Food Engineering Reviews

https://doi.org/10.1007/s12393-020-09271-8

Bacterial Injury Induced by High Hydrostatic Pressure

Kazutaka Yamamoto1   · Xue Zhang1 · Takashi Inaoka1 · Kazuya Morimatsu2 · Keitarou Kimura1 · Yoshiko Nakaura1

Received: 3 August 2020 / Accepted: 9 November 2020

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Abstract
In food processing, high hydrostatic pressure (HHP) can inactivate microbes, and the inactivation is either lethal or
sublethal, depending on the intensity of HHP-induced stress. Inactivation of bacteria is a key to ensure food safety
by HHP food processing. This manuscript reviews HHP-induced injury of bacteria such as Escherichia coli, Listeria
monocytogenes, and (vegetative) Bacillus subtilis. The stress in the sublethal inactivation depends on HHP level,
holding time, bacterial species/strain, and other environmental factors. The sublethal inactivation induces injury of
bacteria, and the injured bacteria may recover under suitable conditions. The recovery behavior depends on nutrients
surrounding the bacteria and the storage temperature. In the detection of HHP-injured bacteria, detection media and
incubation temperature play important roles. Mechanisms involved in HHP-injured bacteria can be discussed from
several viewpoints including membrane damage, reactive oxygen species, HHP resistance, ribosomes, metabolome, and
colony-forming behavior. HHP-induced injury of molds, yeasts, parasites, and viruses has not been sufficiently studied.

Keywords  High hydrostatic pressure · Bacteria · Injury · Recovery · Mechanism · Detection

Introduction worldwide acceptance, and in the last three decades the

HHP-processed food industry became fully established
Employing high hydrostatic pressures (HHPs) of sev- with increased market share. Although research and
eral thousand atm/several hundred MPa was suggested development in HHP food processing have achieved major
as an opportunity to commercialize novel food products advances since the first commercialization [3], some HHP-
[1]. The first products commercialized in Japan in 1990 specific phenomena remain unclarified, particularly when
were jams and fruit desserts [2]. The technology gained compared with thermal processes which have been studied
intensively for a long time.
HHP food processing has attracted scientists and
* Kazutaka Yamamoto technologists to its characteristics: homogeneous and quasi-
kazutaka@affrc.go.jp spontaneous pressure transfer, suppressed chemical reactions
Xue Zhang leading to minimal losses in food components contributing
yukizhangxue@affrc.go.jp nutrition, flavor, and color in fresh products, inactivation of
Takashi Inaoka microbes and viruses, denaturation of proteins ,
tina2672@affrc.go.jp starch gelatinization, enhanced liquid impregnation and gas
Kazuya Morimatsu dispersion, and shucking of shellfish and crustaceans [4].
morimatsu@agr.ehime‑u.ac.jp HHP processing is often applied to inactivate pathogenic
Keitarou Kimura and/or spoilage bacteria, and HHP-induced bacterial injury
keitarou@affrc.go.jp and recovery have been reviewed [5-8]. This paper reviews
Yoshiko Nakaura HHP-induced injury and subsequent recovery of bacteria,
nakaura@affrc.go.jp introducing some recent reports on E. coli, L. monocytogenes,
1
Food Research Institute, National Agriculture and Food
B. subtilis, and lactic acid bacteria.
Research Organization, 2‑1‑12 Kannondai, Tsukuba,
Ibaraki 305‑8642, Japan
2
Graduate School of Agriculture, Ehime University, 3‑5‑7
Tarumi, Matsuyama, Ehime 790‑8566, Japan

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Factors Affecting HHP‑Induced Bacterial
Inactivation
Conventional heat processing inactivates microbes such
as bacteria and fungi as well as viruses. HHP process-
ing as a nonthermal process can also inactivate them, and
an HHP of 600 MPa for several minutes is often used in
commercial applications [3]. Factors affecting microbial
inactivation in HHP processing are protein denaturation,
membrane damage, oxidation stress, and others [9, 10].
HHP treatment at 600 MPa of microbes may disrupt criti-
cal cell activities and result in lethal inactivation.
HHP-induced damage to proteins and other cellular com-
ponents such as DNA, lipids, and polysaccharides may affect
directly or indirectly multiple cellular functions. It should be
noted that denaturation of proteins functioning as enzymes
and receptors by heat processing may differ from that by
HHP processing. Their macroscopic structures such as
micelles and gels should also be considered when discussing
their functions [11]. In general, proteins may be denatured
by 400 MPa and higher; meanwhile, their oligomeric struc-
tures may be disrupted by 200 MPa and higher, leading to the
dissociation of their monomers [12]. Heat stability is often
improved by HHPs at lower than 200 MPa [13].
In the study of physical events such as membrane damage
[14], the concept of molecular crowding will be indispensa-
ble to discuss biochemical reactions in cells under HHP [15].
Under molecular crowding conditions, biological macromol-
ecules exist at high concentrations of 0.3–0.4 g/ml, where
their activity coefficients drastically increase depending on
their size [16]. On the other hand, HHP increases inter/intra-
molecular and matrix densities and thus reduces molecular
mobility, leading to a reduction in the rate of chemical reac-
tions. The increased densities enhance hydration of the cel-
lular components for protein denaturation/dissociation [17],
phase transition of lipid [18], and disorder of noncanonical
DNA structures [15]. As well as oxidative stress [19], physi-
cal changes in proteins, lipids, DNA, and other molecules
should be considered for their effects on reducing microbial
cellular activities.
Although HHP-induced inactivation of microbes and viruses
as biological hazards, particularly bacteria, has a long history
[20], relevant knowledge has drastically advanced since HHP
food processing became commercially viable in 1990. Many
reports can be found especially on pathogenic bacteria and also
spoilage bacteria. In contrast, less information is available on
HHP effects on fungi including molds and yeasts which may
deteriorate food quality, since they are not generally relevant to
cases of acute food poisoning.
Microbial inactivation is in general affected by biological
species, cell morphology and status, surrounding matrix,
detection method and instrument, and others. Microbial
13
Food Engineering Reviews
resistance to HHP inactivation can in general be listed in
order of bacterial spores > Gram-positive bacteria > Gram-
negative bacteria, yeasts, and molds, and the trend is
similar to that of heat resistance [21]. In terms of bacterial
morphology, rod-shaped bacteria tend to show the lowest
resistance to HHP and spherical cocci tend to be the
most resistant [23]. In fact, the Gram-positive bacterium
Staphylococcus aureus shows a relatively high HHP
resistance [24]. In addition, bacterial cells in the stationary
phase are more resistant to HHP than those in logarithmic
growth phase [25]. HHP resistance depends on the species
and/or strain of foodborne pathogen and spoilage bacteria.
For instance, enterohemorrhagic Escherichia coli shows
strain-dependent HHP resistance [26]. In addition, food
matrix surrounding bacteria may also affect HHP resistance
[7]. Lipids, proteins, minerals, and saccharides may increase
the microbial resistance to HHP [27].
HHP‑Injured Bacteria
Bacteria can be inactivated by HHP treatment which damages
cell membrane, membrane proteins, enzymes, ribosomes,
and nucleoids [6, 9, 10]. Bacterial injury after HHP treatment
is considered to be a consequence of sublethal inactivation,
and further details of HHP-induced bacterial injury have to
be clarified [8] as well as those of injury induced by other
stresses. In general, a high stress may lethally inactivate a
healthy cell population, while a lower stress may only suble-
thally inactivate it [8]. Furthermore, applying a mild heat stress
to HHP-injured bacterial cells made them nondetectable [28],
implicating that HHP-injured cells were lethally inactivated by
mild heat stress. In addition, a mild HHP treatment at 400 MPa
retarded colony formation of bacteria and colony appearance
was much more heterogeneous than their untreated control
[29], suggesting the presence of heterogeneous populations
among the HHP-injured cells. Based on these observations, it
is speculated that sublethally inactivated bacterial cells might
be classified into slightly, moderately/lightly, and severely
injured populations (Fig. 1). It is suggested that slightly-injured
cells may recover relatively independent of the environment,
and moderately/lightly injured cells may either recover or fur-
ther injure depending on the severity of environmental stress,
while severely injured cells will remain inactive [30]. This
paper refers to lethal inactivation and sublethal inactivation as
processes leading healthy cells to inactive and injured cells,
respectively, while referring to recovery as a process to restore
injured cells into healthy cells. In addition, nondetectable cell
populations on nonselective plate media will be described as
an inactive population [31, 32]. However, nondetectable cells
might include viable but nonculturable (VBNC) cells [6, 33,
34]. After exposure to one or more environmental stresses,
Food Engineering Reviews

high

healthy cells
sublethal
inactivation injury recovery severe moderate slight recovery
injury / light injury
stress tolerance

injury
slightly-injured cells
live cells
injury recovery
death injured cells moderately- / lightly-injured cells

injury
lethal recovery
inactivation severely-injured cells
low

death death

dead cells

Fig. 1  Classification of bacterial injury [30]. Considering possible VBNC states, this paper describes “death” and “dead cells” as “(lethal) inacti-
vation” and “inactive cells,” respectively

bacteria may enter a VBNC state which can be detected by
flow cytometry [32, 34]. Therefore, VBNC cells will be
excluded in this work. Recovery is sometimes referred to as
“resuscitation” which implies recovering an organism after
its apparent inactivation. Resuscitation is applied to specific
recovery phenomena from the VBNC state [6, 33]. Literally,
resuscitation should be applied to organisms which have been
judged inactive by conventional methods. In addition, judg-
ing apparent death of organisms would largely depend on the
available technologies at the time. Accordingly, the expression
“resuscitation” is considered to be applicable to the recovery
of VBNC cells whose viability cannot easily be judged by con-
ventional methods. Consequently, resuscitation should not be
applied to slightly and moderately/lightly injured cells which
are apparently neither inactive nor VBNC. These speculations
must be verified by further studies. Although studies on bacte-
rial cells that can be lethally inactivated or lightly to severely
injured have been reported for simultaneous or subsequent
combinations of heat treatment and other treatments such as
ultrasound [35], exposure to an ampholytic surfactant [36], and
electron beam [37], HHP-induced injury and their recovery
and inactivation have not been fully studied.
The effect of HHP treatment on the inactivation of
Escherichia coli, a Gram-negative bacterium, has been
comprehensively studied intensively from the protein
denaturation, oxidative stress, and membrane damage
viewpoints [10]. Therefore, E. coli can be adopted as a model
bacterium to study its viability, injury, and recovery after
HHP treatments and subsequent storage. This knowledge can
be applied to the study of Gram-positive bacteria such as
Listeria monocytogenes and lactic acid bacteria. Since studies
on HHP-induced bacterial injury and recovery have been
previously reviewed [5-8], recent reports will be discussed
next in terms of recovery in systems that are nutrient poor
or rich.
Recovery After HHP‑Induced Injury
Recovery in Nutrient‑Poor System
Escherichia coli cells (approximately 8 ­log10 CFU/ml)
treated at 500–600 MPa and 25°C for 10 min in phosphate-
buffered saline (PBS) were nondetectable during storage at
4°C for 120 h. Thereafter, the number of viable cells was
dependent on the storage temperature, increasing to a plateau
value at 25°C and reaching again nondetectable level at 37°C
(Fig. 2). The authors reported that the plateau cell number
at 25°C was less than one log cycle lower than the inoculum
level, but they provided no explanation for the observation
[38]. In a related study, E. coli cells in PBS (approximately
9 ­log10 CFU/ml) treated with HHP (400 or 500 MPa, 25°C,

13

Fig. 2  Effect of HHP treatment (500–600  MPa, 25°C, 10  min) on the
recovery of Escherichia coli in phosphate-buffered saline [38]. HHP-
treated cells were stored at 4 or 37°C for 120 h and then stored at 25°C
10 min) were subsequently stored (Fig. 3) at 5–25°C [39].
While the number of viable cells after HHP treatment did
not increase at 5 and 10°C, the number of cells increased
after a lag phase during storage at 15°C and increased almost
immediately when stored at 25°C.
In both previous reports, washed cells were suspended in
PBS which solely contains minerals and thus does not support
cell growth. Therefore, inactive cells were the sole nutrient
source for the recovery of HHP-injured cells. To investigate
this cannibalism possibility, E. coli cells in PBS were heat-
inactivated and used as a sole nutrient source for healthy cells
[39]. The number of cells did not increase at 5 and 10°C but
increased at 15 and 25°C, indicating that heat-inactivated
Fig. 3  Effect of storage
(a)
temperature on HHP-treated
Escherichia coli cells in initial counts
viable cell counts (log CFU/ml)
phosphate-buffered saline [39];
a 400 MPa; b 500 MPa at 25°C
for 10 min
0 60 120
storage time (h)
13
Food Engineering Reviews
cells were cannibalized during storage at 15 and 25°C by
healthy cells (Fig. 4). Accordingly, it can be speculated that
HHP-injured E. coli cells in the both reports proliferate to a
plateau level slightly lower than the inoculum level based on
the possible cannibalism of HHP-inactivated cells.
Recovery in Nutrient‑Rich System
Psychrotrophic and psychrophilic Listeria monocytogenes,
a pathogenic strain of genus Listeria [40], was suspended
in nutrient-rich milk (approximately 7 ­log10 CFU/ml) and
subjected to HHP (550  MPa, 25°C, 5  min). As shown
in Fig.  5,  L. monocytogenes cells were nondetectable
immediately after HHP treatment and later recovered or
remained inactive depending on the subsequent storage
temperature at 37°C, 40°C, 45°C, and 50°C for shorter
and longer than 4 h, 3 h, 1 h, and 5 min, respectively [28].
Trypticase soy broth (TSB) was also used as a nutrient-
rich medium to suspend the bacterium (approximately
8.5 ­log10 CFU/ml) for HHP treatment (500 MPa, 25°C,
10 min). Immediately after the treatment, injured cells
were detected at approximately 2 l­og 10 CFU/ml on a
nonselective agar medium while were nondetectable
on a selective medium. Furthermore, the cells became
detectable on the selective medium at approximately 0.5
­log10 CFU/ml and 4–5 l­og10 CFU/ml after 1-day storage
at 0°C and at 5–15°C, respectively [41]. This suggests
that conditions for HHP processing and subsequent storage
should be optimized when inactivating psychrotrophic
and/or psychrophilic bacteria in nutrient-rich foods.
Carrot juice (pH 6.0–6.7) and beetroot juice (pH 4.0–4.2)
inoculated with L. innocua or E. coli were HHP-processed
(300–500  MPa) and then observed during long-term
storage at 5 or 25°C, showing that carrot juice can support
the growth and regeneration of HHP-injured L. innocua
[42].
(b)
initial counts
CFU/ml)
ml)
U/m
g CF
viable
vi c counts
ll c
cell
iable ce oun ( og
ountts (log
(l C
180 240 300 360 0 60 120 180 240 300 360
storage time (h)
Food Engineering Reviews
level of dead cells
viable cell counts (log CFU/ml)
storage time (h)
Fig. 4  Effect of storage temperature on healthy cells of Escherichia
coli (2 ­log10 CFU/ml) in phosphate-buffered saline containing heat-
inactivated cells (9 l­og10 CFU/ml) [39]
Detection of HHP‑Injured Bacteria
Detection Media
Bacterial injury is often evaluated by differential plating
method as the difference between viable cell counts on non-
selective and selective media [43]. Injured bacteria do not
form colonies on selective medium imposing high stress on
them, while nutrient-rich nonselective media with relatively
low stress allow injured cells to form colonies [44].
HHP-treated cells after a storage at 25 °C
㸸 dead (non-detected for 28 days)
㸸 recovered
㸸 1 or 2 recovered among 3 samples
Fig. 5  Inactive and viable cells after mild heating of HHP-treated
(550 MPa, 25°C, 5 min) Listeria monocytogenes in milk [28]
HHP-treated L. monocytogenes cells in PBS (500 MPa,
25°C, 10 min) were nondetectable on selective medium but
detectable at approximately 2 l­og10 CFU/ml on nonselective
medium, indicating that the survivors were not healthy but
an injured population [41]. Performance of selective media
may affect the evaluation of HHP-injured bacteria. Lactic acid
bacteria (Leuconostoc mesenteroides, Enterococcus faecalis,
and Lactobacillus fermentum) were separately HHP-treated in
PBS (400 or 600 MPa, 25°C, 10 min) and plated on selective
media specific to lactic acid bacteria. After incubation at 25
or 30°C under aerobic or anaerobic condition on the tested
media, each strain of HHP-treated bacteria showed different
colony-forming abilities and the difference between the lowest
and highest cell counts observed was to be approximately 3–7
log cycles [45].
Salmonella and Listeria monocytogenes in ground chicken
meat with carvacrol, an essential oil component, were treated
with HHP (350 MPa, 10 min) and stored at 4°C or 10°C,
showing recovery of HHP-injured bacterial cells as a differ-
ence in the viable colony counts on selective and nonselective
media [46], demonstrating that the use of selective media may
overestimate the efficacy of bacterial inactivation by HHP.
Incubation Temperature for Detection
When using the plate count method to assess HHP treatments,
the incubation temperature of the plates for the enumeration of
HHP-treated cells is crucial. Higher incubation temperatures
may result in lower detection efficacy of HHP-treated bacteria
such as E. coli (Fig. 2) [38] and L. monocytogenes (Fig. 5)
[28]. Furthermore, recovery of HHP-injured cells of E.
coli can be promoted at an incubation temperature of 25°C
[29, 38] as compared with the conventional incubation tem-
peratures of 30–37°C, employed to detect bacteria by plate
count methods. For instance, E. coli cells after treatment at
400, 500, or 600 MPa, 25°C for 10 min were detected by 1 log
cycle more on LB agar plate after 3-day incubation at 25°C
than at 37°C (Fig. 6). Conventional incubation temperatures
for bacteria enumeration may underestimate injured cells
including HHP-injured cells. In addition, pour plate methods
using relatively hot agar may inactivate injured cells during
agar pouring.
Characteristics of HHP‑Injured Bacteria
Membrane Damage
Membrane damage is one of the mechanisms for injury
of HHP-treated bacteria [47] and can be detected by flow
cytometry where two fluorescent dyes of different membrane
permeabilities are used to stain healthy and inactive cells in
different manners. For instance, flow cytometric analysis of
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 Food Engineering Reviews

Fig. 6  Effect of incubation
temperature (a 37°C; b 25°C)
and 0.2%w/w pyruvate addi-
tion (+ P) on the number of
detected cells of HHP-treated
(400–600 MPa, 25°C, 5 min)
Escherichia coli in phosphate-
buffered saline [29]

HHP-injured E. coli cells using fluorescent dyes of propidium
iodide and SYTO9 [29] demonstrated a staining pattern differ-
ent from that of healthy or inactive cells (Fig. 7). Propidium
iodide only stains membrane-damaged cells, while SYTO9
stains all cells. The staining pattern of healthy cells was not
observed immediately after HHP treatment but became vis-
ible after recovery.
Membrane permeabilization has been postulated as a
major factor in HHP-induced inactivation of microbes.
However, it is noteworthy that the permeabilization was not
necessarily required for HHP-induced lethal inactivation of
Lactobacillus plantarum in the study where the effect of
Tween® 80 on HHP tolerance was evaluated in terms of cell
viability, injury, metabolic activity, protein release, and pro-
pidium iodide uptake [22].
Reactive Oxygen Species (ROS)
Colony-forming ability of bacteria can be lost due to injury
via environmental stresses such as chemicals, starvation,
and extreme temperatures, and the recovery can be pro-
moted by ROS scavengers such as pyruvate, catalase, and
α-ketoglutarate [48-52]. For example, the number of E. coli
cells after HHP treatment was higher on pyruvate-added agar
plates than on control pyruvate-free plates and even higher
when incubated under anaerobic conditions [29]. LB agar
plates containing 0.2% sodium pyruvate detected injured
cells at levels several log cycles higher than pyruvate-free
plate after 400 MPa or 500 MPa treatment. Furthermore,
pyruvate-added plates incubated at 25°C enabled detection

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Food Engineering Reviews
(a)
dead
(c)
Fig. 7  Flow cytometric analysis of HHP-treated (400–600  MPa,
25°C, 5  min) Escherichia coli in phosphate-buffered saline [29];
a heat-inactivated cells (90°C, 30  min); b healthy cells; c immedi-
ately after HHP treated; d HHP-treated and stored at 25°C for 48 h.
of the cells which were nondetectable after 600 MPa treatment
on pyruvate-free plates at 25 or 37°C (Fig. 6). In addition,
the sample matrix supplemented with 0.2% sodium pyruvate
mitigated the lethal effect of HHP to the cells, indicating a
suppressive function of pyruvate on HHP inactivation. ROS
scavengers in combination with incubation temperature of
25°C may improve the detection sensitivity of agar plates for
counting HHP-injured cells. However, since some of them
(b)
live
(d)
Injured cells were detected at the zones “a” and “b” which overlapped
the inactive (dead) and healthy cell zones, respectively. After stor-
age, injured cells were observed at the zone “c” which overlapped the
zone of healthy cells
are vulnerable to autoclaving, they may have to be added after
filter sterilization.
Mutants of E. coli lacking catalase or superoxide dismutase
as ROS scavengers are significantly more sensitive to HHP
than the wild type, indicating an inactivation mechanism
involving oxidative stress [19]. In contrast, HHP-treated veg-
etative cells of Bacillus subtilis mutants lacking superoxide
dismutase were slightly more sensitive to HHP than the wild
13

type, while those cells lacking catalases were not significantly
inactivated by HHP, indicating no effect of ­H2O2 on the HHP-
induced inactivation of vegetative cells of B. subtilis [53].
Acquired HHP Resistance
Cold shock proteins, heat shock proteins, and chaperons
can be induced by culturing E. coli under medium HHPs of
100 MPa and lower. These proteins play important roles in
cell proliferation under sublethal pressure conditions [54].
Pressure resistance of E. coli can be promoted by inducing
heat shock protein at 150 MPa [55]. On the other hand, it is
presumed that induction of pressure resistance would be neg-
ligible under commercial HHP food processing conditions due
to limited gene expressions within the short time of pressure
come-up time to 600 MPa, the world-wide adopted pressure
level used in commercial applications.
Ribosomes
Effect of HHP on ribosome dissociation was studied using
vegetative cells of sporulation-deficient mutant Bacillus
subtilis [56], revealing that the growth of the cells was
arrested in LB liquid medium after an HHP treatment
(250 MPa, 25°C, 10 min), although their viable cell counts
on LB agar plate were comparable to those of untreated
control cells. During the growth arrest phase, substantial
numbers of ribosomal genes and translation-related genes
(e.g., translation initiation factors) were upregulated,
the dissociated ribosomes were reconstructed, and the
reconstruction was delayed by ­Zn2+ or ­Mn2+ [56]. Free
ribosomal subunits (30S and 50S) are more susceptible
to degradation by RNase(s) than 70S ribosomes [57]. In
addition, 16S and 23S RNAs in the subunits in starved E. coli
cells can be degraded by endonucleases into nucleotides [58].
Therefore, the ribosome dissociation from polysomes via
70S-monosomes into 30S- and 50S-subunits accompanied
by degradations via RNase(s) and endonucleases may
provide starved E. coli cells with nutrients derived from
their ribosomes under nutrient-limiting conditions [57-59].
In HHP-treated cells, the dissociated ribosomal particles may
also be degraded and recycled as a nutrient source.
Cells of E. coli [39] and L. monocytogenes [41] were
suspended in PBS and treated with HHPs of 400 MPa and
500 MPa at 25°C for 10 min, respectively. Injured cells at
approximately 2 l­og10 CFU/ml were detected in each sys-
tem immediately after each of HHP treatment. In addition,
a similar trend was observed in viable cell counts during
storage of each HHP-treated bacterial suspension. Viable
cell counts increased temporarily and then decreased for E.
coli in HHP-treated samples stored at 5, 10, 15, and 25°C
(Fig. 3) and for L. monocytogenes at 0, 5, 10, and 15°C.
Furthermore, viable cell counts for both bacteria drastically
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Food Engineering Reviews
increased during storage at 15°C and higher. Meanwhile,
heat-inactivated cells were suspended in PBS and then inoc-
ulated with healthy cells for each bacterial strain, although
the characteristics of HHP-inactivated cells as a nutrient
source may differ from those of heat-inactivated cells. Cell
proliferation during storage was only observed at 15°C and
higher for both bacterial strains, indicating possible can-
nibalisms. Therefore, the temporary increase in viable cell
counts below 15°C may not reflect cannibalism but recovery
based on ribosome-derived finite nutrients. Their depletion
might have led to injured cell inactivation.
Metabolome Analysis
Metabolome analysis of HHP-injured E. coli [60] revealed that
the amounts of 56 metabolites decreased among the 133 that
were analyzed. Furthermore, central sugar metabolic path-
ways and nucleic acid metabolic pathways were affected by
HHP treatment. For instance, accumulation of pentoses such
as ribose 5-phosphate and ribulose 5-phosphate was observed.
In addition, a bottleneck between fructose 1,6-diphosphate
and 3-phosphoglycerate in the glycolysis system induced
upstream accumulation and downstream depletion of metabo-
lites (Fig. 8). Intercellular activities of phospho-fructokinase,
aldolase, and pyruvate kinase, particularly aldolase, decreased
after HHP treatment. However, a large amount of cellular pro-
teins was released to the extracellular environment by HHP
treatment, and thus, HHP might minimally affect each enzyme
activity per molecule. Accumulation of pentoses should fur-
ther be clarified from a viewpoint of recovery from HHP-
induced injury on ribosome-derived nutrients.
Colony‑Forming Behavior
HHP-injured bacterial cells can be evaluated by differential
plating method using selective and nonselective media. In
addition, colony-forming behavior, such as colony emer-
gence time, and colony size distribution should also be
important. As shown in Fig. 9, colonies of HHP-injured
E. coli cells emerged later than those of control cells and
obvious increase in the heterogeneity of colony size was
observed [29]. This suggests the need for future quantita-
tive studies on the colony-forming behavior.
HHP‑Induced Injury of Other
Microorganisms
HHP-induced injury of molds, yeasts, parasites, and viruses
has not been sufficiently studied. Saccharomyces cerevisiae
cells were treated with HHP (2,250 atm ≈ 228 MPa, 25°C,
30 min) in citrate-buffered solution and spread on potato dex-
trose agar (PDA) and glucose-supplemented PDA (PDAG)
Food Engineering Reviews

Fig. 8  Metabolome analysis
of the central sugar metabolic
pathways in HHP-treated
Escherichia coli cells [60]. The
relative abundances of metabo-
lites are drawn in boxes; left
bar (blue), control; middle bar
(red), 400 MPa treatment; right
bar (green), 600 MPa treatment;
ND, not detected

to evaluate their injury as difference in viable colony counts
[61]. Inactivation at pH 3.0 was more than 2 log cycles higher
than at pH 5.0. The maximal injury observed was a reduc-
tion of 2 log cycles from viable colony counts on PDAG as
compared with those on PDA. It should be noted that there is
no established method to distinguish the healthy and injured
states of the above-mentioned microorganisms. In addition,
since viruses have both biotic and abiotic characteristics, it will
also be difficult to distinguish their healthy and injured states.

13

Fig. 9  Colony emergence of (a) control
HHP-treated Escherichia coli
cells (400 MPa, 25°C, 10 min)
on LB agar plate; a untreated
control; b HHP treatment [29]
24 h
48 h
3d
Conclusions
In this review, some aspects of HHP-induced injury of
prokaryotic bacteria have been discussed focusing on E. coli
and L. monocytogenes: factors affecting HHP inactivation,
HHP-induced injury, recovery in nutrient-poor/-rich media,
detection, and characteristics of injured cells in terms of
membrane damage, reactive oxygen species, HHP resist-
ance, ribosomes, and metabolomic activity. It is necessary
for HHP food processors to understand HHP-induced injury
of bacteria and their recovery more in detail to ensure that
the process condition selected for their products will achieve
the desired food safety and quality level. Therefore, fur-
ther studies are essential to understand the characteristics
of HHP-injured bacteria. In addition, eukaryotes such as
yeasts and molds should be investigated as well as viruses
to clarify their injury and recovery states, if they do exist.
Difference in injury and recovery from other stresses such
as heat and chemicals should be paid attention when seek-
ing to understand injury and subsequent recovery of HHP-
treated bacterial cells.
Compliance of Ethical Standards  Food Technol 43:99–107
Conflict of Interest  The authors declare that they have no conflict of
interest.
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Food Engineering Reviews
(b) 400 MPa
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