Chapter 4

~Microbial growth in foods~

Prepared by:
Ilyanie Hj. Yaacob
 Syllabus content

 Microbial growth characteristics.

 Factors that influence microbial growth in food.
~Microbial growth
characteristics~
 Growth & cell division

 Unicellular organisms growth occurs when the mother cell doubled

in size and duplicated its contents, and divides into two daughter
cells.
 Thus, microbial growth is defined not in terms of cell size but as the
increase in the number of cells, which occurs by cell division.
 Microbes cell division usually occurs by:

i) binary fission
ii) budding
 Growth & cell division
Binary fission:
 During binary fission, the single DNA
molecule replicates.
 Next, the cell membrane begins to
grow between the two DNA molecules.
 Once the bacterium just about doubles
its original size, the cell membrane
begins to pinch inward.
 The daughter cells become independent
when a septum (partition) grows
between them and separate.
 Growth & cell division
Budding:
 In budding process, an offspring grows
out of the body of the parent.
 It results in an identical 'mother' and
'daughter' cell. The offspring organism is
smaller than the parent.
 The buds grow into fully matured
individuals which eventually break away
from the parent organism.
 Budding occurs in yeast and a few
bacteria.
Differentiate binary
fission and budding.
 Phases of growth

 When microbes are introduced into a fresh and nutrient-rich

medium, the growth of microbes can be modeled with four different
phases:
1) lag phase
2) log/exponential phase
3) stationary phase
4) death phase
 Phases of growth

The lag phase:

 During lag phase, microbes adapt
themselves to growth conditions.
 The individuals are maturing but
not yet able to proliferate.
 They do not increase significantly
in number, but they grow in size.
 They synthesis RNA, enzymes and
other molecules.
 Phases of growth

 They produce large quantities of

energy (ATP).
 The length of this phase is
determined by the characteristics
of the bacterial species and
condition of media.
Define
PROLIFERATE.
 Phases of growth

The log/exponential phase.

 The organisms divide at their most
rapid rate – a regular, genetically
determined interval called the
generation time.
 The population of organisms
doubles in each generation time –
this growth is said to be
exponential/logarithmic.
 Phases of growth
 The generation time for most
bacteria is between 20 min to 20
hours.
 However, as the number of
organisms increases, nutrients are
used up, metabolic wastes
accumulate, living space become
limited, and aerobes suffer from
oxygen depletion.
 The organisms become less able to
generate energy and their growth
rate decreases.
 Phases of growth

The stationary phase.

 Stationary phase results from a
situation in which growth rate and
death rate are equal - the number
of new cells created is limited by
the growth factor and as a result
the rate of cell growth matches
the rate of cell death.
 Phases of growth

 The phase is represented by a

horizontal straight line.
 The medium now contain limited
amount of nutrients and may
contain toxic quantities of waste
materials.
 Phases of growth

Death phase.
 The medium become less and
less supportive for the cell
division.
 Cells lose their ability to divide
and thus die.
 Number of living organisms
decreases at a logarithmic rate.
 Phases of growth

 The shape of the growth curve is affected by factors:

a) temperature higher or lower than optimum

b) limitation of nutrients for the microorganism
c) presence of natural antimicrobial compound or disinfectants
Illustrate the growth curve of
two E.coli plates incubated at
room temperature and 37OC,
respectively.
 ~Factors influence
microbial growth in foods~
 Overview

 Microbes can exist in many environment because:

- They are easily dispersed

- They occupy little space
- They need small quantities of nutrients
- They are diverse in their nutritional requirements.
- They can adapt environmental changes
 The growth of microorganisms can be influenced by:

i) intrinsic factors
ii) extrinsic factors.
Define intrinsic factors.
Define extrinsic factors.
i) nutrients
 Nutrients

 Nutrients include carbohydrate, protein, lipid, minerals and vitamins.

 Microorganisms depend on nutrients for:

1) energy: food derived from carbohydrate can be used as energy

sources. Other carbon compounds that can be used are alcohol,
organic acids, amino acids, peptides etc.
2) growth: mainly derived from protein sources, nitrogen sources,
peptides, amino acids, urea, ammonia etc.
3) proper cell functioning: food consist of vitamins and minerals.
 Nutrients

 Microbes found in food vary greatly in nutrient requirement - different

microorganisms possess different enzyme systems which are specific in
breakdown of certain nutrient compounds.
 Microbial growth can be enhanced by enriching the growth medium
with specific nutrients, e.g. adding tryptophan for Salmonella typhi.
 Microbial growth can be retarded using growth inhibitors.
 Thus, creating specific nutrient media is a very useful tool both in
laboratory work and in industry for isolation and growth of certain
microorganisms.
ii) water activity
 Water activity

 Water activity is a measure of the availability of water for biological

functions - relates to water present in "bound" and “free” forms.
 Bound water is water that cannot be extracted easily, which is found in
the integral part of the living tissue. This type of water are vital to all
processes associated with the cell.
 Free water is water that can be extracted easily from foods by
squeezing or cutting or pressing, which exists around the tissue or cell.
This type of water is important for metabolism and survival of
microorganisms such as transport of nutrients, removal of waste
materials and carry out enzymatic reactions.
 Water activity

 Amount of water available for microorganims is expressed in terms of

water activity (aw).

aw = P/Po

P = the vapor pressure of water in the substance.

P₀ = the vapor pressure of pure water at the same temperature.

0.0  aw  1.0
 Water activity

 Example of food and the aw:

Food aw

Cereals, crackers, sugar, salt 0.1 – 0.2

Honey, chocolate < 0.6

Jam, jelly 0.6 – 0.85

Condensed milk 0.85 – 0.93

Tomato paste, fruit juice 0.93 – 0.98

 Water activity

 Each microbial species has an optimum, maximum and minimum a w

level for growth.

microorganism aw

Bacteria 0.9

Molds 0.6

Yeasts 0.85
Why does perishable
foods are easily
spoil?
 Water activity

 aw can be reduced by removing water and can be increased by

absorption of water - this is important in the control of microorganism.
 Preserving techniques are based on making water unavailable for the
microorganisms. As aw is lowered, the ability to grow will reduce, thus
shelf life of the food can be extended.
 Three main methods for making water unavailable:

1. Increasing the solute concentration: removing water or adding of

solutes (salt, sugar).
2. Addition of hydrophilic (water-binding) colloids (gels, pectins,
gums).
3. Bringing water to a solid phase (freezing).
 Water activity

 However, molds and yeast are more tolerant to low aw than the
bacteria - play an important role in spoilage of bread and dried foods.

 Most spoilage bacteria do not able to grow in aw below 0.9.

 The most xerophillic bacteria can only withstand is up to 0.65 whereas

for fungi is up to 0.6.

 If aw is reduced to 0.65 / 0.60 the food cannot spoil for at least one
year (if other parameters are also controlled).
iii) pH
 pH

 i) pH.
 Microbes have an optimum pH – the pH at which they grow best –
usually near neutral.

 Most microbes do not grow at a pH more than 1 pH unit above or

below their optimum pH.
 pH

 Example of microbes and their pH range:

Minimum pH Optimum pH Maximum pH

Bacteria 4.5 6.5-7.5 9.0
Molds 1.5 4.0-6.8 11.0
yeasts 1.5 4.5-6.5 8.5
 pH

 Food can be grouped as:

- low acid foods (pH > 4.6): e.g. meat, fish, milk, soups. For this
group, sterilization is needed to destroy pathogenic microorganisms
and their toxins.
- high acid foods (pH < 4.6): e.g. fruits, juices, salad dressings.
Pasteurization is sufficient for acid foods.
 pH ranges for major foods:

Fruits: 3.0 – 4.5 Vegetables : 4.6 – 6.6

Meat: 5.5 – 6.8 Milk : 6.0 – 6.8
 pH

 Microorgisms are affected by pH of foods, since they do not have

any mechanism to adjust their internal pH.
 pH also affects resistance of microorganisms to destruction during
heating, drying and other processes.
 pH of the food could change due to process done to food, e.g.:

- Pickling: Production of acids in foods by fermentation.

- Acetic-, citric-, lactic-, propionic-, benzoic-, and sorbic acids added
to foods for preservation (acidulants).
Molds are the
principal spoilage
agents of fruits.
State the reason.
Fruits are nutrient rich substrates but the pH of fruits does not favor
bacterial growth.
Yeast often initiate fruit spoilage as it grows faster than molds.
Then molds finish the job by degrading complex polysaccharides in cell
walls and rinds.
iv) redox potential
 Redox potential

 The redox potential is a measure of potential difference in a system/food.

 It can be generated by a couple reaction: (OILRIG)

1) Oxidation: Loss of electron by a substance (the substance is an

electron donor) or known as reducing agent.
2) Reduction: Gain of an electron by another substance (the substance is
an electron acceptor) or known as oxidizing agent.
 The redox potential is measured in electrical units of milivolts designated
as Eh.
 In the oxidized range, the redox potential is + mV.
 In the reduced range, the redox potential is -mV.
 Redox potential

 The redox potential of a food is influenced by:

- its chemical composition

- specific processing treatment given

- storage condition in relation to air (vacuum packed, liquid N2, C02

etc).
 Redox potential
 Fresh foods of plant and animal origin are in a reduced state due to
presence of reducing substances such as sugars, ascorbic acids and
sulfhydryl group of protein.
 Diffusion of oxygen into these fresh food substances can change the
redox potential of the food.
 Processing food such as heating can also alter the Eh (reduced or
oxidized).
 Food stored in air can have a wide Eh range (+Mv) than when stored
under vacuum.
 Redox potential
 Grow of all these microorganisms and their metabolic reactions are
extremely dependent on positive redox potential of food.
 The range of Eh at which different groups of microorganisms grow are:

Aerobes Between +500 and +300 mV

Anaerobes Between +100 and -250 mV

Facultative anaerobes Between +300 and -100 mV

v) Presence of Oxygen
 Presence of oxygen
 The presence or absence (and concentration) of oxygen in the
environment is important in the growth of microorganisms, as oxygen
is the essential elements for metabolic activity.
 Microorganisms can be grouped into categories based on their
requirement on intolerance to oxygen.
Presence of oxygen
 Presence of oxygen
 The presence or absence of oxygen and the Eh of food determine the
growth of a particular microbial group in foods.
 This is important in microbial spoilage of foods (e.g. putrefaction of
meat by Clostridium spp) and in desirable characteristics of fermented
foods (e.g. Penicillium spp. in blue cheese).
vi) osmotic pressure
 Osmotic pressure
 Microbes in hypoosmotic environments gain water and become turgid
– the rigid cell wall prevents cell from swelling and bursting.
 Microbes in hyperosmotic environments lose water and undergo
plasmolysis – the plasma membrane separates from the cell wall.
 As the solute concentration is higher, osmotic pressure is higher, thus
more inhibition to growth.
 Osmotic pressure
 Most microorganisms are not able to tolerate high osmotic pressure
except:
i) Halophiles: salt-loving organisms that require moderate to large
quantities of salt. Typically found in the ocean. e.g. Pseudomonas spp.,
Vibrio spp. (0.5% - 0.3% salt concentration).
ii) Osmophiles: microorganisms adapted to environments with high
osmotic pressures, such as high sugar concentrations. e.g.
Leuconostoc spp.
 Spoilage of food with higher sugar and salt content is usually caused
by osmophiles and halophiles respectively.
Differentiate
halophiles and
osmophiles.
vii) temperature
 Temperature
 The temperature range of each microbes is determined largely by the
temperatures at which its enzymes function - most bacteria can grow
over a 30oC temperature range.
 With respect of temperature, microorganisms can be divided into:
a) Psychrophiles: grow best at low
temperatures (<20°C).
b) Mesophiles: grow best at
moderate temperatures (20 - 40°C).
c) Thermophiles: grow best at high
temperatures (> 45/50°C).
d) Hyperthermophiles: grow best at
extremely high temperatures.
 Temperature
 Three different effects of temperature contribute to the death of
microbial cells:
1. Denaturation of proteins (enzymes) by heat.
2. Intoxication due to accelerated metabolic reactions.
3. Changes in essential lipids - melting points of the fats found in the
organisms and temperature ranges of death are related.
 Temperature
 However, each microorganisms have their own heat resistance value.
 Factors affecting heat resistance of microorganisms:

i) Type of microorganisms.
- heat resistance is related to the optimal growth temperature of the
microorganisms e.g. psychrophiles grow well in refrigeration
temperature, thus as temperature is increased, death occur rapidly.
- spores are more resistant than the vegetative cells.
- gram positive bacteria are more resistant than gram negative
bacteria.
- molds and yeast are not heat resistant. They can be killed at 70 – 80
o
C.
 Temperature
ii) Number of microorganisms.
- The higher the number of microorganisms, the higher is the degree
of heat resistance due to:
a) Higher production of protective substances / protein
extrcellular component.
b) More varieties of microorganisms with different heat resistant.

iii) Time and temperature.

- as time and temperature increase, the death rate increases.
 Temperature
iv) pH.
- Microorganisms are most resistant at their optimal pH.
- If pH is increased or decreased from the optimum, the heat
resistance value is reduced.
v) Water.
- High humidity (more water) will cause better heat penetration thus
less heat resistant. Denaturation of protein occur at faster rate and
microorganisms will be killed in shorter time.
- Less water will cause microorganism to be more heat resistant.
 Temperature
vi) Food ingredient / medium / substrate.
- A microorganism in food with high fat is more resistant due to fat
protection - long chain fatty acids provide better protection than the
short chain.
- Microorganisms with high proteins are more resistant - proteins are
colloids, which also provide better protection.
Which of these factors are:
intrinsic?
Extrinsic?