Factors affecting microbial growth in Livestock products

Factors Affecting Microbial
Growth in Livestock Products
Dr Ravi Kant Agrawal, MVSc, PhD
Senior Scientist (Veterinary Microbiology)
Food Microbiology Laboratory
Division of Livestock Products Technology
ICAR-Indian Veterinary Research Institute
Izatnagar 243122 (UP) India

Factors affecting the growth and survival of micro-organisms in
foods

Intrinsic factors: Biological Structure
 Outer barriers against the invasion of MOs-Some foods have
biological structures that prevent microbial entry (e.g. the skin
of fruits and vegetables form a protective layer to invasion by
microorganisms; meat has fascia, skin and other membranes
that prevent microbial entry; Eggs have shell and inner
membranes that prevent yolk and egg white from infection.
 Inner parts of whole healthy tissues are sterile
 Damages during harvesting or processing (peeling, skinning,
chopping) expose tissues and increase microbial loads
throughout the product - Ground meat spoils faster than whole
meat cuts (grinding distributes surface microorganisms
throughout)
 Eggs are usually sterile inside but heavily contaminated on the
shell, crack in the shell allows microbes to enter
 Milk has no protective barrier

Intrinsic factors: Nutrients
 When a microbial cell is growing in a food, the nutrients supplied by the
food include: carbohydrates, proteins, lipids, minerals and vitamins.
 All foods contain these 5 major nutrients but nutrients vary greatly with the
type of food. e.g. meat is rich in proteins, lipids, minerals and vitamins but poor in
carbohydrates. Foods from plant sources are rich in carbohydrates but poor in proteins,
lipids, minerals and some vitamins.
 Microorganisms normally found in food vary greatly in nutrient
requirement with bacteria requiring the most followed by yeast and molds.
 Why is nutrition important? The hundreds of chemical compounds present
inside a living cell are formed from nutrients.
 Nutrition is the provision, to cells and organisms, of the materials necessary
to support life.
 Growth of any MO depends on a suitable physical environment, as well as
an available source of chemicals to use as nutrients (Nester et al. 2004).
 Macronutrients: Elements required in fairly large amounts e.g. carbon,
oxygen, hydrogen, nitrogen, phosphorus, potassium, sulfur, calcium, iron,
sodium, chlorine, magnesium and a few other elements.
 Micronutrients: Metals and organic compounds needed in very small
amounts e.g. Mn, Co, Zn, Cu, Ni and Mo.

 Microbes do have significant variances when it comes to the source,
chemical form and the amount they will need.
 Most organisms require CO2 for certain biosynthetic reactions.
 Some organisms require high concentration (5-10%) to grow well.
 H2O = 80-90%, thus a major nutrient.
 Growth ceases whenever an essential nutrient is exhausted, be it energy
source, nitrogen sources or growth factor.
Macronutrients:
Carbon: (C, 50% of dry weight)
 Source of carbon required for basic structures
 Autotrophs are able to build all of their cellular organic molecules from
carbon dioxide
 Source of cellular energy (ATP or related compounds) to drive metabolic
reactions
 Source of high energy electrons/H, reducing power, typically in form of
NADH/NADPH.

6
Microbial Growth requirements - Main Macronutrients
Nitrogen: (N, 12% of dry weight)
 Nitrogen mainly incorporated in proteins, nucleic acids
 Most Bacteria can use Ammonia -NH3 and many can also use nitrates - NO3-
 Although many biological components within living organisms contain N,
and N2 is the most abundant component of air, very few organisms can “fix”
or utilize N2 by converting it to NH3
 Nitrogen fixers can utilize atmospheric nitrogen (N2)
 N is often growth limiting as organisms must find source as ammonium ion
- NH4
+
for biosynthesis
 Photosynthetic organisms and many microbes can reduce NO3
-
to NH4
+

9
Micronutrients: Need very little amount but critical to cell
function. Often used as enzyme cofactors

11
Growth factors: Organic compounds, required in very small
amount and then only by some cells

Classification of organisms based on sources of C and energy
used
Carbon
Autotrophs: source is CO2
Heterotrophs: source is reduced organic compounds
Energy
Phototrophs: energy source is light
Chemotrophs: energy source if from redox reactions involving inorganic
and organic compounds (aerobic respiration, anaerobic respiration,
fermentation)
Electrons or hydrogen ions
Organotrophs: from organic compounds - most organisms
Lithotrophs: from inorganic compounds - rarer

Classification of organisms based on sources of C and
energy used

Effect of nutrients concentration on growth rate and total
yield
 At low sugar concentration
growth rate increases with
increased substrate
 Above threshold concentration
growth rate is constant and
independent of substrate
 At high concentration substrate
limits growth by limiting water
availability
 Substrate concentration limits
total yield at stationary phase

Responses of microbes to nutritional deficiency
 Extracellular molecules collect nutrients
o Siderophores, hemolysins collect iron
o extracellular enzymes break down polymers
 Cells enter Semi-starvation state:
o slower metabolism, smaller size.
 Sporulation and “resting cells”:
o cells have very low metabolic rate
o Some cells change shape, develop thick coat
o Endospores form within cells; very resistant.
o Spores are for survival, triggered by low nutrients

Intrinsic factors: Water activity (Aw)

Aw and microbial growth
 Free water in a food is necessary for microbial growth.
 It is needed to transport nutrients, remove waste materials, carry out
enzymatic reactions, synthesize cellular materials and take part in other
biochemical reactions.
 Each microbial species has an optimum, maximum and minimum Aw level
for growth.

Minimum water activity that supports growth of some
microorganisms
Microorganism Water activity
Clostridium botulinum,
Bacillus cereus,
Pseudmonas aeroginosa,
Salmonella spp.
0.95
0.95
0.95
0.95
Staphylococcus aureus (anaerobic),
Candida spp., Saccharomyces
0.90
Staphylococcus aureus (aerobic) 0.86
Penicillium spp. 0.82
Most spoilage yeast 0.88
Most spoilage molds 0.80
Osmotic yeast 0.70

 Growth of microorganisms is greatly affected by the level
of water activity (Aw) in the food.
 No growth of any microbe below aw = 0.60.
 Exceptions are: Halophilic bacteria (min. aw = 0.75 e.g.
Halobacter spp), Xerophilic molds (min. aw = 0.60 e.g.
Xeromyces bisporus) and Osmophilic yeasts (min. aw = 0.60
e.g. Zygosaccharomyces rouxii).
 Inhibition of growth occurs if the water activity for food is
lowered beyond an organism’s minimum level of water
activity that is necessary for growth.
 Microorganisms have varied minimum water activity
requirements that supports their growth in food.
 Microorganisms can be controlled by reducing the Aw of
food.
 Aw of foods can be reduced by removing water
(desorption) and increased by the adsorption of water.
 Aw can be reduced by adding solutes, ions, hydrophilic
colloids, freezing and drying.

Intrinsic factors: pH
 Foods can be grouped as high acid foods (pH below 4.6) and low acid food
(pH 4.6 and above).
Fruits, fruit juices, fermented foods
from fruits, vegetables, meat and
milk and salad dressings
(HIGH ACID FOODS)
Most vegetables, meat, fish, milk and
soups
LOW ACID FOODS

pH values of some food products
Food type Range of pH values
Beef 5.1 - 6.2
Chicken 6.2 – 6.4
Fish 6.6 - 6.8
Oyester 4.8 - 6.3
Milk 6.3 – 6.8
Cheese 4.9 - 5.9
Fruits < 4.5 (most < 3.5)
Vegetables 3.0 – 6.1

• pH has profound effect on the growth of microbial
cells.
• Each species has an optimum and a range of pH for
growth:
o Molds and yeasts - able to grow at lower pH
than bacteria.
o Gram negative bacteria are more sensitive to
low pH than Gram positive bacteria
o Most bacteria grow best at neutral or weakly
alkaline pH usually between 6.8 and 7.5.
o Some bacteria can grow within a narrow pH
range of 4.5 and 9.0, e.g. Salmonella
o Other microorganisms especially yeasts and
molds and some bacteria grow within a wide pH
range, e.g. molds grow between 1.5 to 9.0,
while yeasts grow between 2.0 and 8.5.
 However, ACID TOLERANT STRAINS (Pediococcus
acidilactici) can acquire resistance to lower pH
compared with other strains e.g. Salmonella.
 When pH is reduced below the lower limit, microbial
cells stop growing and lose viability.
 Information on the influence of pH on growth and
viability of microorganisms is important in developing
methods to prevent the growth of undesirable
microorganisms in food.

Microoganisms Min. pH value Opt. pH value Max. pH value
Gram +ve bacteria 4.0 7.0 8.5
Gram –ve bacteria 4.5 7.0 9.0
Molds 1.5 7.0 9.0-11.0
Yeasts 2.0 4.0- 6.0 8.5- 9.0
pH and Microbial Growth
pH – measure of [H+
]:- each organism has a pH range and a pH optimum
ACIDOPHILES - optimum in pH range 1-4
ALKALOPHILES - optimum in pH range 8.5-11
NEUTROPHILES - optimum in pH range 6-8

25
 The acidity or alkalinity of an environment can
greatly affect microbial growth.
 Most organisms grow best between pH 6 and 8,
but some organisms have evolved to grow best
at low or high pH.
Examples:
Acidophiles: Helicobacter pylori, Thiobacillus
thiooxidans, Lactic acid bacteria (pH 3.3 – 7.2)
and acetic acid bacteria (pH 2.8 – 4.3).
Alkaliphiles: Vibrio cholerae, Vibrio
parahaemolyticus (pH 4.8- 11.0) and Enterococcus
spp (pH 4.8- 10.6).
Fungi – 4-6
 The internal pH of a cell must stay relatively
close to neutral even though the external pH is
highly acidic or basic.
 Internal pH regulated by BUFFERS and near
neutral adjusted with ion pumps
MOST OF PATHOGENIC BACTERIA ARE
NEUTROPHILES

Increasing the acidity of foods either through fermentation or
the addition of weak acids could be used as a preservative
method.

Intrinsic factors: Redox potential, Oxygen and growth
 Redox potential (Eh) measures the oxidation-reduction
potential in a system whereby a substance is oxidized and the
other reduced, simultaneously.
Process involves:
 loss of electrons from a reduced state (oxidation)
 gain of electrons by an oxidized substance (reduction)
 electron donor itself gets oxidized and reduces oxidized
substance (reducing agent).
 electron recipient itself gets reduced and oxidizes reduced
substance (oxidizing agent).
 Redox potential is measured as units of millivolts (mV).
Oxidized range: + mV Reduced range: - mV

 This is the ratio of the total oxidizing (electron accepting)
power to the total reducing (electron donating) power of
a substance.
 Eh is a measurement of the ease by which a substance
gains or losses electrons.
 Eh is measured in millivolts (mV)
 The more oxidized substances, the higher the Eh; the
more reduced substances, the lower the Eh.
 Microorganisms that grow at:
 high Eh or +ve Eh (require oxygen) – Aerobes
 low Eh or –ve Eh (oxygen is toxic)- Anaerobes
 high and low Eh (+ve /-ve Eh) – Facultative anaerobes
 relative low Eh values – Micro-aerophilic
Oxidation- Reduction potential (O/R or Eh)

Redox potential in food
 Is influenced by its chemical composition, specific processing treatment
given and storage condition in relation to air.
 Fresh foods of plants and animal origin are in their reduced stage due to the
presence of reducing substances e.g. ascorbic acid, reducing sugars and the –
SH group of proteins.
 Once respiration of cells has been stopped, O2 will diffuse inside and change
the redox potential.
 Processes such as heating, can increase or decrease reducing compounds
and alter the Eh.
Redox potential of some foods

Redox potential and microbial growth
 On the basis of the growth in presence and absence of free oxygen,
microorganisms have been grouped as aerobes, anaerobes, facultative
anaerobes or microaerophiles.
 Growth of microorganisms and their ability to generate energy by the
specific metabolic reactions depend on the redox potential of foods.
 Presence or absence of oxygen and the Eh of food determine the growth
capability of a particular microbial group in a food and the specific metabolic
pathways used during growth.
 Aerobes-need free O2 for energy generation as the final electron acceptor
through aerobic respiration.
 Facultative anaerobes can generate energy if free O2 is available or they can
use bound O2 in compounds e.g. NO3 or SO3 as final electron acceptors
through anaerobic respiration.
 If O2 is not available then other compounds are used to accept the electron
through fermentation (anaerobic).
 Anaerobic and facultative anaerobes can only transfer electrons through
fermentation.
 Anaerobes such as obligate or strict cannot grow in the presence of even
small amount of O2 as they lack various enzymes to scavenge the toxic
oxygen free radicals.
 Aerobic species - molds, yeasts, Bacillus, Pseudomonas, Micrococcus
 Anaerobic species - lactic acid bacteria, Clostridium
 Facultative anaerobic species - Enterbacteriaceae

Technologies to control the redox potential of food in
order to control the growth of microorganisms

Intrinsic Factors-Antimicrobial constituents
Various foods have inherent antimicrobial substances that prevent (inhibit)
microbial growth/attack.
Antimicrobial substances
 Coumarins – fruits and vegetables
 Lysozyme – eggs
 Aldehydic and phenolic compounds – herbs and spices
 Allicin – garlic
 Polyphenols – green and black teas
 lactinin, lactoferrin and anti-coliform factors – in milk
 Lactoperoxidase e.g. Cow’s milk
 Conglutinin e.g. Cow’s milk

Extrinsic factors
Extrinsic factors important for microbial growth in a food include
the environmental conditions in which it is stored.
These include :
1.Temperature
2.Relative humidity
3.Gaseous Environment
The relative humidity and gaseous conditions affect of storage
influence the Aw and Eh of the food, respectively .

Extrinsic factors: Temperature
• Foods are exposed to different temperatures from time of production until
the time of consumption.
• Microbial growth is accomplished through enzymatic reactions which is
depended on temperature.
Remember psychrophiles, mesophiles and thermophiles
• Every 10o
C rise doubles the catalytic rate of enzyme and every 10o
C decrease
reduces it to half.

Temperature Classes of Organisms
Psychrophiles ( 0-20C)
 Cold temperature optima
 Most extreme representatives inhabit permanently cold environments
 These grow best at about 20o
C but also down to -10o
C in unfrozen media.
 Psychrophilic bacteria can cause food spoilage at low temperatures.
 Several of the microorganisms found in the soil and water belong to this group.
Mesophiles ( 20 – 45C)
 Midrange temperature optima
 Found in warm-blooded animals and in terrestrial and aquatic environments in
temperate and tropical latitudes.
 These organisms grow between 25o
C and 40o
C, with an optimum growth temperature
close to 37o
C
 Some such as Pseudomonas aeroginosa may grow at even lower temperatures
between 5-43o
C
 None of the mesophilic bacteria are able to grow below 5o
C or above 45o
C.
 Most pathogenic bacteria belong to this group.
Thermophiles ( 45- 80C)
 Growth temperature optima between 45ºC and 80ºC .
 Bacteria in this group are mainly spore formers and are of importance in the food
industry especially in processed foods.
Hyperthermophiles
Optima greater than 80°C
These organisms inhabit hot environments including boiling hot springs, as well as
undersea hydrothermal vents that can have temperatures in excess of 100ºC

Temperature optima of bacteria

Extrinsic factors: Relative Humidity
• Relative humidity and water are interrelated.
• Relative humidity is a measure of water activity of the gas phase.
• When food commodities have low Aw are stored in high relative humidity,
water transfers from gas phase into the food. This causes the otherwise
dormant spores of bacteria or fungi to germinate. Once they are actively
growing, they produce water as an end product of respiration. Hence they
increase the Aw of their own, this favors the growth of high Aw requiring
bacteria and increase in spoilage of food.

Extrinsic factors: Gases in atmosphere
 Oxygen influences the redox potential of microbial associations.
 Carbon dioxide regulates cell growth of some bacteria.
 If partial pressure of carbon dioxide increases over a critical
level, metabolic activity will be retarded.
 Retarding effect of CO2 increases with increase in
concentrations.
 CO2 is used in packaging of some food items in order to control
the growth of microorganisms.

42
Classification of organisms based on O2 utilization
HydrogenHydrogen
peroxideperoxide
SuperoxideSuperoxide

43
Classification of organisms based on O2 utilization
 Obligate (strict) aerobes require O2 in order to grow
 Obligate (strict) anaerobes cannot survive in O2
 Facultative anaerobes grow better in O2
 Aerotolerant organisms don’t care about O2
 Microaerophiles require low levels of O2

Oxygen and growth
Environment
Group Aerobic Anaerobic O2
Effect
Obligate Aerobe Growth No growth Required (utilized for
aerobic respiration)
Microaerophile
Growth if
level not
too high
No growth
Required but at levels
below 0.2 atm
Obligate Anaerobe No growth Growth Toxic
Facultative
(An)aerobe
Growth Growth
Not required for growth
but utilized when available
Aerotolerant
Anaerobe
Growth Growth Not required and not
utilized

46
Test for Oxygen Requirements of Microorganisms
Thioglycolate broth :
contains a reducing
agent and provides
aerobic and anaerobic
conditions
a) Aerobic
b) Anaerobic
c) Facultative
d) Microaerophile
e) Aerotolerant

Foods affected by various groups

Food preservation
 Food preservation is a process through which physical and /or
chemical agents are used to prevent microbial spoilage of food.
 Food preservation aims at treating food in a manner to prolong
its storage life
 In food preservation, efforts are made to destroy organisms in
the food or Increase the period taken by microorganism to
adapt to the food environment before they start to spoil the
food.

Food preservation principles
Two general principles are employed in food
preservation.
(1) Inhibition priciple
(2) Killing principle

(1) Inhibition principle
 In this principle, food preservation is achieved by inhibition of
growth and multiplication of microorganisms.
 Preservation of food by inhibition methods does not necessarily
imply the destruction of organisms.
 On removal of the inhibiting influence, the food will undergo
spoilage as the microorganism present will grow and multiply to
cause spoilage.
The inhibition principle can be achieved by any of the following
methods:
1. Reduction of water activity e.g. By drying and salting
2. Reduction in pH e.g. by fermentation and addition of acids.
3. Use of preservatives e.g. sodium benzoate
4. Use of low temperatures e.g. chilling or freezing
5. Smoking – which has a drying and preservative effect

Food preservation by lowering pH
 Many food products can be preserved by lowering pH
so that the growth of pathogenic bacteria and
spoilage is prevented.
 The lowering of pH can be achieved by addition of
acids and fermentation.
 Fermentation is the breakdown of carbohydrates
under anaerobic conditions into alcohol or lactic acid
and carbon dioxide.

Food preservation by lowering water activity

Preservation of food by addition of high content
of sugar

Food preservation by use of low
temperatures

Effect of low temperatures
 Low temperatures are used to retard chemical
reactions and actions of food enzymes and to slow
down or stop the growth and activity of
microorganisms in the food.
 A low enough temperature will prevent growth of
any microorganisms.
 Spores are not usually injured at all by freezing.
However, most parasites are killed by freezing.

(2) Killing principle
 In this principle, spoilage microorganisms are
destroyed (Killed) in the food, and the food is
protected against subsequent contamination by being
enclosed in an air tight container.

Methods employed to achieve the killing principle

Pasteurization
 Is the process of heat treatment at specific
temperatures and times.
 Pasteurization is aimed at destroying all pathogenic
microorganisms without affecting the nutritive value
of the food.
 Three methods of pasteurization
a. Low temperature long time (63o
C for 30 min)
b. High Temperature short time (72o
C for 15 seconds)
c. Flash method (80o
C for 1-2 seconds)

Sterilization
 Is the use of physical or chemical means to destroy all
microorganisms that are present in the food.
 Sterilization can be achieved by:
 Heating at high temperatures, e.g. 100-140o
C
 Irradiation: Irradiation kills bacteria, spores, and
insects as well as inactivates enzymes.

Applications
 In practice, often a combination of inhibition and
killing principles and the various methods are used
depending on the food type. e.g.
 use of pasteurization and chilling of milk
 lowering of water activity and low temperature
storage
 use of preservatives and low temperature etc.

Measuring Heat-Killing Efficiency
To develop standards for killing efficiency: specially
important for industrial settings to develop SOPs.
 D- value
 Z- value
 F-value

Decimal reduction Time (D-Value)
 The D-value is defined as the decimal (or decadal) decay (or
reduction) time: i.e. it is the time required, at a specified
temperature T, to reduce the microbial population being
considered by one logarithmic value, i.e. from 100% to 10% of the
initial value or it is the time required at any specified
temperature to destroy 90% of the spores or vegetative cells of
a given organism.
 D- value is numerically equal to the number of minutes required
for the survivor curve to trasverse one log cycle.
 The higher the temperature, the faster is the rate of
destruction and the shorter it takes to kill 90% of the cells.
 The larger the initial number of vegetative cells or spores, the
longer it will take to destroy 90 % of the cells at a given
temperature.
 For example, D-value for Clostridium sporogenes in a given food
at 120o
C is 1 minutes, at 115o
C is 4 minutes, at 110o
C is 10 minutes.

Kinetics of thermal reduction
#Bacteria
DT=
Δt
log N1-logN2
D is the time required for one log reduction (90% kill)
Can be calculated using:
Δt: total exposure time
N1: initial population
N2: population size after treatment
Time
106
105
104
103
100o
C
D100
1 log
T= applied Temperature

Example 1:
DT=
Δt
log N1-logN2
 Calculate the D value for a bacterial suspension of 109
cfu/ml
that was subjected to 85˚C for 15 minutes at which point its
density was reduced to 106
cfu/ml.
Δt: 15 minutes
N1: 109
cfu/ml
N2: 106
cfu/ml
T= 85˚ C
D85=
15
log 109
-log106
D85=
15
9- 6
D85= 5 minutes

Example 2:
DT=
Δt
log N1-logN2
 The D90 value for a bacterium is 2 minutes. If starting culture has
108
cfu/ml, how long should this suspension be kept at 90C
to kill the entire population?
Δt: ? minutes
N1: 108
cfu/ml
N2: 1 cfu/ml
T= 90˚ C
2=
Δt
log 108
-log100
2=
Δt
8- 0
Δt = 16 minutes

The D value: an index for sensitivity to thermal killing
Time
#Bacteria
• Which one is more sensitive to heat killing at
100˚C? Bacillus subtilis or E.coli?
• At 100C the time required to reduce Bacillus
population is longer than that required for E.coli
106
105
104
103
100o
C
DE.coli
DB.subtilis

The D value is temperature dependent
D value decreases as the temperature increases
ie. there is less time required to reduce the
population by one log at higher temperatures
Time
106
105
104
103
#Bacteria
120o
C 110o
C 100o
C
D110 D100D120

Z-value
 Z value: Is the number of degrees (0
C) the temperature has
to be increased in order to reduce the thermal death time,
tenfold under specified conditions.
 The z value is relatively constant and depends very little
upon the environment.
 The spore killing effect of a heat treatment can be
expressed as a function of temperature and the time the
material has been exposed to that heat.
 For spores of bacteria, the z - value used is 10o
C.
 For example, when it takes 1 min to kill 90% of the
remaining spores at 120o
C, it will take 10 min to obtain the
same effect at 110o
C, and it will take 100 min at 100o
C.

Kinetics of thermal reduction: the Z value
Dvalue(min)
Z value
increase in temperature required to
reduce D by 1/10 (one log reduction)
Temperature (T)
100
10
1
1 log
100 105 110 115 120
Z =10˚C
Z =
ΔT
log D1-logD2
ΔT: Temperature change
D1: initial D value
D2: secondary D value

The use of Z value
 Example:
 A food processing company produces canned meat. Prevention of
Clostridium botulinum spores from growing is important. The D121 for
botulinum spores is 0.2 minutes and the Z value is 10˚C. The company
wants to sterilize the canned food at 111˚C. what should be the length of
sterilization if they consider to kill 1012
spores per can content.
 Since every10˚C decrease in treatment causes 10-fold increase in D value
then:
 D111= D121x10 ie. D111= 0.2x10 = 2 minutes

 using,
D111=
Δt
log1012
-log100
2 =
Δt
12- 0
Δt= 2x12= 24 minutes
They should heat treat their product at 111˚C for 24 minutes.

F-value
 F-value: The time in minutes at a specific temperature
(usually 121.1°C or 250 °F) needed to kill a population of cells
or spores.
 The F-value express the time taken to expose food to the
same amount of heat required to destroy spores and
vegetative cells of a particular organism using different
temperatures.
 This means that one can obtain the same killing effect of
spores and /or vegetative cells at a lower temperature,
provided the time of exposure is longer.
 For example, food heated at 121.1o
C for 2 minutes will give a
value F=2. To get the same F-value of 2 using 111.1o
C, one
needs to heat the food for 20 min. Heating such a food at
111.1o
C for 2 minutes will give F value of 2/10 = 0.2.
 Thus, F-value shows the heat treatment given to a food
product to destroy bacteria.
 As far as spore killing is concerned, F=1 is equal to 1 min at
121.1o
C (or 10 min at 111.1o
C or 100 min at 101.1o
C).

Thanks
Acknowledgement: All the material/presentations available online on the
subject are duly acknowledged.
Disclaimer: The author bear no responsibility with regard to the source and
authenticity of the content.
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