods products
Salmonella (salmonellosis)
• Based on DNA-DNA hybridization, only one species – Salmonella enterica with
subspecies: enterica, salamae, arizonae, diarizonae, houtenae, bongori
• Carrier state in humans and animals – infected but symptomless
• Cells invade intestinal mucosa due to cytotoxic factor, produce toxin causing tissue
inflammation and fluid accumulation
• Symptoms last for 2-3 days; abdominal cramps, diarrhoea, nausea, vomiting, chills,
fever, body weakness
• Prevention – avoid raw meat, heating of ready-to-eat foods
• Detection – selective media
Listeria monocytogenes (listeriosis)
• Opportunistic pathogen – healthy individual show mild enteric disease but fatal (30-
40% of cases) to fetuses, newborns, infants, elderly, pregnant women,
immunocompromized people
• Psychrotrophic – can grow on many food at 5-10 OC
• Cells invade blood stream and vital organs, e.g., CNS, multiply intracellularly,
release toxin causing cell death
• Symptoms appear 1-7 days after eating contaminated food; slight fever, abdominal
cramps, diarrhea which subside after few days
• Prevention – detection from ready-to-eat foods, HACCP system
• Detection – pre-enrichment and enrichment isolation
FOOD-BORNE INFECTION
Feature of Organism Campylobacter enteritis, Yersinia enterocolitica
C. jejuni
Cell morphology Spiral rod Short rod
Gram reaction Negative Positive
Spore formation Absent Absent
Natural habitat GIT GIT
Growth conditions:
Oxygen Microaerophile Facultative anaerobe
Temperature (OC) 32 to 45 0 to 44
pH 4.9 to 9.3 > 4.5
NaCl (%) 10
Dose (cells/g) 5000 107
Toxin heat stability Thermostable invasive Invasive factor
factor
Thermostable enterotoxin
Food association Many foods especially raw Raw milk, dairy products,
foods meat
Campylobacter enteritis
• Grow better on amino acids than on carbohydrates
• Survive in frozen state for months
• Toxins – invasive factor enables cells to invade and colonize intestinal epithelial
cells, enterotoxin cross-reacts with cholera toxin
• Symptoms occur in 2-5 days and last for 2 days to 2 weeks; abdominal cramps,
profuse sometimes bloody diarrhea , nausea, fever, headache and chills; with
relapse
• Prevention – sanitation, avoid raw meats, heat treatment of food
• Detection – selective media

Yersinia enterocolitica (yersiniosis)

• Emerging foodborne pathogen but rarely fatal
• Psychrotophic
• Invasive factor enables cells to colonize intestinal epithelial cells and lymph nodes
• Symptoms – appear 24-30 h after eating contaminated food and last for 2-3 days;
sever pain at lower quadrant of abdomen, diarrhea, nausea, vomiting and fever
• Prevention – sanitation, heat treatment of food, refrigeration of food not effective
because of its being psychrotrophic
FOOD-BORNE INFECTION
Feature of Organism Shigella Vibrio
Cell morphology Rod Curved rod
Gram reaction Negative Negative
Spore formation Absent Absent
Natural habitat GIT GIT
Growth conditions:
Oxygen Facultative anaerobe Facjultative anaerobe
Temperature (OC) 7 to 46 30 to 35
Aw > 0.95 < 0.93
pH 4.9 to 9.3 <5
Dose (cells/g) 10 to 1000 106
Toxin heat stability Shiga toxin Thermostable cytotoxic
protein
Food association Many foods, especially Improperly cooked
Shigella (shigellosis, bacillary dysentery)
• Shigella dysenteriae, S. flexneri, S. boydii, S. sonnei
• Have high fatality rate among ≤ 5 years in third world
• DNA homology suggests Shigella and Escherichia belong to one genus; Shigella spp
are pathogenic variants of E. coli
• Bacterial cells engulfed by epithelial cells kill them and attack fresh epithelial cells
• Symptoms – abdominal pain, bloody diarrhea with mucus and pus, fever, chills,
headache
• Prevention – sanitation, chlorinated water for washing salads, blanching vegetables
• Detection – selective media

Vibrio (gastroenteritis, cholera-el tor)

• Vibrio cholerae, V. parahaemolyticus
• Halophilic bacteria; marine environment is natural reservoir
• Symptoms – appear 10-24 h after eating food or drinking water contaminated with
bacteria; nausea, vomiting, abdominal cramps, rice water diarrhea, headache, fever,
chills
• Immediate rehydration of patients
• Prevention – refrigeration and freezing of raw food before cooking, avoid raw
seafoods
Pathogenic E. coli strains
• Enteropathogenic E. coli (EPEC)
 Disease – gastroenteritis, infants’ diarrhea
 Infective dose – 106-109 cells
 Carrier state present
• Enterotoxic E. coli (ETEC)
 Disease – travelers’ diarrhea, infants’ diarrhea
 Infective dose – 108-109 cells
 Toxin – invasive factor, thermostable and thermolabile enterotoxins
 Carrier state present
• Enteroinvasive E. coli (EIEC)
 Disease – dysentery
 Infective dose – 106 cells
 Toxin – Invasive factor
 Carrier state present
• Enterohemorrhagic E. coli (EHEC)
 Disease – dysentery, hemorrhagic colitis, uremic disease, thrombocytopenic
purpura (often fatal)
 Infective dose – 10 to 100 cells
 Toxin - verotoxin
FOOD-BORNE PARASITIC AND VIRAL INFECTIONS
Foodborne parasitic and viral infections are results of presence of
parasites or viruses in food due to direct contamination or to a prior
infection of the living host before its slaughter
• Mainly the result of poor sanitary practices of food handlers and/or
improper cooking of food
• Parasites and viruses do not grow in food (Why so?)

Protozoan infections
• Giardia lamblia (= G. duodenalis) cause of giardiasis
 Ingestion of cyst in food, excyst to trophozoites in upper small
intestines
 Incubation – 7-13 days, cysts appear in stool after 3-4 weeks
 Symptoms – abdominal cramps and distension, nausea, weight
loss
 Food association – raw vegetables, salads
• Entamoeba histolytica cause of amoebic dysentery
 Ingestion of cyst, excyst to trophozoites in intestines
 Incubation – 2-4 weeks
 Symptoms – fever severe diarrhea, vomiting, weakness
 Food association – raw vegetables

Helminthic parasitization
Ingestion of worm in improperly cooked liver or meat
• Fasciola hepatica (liver fluke)
• Taenia saginata (cattle tape worm)
• Taenia solium (swine tape worm)
• Trichinella spiralis (found in striated muscle of animal host)
Viruses:
• Polio virus – ssRNA, incubation 3-5 days
 Ingestion of viral particle in water or food due to unsanitary
practice of food handlers or improperly cooked food
 First stage of infection – headache, fever, sore throat
 Second stage – invasion of meninges; head ache, back pain
 Uncommon third stage – invasion of spinal cord; paralysis of legs
Food association – raw foods, salads, raw meat

• Hepatitis A and E viruses – infectious hepatitis; incubation 2-6

weeks
 Ingestion of viral particles in raw or improperly cooked food
 Symptoms – anorexia, fever, malaise, nausea, vomiting, liver
damage (dark urine and jaundice)
Food association – vegetables salads, milk, fruits such as berries
• Gastroenteritis viruses
 Astrovirus – ssRNA virus; causes mild gastroenteritis
 Calicivirus – ssRNA virus; gastroenteritis
Ingestion of viral particle in contaminated food
Food association – salads, fruits especially berries
 CHAPTER 7. LABORATORY METHODS FOR
MICROBIOLOGICAL AND TOXICOLOGICAL
ANALYSIS OF FOOD

SAMPLING OF PRODUCT

SAMPLING PLAN
Sampling plan is statement of criteria of acceptance applied to a lot or
batch based on appropriate examination of required number of
sample units by specified method
• Consists of sampling procedure and decision criteria
• Two types of sampling plan: two-class or three-class plan
SAMPLING SPECIFICATIONS
• n – number of sample units (packages, cans, bottles, etc)
• c – maximum acceptable number that may exceed the
microbiological criterion, m
 Note: when c is exceeded, the lot or batch is rejected.
• m – maximum number of bacteria/g or ml
 Values above m is either marginally acceptable or unacceptable
quality of product
 Used to separate acceptable from unacceptable or marginally
acceptable quality of the product
• M – microbial count used to separate marginally acceptable from
unacceptable quality of the product
Examples of two-class plan
• Accept/reject a lot or batch based on presence/absence of Salmonella:
n = 5, c = 0
 n = 5 means 5 individual sample units to be examined
 c = 0 means that all 5 sample units must be free of Salmonella in
order for the lot to be acceptable; if even 1 sample is positive for
Salmonella, the whole lot is rejected.
• Presence/absence decision for coliforms: n = 5, c = 2
 If more than 2 sample units contain coliform, the whole lot will be
rejected.
• Allowable limits for coliforms in a lot: n = 5, c = 2, m = 102/g
 If no more than 2 sample units contain ≤ 102/g coliform, lot is
acceptable.
 This plan can be made more strict by increasing n to 10 (n = 10, c
= 2, m = 102/g) or by reducing c to 1 (n = 10, c = 1, m = 102/g).
Example of a three-class plan
Acceptable, marginally acceptable, unacceptable decision for coliforms:
n = 5, c = 2, m = 105/g, M = 106/g
• If any of the 5 sample units exceeds 106/g coliforms, entire lot is
rejected (unacceptable)
• If no more that 2 sample units yield counts above m but less than M,
the lot is acceptable
 This plan distinguishes between marginally acceptable from
unacceptable

Standards:
• Sugar (US National Canners Association)
 Total thermophilic spore count: n = 5, c = 0, m = 150 spores/g
 Thermophilic anaerobe spores: n = 5, c = 3
 Sulfide spoilage spores: n = 5, c = 2, m = 5/10 g
• Raw chicken: aerobic plate count: n = 5, c = 3, m = 5x 105,
M = 107
• Pasteurized milk: ≤ 20,000 bacteria/ml and ≤ 10 coliforms/ml
• Pasteurized culture product (e.g., yogurt): ≤ 10 coliforms/ml
• Dry milk:
 Mesophilic count: n = 5, c = 2, m = 5x 104, M = 2x 105
 Coliforms: n = 5, c = 1, m = 10, M = 100
 Salmonella: n = 15, c = 0, m = 0
• Ice cream:
 Coliforms: n = 5, c = 1, m = 10, M = 103
 Aerobic count: n = 5, c = 2, m = 105, M = 106
• Cereals:
 Molds: n = 5, c = 2, m = 102–104, M = 105
 S. aureus: n = 5, c = 1, m = 103, M = 104
• Bottled water, coliforms: n = 5, c = 0, m = 0
MICROBIOLOGICAL METHODS OF FOOD ANALYSIS

DIRECT MICROSCOPY
Helpful in determining types and approximate number of microbes
present in samples before actual microbial count/enumeration
• Gram staining for bacteria:
 Sample homogenization: 25 g sample in 225 ml of maximum recovery diluents
(MRD; 0.1% peptone and 0.85% sodium chloride) to give dilution of 1:10 (or 10–)
using a blender or stomacher
 Prepare smear of liquid or homogenized solid food
 Fix smear over flame or heat
 Stain with Gram stain, dry slide and examine under the oil immersion
objective (100x) of microscope
• Wet mount for fungi:
 Emulsify small amount of sample in a drop of distilled water on a slide and cover
the smear with cover slip
 Add one drop of lactophenol cotton blue to edge of cover slip (stain will disperse
to the smear)
 Examine under high power objective (40x) of microscope
TOTAL MICROBIAL COUNT
• Epifluorescent stain method
 Filtrate of liquid or homogenate (through 5 µm membrane filter) is
treated with Triton X-100 and 0.5 ml trypsin solution to lyse meat
somatic cells
 After incubation, filtrate is passed through 0.6 µm membrane filter
 Membrane is stained with acridine orange and then dried
 Stained cell counted under UV microscope
• Tetrazolium stain method:
 Membrane filtered diluted liquid or homogenate sample treated
with sterile tetrazolium [(p-iodophenyl-3-p-nitrophenyl)-5-phenyl
tetrazolium chloride] for 10 min at 37OC in water bath
 After drying membrane at 50OC for 10 min, it is mounted in cotton
seed oil, overlaid with cover slip and viewed under UV microscope
for cell counting
 Viable cells highly colored green, dead cells are red-colored
VIABLE CELL COUNT

Standard plate count (SPC)

• Spread plate method (for aerobic microbes)
 Convenient because use of pre-poured plates
 Eliminates possible heat stress to organism from molten agar
(agar solidifies at about 40OC)
 Provides fully aerobic conditions for growth and facilitate
identification of organism present (many microbes found in food
are aerobic)
 Preferable when selective media are used; it allows observation
of colony characteristics for preliminary identification
• Pour plate method (for anaerobic microbes)
 Require use of clear growth medium to allow counting of
colonies below medium surface
 Depth helps maintain anaerobic condition
 Need to maintain molten agar medium at 44 – 47OC and used
within 8 h
 Relative anaerobic condition in depths of agar may result in
under-recovery if aerobic microbes are being enumerated
Table 6. Some culture media used for microbiological
examination of foods.
Medium Use
Plate count agar Aerobic mesophiles
MacConkey broth MPN of coliforms in water
Brilliant green/lactose/bile broth MPN of coliforms in food
Violet red/bile/glucose agar Enumeration of Enterobacteriaceae
Crystal violet/azide/blood agar Enumeration of faecal streptococci
Baird-Parker agar Enumeration of Staphylococcus aureus
Rappaport-Vassiliadis broth Isolation of Salmonella
Thiosulfate/bile/citrate/sucrose agar Isolation of Vibrio
Dichloran/18% glycerol agar Enymeration of molds
Rose Bengal/chloramphenicol agar Enumeration of yeasts
Cifeximine/tellurite/sorbitol, E. coli O1578
MacConkey agar
Plating and colony counting procedure
• Plates of agar medium are replicated at least two times.
• Plating is done either by pour or spread plate depending on the
purpose of the enumeration.
• Incubate inoculated plates upside down; Why?
• Count colonies in plates containing 30 to 300 colonies.

Theoretically, one viable cell gives rise to one colony; however two or
more cells adhering together give rise to also one colony so the
counts are called colony forming units (cfu)/ml or /g of sample.
Calculation of colony number:

N = (∑mean count x reciprocal of dilution in each dilution) ÷ 2

1st dilution (10–3): 171 and 194 colonies

2nd dilution (10–4): 14 and 20 colonies
Volume added to each plate: 0.1 ml

First dilution
171 + 194 = 365
365/2 = 182.5, then 182.5 X 103 or 18.25 X 104
Second dilution
14 + 20 = 34
34/2 = 17, then 17 X 104

N = 18.25 + 17 = 35.25
35.25/2 = 17.62 X 104 or 1.8 X 105
Most probable number (MPN) or multiple tube method
This method is based on probability principle on the assumption that
the microbes are distributed evenly in and on food.
Only for specific microbes (e.g., E. coli) and cannot be used for total
count.

Method for ten tubes (ten by 1 ml or g)

• Add 1 ml of liquid sample or equivalent of 1 g homogenate to each of
ten tubes containing 10 ml medium.
• Incubate overnight at appropriate temperature.
• Observe for positive growth characteristics.
• Find the most probable number from the table.
Table of most probable numbers for 10-tubes
method
Number MPN 95% CI Number MPN 95% CI
of per 100 of per 100
+ tubes ml + tubes ml
0 0 0 – 37 6 92 30 – 211
1 10 0.25 – 59 7 120 43 – 270
2 22 3 – 81 8 160 59 – 368
3 36 7 – 106 9 230 81 – 600
4 51 13 – 134 10 >230 118 - >600
5 69 21 – 168
Epifluorescent
staining

MPN method
Standard plate count
Membrane filtration method
• Prepare serial dilution of sample of liquid food or homogenate
• Using membrane filter with pore sizes of 0.2 to 0.45 µm, filter the diluted sample
• Transfer the membrane onto plate of medium or absorbent pad saturated with
medium of choice and incubate at appropriate temperature
Dye reduction test for milk
Live microbes reduce (donate electrons to) methylene blue to
colorless leucomethylene blue and resazurin (blue) to resorufin
(pink) to dihydroresorufin (colorless) in milk.

However, because milk is usually refrigerated, it has been shown

that methylene blue reduction has little correlation with the number
of psychrotrophic microbes.

Methylene blue reduction has become unreliable and thus not

anymore widely used.

Since resazurin reduction occurs in two stages, it has wider change

in color that can be scored. The 10-minute test is still useful in
assessing the quality of raw milk.
10-minute resazurin reduction test
• One milliliter of resazurin solution (10 mg in 200 ml distilled
water) is added to 10 ml milk in screw-capped test tube and
mixed gently. Milk without resazurin serves as control
• Incubate for ten minutes in a water bath at 35OC.
• Compare the color of the milk with a comparator (Lovibond) as
follows

Resazurin disc No. Color Grade of milk

6 Blue Excellent
5 Light blue v. good
4 Purple Good
3 Purple pink Fair
2 Light pink Poor
1 Pink Bad
0 White Very bad
Immunological method for examination of microbes and toxins
Enzyme-linked immunosorbent assay (ELISA)
• Antibody (globulin) against a purified organism or toxin is raised from experimental
animals such as Guinea pig and rabbit.
• Antibody dissolved in buffer is coated on microtiter plate.
• Uncoated sites on microtiter plate are blocked using bovine serum albumin to avoid
nonspecific binding of antigen.
• Sample food extract in buffer, presumed to contain the target organism or toxin
(antigen), is added to the bound antibody. Excess antigen is washed away with
buffer
An enzyme (horse radish peroxidise or alkaline phosphatise)–labelled antiglobulin
(antibody against globulin) is added forming a sandwich (antibody–antigen–antibody).
Excess antiglobulin is washed with buffer.
A chromogenic substrate (substance that produce a color) such as 4-chloronaphthol
(for peroxidise) that binds to the antiglobulin is added. Excess chromogen is washed
away.
The light emitted by the chromogen is measured by special spectrophotometer.
Intensity of color is proportional to concentration of antigen.
Enzyme-linked immunosorbent assay
DNA methods
• Probe method
 DNA is isolated from food sample, cut (restricted with specific
enzymes and separated according to size by electrophoresis
 The double-stranded DNA is denatured and adsorbed to a
membrane and fixed with alkali treatment or heat.
 Blocking agent is added to prevent nonspecific binding of DNA
probe
 Biotin- or radioactive-labelled probe (short-chain DNA specific
for the target organism) is added to membrane and incubated.
Unabsorbed probe is washed away with buffer.
 The membrane is overlaid with x-ray film which record the
light emitted by biotin or radiolabel. Presence of exposed
sections of x-ray film indicate presence of the organism of
interest
DNA probe method
• Polymerase chain reaction (PCR) method
 DNA is isolated from food sample
 Sample DNA is loaded into PCR tubes containing activated nucleotides, DNA
polymerase, ligase and primers (forward and reverse).
Primers are short DNA sequences specific to the organism of
interest.
 The temperature is raised to 95OC to separate the double stranded DNA.
 The temperature is lowered to 50 to 65 OC to allow primers to bind (anneal)
to the single stranded DNA.
 Then, the temperature is raised to 72OC to allow the synthesis of the
complementary strands.
 The temperature is again raised to 95OC to separate the doubles strands of
DNA followed by lowering to 50 – 65OC and then raising to 72OC
 This cycle is repeated for more than 20 times to obtain sufficient copies of
the synthesized target DNA.
 The resulting DNAs are separated in an electrophoresis system and the
bands of different sizes viewed after staining.