Advanced Food Microbiology

Effect of pH, Acid and Low Temperature on

Microbial Growth: Mechanism and Application
on Food Product

Harsi D. Kusumaningrum
Department of Food Science and Technology
Faculty of Agricultural Engineering
Bogor Agricultural University

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 Hydrogen ion concentration (pH) and microbial growth

pH: hydrogen ion concentration,

relative acidity or alkalinity

pH range of a microorganism is
defined by a minimum value (at
the acidic end of the scale) and
a maximum value (at the basic
end of the scale).

Moving away from the pH

optimum in either direction slows
microbial growth.

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 A food may start with a pH which precludes bacterial
growth, but as a result of metabolism of other microbes
(e.g. yeasts or molds), pH shifts may occur and permit
bacterial growth
Organisms are in general more sensitive to changes in
the pHi than to changes in external pH, although
significant changes in either will lead to loss of viability

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 Effect of hydrogen ion concentration (pH)
on microbial growth

Increasingly acidic conditions can affect the

growth of microorganisms by:
interfering with the synthesis of cellular
components
inducing cell death as a result of damage
to the outer membrane
disruption of the cytoplasmic pH
homeostasis
subsequent damage to DNA and
enzymes

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Although microbes are sensitive to hydrogen ion concentration,
the acid type and concentration of the acid are both important.

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 Effect of strong acids on microbial growth

Strong acids, such as hydrochloric acid, have a much

lower pKa value than weak acids. At a pH between 3 and
6, strong acids will be dissociated
Strong non-permeant acids do not affect the pH of the
cytoplasm to the same extent as weak permeable acids
Relatively large changes in external pH are required for
effective preservation
In many cases, acidification that detrimental to the
sensory quality of foods is acceptable as the only means of
controlling microbial growth

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 Modes of action

Strong acids lower the external pH (pHo) but are not able
to permeate through the cell membrane
Exert antimicrobial effect by
denaturing enzymes present on the cell surface
lowering the cytoplasmic pH due to increased proton
permeability when the pH gradient is very large
usually, the catalytic properties of the enzymes are lost
and metabolism is halted.

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 Modes of action strong acids (cont’d)

Resulting in reduced growth rate and cause an

extension to the lag phase.
Growth may be limited by a reduction in the
activity of ion transport systems in which case
essential ions and nutrients will not be taken up

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 Effect of weak acid on the microbial growth

In solution, weak acids exist in a pH-dependent

equilibrium between the undissociated and dissociated
state

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 Effect of pH on the antimicrobial action of weak acid
preservatives

Minimum inhibitory concentrations to inactivate various

microorganisms may vary considerably
The pH of the environment and the dissociation
constant (pKa) of the weak acid determine the
proportion of the hydrophobic (undissociated) form in
the medium and thus the effectiveness of the weak acid
The strength of an acid is defined by its dissociation
constant (pKa). This is the pH value when the
dissociated and undissociated forms of the acid are in
equal amounts.

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 Weak organic acids

Inhibition of growth by weak acid preservatives due to a

number of actions including:
membrane disruption,
inhibition of essential metabolic reactions,
stress on intracellular pH homeostasis
the accumulation of toxic anions
interference with protein synthesis system or genetic
material

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 Modes of action of weak acid At low pH, acetic acid (pKa
4·75), sorbic acid (pKa 4·76)
or benzoic acid (pKa 4·19)
will exist substantially in the
undissociated state
(XCOOH;), a form in which
they are potent growth
inhibitors.
The undissociated acid,
being uncharged, readily
diffuses across the cell
membrane only to dissociate
in the higher pH
environment of the cytosol.
Such dissociation generates
protons and the acid anion
(XCOO- ).
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 The acid anion will tend to
accumulate intracellularly to
very high levels as being
charged, it cannot very
readily diffuse from the cell.
This high anion
accumulation may generate
an abnormally high turgor
pressure.
The proton release can
potentially acidify the
cytosol. This acidification, if
it occurs, will inhibit many
metabolic functions

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 Effect of weak acid on the microbial growth

Undissociated lipid-permeable weak acids can diffuse

freely through the cell membrane and ionize in the cell to
yield protons that acidify the interior of the cell; this fall
in pHi is a major cause of growth inhibition by weak
acids.

Changes in pHi also seem to be important in controlling

the cell cycle; rates of DNA and RNA synthesis appear to
increase with higher pHi within the normal physiological
range.

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 Effect of weak acid on the microbial growth
Weak acid preservatives may also affect the cell yield, ATP levels,
and the cells’ ability to maintain pH homeostasis, therefore
disrupting substrate transport and oxidative phosphorylation.
Generally, Gram-negative bacteria are more resistant to weak acid
preservatives than are Gram-positive bacteria; one reason is to be
found in the different structural and chemical composition of the
outer layers of the cells.

Gram negative Gram positive

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 Common food preservatives and their uses

Preservative Effective Uses

Concentration
Propionic acid 0.32% Antifungal agent in breads,
and propionates cake, Swiss cheeses
Sorbic acid and 0.2% Antifungal agent in cheeses,
sorbates jellies, syrups, cakes
Benzoic acid 0.1% Antifungal agent in margarine,
and benzoates cider, relishes, soft drinks
Lactic acid unknown Antimicrobial agent in cheeses,
buttermilk, yogurt and pickled
foods

The antimicrobial effects of weak organic acids at low pH have

often been attributed to intracellular acidification and anion
accumulation
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 Effect of pH on the antimicrobial action of weak acid preservatives

The sorbic acid: binds covalently with the sulphydryl groups of

the enzymes and inactivates this part of the enzyme.
It may also be that more than one target is involved and that
growth inhibition may be a result of the combined load on the
cell.

Recent studies: the undissociated form of sorbate (pKa = 4.74)

was 10 to 600 times greater at inhibiting microbial growth than
was the dissociated acid, but dissociated acids also possessed
antibacterial activity, which was a major factor in the growth
inhibition at pH 6 and above.

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 Effect of low temperature on the
microbial growth

Storage temperature is one of the most important

parameters regulating the activities of
microorganisms in food systems.
Sensitivity of cells to cold stress is dependent on
several factors including:
temperature
rate of cooling/freezing
culture medium
Reading:
microbial strain Chapter 16, Protection of Food with
Low Temperatures. In: Modern
duration of storage. Food Microbiology, 7th ed. 2005

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 Effect of low temperature on the microbial growth

Temperature values for microbial

growth have a minimum and
maximum range with an optimum
temperature for maximal growth.
The rate of growth at extremes of
temperature determines the
classification of an organism (e.g.,
psychrotroph, thermotroph).
The optimum growth temperature
determines its classification as a
thermophile, mesophile, or
psychrophile.

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 Physiological and structural adaptations related to temperature:
Psychrophiles produce enzymes with lower temperature optima.
They often denature at room temperatures.
Psychrophiles have higher unsaturated fatty acids in membrane
lipids, keeps membranes fluid at lower temperatures.

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 Effect of low temperature on the microbial growth

At temperatures below the optimum for growth can

cause a number of physiological and morphological
changes
Changes to metabolic products can occur as a result
of delays in enzyme activity.
Reduced temperature during growth can also lead to
metabolic imbalance and growth cessation due to
the sensitivity of some metabolic regulatory
processes

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 Effect of low temperature on the microbial growth

Incubation at low temperatures can also change the

lipid composition of microbial cells. Both bacteria
and yeasts have been reported to contain an
increasing proportion of unsaturated fatty acids as
the growth temperature decreases.
This increase in the proportion of unsaturated fatty
acids with decreasing temperature is believed to be
essential for membrane function at low
temperatures.

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 Effect of low temperature

Temperature can influence the response of microorganisms on:

directly:
• the growth rate,
• enzyme activity,
• cell composition,
• nutritional requirements,
indirectly:
solubility of solute molecules,
ion transport and diffusion,
osmotic effects on membranes,
surface tension
density
http://www.bact.wisc.edu/Microtextbook
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 Effect of low temperature

• Slows the rate of all chemical reactions, including those

catalyzed by enzymes
• Decreases the fluidity of the cell membrane.
• “Freezing” the membrane thus prevents much of
cellular metabolism by preventing enzymes in the
membrane from functioning properly.

http://www.bact.wisc.edu/Microtextbook
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 Effect of low temperature

Freezing a sample at or below –20 °C stops all microbial

growth.
Low temperature, even freezing, is not damaging to most
microorganisms and, when brought up to suitable
temperatures, the microbes will begin growing again.
In fact, microorganisms are well preserved in liquid
nitrogen (-196°C) and this is a common method of
preserving bacterial strains in research laboratories.

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 http://vetsci.co.uk/2011/01/20/surviving_sub_zero/

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 Modes of action of low-temperature stress
As the temperature decreases, the lag phase before growth
extends,the growth rate decreases, and the final cell
numbers may decrease.
During the lag phase at low temperature, many
physiological changes occur, including a decrease in the
saturation of fatty acids and inhibition of DNA, RNA, and
protein synthesis
Growth of microorganisms at temperatures below their
growth optimum can also cause a number of structural
changes

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 Terimakasih

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