MICROBIAL BEHAVIOUR AGAINST

NEWER METHODS OF FOOD

PROCESSING, ADAPTATION AND
RESISTANCE DEVELOPMENT
 INTRODUCTION

Food Processing is the conversion of raw materials or

ingredients to a final product.

Acc. To Connor (1988) food processing is that branch

of manufacturing that starts with raw animal, plant or
marine materials and transforms them into intermediate
foodstuffs or edible products through the application of
labor, machinery, energy and scientific knowledge.
CONTD

A processing unit operation aims at microbial

destruction to ascertain safety and stability of food.

Heat treatments are traditionally applied to pasteurize

or sterilize food, generally at the expense of its sensory
and nutritional qualities.

As consumers increasingly perceive fresh food as

healthier than heat treated food, the industry is now
seeking alternative technologies to maintain most of
the fresh attributes, safety and storage stability of food.
(Lado and Yousef, 2002)
THERMAL DESTRUCTION OF MICROORGANISMS

Heat is lethal to microorganisms, but each species has its own

particular heat tolerance.
During a thermal destruction process, such as pasteurization, the
rate of destruction is logarithmic, as is their rate of growth.
Thus bacteria subjected to heat are killed at a rate that is
proportional to the number of organisms present.
The process is dependent both on the temperature of exposure
and the time required at this temperature to accomplish to
desired rate of destruction.
 RESISTANCE OF MICROORGANISMS
The extent of the pasteurization treatment required is
determined by the heat resistance of the most heat-resistant
enzyme or microorganism in the food e.g. Mycobacterium
tuberculosis and Coxiella burnetti.
A thermal death curve for this process is:
 It is a logarithmic process, meaning that in a given time interval
and at a given temperature, the same percentage of the bacterial
population will be destroyed regardless of the population present.
For example, if the time required to destroy one log cycle or 90%
is known, and the desired thermal reduction has been decided (for
example, 12 log cycles), then the time required can be calculated.
If the number of microorganisms in the food increases, the heating
time required to process the product will also be increased to
bring the population down to an acceptable level.
The heat process for pasteurization is usually based on a 12 D
concept, or a 12 log cycle reduction in the numbers of this
organism.
Several parameters define the rate of thermal lethality:
D value (Decimal Reduction Time): It is a measure of the heat
resistance of a microorganism. It is the time in minutes at a given
temperature required to destroy 1 log cycle (90%) of the target
microorganism.
Z value: It reflects the temperature dependence of the reaction. It
is defined as the temperature change required to change the D value
by a factor of 10. In the illustration below the Z value is 10C.
 The sterilizing effect, which is also called lethality or death rate,
indicates the effect of a heat treatment, expressed as the number
of decimal reductions in the number of microorganisms.
F-value: It is the number of minutes required to kill a known
population of microorganisms in a given food under specified
conditions.
This F value is usually set at 12 D values to give a theoretical 12
log cycle reduction of the most heat-resistant species of
mesophilic spores in a can of food.
For example, if there were 10,000 spores of a species of spore in
a can of food and a 12 D process was given, the initial 10,000
spores (10 4 spores) would be reduced to a theoretical 10-8 living
spores per can.
(Potter, 2007)
 NEWER METHODS OF FOOD PROCESSING

These technologies are named according to the main

processing parameter leading to cell inactivation.

Process Range of Solid food Fluid food Approval

intensity
Ionizing 2-10 kGy Raw poultry Liquid In 41
radiation meat, raw eggs countries
red meat,
seafood,
spices
High 100-1000 Ham, Fruit juice, In Japan,
pressure MPa seafood guacamol north
processing e, America,
jam, salad Europe
dressing,
milk
 CONTD

Pulsed 5-55 kV/cm - Fruit juice, Limited

electric field liquid eggs approval in
the US
Ultraviolet 0.5-20 J/M2 Meat Orange juice Approval
radiation surface, pending in
(200-280nm) shell egg the US
surface
HPP and gamma radiation are more suitable than other
alternative technologies for application in solid foods.

The current design of PEF treatment chambers does not

allow processing of solid foods.

The shielding effect of solid particles restricts

applications of UV radiation and electron beams to the
treatment of food surfaces
 LETHALITY OF IRRADIATION (IR), MANOSONICATION (MS), HIGH
HYDROSTATIC PRESSURE (HHP) AND PULSED ELECTRIC FIELD (PEF)
TREATMENTS
 MICROBIAL BEHAVIOUR

a. Inactivation: structural damage, altered cell

functions
b. Resistance development
c. Stress adaptation
 HIGH PRESSURE PROCESSING

HHP is the technology by which a product is treated

at or above 100 MPa.

Microbial growth is retarded at pressures in the range

of 20-180 MPa; these pressures also inhibit protein
synthesis.

Loss of cell viability begins at approximately 180

MPa, and the rate of inactivation increases
exponentially as the pressure increases.
CONTD
The irreversible denaturation of proteins above 300 MPa
corresponds to the range of pressure necessary for the
inactivation of most vegetative cells and bacteriophages, i.e.
protein-coated viruses.

The formation of pores in spore coats during treatment at 50-300

MPa may indicate that HPP induces spore germination.

No germination was observed at higher pressures.

Structural and functional changes in microorganisms at various pressures
 MECHANISM OF INACTIVATION

Bacterial membranes being a target for HHP inactivation.

High pressure causes tighter packing of the acyl chains within the
phospholipid bilayer of membranes and promotes membrane
transition from liquid crystalline to gel phase, in a similar way as
a temperature downshift.
CONTD

Phase transition of membrane lipids is not necessarily lethal to

bacteria, it has been demonstrated that the composition and state
of the bacterial cell membrane prior to pressure treatment affect
bacterial resistance to HHP (Casadei et al. 2002).

Cells with a more fluid membrane, i.e. with a higher degree of

unsaturation are more barotolerant.

Damage to the cytoplasmic membrane after pressurization has

also been repeatedly reported, through loss of osmotic
responsiveness.
CONTD...

HHP inactivation seems to be multitarget in nature.

Membrane is a key target, but in some cases additional damaging
events occur such as :-
extensive solute loss during pressurization,
protein coagulation,
key enzyme inactivation and ribosome conformational changes,
together with impaired recovery mechanisms, seem also needed to
kill bacteria.
CONTD

Factors affecting rate of inactivation:

i.Strain of microorganism
ii.Pressure range
iii.Time
iv.Chilling or ambient conditions
A. STRAIN OF MICROORGANISM AND TIME:
B. PRESSURE RANGE
 PULSE ELECTRIC FIELD

PEF technology is based on a pulsing power delivered to the

product placed between a set of electrodes (Gongora-Nieto et al.,
2002).

Micro-organisms are destroyed by the application of short high-

voltage pulses between the set of electrodes, causing disruption
of microbial cell membranes.
FACTORS AFFECTING MICROBIAL INACTIVATION

i. Process parameters:- voltage applied, pulse duration etc.

ii. Type of medium in which the treatment was
performed
iii. Electrical conductivity of the medium
iv. Other stressing factors such as antimicrobials
 MECHANISM OF INACTIVATION

Two mechanisms have been proposed by which microorganisms are

inactivated:- electroporation and electrical breakdown of cells.
Membrane structural or functional alteration is generally accepted
as the cause of cell death by PEF.

Hamilton (1967) demonstrated that the inactivation was the result

of the direct effect of PEF on the membrane (electroporation) rather
than because of the temperature increase or the electrolysis
products.

Electroporation can be defined as the formation of pores in cells

and organelles.
CONTD..

The cell membrane has been compared to a capacitor.

Free charges tend to accumulate in the inner and outer surface of

the membrane generating a transmembrane potential of about 10
mV.

When an external electric field is applied, as in PEF treatment, a

higher amount of free charges of opposite charge accumulate at
both membrane surfaces, resulting in compression of the
membrane.

When the external electric field exceeds a critical value or

threshold, the membrane is unable to withstand the electro
compression and pores are formed.
CONTD..

Electroporation in protein channels and lipid domains results in

osmotic swelling of the cell and thus membrane weakening until
the cell bursts.

Bacteria and yeasts have shown morphological alterations like

surface roughness, disruption of organelles, ruptures in the
membrane, etc. (Dutreux et al. 2000).

Electric discharge in liquid media may generate small amounts of

microbicidal agents such as chlorine, free radicals and Hydrogen
peroxide that alter the DNA and cytoplasmic activity during the
treatment
 IRRADIATION
Irradiation preserves the food by the use of ionizing radiation (-rays, from
electrons and X-rays).
The effects of ionizing irradiation on bacterial cells are classified as direct
and indirect.

DIRECT ACTIONS INDIRECT ACTIONS

caused by the derived from the

absorption of radiation
energy by the target
molecules.
interaction between the
reactive species formed
by the radiolysis of
water, such as the
hydroxyl radical, and
the target molecules.
 MECHANISM OF ACTION
The hydroxyl radical OH is able to react with the sugar-
phosphate backbone of the DNA chain giving rise to the
elimination of hydrogen atoms from the sugar.

This causes the scission of the phosphate ester bonds and

subsequent appearance of single strand breaks.

Double strand breaks occur when two single strand breaks take
place in each chain of the double helix at a close distance.
CONTD..

Ultra-violet radiations:-

i. UV radiation cross-links aromatic amino acids at their carbon

carbon double bonds.

ii. The resulting denaturation of proteins contributes to membrane

depolarization and abnormal ionic flow .

iii. Irradiation with long-wave UV (320-400 nm) causes the

formation of hydroperoxide radicals in the membranes
unsaturated fatty acids, which induces changes in membrane
permeability.
 ULTRASOUND

Ultrasound waves have a frequency that is above 16 KHz and

cannot be detected by the human ear.
Mechanism:-
Cavitation phenomenon is responsible for the lethal effects of
ultrasound.

When bubbles implode under an intense ultrasonic field, very

high pressures and temperatures are generated, and consequently
strong mechanical forces and free radicals are formed.

Free radicals could therefore inactivate bacterial cells.

a. High frequency sound waves cause the formation of microscopic bubbles
in the fluid.

b. As the bubbles collapse, shock waves are created in the fluid.

c. Alternating high and low- pressure areas impinge upon microorganisms and
destroys them.
 STRESS ADAPTATION AND RESISTANCE

It depends upon:-
a.Type of organism
Bacterial spores are generally the most resistant to inimical
processes, followed by Gram-positive and Gram-negative bacteria.
This is due to the rigidity of the teichoic acids in the
peptidoglycan layer of the Gram-positive cell wall.

b. Size of organism
a.Bacteria of small size and coccoid in shape are generally more
resistant to HPP than the large rod-shaped ones.
Reduced cell surface area in contact with the environment may
limit cell leakage at a given treatment intensity, and thus minimize
the effect of the treatment.
CONTD..

c. Nature of cell membrane

High membrane fluidity increases the resistance to HPP and low

temperatures.
Membranes with relatively high fluidity are rich in unsaturated
fatty acids.
Pressure resistant cells also have low-diphosphatidylglycerol
content. A specific porin has been associated with increase in
pressure resistance .
The enhanced resistance of bacterial spores to physical stress,
solvation and ionization is linked to the protective effect of the
membranes and coat layers surrounding the core.
CONTD..

Adaptation of microorganisms to stress during processing

constitutes a potential hazard.

Sub-lethal stress induces the expression of cell repair systems .

For instance, exposure of cells to UV induces enzymatic

photorepair and expression of excision-repair genes that may
restore DNA integrity.

The production of high levels of shock proteins is typical of gene-

controlled resistance to stress and may contribute to the bacterial
stress hardening effect, i.e. the expression of cross-protection
mechanisms against multiple types of environmental stresses .
CONTD..

Reversible DNA supercoiling protects cells against osmotic

pressure.
Slow replication under sub-optimal growth conditions increases
the time allowed for cell repair activity and thus favors recovery
of sub-lethally injured microorganisms.
 SIGMA FACTOR
The modification of sigma factors (r) bound to core RNA polymerase, conferring
promoter specificity, is possibly the most important regulatory mechanism in bacterial
cells.

Sigma factor regulates, in Gram-negative bacteria, the transcription of more than 50

genes involved in resistance to osmotic, heat, oxidative and acid stress, among others.

The induction of this sigma factor occurs in response to starvation, generally when cells
enter the stationary phase of growth, and also when exponentially growing cells are
subjected to stresses other than starvation.

It is known that the higher resistance to HHP of stationary phase cells is partly the
result of the presence of the RpoS protein in E. coli and sigB in L. monocytogenes.
 BACTERIAL INACTIVATION AND RESISTANCE TO
IRRADIATION (IR), MANOSONICATION (MS), HIGH HYDROSTATIC
PRESSURE
(HHP) AND PULSED ELECTRIC FIELD (PEF)
 FACTORS AFFECTING MICROBIAL INACTIVATION BY IRRADIATION
(IR), MANOSONICATION (MS), HIGH HYDROSTATIC PRESSURE (HHP)
AND PULSED ELECTRIC FIELD
(PEF)
THANK YOU..