Journal of Applied Microbiology 2005, 98, 1387–1399
A REVIEW
Microbial inactivation by new technologies of food
preservation
P. Mañas and R. Pagán
Tecnologı´a de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, Zaragoza, Spain
2004/0771: received 5 July 2004, revised and accepted 26 November 2004
1. Summary, 1387
2. Introduction, 1387
3. Mechanisms of inactivation, 1388
3.1 Inactivation targets and mode of action, 1388
3.2 Sublethal injury, 1390
3.3 Stress adaptation and resistance, 1391
4. Factors affecting microbial resistance, 1392
4.1 Process parameters, 1392
1. SUMMARY
The increasing consumer demand for
fresh-like foods has
led to much research effort in the last 20 years to develop
new mild methods for food preservation. Nonthermal
methods allow micro-organisms to be inactivated at suble-
thal temperatures thus better preserving the sensory,
nutritional and functional properties of foods. The aim of
this review is to provide an overview of the microbiological
aspects of the most relevant nonthermal technologies for
microbial inactivation currently under study, including
irradiation, high hydrostatic pressure, pulsed electric field
and ultrasound under pressure. Topics covered are the
mechanisms of inactivation, sensitivity of different microbial
groups and factors affecting it and kinetics of inactivation.
2. INTRODUCTION
Micro-organisms are the main agents responsible for food
spoilage and food poisoning and therefore food preservation
procedures are targeted towards them. Food preservation
methods currently used by the industry rely either on the
inhibition of microbial growth or on microbial inactivation.
Methods which prevent or slow down microbial growth
cannot completely assure food safety, as their efficacy
depends on the environmental conditions such as, for
Correspondence to: Pilar Mañas, Tecnologı́a de los Alimentos, Facultad de
Veterinaria, Universidad de Zaragoza, C/Miguel Servet, 177, 50013 Zaragoza,
Spain (e-mail: manas@unizar.es).
ª 2005 The Society for Applied Microbiology
doi:10.1111/j.1365-2672.2005.02561.
4.2 Microbial characteristics, 1393
4.3 Product parameters, 1394
5. Kinetics of inactivation, 1395
6. Concluding remarks, 1396
7. References, 1397
instance, the maintenance of the chill chain. Thermal
treatment is the most widely used procedure for microbial
inactivation in foods. However, heat causes unwanted side-
effects in the sensory, nutritional and functional properties
of food. This limitation together with increasing consumer
demand for fresh-like foods has promoted the development
of alternative methods for microbial inactivation, among
which ionizing irradiation, ultrasound under pressure, high
hydrostatic pressure (HHP) and pulsed electric field (PEF)
are attracting much interest.
The irradiation process involves the application of electro-
magnetic waves or electrons to foods. Radiation sources are
either gamma rays from cobalt-60, electron beams or X-rays,
and the amount of irradiation absorbed by a food is measured
in kGy (1 Gy ¼ 1 J kg)1). Commercial application of
ionizing radiation (IR) treatment on foods was started at the
beginning of the 1980s, but its success has been prevented by
consumer concerns. Nowadays, social perception of IR is
changing and this technology is being re-examined.
Ultrasound is defined as sound waves with frequencies
above the threshold for human hearing (>16 kHz).
Although ultrasound was initially discarded for food
preservation because of its weak lethal action, the application
of an external hydrostatic pressure of up to 600 kPa
[manosonication (MS)] increases substantially the lethality
of the treatment. In addition, a combination of MS with
temperature [manothermosonication (MTS)] has been pro-
posed (Raso et al. 1998a).
The HHP involved the application of pressures from 100
to 1000 MPa. The first studies on the lethal effect of HHP
1388 P . M A Ñ A S A N D R . P A G Á N
were conducted at the end of the 19th century, but it has
been in recent years when commercial applications of this
procedure have started. Finally, PEF technology consists in
the application of short duration (1–100 ls) high electric
field pulses (10–50 kV cm)1) to a food placed between two
electrodes. Like ultrasound under pressure, PEF technology
is not yet being used to preserve food commercially.
This review discusses the state of the art of the research of
the microbial aspects of the four technologies described
above.
3. MECHANISMS OF INACTIVATION
The successful implementation of a novel technology for
food preservation relies on the progress in the field of
mechanisms of inactivation. An adequate knowledge of the
physiological behaviour of micro-organisms towards inacti-
vation agents is essential for the development of safe foods.
It is necessary for understanding the effect of environmental
factors on resistance and also for identifying critical factors.
It would help to interpret kinetics of inactivation and to
develop mathematical models based on parameters with a
biological meaning and therefore more useful and able to
predict microbial inactivation in a wider range of conditions.
A better knowledge of the effect of the preservation agents
on the micro-organisms would lead to a more rational design
of processes.
3.1 Inactivation targets and mode of action
Cell death has been associated with either structural damage
or physiological dysfunctions. Among structural damage,
disruption of the envelopes, DNA conformational changes,
ribosome alterations or protein aggregation are the most
frequently described. Also physiological disorders, such as
membrane selective permeability alterations or loss of
function of key enzymes have been proposed as events
leading to cell death (Gould 1989). Perhaps the most
important difficulties that researchers encounter in this area
is that several of these lesions may occur simultaneously
when the cells are subjected to an agent and it is therefore
difficult to attribute the loss of viability of the cell to a single
event. Nevertheless, it must be kept in mind that also
multitarget inactivation is feasible, this being the addition of
several lesions that together cause death. It is also possible
that the key target is only affected when a secondary
structure is previously damaged.
For instance, heat causes membrane damage, loss of
nutrients and ions, ribosome aggregation, DNA strand
breaks, inactivation of essential enzymes, protein coagula-
tion, etc. (Gould 1989). In other words, almost every cellular
structure is somehow affected by elevated temperatures, and
it is very difficult to discern which events are leading to cell
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 1387–1399, doi:10.1111/j.1365-2672.2005.02561.x
Table 1 Events related to bacterial inactivation and resistance to
irradiation (IR), manosonication (MS), high hydrostatic pressure
(HHP) and pulsed electric field (PEF)
Novel method
Event IR MS HHP PEF
Occurrence of damage to
DNA +++ +
Envelopes + +++ ++ +++
Ribosomes ++
Other proteins + +*
Occurrence of sublethal injury +* ) +++ ++
Occurrence of stress adaptation +* ) +++ +*
*Observations based on few data.
Depending on micro-organism and treatment conditions.
death. In fact, this is only possible if a direct relationship
between the degree of inactivation and the degree of
modification of a given target under different environmental
conditions is found.
Regarding novel inactivation technologies, progress on
inactivation mechanisms research is heterogeneous. In this
way, sound hypotheses have been put forward to describe
the effect of IR and ultrasound on micro-organisms, but
much research is still needed to completely explain the way
PEF and HHP can inactivate bacterial cells. Table 1
summarizes the relevant events related to bacterial inacti-
vation by the four technologies.
It is well established that the critical target for irradiation
is the chromosome (Moseley 1989). The effects of ionizing
irradiation on bacterial cells are classified as direct and
indirect. Direct actions comprise the events caused by the
absorption of radiation energy by the target molecules,
whereas indirect actions are those derived from the inter-
action between the reactive species formed by the radiolysis
of water, such as the hydroxyl radical, and the target
molecules. 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. Bases
are also attacked by the free radicals generated by radiolysis,
but it is not clear whether this is relevant to cell death
(Moseley 1989).
The mechanism of inactivation of bacterial cells by
ultrasound under pressure has also been described. Most
authors agree that the cavitation phenomenon is responsible
for the lethal effects of ultrasound (Kinsloe et al. 1954; Raso
et al. 1998a). 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 (Suslick 1990). Free radicals could
therefore inactivate bacterial cells in a similar mode as that
described for IR. However, experimental data using free
radical scavengers have lead to the conclusion that the
possible effect of free radicals is negligible in comparison
with that of the strong mechanical effects generated by
cavitation (Allison et al. 1996; Raso et al. 1998a). Raso et al.
(1998a) studied the effect of equivalent heat, MS and MTS
treatments (99% of inactivation) on the degree of cell
disruption evaluated through phase contrast microscopy and
they observed that whereas heat-treated cells maintained full
cellular integrity, MS treated cells were completely broken.
MTS treated cells showed a medium degree of disruption.
These results confirmed that ultrasound inactivate microbial
cells through envelope breakdown in an
all or nothing type
phenomenon.
There are however some unclear aspects still not solved
regarding the mechanism of action of ultrasound. In some
experimental conditions, and with some bacterial species, a
synergistic effect of MS and heat (MTS) has been observed.
This is the case of Enterococcus faecium (Pagán et al. 1999c),
heat-shocked cells of Listeria monocytogenes (Pagán et al.
1999b) and cells suspended in a low water activity media
(Álvarez et al. 2003b). The reasons for the increased
sensitivity of these cells to a combined MS-heat treatment
are still not known. It has been suggested that moderately
elevated temperatures (55–60
C) would cause a weakening
effect on cell envelopes, facilitating the mechanical disrup-
tion of the cell by ultrasonic waves (Álvarez et al. 2003b).
This weakening effect would have no relevance for thermo-
sensitive cells, as they are killed by heat at relatively low
temperatures.
In addition, a synergistic effect of MS and heat has been
described for bacterial spores (Raso et al. 1998b). Ultrasonic
treatments cause the release of some low molecular weight
polypeptides and dipicolinic acid from the spore (Palacios
et al. 1991). It has also been found that MS treatments
sensitize spores of Bacillus subtilis to lysozyme (Raso et al.
1998b). Therefore, it has been suggested that ultrasonic
waves could damage the external layers of the spore,
facilitating its rehydration and consequently reducing its
extreme heat resistance.
In contrast to the clear mechanisms of inactivation
proposed for irradiation and ultrasound, a much more
complicated picture emerges for high hydrostatic pressure
inactivation. Much research has been carried out in this
matter, but there is still controversy about the key target
leading to cell death by high pressure.
Initial investigations on mechanism of inactivation of
HHP suggested the cytoplasmic membrane as the key target
(Cheftel 1995; Smelt 1998; Patterson 1999), and most
researchers still agree with that hypothesis despite some
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MICROBIAL INACTIVATION BY NEW METHODS 1389
apparent inconsistencies having been reported. Evidences of
bacterial membranes being a target for HHP inactivation are
clear. 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 (MacDonald
1993). Although 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 (Casadei et al. 2002). It is not clear how a
more fluid membrane renders a more resistant cell to HHP.
The pressure at which phase transition occurs would be
higher in cells with a more fluid membrane, but it is not
known in which circumstances, if any, cell damage is linked
to phase transition.
Damage to the cytoplasmic membrane after pressurization
has also been repeatedly reported, through loss of osmotic
responsiveness (Pagán and Mackey 2000; Mañas and
Mackey 2004), uptake of vital dyes (Shigehisa et al. 1991;
Benito et al. 1999; Pagán and Mackey 2000; Mañas and
Mackey 2004), loss of intracellular material, and formations
of buds and vesicles of lipidic origin (Perrier-Cornet et al.
1999; Ritz et al. 2000; Mañas and Mackey 2004). The loss of
function of some proteins including the F1–F0 ATPase or
multidrug efflux pumps has also been described (Smelt et al.
1994; Wouters et al. 1998; Molina-Hoppner et al. 2004).
Even a direct relationship between loss of membrane
integrity and loss of viability has been found for pressure-
treated exponentially growing cells (Pagán and Mackey
2000; Mañas and Mackey 2004). However, it has also been
demonstrated that both outer and cytoplasmic membrane
permeabilization is transient to some extent (Hauben et al.
1996; Pagán and Mackey 2000; Ganzle and Vogel 2001;
Mañas and Mackey 2004) and that pressure-treated station-
ary-phase cells of Escherichia coli may maintain a physically
intact cytoplasmic membrane upon decompression even in
dead cells (Pagán and Mackey 2000; Mañas and Mackey
2004). Therefore, other structures inside the cell have also
been proposed as potential key targets for inactivation by
HHP.
Some authors have reported similarities between cell
inactivation and protein denaturation kinetics by HHP
(Sonoike et al. 1992), and changes in the conformation of the
nucleoid, ribosomes and cytoplasmic protein have been
described (Mackey et al. 1994; Niven et al. 1999; Mañas and
Mackey 2004). Niven et al. (1999) found a direct relation-
ship between loss of viability in E. coli and ribosome
damage, evaluated by differential scanning calorimetry.
Further incubation of treated cells in a magnesium-rich
medium, which is known to have a stabilizing effect on
1390 P . M A Ñ A S A N D R . P A G Á N
ribosome structure, aided the ribosomes to recover the initial
conformation. The authors concluded that other factors
together with ribosome initial destabilization accounted for
cell death and suggested that the loss of essential ions like
magnesium through a damaged membrane could be the
event triggering ribosome destabilization. Moreover, Per-
rier-Cornet et al. (1999) correlated loss of viable cells of
yeasts of the genus Saccharomyces with the loss of internal
solutes caused by the pressure-induced cell permeabilization
during HHP treatment.
It seems clear that some of these cellular lesions like DNA
and protein condensation are not necessarily lethal (Mañas
and Mackey 2004) and are repairable if the cell keeps a
functional membrane and the environmental conditions are
suitable.
In conclusion, HHP inactivation seems to be multitarget in
nature. Membrane is a key target, but in some cases additional
damaging events 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.
Bacterial spores are extremely resistant to HHP, being
able to withstand up to 1000 MPa for long treatment times,
unless they are in the germinated state (Cheftel 1995).
Pressure itself at a moderate level induces spore germination
(Gould and Sale 1970). This has been the basis for the
design of a cyclic combined treatment in which spores are
induced to germinate in a first step and inactivated in a
second step, generally by a combination of mild heat and
pressure (Raso and Barbosa-Cánovas 2003).
Membrane structural or functional alteration is generally
accepted as the cause of cell death by PEF. Sale and
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, and
the most accepted theory to explain it is that proposed by
Zimmermann et al. (1974). Zimmermann compares the cell
membrane 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
electrocompression and pores are formed. The size and
amount of pores depend on the electric field strength and
the duration of the treatment. Alternative theories propose
that the permeabilization is the consequence of dipolar
reorientation of the membrane phospholipids under an
electric field (Tsong 1991). Tsong (1991) has also suggested
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 1387–1399, doi:10.1111/j.1365-2672.2005.02561.x
that the formation of hydrophilic pores would lead to a
localized Joule heating phenomenon that could be respon-
sible for the denaturation of proteins and phase changes in
the membrane.
Studies on electron microscopy of several bacteria and
yeasts have shown morphological alterations like surface
roughness, disruption of organelles, ruptures in the mem-
brane, etc. (Jayaram and Castle 1992; Pothakamury et al.
1997; Dutreux et al. 2000). However, no correspondence
between the frequencies of appearance of morphological
alterations with loss of viability has been proven (Aronsson
and Rönner 2001). Many attempts have been made to
establish whether there is a relationship between membrane
damage and microbial inactivation by PEF. Using vital
staining and detection of UV-absorbing material leakage
various researchers have shown the occurrence of membrane
permeabilization in relation to cell death (Hamilton and Sale
1967; Simpson et al. 1999; Wouters et al. 2001b). Wouters
et al. (2001b) have described a linear relationship between
the percentage of permeabilized cells and the intensity of the
PEF treatment, supporting the hypothesis of membrane
permeabilization being the cause of cell inactivation.
An interesting and almost nonexplored aspect of PEF
treatments is the occurrence of reversible pores. The same
way as it happens with high pressure (Pagán and Mackey
2000), a proportion of cell membranes could become leaky
during PEF treatment but reseal to a certain extent after it.
Experiments carried out in our laboratory using the
addition of propidium iodide to the treatment medium as
a marker of nonpermanent permeabilization of the cyto-
plasmic membrane, have shown that the degree of staining
of Salmonella serotype Senftenberg 775 W cells was
approximately twice as that observed when the propidium
iodide was added after PEF treatment. These results would
indicate that a percentage of cells were able to reseal their
pores just after PEF treatment.
3.2 Sublethal injury
Micro-organisms surviving the lethal action of preservation
agents may be sublethally injured: able to repair the damage
and outgrow only if the environmental conditions are
suitable (Mackey 2000). The occurrence of sublethal injury
has two main consequences. First, injured cells might not be
detected when selective conditions are used for enumeration
of survivors. This can lead to an overestimation of the
lethality of the treatment. Secondly, the occurrence of
sublethal injury means in practice that a delay time between
treatment and outgrowth of survivors takes place. Alternat-
ively, if repair is adequately prevented by the combination of
additional preservation agents (hurdles) that interfere with
cellular homeostasis maintenance, the cell might not be able
to outgrow, and the inactivation level attained might be

higher (Mackey 2000). Thus, detection and characterization
of sublethal injury by novel preservation technologies is
essential for the optimization of mild combined methods
with a higher lethal effect on microbes.
Among the four methods for microbial inactivation
reviewed here, only in the case of ultrasound under pressure
have no sublethally injured cells been detected. Survival
curves of MS treatments of Gram-positive and Gram-
negative cells recovered in media with sodium chloride
added are virtually identical to those recovered in a
nonselective medium (Pagán et al. 1999a). This indicates
the total absence of repairable membrane damage and is in
concordance with the hypothesis of the all or nothing lysis of
the cell as the mechanism of inactivation of ultrasonic waves
on bacterial cells.
Sublethal or repairable injury has been detected for
irradiated, pressurized and PEF-treated cells. For each agent
the mechanism of inactivation is different, and so is the
nature and the magnitude of the sublethal injury.
Studies on sublethal cell injury and repair of irradiated
cells have focused on DNA. It seems clear that the relative
sensitivity of the different microbial groups depends not only
on their susceptibility to the direct and indirect action of
irradiation itself, but also on their capability of repairing the
single and double strand breaks through several enzymatic
actions (Moseley 1989). Irradiation, however, does not
discriminate among molecules in a sample, and virtually all
the molecules in an irradiated cell may be affected. Irradiation
damage to other structures has been scarcely studied. Some
authors have reported the sensitization of irradiated cells to
selective media (Patterson 1989; Tarté et al. 1996; Buchanan
et al. 1999) but Kim and Thayer (1996) showed that
irradiation did not induce membrane damage as detected
by vital dye staining. It is not clear whether the secondary
hurdle added (salt, acid, CO2) would exert its inhibitory
action either towards the damaged DNA as suggested by Kim
and Thayer (1996) with heat, interfering with DNA repair
systems or at any other level.
Bacterial membranes are the main target for sublethal
injury in HHP treated cells. Permeabilization of the outer
and cytoplasmic membrane has been described (Pagán and
Mackey 2000; Ganzle and Vogel 2001) and combined
treatments of HHP and antimicrobial peptides in the
pressurizing medium, such as lysozyme, nisin, pediocin
AcH, lacticin, lactoferrin and lactoferricin with increased
efficacy for both Gram-positive and Gram-negative micro-
organisms have been proposed (Hauben et al. 1996; Kalc-
hayanand et al. 1998). However the permeabilization of the
outer membrane of Gram-negative cells is transient and a
fully functional membrane is recovered automatically shortly
after decompression (Chilton et al. 2001). On the contrary,
cytoplasmic membrane damage repair is highly demanding,
and requires energy, RNA and protein synthesis (Chilton
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MICROBIAL INACTIVATION BY NEW METHODS 1391
et al. 2001). An intact cytoplasmic membrane is essential for
the maintenance of the homeostasis under unfavourable
environmental conditions, and thus, from a practical point of
view, combinations of HHP with alkali, salt or acidic
environment also attain a higher lethal effect on micro-
organisms (Garcı́a-Graells et al. 1998; Pagán et al. 2001;
Wuytack et al. 2003; Sherry et al. 2004).
The occurrence of sublethal cell damage in PEF-treated
cells is a matter of controversy. Most authors have not
observed the occurrence of sublethal injury using the
selective medium plating technique (Simpson et al. 1999;
Dutreux et al. 2000; Ulmer et al. 2002; Wuytack et al. 2003)
so it was generally accepted that bacterial inactivation by
PEF was an all-or-nothing effect. However, recent results
obtained in our laboratory (Garcı́a et al. 2003) have shown
the occurrence of sublethal cytoplasmic membrane damage
in a large proportion of the population of Gram-negative
cells (‡99Æ9% of survivors) estimated by differential plating.
Unpublished results of our research group indicate that the
discrepancies in published data may arise from the fact that
the occurrence of sublethal membrane damage by PEF
treatments depends on the pH of the suspending medium
and on the bacterial species. In this way, several Gram-
negative bacteria showed a higher resistance to PEF at acidic
pH (when compared with neutral pH) that was correlated to
the capability to repair the cytoplasmic membrane, which
was extensively damaged. For Gram-positive cells the
opposite was true, the greater resistance to PEF, the
occurrence of membrane damage and the subsequent repair
ability was detected at neutral pH. At acidic pH the cells
became more sensitive and irreversibly damaged. It is
worthy of note that PEF-sublethally injured cells stored
under acidic conditions lost viability during storage time.
This means, from a practical point of view, that if adequate
post-treatment holding conditions are selected, the intensity
of treatments, both for HHP and PEF, could be diminished
without affecting the microbial quality of the product.
Alternatively, a higher degree of safety could be attained.
We have also detected the occurrence of sublethal damage
to the outer membrane in PEF-treated Psychrobacter immobilis
by differential plating techniques (P. Mañas, unpublished
data). The role of the outer membrane in PEF inactivation
needs further attention. These observations provide new
useful data that contribute to the understanding of mecha-
nisms of membrane electroporation in bacterial cells. They
also contribute to clarify the environmental circumstances
under which PEF might act synergistically with other hurdles
for food preservation. Further work is in progress.
3.3 Stress adaptation and resistance
It is well known that micro-organisms can develop adaptive
responses and resistances when exposed to sublethal stresses
1392 P . M A Ñ A S A N D R . P A G Á N
(Abee and Wouters 1999), which may have serious impli-
cations for food safety. In the last 15 years much research
interest has been directed towards the elucidation of
bacterial stress adaptation mechanisms and gene regulation
behind it. The modification of sigma factors (r) bound to
core RNA polymerase, conferring promoter specificity, is
possibly the most important regulatory mechanism in
bacterial cells (Abee and Wouters 1999). Sigma factor rs
regulates, in Gram-negative bacteria, the transcription of
more than 50 genes involved in resistance to osmotic, heat,
oxidative and acid stress, among others (Huisman et al.
1996). 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 (Dodd and
Aldsworth 2002). In Gram-positive bacteria (B. subtilis, L.
monocytogenes and Staphylococcus aureus) an alternative
sigma factor with equivalent physiological functions has
been described (sigB) (Abee and Wouters 1999; Hill et al.
2002). Therefore, it seems that a parallel mechanism for the
acquisition of multiple stress resistance exists in Gram-
positive and Gram-negative cells. The influence of the
activation of these regulatory networks on bacterial resist-
ance to novel preservation processes has not yet been
sufficiently studied. Nevertheless, 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 (Robey et al. 2001; Wemekamp-
Kamphuis et al. 2004). A range of morphological and
physiological changes dependent on RpoS expression has
been described for Salmonella and Escherichia cells that
might possibly account for the increase in pressure resist-
ance (Huisman et al. 1996). It can be foreseen that these
changes might also have an influence on bacterial resistance
to PEF, ultrasound and irradiation, but up to now no data
are available on the influence of the RpoS/sigB regulon on
the resistance to these technologies.
Sigma factor r32, encoded by the rpoH gene, controls the
heat shock response, which consists of a rapid and transient
overexpression of chaperons and proteases. The application
of sublethal HHP treatments promotes the expression of
several heat shock proteins (Welch et al. 1993), suggesting a
direct role of the heat shock response in HHP resistance. In
fact, it has been reported that a previous heat shock may
protect cells against HHP (Pagán and Mackey 2000; Aersten
et al. 2004). In addition, Aersten et al. (2004) have described
that the basal expression of several heat shock proteins like
dnaK, lon or clp is increased in pressure-resistant mutants
(Hauben et al. 1997). The influence of a sublethal heat shock
on PEF resistance has been scarcely investigated but
Evrendilek and Zhang (2003) have shown that survival
seems to be increased. Preliminary results obtained in our
laboratory suggest that the ability to recover from PEF
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 1387–1399, doi:10.1111/j.1365-2672.2005.02561.x
damage is also increased. More research is needed in this
field.
The application of a heat shock does not protect bacteria
to a subsequent MS treatment (Pagán et al. 1999b). This
indicates that the possible changes that heat shock may
induce in cell envelopes, if any, are not relevant to
ultrasound resistance.
Although scattered information is available about the
influence of other types of stresses on bacterial resistance to
novel preservation methods, from published data it can be
deduced that in most cases cross resistance is induced.
Palhano et al. (2004) have described how the exposure to
hydrogen peroxide, ethanol and cold-shock induces barore-
sistance in Saccharomyces cerevisie. Buchanan et al. (2004)
have reported the increase of irradiation resistance of E. coli
O157:H7 when cells were previously acid adapted. Likewise,
acid, cold and heat adaptation also seems to protect this
micro-organism to PEF treatments (Evrendilek and Zhang
2003).
In summary, adaptation of micro-organisms to adverse
environmental conditions during processing poses a risk that
should not be underestimated. Extensive research is still
needed to characterize the physiology and genetics of
microbial stress responses involved in survival to food
preservation processes.
4. FACTORS AFFECTING MICROBIAL
RESISTANCE
Microbial inactivation by irradiation, ultrasound under
pressure, HHP and PEF has been found to depend on
many factors. Effective comparison of data published in
literature is hampered by the diversity of equipments and
experimental conditions employed by the different authors.
Nevertheless, this section tries to give an overview on the
most relevant factors affecting resistance to novel technol-
ogies. The factors are classified into three groups: process
parameters, microbial characteristics and product para-
meters. A summary of the most important factors for each
technology is given in Table 2.
4.1 Process parameters
Some process parameters are intrinsic to each technology
and no general conclusions can be drawn. For instance, the
intensity of an irradiation treatment is given by the
irradiation dose absorbed, as the radiation energy is normally
fixed (van Gerwen et al. 1999). Critical inherent parameters
for ultrasound under pressure are treatment time, amplitude
of the ultrasonic waves and external pressure applied (Raso
et al. 1998a). Whereas HHP efficacy depends on treatment
time and pressure (Smelt et al. 2002), PEF lethality varies
with parameters such as electric field strength, pulse
 MICROBIAL INACTIVATION BY NEW METHODS 1393

Table 2 Factors affecting microbial inactivation by irradiation (IR), manosonication (MS), high hydrostatic pressure (HHP) and pulsed electric field
(PEF)

Factors IR MS HHP PEF

Process parameters Dose (kGy) Treatment time (min) Treatment time (min) Treatment time: number of
pulses · pulse width (ls)
Temperature Amplitude (60–150 lm) Pressure (100–600 MPa) Electric field strength (9–50 kV cm)1)
Pressure (0–300 KPa) Temperature Pulse specific energy (J ml)1)
Temperature Temperature
Microbial characteristics
Resistance V>S>Y&M> S > G+ > G) S > G+ > G), Y & M S > G+ > G) > Y & M
G+ > G)
Spore inactivation At high dose At high intensity Cyclic treatments Not possible
Intraspecies variation Medium Low Large Medium
Product parameters Oxygen Water activity Composition Composition
Composition Water activity Water activity
Water activity pH (recovery) pH (treatment/recovery)
Preservatives Preservatives
V, viruses; S, spores; Y & M, yeasts and moulds; G+, Gram-positive vegetative cells; G), Gram-negative cells.

characteristics and frequency, apart from treatment time

4.2 Microbial characteristics
(Wouters et al. 2001a).
There are, however, some process parameters that are Data on the resistance of representative micro-organisms to
common for the four technologies. This is the case for medium-intensity irradiation, ultrasound under pressure,
treatment temperature. As a general conclusion, as the HHP and PEF treatments are shown in Table 3. Maximum
temperature is raised, the lethality of the four technologies inactivation levels attained with each technology will depend
increases (Raso and Barbosa-Cánovas 2003). The lethal on factors such as equipment technical developments and
effects of irradiation on micro-organisms are more pro- food characteristics. We would like to point out that
nounced when the treatment is carried out at elevated comparison of data is hampered by the different equip-
temperatures. When irradiation takes place in the frozen ments, treatment media, strains, etc. Therefore, data in the
state the sensitivity of the micro-organisms in reduced by a table are for illustrative purposes.
factor of 2–5 (van Gerwen et al. 1999), and this has been As a general rule, bacterial spores are the most resistant
attributed to the reduced mobility of free radicals. The micro-organisms to physical stresses (Grahl and Markl 1996;
lethality of ultrasound under pressure treatments is almost Smelt 1998; van Gerwen et al. 1999; Pagán et al. 1999c;
not modified by an increase in temperature unless lethal Patterson 1999; Wouters et al. 2001a). Gram-positive bac-
temperatures are reached (MTS treatments), in which case teria are more resilient than Gram negatives, and this has
an additive lethal effect is generally attained although in been attributed to the greater rigidity of their envelopes.
some cases the total lethal effect has been found to be Resistance of yeasts and moulds is quite variable. In general,
synergistic (Pagán et al. 1999b,c; Álvarez et al. 2003b) (see they are more resistant to irradiation than nonsporulated
section 3.1). The effect of temperature on high pressure prokaryotic cells (Patterson and Loaharanu 2000). On the
inactivation is complex. Combinations of HHP with mild
treatment temperature lead to a higher lethal effect (Patter- Table 3 Lethality of irradiation (IR), manosonication (MS), high
son and Kilpatrick 1998; Alpas et al. 2000), and this has hydrostatic pressure (HHP) and pulsed electric field (PEF) treatments
been attributed to a higher degree of damage on proteins
(Sonoike et al. 1992). Also treatment temperatures below Microbial group IR* MS HHP PEF§
20–30
C inactivate cells faster (Casadei et al. 2002), and this Bacterial spores 0–1 0–1 0 0
effect has been proposed to be the result of the reduced Gram positives (vegetative cells) 1–4 1–3 <1–2 <1–3
fluidity of the membrane. Regarding the effect of treatment Gram negatives 3 to >9 4–6 1–7 3–4
temperature on PEF lethality, increases in temperature, both Yeasts and moulds 0–1 ND 2 to >8 3–5
at nonlethal and lethal values, improve the efficacy of the *Log cycles after 1Æ5 kGy.
treatment. This effect has been related to a higher fluidity of Log cycles after 5-min treatment at 117 lm and 200 KPa.
the membrane that would make cells more susceptible to Log cycles after 15-min treatment at 300 MPa.
pore formation (Jayaram and Castle 1992). §Log cycles after 200 ls treatment at 22 kV cm)1.

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 1387–1399, doi:10.1111/j.1365-2672.2005.02561.x
1394 P . M A Ñ A S A N D R . P A G Á N
contrary, yeasts are more sensitive to PEF than prokaryotic
cells, and this difference is specially evidenced at low electric
field strengths, as the minimal electric field strength
threshold needed to inactivate yeast is lower (Sale and
Hamilton 1967). Yeasts and moulds are considered to be
sensitive to HHP, but wide differences among genus have
been detected.
Regarding viruses inactivation, they are among the most
radiation resistant micro-organisms, and within the practical
limits irradiation is considered not to be effective in virus
elimination (Patterson and Loaharanu 2000). According to
Smelt (1998), the HHP resistance of viruses depends on
their structure, which is heterogeneous. Human rotavirus
and hepatitis A virus are inactivated to safe levels with
treatments of 300 MPa/2 min and 450 MPa/5 min,
respectively (Khadre and Yousef 2002; Kingsley et al.
2002). The inactivation of viruses by PEF has been scarcely
studied, but Khadre and Yousef (2002) reported no decrease
in infectious dose of human rotavirus after PEF treatments
of 20–29 kV cm)1 for 146 ls. No data are available on the
resistance of viruses to ultrasound.
In addition, cell size and shape seem to have a role in
resistance to physical stresses. The smaller the cell size, the
higher the resistance to ultrasound, HHP and PEF (Cheftel
1995; Pagán et al. 1999c; Heinz et al. 2001). Coccoid cells
tend to be more resilient than rod-shaped cells. Nevertheless
there are many exceptions to these general rules.
It should be noted that in some cases intraspecific
variation is of great importance. Benito et al. (1999)
reported differences in survival to HHP treatments of
more than 6 log cycles among natural isolates of E. coli
O157. Sherry et al. (2004) found between 1 and 2 log
cycles of difference in the fraction of surviving cells among
40 Salmonella strains subjected to irradiation and pressure
treatments. Intraspecies variability in resistance to PEF has
also been demonstrated with L. monocytogenes (Lado and
Yousef 2003) and Salmonella enterica (Álvarez et al.
2003b). Moreover, the isolation of pressure-resistant
strains has been reported (Hauben et al. 1997). These
data raise concern as an adequate target strain should
always be selected for designing safe processes. In
addition, the lack of relationship between the resistances
of a given strain to various stresses emphasizes the
necessity of carefully choosing the most appropriate strain
for each agent.
This wide variation of resistance among strains of the
same species has not been found for ultrasound under
pressure treatments. Salmonella strains that differed greatly
in their heat resistances were almost identical in their
resistance to MS treatments (Mañas et al. 2000).
Microbial resistance to different physical agents depends
not only on the intrinsic resistance of the micro-organisms
but also on their physiological state. It is well known that
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 1387–1399, doi:10.1111/j.1365-2672.2005.02561.x
bacterial heat resistance varies widely depending on the
growth phase, growth temperature and exposure to previous
stressing environments (Tomlins and Ordal 1976). Cells in
the exponential phase are generally more sensitive to all
types of agents (see section 3.3). Data about the influence of
growth temperature are scattered and in many cases
inconclusive. It seems to be an important factor determining
HHP resistance, but its effect is different in exponential and
stationary phase cells (Casadei et al, 2002).
4.3 Product parameters
Environmental factors, such as composition of the treatment
medium, pH, water activity or addition of preservative
substances, strongly affect the resistance of micro-organisms
to heat. The relative influence of such factors on microbial
resistance to novel technologies depends on their mecha-
nisms of action. Additional factors of special relevance in a
particular technology have been identified (see Table 2). For
instance, one of the most important factors influencing
irradiation sensitivity is the composition of the treatment
atmosphere. The presence of oxygen during irradiation has
been found to enhance lethal effect because of oxygen radical
formation (van Gerwen et al. 1999).
As a general rule, rich media exert a protective effect on
bacteria against physical agents. This has repeatedly repor-
ted for heat treatments and similar results have been found
for new technologies, except ultrasound under pressure,
where, as for the rest of factors studied, a very small
influence of medium composition has been described
(Mañas et al. 2000).
Water activity of the suspending medium is possibly the
environmental factor that modifies heat resistance in largest
magnitude. It has been reported that the survival of
Salmonella serovar Typhimurium to irradiation is increased
in low water activity meat (van Gerwen et al. 1999). Water
activity also affects resistance to ultrasound under pressure.
Listeria monocytogenes suspended in a medium with aw of
0Æ93 showed a decimal reduction time to a MS treatment
twofold greater as that obtained in a medium with aw 0Æ99
(Pagán et al. 1999a). The magnitude of this protective effect
is very small when compared with other technologies, but it
is worthy of note that a decrease in the aw of the treatment
medium is practically the unique environmental factor
capable of protecting bacteria against MS. Regarding HHP
inactivation, Molina-Hoppner et al. (2004) have suggested
that the protective effect of media of low aw in Lactococcus
lactis depends on the solute added. Sucrose protects cells
against HHP inactivation through stabilization of membrane
protein functionality. However, the addition of sodium
chloride exerts a physical effect, increasing the phase
transition of membrane phospholipids. Studies on the
influence of aw on PEF microbial inactivation are very

limited but all agree that reduced aw media increase PEF
tolerance (Aronsson and Rönner 2001; Álvarez et al. 2003c).
Álvarez et al. (2003c) have observed that aw decrease from
0Æ99 to 0Æ93 increased the PEF survival of Yersinia
enterocolitica by 3.5 log cycles after an 800 ls at
22 kV cm)1. The reasons for this protection are unknown.
The pH of the treatment medium is one of the factors
modifying bacterial resistance with a more obvious practical
side. The combination of acidification with heat has been
used for decades to reduce the intensity of sterilization
treatments for canned products, as acid pH decreases
thermal resistance of micro-organisms (Tomlins and Ordal
1976). On the contrary, it has been demonstrated that acid
pH has little effect on bacterial resistance to irradiation
(Buchanan et al. 2004). It does not affect ultrasound under
pressure resistance either (Pagán et al. 1999a). Regarding
HHP inactivation, from published results it seems that
bacterial cells are more sensitive to pressure in acidic media
(Alpas et al. 2000), but the magnitude of the effect is not
remarkable. On the contrary, the pH of the treatment
medium has a great influence on PEF resistance, but it
depends on the species studied (see section 3.2) (Wouters
et al. 2001a; Garcı́a et al. 2003).
5. KINETICS OF INACTIVATION
The application of any new technology in food preservation
requires a reliable model that accurately describes the
inactivation rate. The model should be able to establish
appropriate treatment conditions to achieve known levels of
microbial inactivation, allowing the production of stable and
safe foods. Models should be simple, and ideally, they
should be built on parameters based on the physiological
mechanism of inactivation (Smelt et al. 2002).
Thermal processing parameters have generally been
calculated through the first order kinetics model. This
model assumes that microbial inactivation results from the
chance of the lethal agent striking the key target. As a result,
a straight survival curve (plot of the logarithm of the fraction
of survival cells vs treatment time) is obtained, and decimal
reduction times (D values) and z values are used to calculate
treatment parameters.
However in the last 10 years the suitability of first order
kinetics for the modelling of heat inactivation, as well as for
novel technologies, is being reconsidered. This is because of
the frequent occurrence of deviations, such as shoulders and
tails (Humpheson et al. 1998; Benito et al. 1999; Raso et al.
2000; Wouters et al. 2001a,b; Smelt et al. 2002). Several
theories have been proposed to explain these deviations,
which may be different for spores and for vegetative cells.
Shoulders have been mainly attributed to the occurrence of
sublethal injury, multitarget inactivation, cell clumping or
activation phenomena for spores. Tails are considered to be
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 1387–1399, doi:10.1111/j.1365-2672.2005.02561.x
MICROBIAL INACTIVATION BY NEW METHODS 1395
the reflection of resistance heterogeneity within the popu-
lation, either inherent to the bacterial cells or acquired
during the treatment. They are commonly detected when
survival curves go beyond 4–5 log cycles (Smelt et al. 2002).
Typical survival curves of irradiation, ultrasound under
pressure, HHP and PEF are shown in Fig. 1. Note that
irradiation survival curves represent the log fraction of
survivors vs irradiation dose, instead of treatment time.
Shoulders have been reported for bacterial inactivation by
irradiation, and it has been attributed to repairable cellular
damage in the low dose range (Moseley 1989; van Gerwen
et al. 1999). Tails have also been detected, especially in
complex media such as fishmeal and crabmeat (Patterson and
Loaharanu 2000). First order kinetics inactivation is
normally described for ultrasound under pressure. This is
in agreement with the existence of a single key target and an
all-or-nothing type inactivation mechanism. It also fits with
the absence of adaptation phenomena and with the small
intra- and interspecies variation in resistance to MS. Survival
curves for HHP and PEF treatments show pronounced tails,
concave upwards in shape (Benito et al. 1999; Raso et al.
2000; Wouters et al. 2001a,b; Smelt et al. 2002). It is
noteworthy that HHP and PEF treatments are homogeneous
throughout the whole sample, and therefore every cell is
subjected to the same stress.
Several approaches have been proposed to explain the
reasons for the upward concavity of survival curves. Some
authors have considered that this kind of curves are biphasic
and reflect the first order inactivation of two distinct
microbial subpopulations, each one homogeneous in resist-
ance (Humpheson et al. 1998). A concave upwards curve
could also be justified by a continuous distribution of
resistance within the microbial population (Smelt et al.
2002).
A number of models have been developed to describe
nonlinear curves, such as the log-logistic model, the
Hülsheger model, and models based on the Weibull
distribution, among others (Smelt et al. 2002). These later
have been by far the most used by several laboratories to
describe nonlinear microbial inactivation by heat, PEF and
HHP. Models based on the Weibull distribution are
characterized by their simplicity, as only two parameters
determine the curve course, and also their versatility, as
they can accurately fit either straight, concave upwards or
concave downwards survival curves. Probably their most
important drawback is the lack of biological significance
of the parameters of the models, which limits their
application.
Nowadays there are considerable amounts of data on
bacterial inactivation by PEF that have been accurately
described through the Weibull model. A secondary model
that describes the variation of the kinetic parameters of the
Weibull model with the electric field strength has been
1396 P . M A Ñ A S A N D R . P A G Á N
(a) (b)
Log (Nt /N0)
Log (Nt /N0)
Dose (kGy)
(c) (d)
Log (Nt /N0)
Log (Nt /N0)
Time (min)
described for several micro-organisms (Álvarez et al. 2003a).
From this secondary model, a zPEF parameter, equivalent to
the z-value for thermal treatments, can be defined that
allows the comparison of relative microbial resistances at
different electric field strengths. Álvarez et al. (2003a) have
also developed a tertiary model that enables treatment
conditions necessary to achieve a certain level of inactivation
of the bacterial population to be determined.
On the contrary, microbial inactivation by HHP has been
studied since a long time; but very few kinetic studies have
been carried out. This is partly because of the fact that
adiabatic heating during compression interferes with HHP,
and under some experimental conditions the inactivation is
the result of the combined effect of pressure and tempera-
ture. Therefore data analysis becomes rather difficult.
Pressure rigs provided with temperature control devices
are available in some laboratories, and a few studies
published have shown the good fitting of the Weibull model
to HHP kinetics (Chen and Hoover 2003). Even secondary
models that allow the prediction of the lethality at different
pressures and temperatures have been proposed (Chen and
Hoover 2003). However data are scarce and a thorough
kinetics study should be carried out.
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 1387–1399, doi:10.1111/j.1365-2672.2005.02561.x
Time (min)
Fig. 1 Typical survival curves for irradiation
(a), manosonication (b), high hydrostatic
pressure (c) and pulsed electric field
Time (µ s) (d) treatments
As a summary, a number of nonlinear models have been
developed in the last years that can be used to adequately
describe inactivation curves, and their application is highly
simplified by current computer resources. We strongly
believe that more research in this field is needed to compile
kinetic data on the inactivation of spoilage and pathogenic
micro-organisms for each technology. In addition, basic
research to identify the causes and circumstances that favour
the appearance of nonlinearity should be carried out.
6. CONCLUDING REMARKS
Irradiation, ultrasound under pressure, HHP and PEF are
effective procedures to inactivate vegetative micro-organ-
isms in foods, but the high resilience of spores limits their
use as a sole method for food preservation. Therefore, these
novel technologies are finding applications as hurdles that
assure food safety through microbial inactivation in minim-
ally processed high quality products. To fully exploit their
potential, more research effort is needed to clarify mecha-
nisms of inactivation, especially for HHP and PEF, to better
understand the effect of environmental factors, and the
occurrence of stress adaptation and sublethal injury, aspects

of great relevance regarding food safety. Characterization of
resistant strains may be useful in mechanistic studies.
Identification and selection of target strains for each
technology is also necessary. Finally, development of
mathematical models based on physiological facts to estab-
lish treatment conditions is urgently needed. Ideally, models
should consider the lethal event or events leading to death,
the heterogeneity within the bacterial population and
possible phenomena of adaptation and damage.
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