International Journal of Food Microbiology 197 (2015) 4552

Contents lists available at ScienceDirect

International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Carvacrol suppresses high pressure high temperature inactivation of

Bacillus cereus spores
Hue Luu-Thi a, Jorinde Corthouts a, Ioannis Passaris a, Tara Grauwet b, Abram Aertsen a,
Marc Hendrickx b, Chris W. Michiels a,
a
Laboratory of Food Microbiology, Leuven Food Science and Nutrition Research Center (LFoRCe), Department of Microbial and Molecular Systems (M2S), KU Leuven, Kasteelpark Arenberg 22,
B-3001 Heverlee, Belgium
b
Laboratory of Food Technology, Leuven Food Science and Nutrition Research Center (LFoRCe), Department of Microbial and Molecular Systems (M2S), KU Leuven, Kasteelpark Arenberg 22,
B-3001 Heverlee, Belgium

a r t i c l e

i n f o

Article history:
Received 7 July 2014
Received in revised form 20 October 2014
Accepted 14 December 2014
Available online 17 December 2014
Keywords:
Bacillus cereus spores
High pressure high temperature treatment
Dipicolinic acid
Spore refractility
Germination and inactivation
Carvacrol

a b s t r a c t
The inactivation of bacterial spores generally proceeds faster and at lower temperatures when heat treatments
are conducted under high pressure, and high pressure high temperature (HPHT) processing is, therefore, receiving an increased interest from food processors. However, the mechanisms of spore inactivation by HPHT treatment are poorly understood, particularly at moderately elevated temperature. In the current work, we studied
inactivation of the spores of Bacillus cereus F4430/73 by HPHT treatment for 5 min at 600 MPa in the temperature
range of 50100 C, using temperature increments of 5 C. Additionally, we investigated the effect of the natural
antimicrobial carvacrol on spore germination and inactivation under these conditions. Spore inactivation by
HPHT was less than about 1 log unit at 50 to 70 C, but gradually increased at higher temperatures up to about
5 log units at 100 C. DPA release and loss of spore refractility in the spore population were higher at moderate
(65 C) than at high ( 70 C) treatment temperatures, and we propose that moderate conditions induced
the normal physiological pathway of spore germination resulting in fully hydrated spores, while at higher temperatures this pathway was suppressed and replaced by another mechanism of pressure-induced dipicolinic acid
(DPA) release that results only in partial spore rehydration, probably because spore cortex hydrolysis is inhibited.
Carvacrol strongly suppressed DPA release and spore rehydration during HPHT treatment at 65 C and also
partly inhibited DPA release at 65 C. Concomitantly, HPHT spore inactivation was reduced by carvacrol at
6590 C but unaffected at 95100 C.
2014 Elsevier B.V. All rights reserved.

1. Introduction
High pressure treatment at elevated temperature (HPHT) has gained
an increasing interest as an alternative for thermal appertization (HT) of
low acid foods. In the HPHT process, products are rst preheated to a
moderate initial temperature (typically 7090 C), and subsequently
pressurized for a few minutes at 500800 MPa. Because pressure buildup is rapid, the products heat up by compression heating and reach process temperatures of 100120 C. Similarly, product temperature
rapidly drops to approximately the initial temperature during pressure
release. Because adiabatic heating and cooling are volumetric, solid
products can be heated more rapidly by HPHT than by conventional
heating, and, when considering the thermal component only, HPHT processes will have a lower total thermal impact on the product than HT
processes with the same process value (F0). As a result, HPHT processing
is generally advantageous for the retention of avor, color, texture, and

Corresponding author. Tel.: +32 16 321578; fax: +32 16 321960.

E-mail address: Chris.michiels@biw.kuleuven.be (C.W. Michiels).

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.12.016
0168-1605/ 2014 Elsevier B.V. All rights reserved.

nutritional value. Besides a thermal component, HPHT processes also

have a pressure component that potentially contributes to bacterial
inactivation or has an inuence on the rate of thermal inactivation.
While most studies report increased bacterial spore inactivation by
HPHT compared to HT treatment with the same thermal impact
(Olivier et al., 2011; Patazca et al., 2006; Zhu et al., 2008), some studies
report an opposite effect (Margosch et al., 2006), and it is currently
unclear which factors are at the origin of a synergistic or antagonistic
interaction of HP and HT, respectively.
An issue of interest is the mechanism of spore inactivation. It has been
well documented that high pressure (HP) treatment at ambient or slightly elevated temperature induces spore germination in Bacillus subtilis
and some other Bacillus species (Paidhungat et al., 2002; Setlow, 2003;
Wuytack et al., 2000). At moderate pressures between 50 MPa and
300400 MPa, this germination requires the spore's germinant receptors
although it occurs even in the absence of nutrients (Black et al., 2005).
Germination induced by moderate pressure proceeds through the
same steps as the physiological germinant-induced germination
process. Activation of the germinant receptors is followed by release
of dipicolinic acid (DPA), which activates cortex lytic enzymes (CLEs).

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H. Luu-Thi et al. / International Journal of Food Microbiology 197 (2015) 4552

Cortex hydrolysis leads to core rehydration, which in turn allows the degradation of small acid soluble proteins (SASPs), ATP generation and, nally, resumption of DNA, RNA and protein synthesis when the spore core is
fully hydrated. During germination, spores gradually lose their extreme
resistance and they can subsequently be inactivated by UV radiation, hydrogen peroxide, heat or pressure (Paidhungat et al., 2002; Reineke et al.,
2013c; Wuytack et al., 1998).
HP treatment at 500 MPa and moderate temperature also triggers
spore germination but, at least in B. subtilis, this germination process
differs in several features from that induced at moderate pressures.
One important difference is that spores lacking nutrient receptors also
germinate at very high pressures. The earliest detectable event under
these conditions is the release of DPA, and the current view is that
pressures N500 MPa induce the direct opening of DPA channels in
the spore's inner membrane by a physicochemical mechanism as opposed to the physiological process that takes place at low pressures
(Paidhungat et al., 2002; Reineke et al., 2011, 2012; Setlow, 2013).
Cortex hydrolysis and spore core rehydration also take place, but
spore germination is not complete or is delayed, since no degradation
of SASPs and ATP production are observed immediately after treatment,
possibly because of inactivation of Gpr, the germination protease
that normally degrades the SASPs (Black et al., 2005; Reineke et al.,
2013c; Wuytack et al., 1998). Moderately increased temperatures
(up to 60 C) generally stimulate HP-induced spore germination, in particular the physiological germination induced by moderate pressures
(Wuytack et al., 1998). On the other hand, higher temperatures, like
those prevailing in HPHT processes (typically 90 C) probably suppress physiological spore germination because they may irreversibly
denature proteins of the germination system like Gpr and the cortexlytic enzymes. Nevertheless, even at very high temperature and pressure, HPHT treatment triggers Ca-DPA release and a partial rehydration
of the spores, and this is believed to be the reason for the higher spore
inactivation efciency of HPHT treatment compared to HT treatment
(Margosch et al., 2004; Reineke et al., 2013a, 2013b, 2013c).
The combination of HP treatment with natural antimicrobial compounds has been explored as a strategy to enhance microbial inactivation, and several studies have reported synergistic effects. While most
of these studies have been conducted with vegetative bacterial cells,
synergistic effects have also been observed with spores. Nisin enhanced
HP inactivation of spores from Alicyclobacillus acidoterrestris, B. subtilis,
Bacillus cereus, Bacillus amyloliquefaciens and Clostridium sporogenes,
while for some bacteria, no enhancement of spore inactivation was
seen (Black et al., 2008; Hofstetter et al., 2013a; Sokolowska et al.,
2012). The effect of reutericyclin was also species dependent, with an
enhanced inactivation in Clostridium beijerinckii, but reduced inactivation in C. sporogenes (Hofstetter et al., 2013a). Olive powder also slightly
reduced spore inactivation by HP treatment in B. cereus (Marco et al.,
2011). A group of natural antimicrobials that have received growing
attention is composed of essential oils from herbs and spices, because
of their ability to control spoilage bacteria, inhibit foodborne pathogens
and extend shelf-life (Gayan et al., 2012). Essential oils have been
reported to increase the inactivation of bacterial spores by heat. For
instance, the time needed for a 6 log reduction of Bacillus coagulans
spores at 100 C was reduced from 3.25 min to 1.67 min in the presence
of 400 g/g oregano essential oil in nutrient broth (Haberbeck et al.,
2012). However, to our knowledge, no information is available on the
combination of essential oils with HPHT treatment to enhance bacterial
spore inactivation.
In the present study, we investigate the effect of carvacrol on the
HPHT inactivation of B. cereus spores. Besides quantifying spore inactivation and germination by plating methods, we also report the release
of DPA and the loss of spore refractility. Together, these data provide
novel insights in the mechanisms of B. cereus spore inactivation and germination at different treatment conditions (600 MPa, 50100 C with
holding time of 5 min), and in the way by which carvacrol interferes
with these mechanisms.

2. Material and methods

2.1. Bacterial strain and preparation of spores
B. cereus F4430/73, a strain that was isolated from a diarrheal outbreak and belongs to phylogenetic group IV of B. cereus sensu lato,
was kindly provided by M. H. Guinebretire (INRA, Avignon, France).
To induce sporulation, a loopful of cells from a 80 C glycerol stock
was streaked on a BHI plate and incubated at 30 C for 24 h. A single colony was then grown for 48 h at 30 C with shaking (200 rpm) in Brain
Heart Infusion broth (BHI, Oxoid, Basingstoke, United Kingdom). A hundred microliters of this culture was diluted into 1 ml of sterile distilled
water. From that, 50 l was surface-plated on nutrient agar CM0003
(Oxoid) using sterile glass beads and incubated at 30 C. Samples were
examined microscopically daily until more than 95% of the cells
consisted of phase bright spores, which was after about 45 days of incubation. The plates were then ooded with 3 ml sterile cold deionized
water and the spores were dislodged and suspended with a sterile bent
glass rod. Pooled suspensions from several plates were washed and resuspended in one fth of the original volume in cold sterile deionized
water. To separate spores from cell debris and vegetative cells, 10 ml
of this suspension was gently deposited on top of 50 ml 40% sucrose
and centrifuged at 4000 g for 10 min at 4 C (procedure adapted
from Sorg and Sonenshein (2010)). The pellet was resuspended in sterile cold deionized water, washed four additional times, and stored at
20 C. These spore suspensions contained no more than 1% vegetative
cells and were used within a week after harvesting for thermal and
HPHT treatments. No changes in microscopical spore appearance and
in the fraction of heat-sensitive spores occurred during storage.
2.2. Thermal treatment
Before use, spores were collected by centrifugation at 4000 g for
10 min and resuspended in 0.1 M 2-(N-morpholino) ethanesulfonic
acid (MES) buffer (pH 6.1) at approximately 108 cfu ml1. MES buffer
was chosen for both HT and HPHT experiments because its pH varies
only slightly with temperature and pressure (pKa / C = 0.011;
V = 3.9 cm3 mol1) (Bruins et al., 2007). Therefore, the pH of the
spore suspensions is predicted to be in the range of 5.35.9 under the
conditions of our HPHT and HT experiments. Moreover, MES is nontoxic, does not permeate membranes and does not form complexes with
metal ions like Ca2+ from the spores.
Where applicable, 5 mM carvacrol (Sigma-Aldrich, Diegem,
Belgium) was added to the cell suspensions using a 1 M carvacrol
stock solution in 97% ethanol. This concentration is about three times
lower than the minimal inhibitory concentration for B. cereus
ATCC11778 (Rosato et al., 2007). Eighty microliters of the spore suspensions was transferred into a glass capillary, which was then heat-sealed,
immersed in an oil bath at 50100 C (with interval of 5 C) for 5 min
and put in ice-water slurry immediately after treatment. The treated
spore suspensions were serially diluted in sterile potassium phosphate
buffer (pH 7.0), plated on Tryptone Soya Agar (TSA, Oxoid) and incubated at 30 C for 24 h to enumerate survivors. Prolonged incubation time
did not yield higher spore viability. All thermal experiments were
conducted in triplicate using the same spore suspension.
2.3. HPHT equipment and procedure
HPHT treatment was conducted using a custom-made apparatus
consisting of six parallel vertical vessels equipped with an external
heating coil (HPIU-10.000, maximum temperature 120 C, maximum
pressure 800 MPa, Resato, The Netherlands), and using propylene glycol
as pressure-transmitting medium. One of the six vessels was used for a
dummy sample with a thermocouple, prepared in the same way as the
real samples, to allow inline temperature registration inside the sample

H. Luu-Thi et al. / International Journal of Food Microbiology 197 (2015) 4552

holder during processing. Pressure was also continuously recorded for

all individual vessels during HPHT treatment.
Three hundred microliter aliquots of the spore suspensions (with
or without carvacrol 5 mM) were sealed in a sterile polyethylene
pouch. All high pressure treatments were done in triplicate from
the same spore suspension. The samples were placed in cylindrical
polytetrauoroethylene (Teon) sample holders with an inner diameter of 12 mm, length of 85 mm and wall thickness of 4 mm (Vink,
Belgium). The sample holders were further lled with water while
avoiding inclusion of air bubbles, closed with moveable caps and vacuum sealed in double plastic pouches to prevent contamination of the
equipment in case of leakage.
The following procedure was used to ensure that isothermal and isobaric conditions were rapidly reached and stably maintained (Grauwet
et al., 2011; Vervoort et al., 2011). After being pre-equilibrated at 4 C,
the lled sample holders were loaded into the HP vessels that were
preheated at the target process temperature. When the samples had
warmed up to a predetermined temperature Ti (period t1, Fig. 1), pressure was built up immediately to 150 MPa, and then further to 600 MPa
at a rate of 10 MPa/s. Based on preliminary experiments, the temperature Ti for each treatment was selected in the way that the heat generated by compression raised the temperature inside the sample holders
to the desired value (period t2, Fig. 1). After reaching 600 MPa, an isolation time of 90 s was used to allow further temperature equilibration in
order to reach more accurately the target temperature (period t3, Fig. 1).
From that point onwards, individual vessels were isolated from the high
pressure circuit and held for 5 min of holding time (period t4, Fig. 1.),
after which pressure was released instantaneously, resulting in an
immediate temperature drop. As can be seen for the treatment at
600 MPa, 80 C in Fig. 1, this procedure ensured stable isothermal and
isobaric conditions.
After decompression, the samples were immediately removed from
the vessels and cooled in ice-water slurry for further analysis. A portion
of this spore suspension was taken immediately for examination by

47

phase contrast microscopy. A second and a third portion were used to

enumerate the survivors and the ungerminated spores among the survivors by performing a plate count, directly or after an additional heat
treatment at 70 C for 10 min, respectively. The enumeration of spores
was done as described above for thermal treatments. Finally, the last
portion was centrifuged (10,600 g for 4 min) to remove the spore pellet and the supernatant was diluted 1:10 in TrisHCl buffer (pH 7.5) and
stored at 20 C for DPA analysis.
2.4. Analysis of loss of spore refractility by phase contrast microscopy
Immediately after HPHT treatment, spore samples were immobilized
on thin pads of 2% agarose (Eurogentec, Belgium) on a microscopy slide
and mounted with a cover glass. Phase contrast microscopy (Eclipse Ti
inverted microscope, Nikon, Champigny-sur-Marne, France) was used
to acquire images of ve different elds from each sample which
contained a sufcient number of well isolated spores. Images were visualized using NIS-Elements software (Nikon). To quantify the loss of spore
refractility, pixel intensity was measured in a square of 3 3 pixels
(or 0.22 0.22 m) in the center of 80 individual randomly picked spores
for each sample, using the open source software ImageJ. The center of the
spore was chosen because the light path through the spore core is longer
at the center than at the edges, and consequently the pixel intensity at the
center is most representative for the state of the spore core. From these
measurements, a distribution of spore refractility was constructed for
each treated sample (and for an untreated control sample) using ten
equally sized intervals of pixel intensities ranging between the lowest
and the highest values observed.
2.5. DPA measurement
DPA measurement was based on the formation of a uorescent complex with Tb3+ (Kort et al., 2005). The spore supernatants from 20 C
were diluted 1:25 in TrisHCl buffer (pH 7.5), and 100 l of this suspension was transferred into a 96-well black microtiter plate (Greiner
Bio-one, Belgium) and mixed with an equal volume of 20 mM TbCl3 in
TrisHCl buffer (pH 7.5). Fluorescence measurements were done in a
spectrouorometer (Synergy Mx-biotek, USA) with excitation and
emission wavelengths of 270 and 545 nm, respectively. DPA release
from the spores by HPHT or HT treatment was expressed relative to
the value obtained for the supernatant of an autoclaved spore suspension, which is assumed to have released its entire DPA depot. The presence of 5 mM carvacrol did not interfere with the quantication of DPA
by this method (data not shown).
2.6. Statistical analysis
Spore inactivation and DPA release data were analyzed statistically
using one-way analysis of variance (ANOVA), followed by Tukey
Kramer's post-hoc test for multiple comparison with a 5% level of significance (p b 0.05).
3. Result and discussion
3.1. Loss of viability and heat resistance after HPHT and HT treatment

Fig. 1. Recorded pressure and temperature history of sample subjected to (A) 600 MPa, 50 C,
5 min and (B) 600 MPa, 95 C, 5 min: t1: preheating time at atmospheric pressure from 4 C to Ti
(only part of t1 shown); t2: time of pressure build-up; t3: isolation time (90 s); t4: isobaric and
isothermal treatment time. Inactivation is studied from the start of period t4, indicated as
t = 0 min.

In a rst experiment, we determined spore inactivation and germination after HPHT treatments at a single pressure (600 MPa) but over
a wide range of different temperatures (50100 C). In these experiments, germination is operationally dened as the loss of resistance to
a 10 min/70 C heat treatment, and it should be emphasized that this
does not necessarily imply involvement of all the germination proteins
of the physiological germination process induced by germinants. When
we rst consider the results in the absence of carvacrol, a number of
observations can be made (Fig. 2). The treatments at 5060 C cause a
small reduction of 0.40.7 log units that could be due to the residual

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H. Luu-Thi et al. / International Journal of Food Microbiology 197 (2015) 4552

9
8
7

Log N

6
5
4
3
2
1
0
ab c d
Initial HP50C

ab c d
HP55C

ab c d
HP60C

ab c d
HP65C

ab c d
HP70C

ab c d
HP75C

ab c d
HP80C

ab c d
HP85C

ab c d
HP90C

ab c d

ab c d

HP95C HP100C

HP treatment at 600 MPa

Fig. 2. Surviving heat resistant and heat sensitive spores (log N) after HPHT treatment at 600 MPa, 50100 C for 5 min. (a) HPHT treatment; (b) HPHT treatment with additional heating at
70 C for 10 min; (c) HPHT treatment in the presence of 5 mM carvacrol; (d) HPHT treatment in the presence of 5 mM carvacrol with additional heating at 70 C for 10 min. Initial refers to
the spore count of the untreated spore suspension. Error bars represent standard deviation (N = 3).

Log N

presence of vegetative cells or spores that have not developed full resistance. Additional heat treatment before plating further reduced the
number of survivors by 0.81.1 log after HPHT at 55 and 60 C, indicating that these treatments induced spore germination. The HPHT treatments at 65 and 70 C also caused germination, but at the same time
they also caused more inactivation, probably because the germinated
spores were partially killed during the treatment. At still higher temperatures (75100 C), there was progressively more inactivation, up to
about 5 log at 95 and 100 C. As expected, heat sensitive survivors are
no longer detected after these treatments, and whether or not inactivation is preceded by germination under these conditions cannot be
deduced from the data. Treatment at 100 C did not enhance spore inactivation compared to at 95 C, probably due to the presence of a
superdormant spore fraction.
Carvacrol had a pronounced effect on spore germination and inactivation, except at the highest process temperatures (95 and 100 C).
First, it completely inhibited spore germination by HPHT at 5570 C.
As a result, spore inactivation under these process conditions remained
very low, and can probably be entirely ascribed to the residual vegetative cells. Interestingly, also at 7590 C, carvacrol suppressed spore
inactivation by 0.81.4 log units, and this suggests that inactivation
at these temperatures is also dependent on germination. At 95 and
100 C, nally, carvacrol had no effect on HPHT inactivation. This
could be because the mechanism of germination under these conditions
is different, or because the mechanism by which carvacrol suppresses
germination is not working.
For comparison, we also conducted thermal treatment at the
same temperatures (Fig. 3). In line with the high spore heat resistance
of B. cereus F4430/73, a considerable level of spore inactivation
was only observed at 100 C (4.2 log). It can also be seen that HPHT

treatment is more effective than HT treatment at the same temperature

(p b 0.05), as is the case for most bacterial spores, at least at temperatures below about 100 C (Olivier et al., 2011; Patazca et al., 2006; Zhu
et al., 2008). However, the effect of carvacrol was opposite to what
was observed for HPHT treatment, since carvacrol increased inactivation by HT, particularly at high temperatures. For example, at 95 and
100 C, inactivation was increased by 3.4 and 1.8 log units, respectively.
This is in line with ndings in other studies where essential oils or plant
extracts have been combined with heat treatment to inactivate spores
(Bevilacqua et al., 2009; Haberbeck et al., 2012). As a result, the difference in spore inactivation between HT and HPHT treatments at the
same temperature became much smaller in the presence of carvacrol
and, in fact, inactivation by HT is even signicantly higher than by
HPHT at 100 C in the presence of carvacrol (p b 0.05).
3.2. DPA release from HPHT and HT treated spores
With few exceptions, spores are more efciently inactivated by
HPHT than by HT treatment at the same temperature. This has been attributed to the release of the spore's large depot of Ca-DPA under pressure, which leads to a partial rehydration and consequently a partial loss
of heat resistance of the spores (Black et al., 2007; Paidhungat et al.,
2002; Reineke et al., 2011; Setlow, 2013). Therefore, to further investigate why carvacrol inhibited spore germination and inactivation by
HPHT treatment, we analyzed the amount of DPA in supernatants prepared from spore suspensions immediately after the HPHT and HT treatments (Figs. 4 and 5). The result for HPHT treatment is a relatively
complex trend of DPA release exhibiting two distinct maxima as a function of temperature. DPA release rst increased from 75 to almost 100%
(5065 C), then abruptly decreased to 56% (75 C), and steadily

9
8
7
6
5
4
3
2
1
0
a b

a b

a b

a b

a b

a b

a b

a b

a b

a b

Initial HT 50C HT 55C HT 60C HT 65C HT 70C HT 75C HT 80C HT 85C HT 90C HT 95C

a b
HT
100C

HT treatment at 0.1 MPa

Fig. 3. Surviving spores (log N) after HT treatment at 50100 C for 5 min. (a) Treatment in the absence of carvacrol; (b) treatment in the presence of 5 mM carvacrol. Initial refers to the
spore count of the untreated spore suspension. Error bars represent standard deviation (N = 3).

H. Luu-Thi et al. / International Journal of Food Microbiology 197 (2015) 4552

49

% of DPA release

100%
80%
60%
40%
20%
0%
a b

a b

a b

a b

a b

a b

a b

a b

a b

a b

a b

HP 50C HP 55C HP 60C HP 65C HP 70C HP 75C HP 80C HP 85C HP 90C HP 95C

HP
100C

HP treatment at 600 MPa

Fig. 4. Percentage of DPA release from spores after HPHT treatment at 600 MPa, 50100 C for 5 min, compared to control sample of autoclaved spores (121 C for 20 min): (a) in the
absence of carvacrol; (b) in the presence of 5 mM carvacrol. Error bars represent standard deviation (N = 3).

increased again up to 90% at 100 C. DPA release was signicantly higher

at 65 C than at 75 and 80 C and, vice-versa, DPA release at 75 C was
signicantly lower than at 60 and 65 C (p b 0.05). This trend is different
from that of B. subtilis spores, for which the rate of DPA release was reported to increase with increasing HPHT (600 MPa) process temperature from 40 to 80 C (Reineke et al., 2013b). The reason for this
difference is not clear, but the results suggest that two different mechanisms of DPA release are active in B. cereus as opposed to only one
mechanism in B. subtilis. The rst mechanism is dominant in the mild
temperature range (5070 C) and includes a heat sensitive step or
component because it is strongly suppressed at 75 C. The second
mechanism becomes gradually more important as the process temperature increases, and dominates at 75 C. These results may indicate
that the physiological germination process can be induced in B. cereus
F4430/73 by mild temperature HPHT conditions, and it would be worthwhile to investigate whether this induction proceeds through activation
of the germinant receptors by making use of receptorless mutants. In
B. subtilis, in contrast, only pressures 200 MPa induce germination
by activation of germinant receptors, while the pressure and temperature limits to activate these receptors in spores of other sporeformers
have not been investigated.
In the presence of carvacrol, DPA release was strongly suppressed in
the moderate temperature range (5070 C) (p b 0.05). This is compatible with the above stated hypothesis that physiological germination
takes place under these conditions, and with reports in the literature

that carvacrol inhibits physiological germination induced by germinants

(Juneja et al., 2006; Juneja and Friedman, 2007; Periago et al., 2006).
DPA release in the presence of carvacrol increased with increasing process temperature up to about 68% at 75 C, and then remained at about
the same level at higher temperatures, except for one deviating value
(46%) at 90 C. DPA release was signicantly reduced by carvacrol at
90100 C (p b 0.05), suggesting that carvacrol also at least partially
suppresses the germinant receptor independent mechanism of DPA release which was proposed to proceed by direct opening of membrane
DPA channels or creation of pores in the membrane (Black et al.,
2007). Comparison of Figs. 4 and 2 also shows that spore inactivation
cannot be predicted by DPA release alone. For example, although DPA
release at 75 and 80 C was about equal in the absence and in the presence of carvacrol, inactivation was about tenfold higher in the absence
of carvacrol.
Compared to HPHT treatment, HT treatment caused much less DPA
release, and this DPA release was not affected by carvacrol (Fig. 5).
Therefore, the mechanism of DPA release by HT treatment is probably
different from that by HPHT treatment in the investigated temperature
range. This could be related to the different effects of HPHT and HT
treatment on the spore membrane (Hofstetter et al., 2013a, 2013b).
Comparison of Figs. 5 and 3 also shows that DPA release is not correlated
to HT inactivation and thus DPA release is not contributing to the lethal
effect of HT treatment on spores. This conclusion conrms earlier ndings of Coleman et al. (2007, 2010), who showed that heat-treated

100%

% of DPA release

80%

60%

40%

20%

0%
a b

a b

a b

a b

a b

a b

a b

a b

a b

a b

HT 50C HT 55C HT 60C HT 65C HT 70C HT 75C HT 80C HT 85C HT 90C HT 95C

a b
HT
100C

HT treatment at 0.1 MPa

Fig. 5. Percentage of DPA release from spores after HT treatment at 50100 C for 5 min, compared to control sample of autoclaved spores (121 C for 20 min): (a) in the absence of
carvacrol; (b) in the presence of 5 mM carvacrol. Error bars represent standard deviation (N = 3).

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H. Luu-Thi et al. / International Journal of Food Microbiology 197 (2015) 4552

spore suspensions of B. subtilis, B. cereus and Bacillus megaterium

contained a large fraction of killed spores that had retained their DPA.
These authors concluded that wet heat inactivated spores through protein denaturation and that DPA release took place after the spores were
killed.
3.3. Spore refractility distribution after HPHT treatment
Spore suspensions subjected to HPHT treatment were also analyzed
by phase contrast microscopy to assess the loss of refractility, which results from the release of solutes and the uptake of water in the spore
core. As such, loss of refractility is also a marker of spore germination
(Paidhungat and Setlow, 2002). Microscopical analysis has the advantage that it allows to determine not only the average, but also the distribution of the loss of refractility in the spore population.
In the absence of carvacrol, treatment at 50 C already caused an important loss of refractility, with the average pixel intensity decreasing
from 11,126 to 6879 pixel units (Fig. 6). With increasing process temperature, the loss of refractility rst rapidly increased (5060 C), then
decreased (6085 C), and nally remained at approximately the
same level (85100 C). In the presence of carvacrol, the loss of
refractility after HPHT treatment was always lower than in its absence,

but the effect of carvacrol was larger at lower than at higher process
temperatures. At 50 and 55 C, the loss of spore refractility was
completely inhibited. At higher temperatures, the trend was similar to
that observed in the absence of carvacrol, with an increase (6065 C)
followed by a decrease (6585 C) and a plateau (85100 C).
A comparison of the patterns of DPA release and spore refractility
loss (Figs. 4 and 6) shows that, although the general trends show
some resemblance, both indices are not strictly correlated. Most notably, spores treated at 5560 C incurred a much larger refractility loss
than spores treated at 95100 C, despite a similar degree of about
80% DPA release. Loss of spore refractility during spore germination is
due to replacement of solutes by water. The rst event during spore germination that results in loss of refractility is the release of Ca-DPA and its
replacement by water, but this results only in a partial rehydration of
the spore core and thus a partial loss of refractility. Ca-DPA subsequently
activates the cortex hydrolases and the hydrolysis of the spore cortex
leads to further spore rehydration (Kong et al., 2010; Reineke et al.,
2013b, 2013c). Therefore, our data seem to indicate that all the HPHT
treatments trigger DPA release, but the released DPA triggers cortex
hydrolysis only at moderate temperatures. A possible explanation is
that the cortex lytic enzymes are inactivated during HPHT treatment
at N 65 C. The spore refractility data therefore further support the

Fraction of spores at different intensity

intervals

A)
1.0

13195-14566

0.9

11825-13195

0.8

10454-11825

0.7

9084-10454

0.6

7713-9084

0.5

6342-7713

0.4
4972-6342

0.3

3601-4972

0.2
0.1

2231-3601

0.0

860-2231

HP treatment at 600 MPa

Fraction of spores at different
intensity intervals

B)
1
13195-14566

0.9
0.8

11825-13195

0.7

10454-11825

0.6

9084-10454

0.5

7713-9084

0.4
6342-7713

0.3
0.2

4972-6342

0.1

3601-4972

2231-3601
860-2231

HP treatment at 600 MPa

Fig. 6. Microscopically determined pixel intensity distributions of spores in the absence (A) and presence (B) of 5 mM carvacrol after HPHT treatment at 600 MPa, 50100 C for 5 min and
nonpressurized control sample. Eighty spores were analyzed for each treatment and for the control sample. Minimum and maximum pixel intensities recorded were 860 and 14,566 for
the darkest and the brightest spore, respectively.

H. Luu-Thi et al. / International Journal of Food Microbiology 197 (2015) 4552

hypothesis formulated above that HPHT treatment at mild temperatures may induce a physiological germination process by activating
the germinant receptors.
The spore refractility data also conrm that spore germination by
HPHT at mild temperatures is strongly inhibited by carvacrol. The effect
of various organic compounds on spore germination by nutrient and
non-nutrient germinants has been investigated previously (Cortezzo
et al., 2004; van Melis et al., 2011a, 2012), and carvacrol and other essential oil components can inhibit nutrient-induced germination of
spores from some Bacillus and Clostridium spp. (Bevilacqua et al.,
2011; Juneja et al., 2006; Periago et al., 2006). Interference with HP induced germination has been studied for sorbic acid (van Melis et al.,
2011b). In this study, undissociated sorbic acid (3 mM, pH 5.5) blocked
the germination of B. cereus by various nutrient germinants as well as
by mild HP treatment (150 MPa), but not by severe HP treatment
(500 MPa) and Ca-DPA. Since only the former triggers germination via
the germinant receptors, this led the authors to propose that sorbic
acid may interfere with the signaling pathway initiated by activation
of a germinant receptor and leading to the opening of the Ca-DPA channels. Unfortunately, DPA release was not reported in this study. At rst
sight, our results with carvacrol are different, since we observed strong
inhibition of germination at 600 MPa (5065 C). However, as discussed
above, the patterns of DPA release and loss of refractility suggest that
germination under these conditions may proceed through activation
of the germinant receptors. Why this was not the case in the study of
van Melis et al. (2011b) is not clear, but it could be because of strainspecic behavior or differences in experimental conditions such as
treatment temperature or medium. In any case, if we accept the hypothesis of receptor-dependent germination by HPHT at 65 C, carvacrol
may have the same mode of action as sorbic acid, i.e., it may accumulate
in the spore inner membrane and interfere with the signaling between
nutrient receptors and the Ca-DPA channel (van Melis et al., 2011a,
2011b). Also the effects of nisin and reutericyclin on DPA release and
spore inactivation by HPHT treatment may be related to their effects
on the spore membrane (Hofstetter et al., 2013a, 2013b).
At higher temperatures, in the absence of carvacrol, there is less DPA
release and the mechanism probably does not involve germinant receptors and cortex hydrolases, as discussed above. Our observations are in
line with previous studies with B. subtilis spores (Reineke et al., 2013a,
2013c) reporting that HP treatment caused full spore hydration when
it was conducted at lower temperature (e.g., 37 C) but only partial hydration when it was conducted at higher temperature (e.g., N 60 C).
This was explained by assuming a delayed onset of stage II germination
as well as the inactivation of cortex lytic enzymes at high temperatures.
Despite the different mechanisms, carvacrol also partially inhibited DPA
release and spore rehydration under the high temperature conditions,
probably because DPA release is affected by the impact of carvacrol on
the inner membrane. This may directly increase membrane permeability for Ca-DPA or indirectly promote opening Ca-DPA channels.
4. Conclusion
Plate counts of total survivors and heat resistant survivors, measurement of DPA release and microscopical analysis of spore refractility loss
have provided insight in the effects of HPHT treatment at 600 MPa and
different temperatures on spores of B. cereus F4430/73. The results suggest that treatment at moderate temperatures ( 65 C) induces the
normal physiological pathway of spore germination by activating the
germinant receptors, and resulting in fully hydrated spores. At higher
temperatures, physiological spore germination is suppressed and another mechanism of pressure-induced DPA release that results only in
partial spore rehydration takes over, probably because spore cortex hydrolysis is inhibited. Carvacrol strongly suppressed the physiological
germination mechanism during HPHT treatment at 65 C, and also
partly inhibited DPA release at 65 C. From an application point of
view, the addition of carvacrol does not increase the efcacy of spore

51

inactivation by HPHT treatment and, at 6590 C, even decreases the

efcacy. This is in contrast to the stimulation of spore inactivation by
carvacrol during thermal inactivation.
Acknowledgments
This work was nancially supported by fellowships from the KU Leuven Research Fund (DBOF/10/043) to Luu-Thi H., from the Research
Foundation Flanders (FWO) to Tara Grauwet, and from the Flemish
Agency for Innovation by Science and Technology (IWT-Vlaanderen)
to Ioannis Passaris. Research grants were obtained from the KU Leuven
Research Fund (METH/07/03, METH/14/03, STRT1/10/36 and IDO/10/
012), and FWO (G.0C77.14). We thank M. H. Guinebretire (INRA,
Avignon, France) for kindly providing the B. cereus strain F4430/73.
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